WO2004023973A2 - Molecules utilisees a des fins diagnostiques et therapeutiques - Google Patents

Molecules utilisees a des fins diagnostiques et therapeutiques Download PDF

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WO2004023973A2
WO2004023973A2 PCT/US2003/028227 US0328227W WO2004023973A2 WO 2004023973 A2 WO2004023973 A2 WO 2004023973A2 US 0328227 W US0328227 W US 0328227W WO 2004023973 A2 WO2004023973 A2 WO 2004023973A2
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polynucleotide
cell
proteins
cells
protein
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PCT/US2003/028227
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WO2004023973A3 (fr
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Jeanette P. Schmidt
Rachel J. Wright
Christopher M. Bruns
Mirjana M. Marjanovic
Fan Shen
Toinette A. Harthshorne
Martin T. Suchorolski
Christina M. Altus
Steven J. Pitts
Linda V. Elder
Elizabeth M. MOONEY
Angelo M. Delegeane
Iqbal S. Panesar
Steven C. Banville
Thirupathi P. REDDY
Kristian A. STEVENS
John L. BLANCHARD
Scott R. Panzer
Xinhao Wang
Alan P. Au
Jr. Edward H. Gerstin
Careyna H. Peralta
Scott B. ANDERSON
Pierre RIOUX
Edward J. SHEN
Mingham C. WU
Laura L. Stuve
Robert E. Lagace
Peter A. Spiro
Elizabeth A. Stewart
James WINGROVE
Ursula A. VITT
Edward S. KIRTON
Yuming Xu
Mary KWONG
Jennifer L. Policky
Bonnie L. Hurwitz
Yan Ma
Jennifer L. Jackson
Darryl Gietzen
Srikanth PATURY
Xiaobing SHI
Charlyn J. SUAREZ
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Incyte Corporation
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Publication of WO2004023973A3 publication Critical patent/WO2004023973A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to human molecules and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases associated with, as well as effects of exogenous compounds on, the expression of human molecules.
  • the human genome comprises thousands of genes, many encoding gene products that function in the maintenance and growth of the various cells and tissues in the body. Aberrant expression or mutations in these genes and their products is the cause of, or is associated with, a variety of human diseases such as cancer and other cell proliferative disorders, autoin_mune/inflammatory disorders, infections, developmental disorders, endocrine disorders, 5 metabolic disorders, neurological disorders, gastrointestinal disorders, transport disorders, and connective tissue disorders.
  • the identification of these genes and their products is the basis of an ever-expanding effort to find markers for early detection of diseases, and targets for their prevention and treatment. Therefore, these genes and their products are useful as diagnostics and therapeutics.
  • genes may encode, for example, enzyme molecules, molecules associated with growth and o development, biochemical pathway molecules, extracellular information transmission molecules, receptor molecules, intracellular signaling molecules, membrane transport molecules, protein modification and maintenance molecules, nucleic acid synthesis and modification molecules, adhesion molecules, antigen recognition molecules, secreted and extracellular matrix molecules, cytoskeletal molecules, ribosomal molecules, electron transfer associated molecules, transcription 5 factor molecules, chromatin molecules, cell membrane molecules, and organelle associated molecules.
  • enzyme molecules molecules associated with growth and o development, biochemical pathway molecules, extracellular information transmission molecules, receptor molecules, intracellular signaling molecules, membrane transport molecules, protein modification and maintenance molecules, nucleic acid synthesis and modification molecules, adhesion molecules, antigen recognition molecules, secreted and extracellular matrix molecules, cytoskeletal molecules, ribosomal molecules, electron transfer associated molecules, transcription 5 factor molecules, chromatin molecules, cell membrane molecules, and organelle associated molecules.
  • cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body.
  • a wide variety of molecules, either aberrantly expressed or mutated, can be the cause of, or involved with, various cancers because tissue growth involves complex and ordered o patterns of cell proliferation, cell differentiation, and apoptosis.
  • Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation.
  • Molecules which directly or indirectly modulate cell cycle progression fall into 5 several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. Aberrant expression or mutations in any of these gene products can result in cell proliferative disorders such as cancer.
  • Oncogenes are genes generally derived from normal genes that, through abnormal expression or mutation, can effect the transformation of a normal cell to a malignant one
  • Oncogenesis Oncoproteins, encoded by oncogenes, can affect cell proliferation in a variety of ways and include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins.
  • tumor-suppressor genes are involved in inhibiting cell proliferation. Mutations which cause reduced function or loss of function in tumor-suppressor genes result in aberrant cell proliferation and cancer. Although many different genes and their products have been found to be associated with cell proliferative disorders such as cancer, many more may exist that are yet to be discovered.
  • Enzyme Molecules Enzyme Molecules
  • oxidoreductases transferases, hydrolases, lyases, isomerases, and ligases.
  • These enzyme classes are each comprising numerous substrate-specific enzymes having precise and well regulated functions. These enzymes function by facilitating metabolic processes such as glycolysis, the tricarboxylic cycle, and fatty acid metabolism; synthesis or degradation of amino acids, steroids, phospholipids, alcohols, etc.; regulation of cell signaling, proliferation, infiamation, apoptosis, etc., and through catalyzing critical steps in DNA replication and repair, and the process of translation.
  • Oxidoreductases Oxidoreductases
  • oxidoreductase dehydrogenase or reductase activity
  • Potential cofactors include cytochromes, oxygen, disulfide, iron-sulfur proteins, flavin adenine dinucleotide (FAD), and the nicotinamide adenine dinucleotides NAD and NADP (Newsholme, E.A. and A.R. Leech (1983) Biochemistry for the Medical Sciences, John Wiley and Sons, Chichester, U.K., pp. 779-793).
  • Reductase activity catalyzes the transfer of electrons between substrate(s) and cofactor(s) with concurrent oxidation of the cofactor.
  • the reverse dehydrogenase reaction catalyzes the reduction of a cofactor and consequent oxidation of the substrate.
  • Oxidoreductase enzymes are a broad superfamily of proteins that catalyze numerous reactions in all cells of organisms ranging from bacteria to plants to humans. These reactions include metabolism of sugar, certain detoxification reactions in the liver, and the synthesis or degradation of fatty acids, amino acids, glucocorticoids, estrogens, androgens, and prostaglandins.
  • SCADs Short-chain alcohol dehydrogenases
  • SCADs are also involved in synthesis and degradation of fatty acids, steroids, and some prostaglandins, and are therefore implicated in a variety of disorders such as lipid storage disease, myopathy, SCAD deficiency, and certain genetic disorders.
  • retinol dehydrogenase is a SCAD-family
  • Retinoic acid a regulator of differentiation and apoptosis, has been shown to down-regulate genes involved in cell proliferation and inflammation (Chai, X. et al. (1995) J. Biol.
  • retinol dehydrogenase has been linked to hereditary eye diseases such as autosomal recessive childhood-onset severe retinal dystrophy (Simon, A. et al. (1996) 0 Genomics 36:424-430).
  • Propagation of nerve impulses, modulation of cell proliferation and differentiation, induction of the immune response, and tissue homeostasis involve neurotransmitter metabolism (Weiss, B. (1991) Neurotoxicology 12:379-386; Collins, S.M. et al. (1992) Ann. N.Y. Acad. Sci. 664:415-424; Brown, J.K. and H. Imam (1991) J. Inherit. Metab. Dis. 14:436-458). Many pathways of 5 neurotransmitter metabolism require oxidoreductase activity, coupled to reduction or oxidation of a cofactor, such as NADVNADH (Newsholme, E.A. and A.R. Leech (1983) Biochemistry for the Medical Sciences.
  • NADVNADH Newsholme, E.A. and A.R. Leech (1983) Biochemistry for the Medical Sciences.
  • neurotransmitter degradation pathways that utilize NAD + /NADH-dependent oxidoreductase activity include those of L-DOPA (precursor of dopamine, a neuronal excitatory compound), glycine (an inhibitory neurotransmitter in the brain and spinal cord), histamine (liberated from mast cells during 5 the inflammatory response), and taurine (an inhibitory neurotransmitter of the brain stem, spinal cord and retina) (Newsholme, supra, pp. 790, 792).
  • L-DOPA precursor of dopamine, a neuronal excitatory compound
  • glycine an inhibitory neurotransmitter in the brain and spinal cord
  • histamine liberated from mast cells during 5 the inflammatory response
  • taurine an inhibitory neurotransmitter of the brain stem, spinal cord and retina
  • Epigenetic or genetic defects in neurotransmitter metabolic pathways can result in a spectrum of disease states in different tissues including Parkinson disease and inherited myoclonus (McCance, K.L. and S.E.
  • o Tetrahydrofolate is a derivatized glutamate molecule that acts as a carrier, providing activated one-carbon units to a wide variety of biosynthetic reactions, including synthesis of purines, pyrimidines, and the amino acid methionine.
  • Tetrahydrofolate is generated by the activity of a holoenzyme complex called tetrahydrofolate synthase, which includes three enzyme activities: tetrahydrofolate dehydrogenase, tetrahydrofolate cyclohydrolase, and tetrahydrofolate synthetase. 5
  • tetrahydrofolate dehydrogenase plays an important role in generating building blocks for nucleic and amino acids, crucial to proliferating cells.
  • 3-Hydroxyacyl-CoA dehydrogenase (3HACD) is involved in fatty acid metabolism. It catalyzes the reduction of 3-hydroxyacyl-CoA to 3-oxoacyl-CoA, with concomitant oxidation of
  • NAD to NADH in the mitochondria and peroxisomes of eukaryotic cells.
  • 3HACD and enoyl-CoA hydratase form an enzyme complex called bifunctional enzyme, defects in which are associated with peroxisomal bifunctional enzyme deficiency. This interruption in fatty acid metabolism produces accumulation of very-long chain fatty acids, disrupting development of the brain, bone, and adrenal glands. Infants bom with this deficiency typically die within 6 months
  • a ⁇ amyloid- ⁇
  • APP amyloid precursor protein
  • 3HACD has been shown to bind the A ⁇ peptide, and is overexpressed in neurons affected in Alzheimer's disease.
  • an antibody against 3HACD can block the toxic effects of A ⁇ in a cell culture model of Alzheimer's disease (Yan, S. et al. (1997) Nature 389:689-695; OMIM, #602057).
  • Steroids such as estrogen, testosterone, corticosterone, and others, are generated from a common precursor, cholesterol, and are interconverted into one another.
  • a wide variety of enzymes act upon cholesterol, including a number of dehydrogenases.
  • Steroid dehydrogenases such as the hydroxysteroid dehydrogenases, are involved in hypertension, fertility, and cancer (Duax, W.L. and D. Ghosh (1997) Steroids 62:95-100).
  • One such dehydrogenase is 3-oxo-5- ⁇ -steroid dehydrogenase (OASD), a microsomal membrane protein highly expressed in prostate and other androgen- responsive tissues.
  • OASD 3-oxo-5- ⁇ -steroid dehydrogenase
  • OASD catalyzes the conversion of testosterone into dihydrotestosterone, which is the most potent androgen.
  • Dihydrotestosterone is essential for the formation of the male phenotype during embryogenesis, as well as for proper androgen-mediated growth of tissues such as the prostate and male genitalia.
  • a defect in OASD that prevents the conversion of testosterone into dihydrotestosterone leads to a rare form of male pseudohermaphroditis, characterized by defective formation of the external genitalia (Andersson, S. et al. (1991) Nature 354:159-161; Labrie, F. et al. (1992) Endocrinology 131:1571-1573; OMM #264600).
  • 17 ⁇ -hydroxy steroid dehydrogenase plays an important role in the regulation of the male reproductive hormone, dihydrotestosterone (DHTT).
  • 17 ⁇ HSD6 acts to reduce levels of DHTT by oxidizing a precursor of DHTT, 3 ⁇ -diol, to androsterone which is readily glucuronidated and removed from tissues.
  • 17 ⁇ HSD6 is active with both androgen and estrogen substrates when expressed in embryonic kidney 293 cells.
  • At least five other isozymes of 17 ⁇ HSD have been identified that catalyze oxidation and/or reduction reactions in various tissues with preferences for different steroid substrates (Biswas, M.G. and D.W. Russell (1997) J. Biol. Chem. 272:15959-15966).
  • 17 ⁇ HSDl preferentially reduces estradiol and is abundant in the ovary and placenta.
  • 17 ⁇ HSD2 catalyzes oxidation of androgens and is present in the endometrium and placenta.
  • 17 ⁇ HSD3 is exclusively a reductive enzyme in the testis (Geissler, W.M. et al. (1994) Nat. Genet.
  • Oxidoreductases are components of the fatty acid metabolism pathways in mitochondria and peroxisomes.
  • the main beta-oxidation pathway degrades both saturated and unsaturated fatty acids, while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids.
  • the auxiliary beta-oxidation enzyme 2,4-dienoyl-CoA reductase catalyzes the removal of 0 even-numbered double bonds from unsaturated fatty acids prior to their entry into the main beta- oxidation pathway.
  • the enzyme may also remove odd-numbered double bonds from unsaturated fatty acids (Koivuranta, K.T. et al. (1994) Biochem. J. 304:787-792; Smeland, T.E. et al.
  • 2,4-dienoyl-CoA reductase is located in both mitochondria and peroxisomes. Inherited deficiencies in mitochondrial and peroxisomal beta-oxidation enzymes are 5 associated with severe diseases, some of which manifest themselves soon after birth and lead to death within a few years. Defects in beta-oxidation are associated with Reye's syndrome, Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum's disease, acyl-CoA oxidase deficiency, and bifunctional protein deficiency (Suzuki, Y. et al. (1994) Am. J. Hum.
  • Peroxisomal beta-oxidation is impaired in cancerous tissue. Although neoplastic human breast epithelial cells have the same number of peroxisomes as do normal cells, fatty acyl-CoA oxidase activity is lower than in control tissue (el Bouhtoury, F. et al. (1992) J. Pathol. 166:27-35).
  • Human colon carcinomas have fewer peroxisomes than normal colon tissue and have lower fatty-acyl-CoA oxidase and bifunctional enzyme (including enoyl-CoA hydratase) activities 5 than normal tissue (Cable, S. et al. (1992) Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 62:221- 226).
  • Another important oxidoreductase is isocitrate dehydrogenase, which catalyzes the conversion of isocitrate to a-ketoglutarate, a substrate of the citric acid cycle.
  • Isocitrate dehydrogenase can be either NAD or NADP dependent, and is found in the cytosol, mitochondria, and peroxisomes. Activity of isocitrate dehydrogenase is regulated developmentally, and by hormones, o neurotransmitters, and growth factors.
  • HPR Hydroxypyruvate reductase
  • a peroxisomal 2-hydroxyacid dehydrogenase in the glycolate pathway catalyzes the conversion of hydroxypyruvate to glycerate with the oxidation of both NADH and NADPH.
  • the reverse dehydrogenase reaction reduces NAD + and NADP + .
  • HPR recycles nucleotides and bases back into pathways leading to the synthesis of ATP and GTP. ATP 5 and GTP are used to produce DNA and RNA and to control various aspects of signal transduction and energy metabolism.
  • Inhibitors of purine nucleotide biosynthesis have long been employed as antiproliferative agents to treat cancer and viral diseases. HPR also regulates biochemical synthesis of serine and cellular serine levels available for protein synthesis.
  • the mitochondrial electron transport (or respiratory) chain is a series of oxidoreductase-type enzyme complexes in the mitochondrial membrane that is responsible for the transport of electrons
  • NADHubiquinone oxidoreductase complex I
  • succinate.-ubiquinone oxidoreductase complex II
  • cytochrome c r b oxidoreductase complex III
  • cytochrome c oxidase 0 complex IV
  • ATP synthase complex V
  • 3-hydroxyisobutyrate dehydrogenase important in valine catabolism, catalyzes the NAD-dependent oxidation of 3-hydroxyisobutyrate to methylmalonate semialdehyde within mitochondria. Elevated levels of 3-hydroxyisobutyrate have been reported in a number of disease states, including ketoacidosis, methylmalonic acidemia, and other disorders associated with deficiencies in methylmalonate semialdehyde dehydrogenase (Rougraff, P.M. et al. (1989) J. Biol. 5 Chem. 264:5899-5903).
  • IVD isovaleryl-CoA-dehydrogenase
  • IVD is involved in leucine metabolism and catalyzes the oxidation of isovaleryl-CoA to 3-methylcrotonyl-CoA.
  • Human IVD is a tetrameric flavoprotein that is encoded in the nucleus and synthesized in the cytosol as a 45 kDa precursor with a mitochondrial 0 import signal sequence.
  • a genetic deficiency caused by a mutation in the gene encoding IVD, results in the condition known as isovaleric acidemia. This mutation results in inefficient mitochondrial import and processing of the IVD precursor (Vockley, J. et al.
  • Transferases 5 Transferases are enzymes that catalyze the transfer of molecular groups. The reaction may involve an oxidation, reduction, or cleavage of covalent bonds, and is often specific to a substrate or to particular sites on a type of substrate. Transferases participate in reactions essential to such functions as synthesis and degradation of cell components, regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Transferases are involved in key steps in disease processes involving these functions. Transferases are frequently 5 classified according to the type of group transferred.
  • methyl transferases transfer one- carbon methyl groups
  • amino transferases transfer nitrogenous amino groups
  • similarly denominated enzymes transfer aldehyde or ketone, acyl, glycosyl, alkyl or aryl, isoprenyl, saccharyl, phosphorous-containing, sulfur-containing, or selenium-containing groups, as well as small enzymatic groups such as Coenzyme A.
  • Acyl transferases include peroxisomal carnitine octanoyl transferase, which is involved in the fatty acid beta-oxidation pathway, and mitochondrial carnitine palmitoyl transferases, involved in fatty acid metabolism and transport. Choline O-acetyl transferase catalyzes the biosynthesis of the neurotransmitter acetylcholine.
  • Amino transferases play key roles in protein synthesis and degradation, and they contribute to 5 other processes as well.
  • the amino transferase 5-aminolevulinic acid synthase catalyzes the addition of succinyl-CoA to glycine, the first step in heme biosynthesis.
  • Other amino transferases participate in pathways important for neurological function and metabolism.
  • glutamine-phenylpyruvate amino transferase also known as glutamine transaminase K (GTK)
  • GTK glutamine transaminase K
  • GTK catalyzes the reversible o conversion of L-glutamine and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine.
  • Other amino acid substrates for GTK include L-methionine, L-histidine, and L-tyrosine.
  • GTK also catalyzes the conversion of kynurenine to kynurenic acid, a tryptophan metabolite that is an antagonist of the N-methyl-D-aspartate (NMD A) receptor in the brain and may exert a neuromodulatory function. Alteration of the kynurenine metabolic pathway may be associated with 5 several neurological disorders.
  • GTK also plays a role in the metabolism of halogenated xenobiotics conjugated to glutathione, leading to nephrotoxicity in rats and neurotoxicity in humans.
  • GTK is expressed in kidney, liver, and brain.
  • Both human and rat GTKs contain a putative pyridoxal phosphate binding site (ExPASy ENZYME: EC 2.6.1.64; Perry, S.J. et al. (1993) Mol. Pharmacol. 43:660-665; Perry, S. et al. (1995) FEBS Lett. 360:277-280; and Alberati-Giani, D. et al. (1995) J. o Neurochem. 64: 1448-1455).
  • a second amino transferase associated with this pathway is kynurenine/ ⁇ -aminoadipate amino transferase (AadAT).
  • AadAT catalyzes the reversible conversion of ⁇ -aminoadipate and ⁇ -ketoglutarate to ⁇ -ketoadipate and L-glutamate during lysine metabolism.
  • AadAT also catalyzes the transamination of kynurenine to kynurenic acid.
  • a cytosolic AadAT is expressed in rat kidney, liver, and brain (Nakatani, Y. et al. (1970) Biochim. Biophys. Acta 198:219- 5 228; Buchli, R. et al. (1995) J. Biol. Chem. 270:29330-29335).
  • Glycosyl transferases include the mammalian UDP-glucouronosyl transferases, a family of membrane-bound microsomal enzymes catalyzing the transfer of glucouronic acid to lipophilic substrates in reactions that play important roles in detoxification and excretion of drugs, carcinogens, and other foreign substances.
  • Another mammalian glycosyl transferase mammalian UDP-galactose- ceramide galactosyl transferase, catalyzes the transfer of galactose to ceramide in the synthesis of
  • UDP-glycosyl transferases share a conserved signature domain of about 50 amino acid residues (PROSITE: PDOC00359, expasy.hcuge.ch/sprot/prosite.html).
  • Methyl transferases are involved in a variety of pharmacologically important processes.
  • Nicotinamide N-methyl transferase catalyzes the N-methylation of nicotinamides and other pyridines, 0 an important step in the cellular handling of drugs and other foreign compounds.
  • Phenylethanolamine N-methyl transferase catalyzes the conversion of noradrenalin to adrenalin. 6- O-methylguanine-DNA methyl transferase reverses DNA methylation, an important step in carcinogenesis.
  • Uroporphyrin-III C-methyl transferase which catalyzes the transfer of two methyl groups from S-adenosyl-L-methionine to uroporphyrinogen III, is the first specific enzyme in the 5 biosynthesis of cobalamin, a dietary enzyme whose uptake is deficient in pernicious anemia.
  • Protein- arginine methyl transferases catalyze the posttranslational methylation of arginine residues in proteins, resulting in the mono- and dimethylation of arginine on the guanidino group.
  • Substrates include histones, myelin basic protein, and heterogeneous nuclear ribonucleoproteins involved in mRNA processing, splicing, and transport.
  • Protein-arginine methyl transferase interacts with o proteins upregulated by mitogens, with proteins involved in chronic lymphocytic leukemia, and with interferon, suggesting an important role for methylation in cytokine receptor signaling (Lin, W.-J. et al. (1996) J. Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H.S. et al. (1998) Genomics 48:330-340).
  • Phosphotransferases catalyze the transfer of high-energy phosphate groups and are important 5 in energy-requiring and -releasing reactions.
  • the metabolic enzyme creatine kinase catalyzes the reversible phosphate transfer between creatine/creatine phosphate and ATP/ADP.
  • Glycocyamine kinase catalyzes phosphate transfer from ATP to guanidoacetate, and arginine kinase catalyzes phosphate transfer from ATP to arginine.
  • a cysteine-containing active site is conserved in this family (PROSITE: PDOC00103).
  • Prenyl transferases are heterodimers, consisting of an alpha and a beta subunit, that catalyze the transfer of an isoprenyl group.
  • An example of a prenyl transferase is the mammalian protein farnesyl transferase.
  • the alpha subunit of farnesyl transferase consists of 5 repeats of 34 amino acids each, with each repeat containing an invariant tryptophan (PROSITE: PDOC00703).
  • Saccharyl transferases are glycating enzymes involved in a variety of metabolic processes.
  • 5 Oligosacchryl transferase-48, for example, is a receptor for advanced glycation endproducts.
  • Coenzyme A (CoA) transferase catalyzes the transfer of CoA between two carboxylic acids.
  • Succinyl CoA:3-oxoacid CoA transferase for example, transfers CoA from succinyl-CoA to a
  • Acetoacetate is essential to the metabolism of ketone bodies, which accumulate in tissues affected by metabolic disorders such as diabetes (PROSITE: PDOC00980).
  • Hydrolysis is the breaking of a covalent bond in a substrate by introduction of a molecule of water.
  • the reaction involves a nucleophilic attack by the water molecule's oxygen atom on a target 0 bond in the substrate.
  • the water molecule is split across the target bond, breaking the bond and generating two product molecules.
  • Hydrolases participate in reactions essential to such functions as synthesis and degradation of cell components, and for regulation of cell functions including cell signaling, cell proliferation, inflamation, apoptosis, secretion and excretion. Hydrolases are involved in key steps in disease processes involving these functions.
  • Hydrolytic enzymes may 5 be grouped by substrate specificity into classes including phosphatases, peptidases, lysophospholipases, phosphodiesterases, glycosidases, and glyoxalases.
  • LPLs o Lysophospholipases
  • LPLs o Lysophospholipases
  • Small LPL isoforms approximately 15-30 kD, function as hydrolases; larger isoforms function both as hydrolases and transacylases.
  • a particular substrate for LPLs, lysophosphatidylcholine, causes lysis of cell membranes. LPL activity is regulated by signaling molecules important in numerous pathways, including the inflammatory 5 response.
  • Peptidases also called proteases, cleave peptide bonds that form the backbone of peptide or protein chains. Proteolytic processing is essential to cell growth, differentiation, remodeling, and homeostasis as well as inflammation and immune response. Since typical protein half-lives range from hours to a few days, peptidases are continually cleaving precursor proteins to their active form, o removing signal sequences from targeted proteins, and degrading aged or defective proteins.
  • Peptidases function in bacterial, parasitic, and viral invasion and replication within a host.
  • peptidases include trypsin and chymotrypsin (components of the complement cascade and the blood-clotting cascade) lysosomal cathepsins, calpains, pepsin, renin, and chymosin (Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New 5 York NY, pp. 1-5).
  • Proteolytic enzymes or proteases either activate or deactivate proteins by hydrolyzing peptide bonds.
  • Proteases are found in the cytosol, in membrane-bound compartments, and in the extracellular space. The major families are the zinc, serine, cysteine, thiol, and carboxyl proteases.
  • the phosphodiesterases catalyze the hydrolysis of one of the two ester bonds in a phosphodiester compound. Phosphodiesterases are therefore crucial to a variety of cellular processes.
  • Phosphodiesterases include DNA and RNA endo- and exo-nucleases, which are essential to cell growth and replication as well as protein synthesis.
  • Another phosphodiesterase is acid sphingomyelinase, which hydrolyzes the membrane phospholipid sphingomyelin to ceramide and phosphorylcholine.
  • Phosphorylcholine is used in the synthesis of phosphatidylcholine, which is involved in numerous intracellular signaling pathways.
  • Ceramide is an essential precursor for the 0 generation of gangliosides, membrane lipids found in high concentration in neural tissue. Defective acid sphingomyelinase phosphodiesterase leads to a build-up of sphingomyelin molecules in lysosomes, resulting in Niemann-Pick disease.
  • Glycosidases catalyze the cleavage of hemiacetyl bonds of glycosides, which are compounds that contain one or more sugar.
  • Mammalian lactase-phlorizin hydrolase for example, is an intestinal 5 enzyme that splits lactose.
  • Mammalian beta-galactosidase removes the terminal galactose from gangliosides, glycoproteins, and glycosaminoglycans, and deficiency of this enzyme is associated with a gangliosidosis known as Morquio disease type B.
  • Vertebrate lysosomal alpha-glucosidase which hydrolyzes glycogen, maltose, and isomaltose
  • vertebrate intestinal sucrase-isomaltase which hydrolyzes sucrose, maltose, and isomaltose
  • the glyoxylase system is involved in gluconeogenesis, the production of glucose from storage compounds in the body. It consists of glyoxylase I, which catalyzes the formation of S-D- lactoylglutathione from methyglyoxal, a side product of triose-phosphate energy metabolism, and glyoxylase II, which hydrolyzes S-D-lactoylglutathione to D-lactic acid and reduced glutathione. 5 Glyoxylases are involved in hyperglycemia, non-insulin-dependent diabetes mellitus, the detoxification of bacterial toxins, and in the control of cell proliferation and microtubule assembly. Lyases
  • Lyases are a class of enzymes that catalyze the cleavage of C-C, C-O, C-N, C-S, C-(halide), P-O or other bonds without hydrolysis or oxidation to form two molecules, at least one of which o contains a double bond (Stryer, L. (1995) Biochemistry W.H. Freeman and Co. New York, NY p.620). Lyases are critical components of cellular biochemistry with roles in metabolic energy production including fatty acid metabolism, as well as other diverse enzymatic processes. Further classification of lyases reflects the type of bond cleaved as well as the nature of the cleaved group.
  • the group of C-C lyases include carboxyl-lyases (decarboxylases), aldehyde-lyases 5 (aldolases), oxo-acid-lyases and others.
  • the C-0 lyase group includes hydro-lyases, lyases acting on polysaccharides and other lyases.
  • the C-N lyase group includes ammonia-lyases, amidine-lyases, amine-lyases (deaminases) and other lyases.
  • lyases Proper regulation of lyases is critical to normal physiology. For example, mutation induced deficiencies in the uroporphyrinogen decarboxylase can lead to photosensitive cutaneous lesions in the genetically-linked disorder familial porphyria cutanea tarda (Mendez, M. et al. (1998) Am. J. Genet. 63:1363-1375). It has also been shown that adenosine deaminase (ADA) deficiency stems from genetic mutations in the ADA gene, resulting in the disorder severe combined immunodeficiency disease (SCID) (Hershfield, M.S. (1998) Semin. Hematol. 35:291-298).
  • SCID severe combined immunodeficiency disease
  • Isomerases are a class of enzymes that catalyze geometric or structural changes within a molecule to form a single product. This class includes racemases and epimerases, cis-trans- isomerases, intramolecular oxidoreductases, intramolecular transferases (mutases) and intramolecular lyases. Isomerases are critical components of cellular biochemistry with roles in metabolic energy production including glycolysis, as well as other diverse enzymatic processes (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co., New York NY, pp.483-507).
  • Racemases are a subset of isomerases that catalyze inversion of a molecules configuration around the asymmetric carbon atom in a substrate having a single center of asymmetry, thereby interconverting two racemers.
  • Epimerases are another subset of isomerases that catalyze inversion of configuration around an asymmetric carbon atom in a substrate with more than one center of symmetry, thereby interconverting two epimers. Racemases and epimerases can act on amino acids and derivatives, hydroxy acids and derivatives, as well as carbohydrates and derivatives.
  • the interconversion of UDP-galactose and UDP-glucose is catalyzed by UDP-galactose-4'-epimerase.
  • Oxidoreductases can be isomerases as well. Oxidoreductases catalyze the reversible transfer of electrons from a substrate that becomes oxidized to a substrate that becomes reduced. This class of enzymes includes dehydrogenases, hydroxylases, oxidases, oxygenases, peroxidases, and reductases.
  • oxidoreductase levels are physiologically important.
  • genetically-linked deficiencies in lipoamide dehydrogenase can result in lactic acidosis (Robinson, B.H. et al. (1977) Pediat. Res. 11:1198-1202).
  • Transferases transfer a chemical group from one compound (the donor) to another compound (the acceptor).
  • the types of groups transferred by these enzymes include acyl groups, amino groups, phosphate groups (phosphotransferases or phosphomutases), and others.
  • the transferase carnitine palmitoyltransferase is an important component of fatty acid metabolism. Genetically-linked deficiencies in this transferase can lead to myopathy (Scriver, CR. et al. (1995) The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York NY, pp.1501-1533).
  • Ligases catalyze the formation of a bond between two substrate molecules. The process involves the hydrolysis of a pyrophosphate bond in ATP or a similar energy donor. Ligases are classified based on the nature of the type of bond they form, which can include carbon-oxygen, carbon-sulfur, carbon-nitrogen, carbon-carbon and phosphoric ester bonds.
  • Ligases forming carbon-oxygen bonds include the aminoacyl-transfer RNA (tRNA) synthetases which are important RNA-associated enzymes with roles in translation. Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA. The aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA.
  • the 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, and each class is characterized by a distinctive topology of the catalytic domain. Class I enzymes contain a catalytic domain based on the nucleotide-binding Rossman fold.
  • Class II enzymes contain a central catalytic domain, which consists of a seven-stranded antiparallel ⁇ -sheet motif, as well as N- and C- terminal regulatory domains. Class II enzymes are separated into two groups based on the heterodimeric or homodimeric structure of the enzyme; the latter group is further subdivided by the structure of the N- and C-terminal regulatory domains (Hartlein, M. and S. Cusack (1995) J. Mol. Evol. 40:519-530). Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and poly ⁇ ryositis, and correlate strongly with complicating interstitial lung disease (JJLD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.
  • JJLD interstitial lung disease
  • Ligases forming carbon-sulfur bonds mediate a large number of cellular biosynthetic intermediary metabolism processes involve intermolecular transfer of carbon atom-containing substrates (carbon substrates). Examples of such reactions include the tricarboxylic acid cycle, synthesis of fatty acids and long-chain phospholipids, synthesis of alcohols and aldehydes, synthesis of intermediary metabolites, and reactions involved in the amino acid degradation pathways. Some of these reactions require input of energy, usually in the form of conversion of ATP to either ADP or AMP and pyrophosphate.
  • a carbon substrate is derived from a small molecule containing at least two carbon atoms.
  • the carbon substrate is often covalently bound to a larger molecule which acts as a carbon substrate carrier molecule within the cell.
  • the carrier molecule is coenzyme A.
  • Coenzyme A is structurally related to derivatives of the nucleotide ADP and consists of 4'-phosphopantetheine linked via a phosphodiester bond to the alpha phosphate group of adenosine 3',5'-bisphosphate. The terminal thiol group of 4'-phosphopantetheine acts as the site for carbon substrate bond formation.
  • the predominant carbon substrates which utilize CoA as a carrier molecule during biosynthesis and intermediary metabolism in the cell are acetyl, succinyl, and propionyl moieties, collectively referred to as acyl groups.
  • Other carbon substrates include enoyl lipid, which acts as a fatty acid oxidation intermediate, and carnitine, which acts as an acetyl-CoA flux regulator/ mitochondrial acyl group transfer protein.
  • Acyl-CoA and acetyl-CoA are synthesized in the cell by acyl-CoA synthetase and acetyl-CoA synthetase, respectively.
  • acyl-CoA synthetase activity i) acetyl-CoA synthetase, which activates acetate and several other low molecular weight carboxylic acids and is found in muscle mitochondria and the cytosol of other tissues; ii) medium-chain acyl-CoA synthetase, which activates fatty acids containing between four and eleven carbon atoms
  • acyl-CoA synthetase activity has been identified from many sources including bacteria, yeast, plants, mouse, and man. The activity of acyl-CoA synthetase may be modulated by phosphorylation of the enzyme by cAMP-dependent protein kinase.
  • Ligases forming carbon-nitrogen bonds include amide synthases such as glutamine synthetase (glutamate-ammonia ligase) that catalyzes the amination of glutamic acid to glutamine by ammonia using the energy of ATP hydrolysis.
  • glutamine synthetase glutmate-ammonia ligase
  • Glutamine is the primary source for the amino group in various amide transfer reactions involved in de novo pyrimidine nucleotide synthesis and in purine and pyrimidine ribonucleotide interconversions.
  • Overexpression of glutamine synthetase has been 0 observed in primary liver cancer (Christa, L. et al. (1994) Gastroent. 106:1312-1320).
  • Acid-amino-acid ligases are represented by the ubiquitin proteases which are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria.
  • UCS ubiquitin conjugation system
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular 5 processes such as gene transcription and cell cycle progression.
  • proteins targeted for degradation are conjugated to a ubiquitin (Ub), a small heat stable protein.
  • Ub is first activated by a ubiquitin-activating enzyme (El), and then transferred to one of several Ub- conjugating enzymes (E2).
  • E2 then links the Ub molecule through its C-terminal glycine to an internal lysine (acceptor lysine) of a target protein.
  • the ubiquitinated protein is then recognized and o degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease.
  • the UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994) Cell 79:13-21).
  • a murine proto-oncogene, Unp encodes a nuclear ubiquitin 5 protease whose overexpression leads to oncogenic transformation of NIH3T3 cells, and the human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D.A. (1995) Oncogene 10:2179-2183).
  • Cyclo-ligases and other carbon-nitrogen ligases comprise various enzymes and enzyme complexes that participate in the de novo pathways to purine and pyrimidine biosynthesis. Because these pathways are critical to the synthesis of nucleotides for replication of both RNA and DNA, 5 many of these enzymes have been the targets of clinical agents for the treatment of cell proliferative disorders such as cancer and infectious diseases.
  • Purine biosynthesis occurs de novo from the amino acids glycine and glutamine, and other small molecules.
  • Three of the key reactions in this process are catalyzed by a trifunctional enzyme composed of glycinamide-ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide 0 synthetase (AIRS), and glycinamide ribonucleotide rransformylase (GART).
  • GAS glycinamide-ribonucleotide synthetase
  • GART glycinamide ribonucleotide rransformylase
  • Adenylosuccinate synthetase catalyzes a later step in purine biosynthesis that converts inosinic acid 5 to adenylosuccinate, a key step on the path to ATP synthesis.
  • This enzyme is also similar to another carbon-nitrogen ligase, argininosuccinate synthetase, that catalyzes a similar reaction in the urea cycle (Powell, S.M. et al. (1992) FEBS Lett. 303:4-10).
  • de novo synthesis of the pyrimidine nucleotides uridylate and cytidylate also arises from a common precursor, in this instance the nucleotide o orotidylate derived from orotate and phosphoribosyl pyrophosphate (PPRP).
  • PPRP phosphoribosyl pyrophosphate
  • ATCase aspartate transcarbamylase
  • carbamyl phosphate synthetase II carbamyl phosphate synthetase II
  • DHOase dihydroorotase
  • Ligases forming carbon-carbon bonds include the carboxylases acetyl-CoA carboxylase and pyruvate carboxylase.
  • Acetyl-CoA carboxylase catalyzes the carboxylation of acetyl-CoA from CO 2 and H 2 0 using the energy of ATP hydrolysis.
  • Acetyl-CoA carboxylase is the rate-limiting step in the biogenesis of long-chain fatty acids.
  • Two isoforms of acetyl-CoA carboxylase, types I and types II, 5 are expressed in human in a tissue-specific manner (Ha, J. et al. (1994) Eur. J. Biochem. 219:297- 306).
  • Pyruvate carboxylase is a nuclear-encoded mitochondrial enzyme that catalyzes the conversion of pyruvate to oxaloacetate, a key intermediate in the citric acid cycle.
  • Ligases forming phosphoric ester bonds include the DNA ligases involved in both DNA replication and repair. DNA ligases seal phosphodiester bonds between two adjacent nucleotides in a
  • DNA chain using the energy from ATP hydrolysis to first activate the free 5 '-phosphate of one
  • Cell division is the fundamental process by which all living things grow and reproduce. In unicellular organisms such as yeast and bacteria, each cell division doubles the number of organisms, 5 while in multicellular species many rounds of cell division are required to replace cells lost by wear or by programmed cell death, and for cell differentiation to produce a new tissue or organ. Details of the cell division cycle may vary, but the basic process consists of three principle events. The first event, interphase, involves preparations for cell division, replication of the DNA, and production of essential proteins, i the second event, mitosis, the nuclear material is divided and separates to o opposite sides of the cell. The final event, cytokinesis, is division and fission of the cell cytoplasm.
  • the sequence and timing of cell cycle transitions is under the control of the cell cycle regulation system which controls the process by positive or negative regulatory circuits at various check points.
  • Regulated progression of the cell cycle depends on the integration of growth control pathways with the basic cell cycle machinery.
  • Cell cycle regulators have been identified by selecting 5 for human and yeast cDNAs that block or activate cell cycle arrest signals in the yeast mating pheromone pathway when they are overexpressed.
  • Known regulators include human CPR (cell cycle progression restoration) genes, such as CPR8 and CPR2, and yeast CDC (cell division control) genes, including CDC91, that block the arrest signals.
  • the CPR genes express a variety of proteins including cyclins, tumor suppressor binding proteins, chaperones, transcription factors, translation factors, and RNA-binding proteins (Edwards, M.C. et al.(1997) Genetics 147:1063-1076).
  • Cdks cyclin-dependent kinases
  • Cdks act by binding to and activating cyclin-dependent protein kinases which then phosphorylate and activate selected proteins involved in the mitotic process.
  • the Cdk-cyclin complex is both positively and negatively regulated by phosphorylation, and by targeted degradation involving molecules such as CDC4 and CDC53.
  • Cdks are further regulated by binding to inhibitors and other proteins such as Sucl that modify their specificity or accessibility to regulators (Patra, D. and W.G. Dunphy (1996) Genes Dev. 10:1503-1515; and Mathias, N. et al. (1996) Mol. Cell Biol. 16:6634-6643).
  • the male and female reproductive systems are complex and involve many aspects of growth and development.
  • the anatomy and physiology of the male and female reproductive systems are reviewed in (Guyton, A.C. (1991) Textbook of Medical Physiology, W.B. Saunders Co., Philadelphia PA, pp. 899-928).
  • the male reproductive system includes the process of spermatogenesis, in which the sperm are formed, and male reproductive functions are regulated by various hormones and their effects on accessory sexual organs, cellular metabolism, growth, and other bodily functions.
  • Spermatogenesis begins at puberty as a result of stimulation by gonadotropic hormones released from the anterior pituitary. Immature sperm (spermatogonia) undergo several mitotic cell divisions before undergoing meiosis and full maturation. The testes secrete several male sex hormones, the most abundant being testosterone, that is essential for growth and division of the immature sperm, and for the masculine characteristics of the male body. Three other male sex hormones, gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle- stimulating hormone (FSH) control sexual function.
  • GnRH gonadotropin-releasing hormone
  • LH luteinizing hormone
  • FSH follicle- stimulating hormone
  • the uterus, ovaries, fallopian tubes, vagina, and breasts comprise the female reproductive system.
  • the ovaries and uterus are the source of ova and the location of fetal development, respectively.
  • the fallopian tubes and vagina are accessory organs attached to the top and bottom of the uterus, respectively.
  • Both the uterus and ovaries have additional roles in the development and loss of reproductive capability during a female' s lifetime.
  • the primary role of the breasts is lactation. Multiple endocrine signals from the ovaries, uterus, pituitary, hypothalamus, adrenal glands, and other tissues coordinate reproduction and lactation. These signals vary during the monthly menstruation cycle and during the female's lifetime. Similarly, the sensitivity of reproductive organs to these endocrine signals varies during the female's lifetime.
  • FSH follicle-stimulating hormone
  • LH luteinizing hormone
  • hCG human chorionic gonadotropin
  • hCS human chorionic somatomammotropin
  • the female breast also matures during pregnancy. Large amounts of estrogen secreted by the placenta trigger growth and branching of the breast milk ductal system while lactation is initiated by the secretion of prolactin by the pituitary gland.
  • Parturition involves several hormonal changes that increase uterine contractility toward the o end of pregnancy, as follows.
  • the levels of estrogens increase more than those of progesterone.
  • Oxytocin is secreted by the neurohypophysis. Concomitantly, uterine sensitivity to oxytocin increases. The fetus itself secretes oxytocin, cortisol (from adrenal glands), and prostaglandins.
  • Menopause occurs when most of the ovarian follicles have degenerated.
  • the ovary then produces less estradiol, reducing the negative feedback on the pituitary and hypothalamus glands. 5
  • Mean levels of circulating FSH and LH increase, even as ovulatory cycles continue. Therefore, the ovary is less responsive to gonadotropins, and there is an increase in the time between menstrual cycles. Consequently, menstrual bleeding ceases and reproductive capability ends.
  • Tissue growth involves complex and ordered patterns of cell proliferation, cell o differentiation, and apoptosis.
  • Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals, such as growth factors and other mitogens, and intracellular cues, such as DNA damage or nutrient starvation. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, 5 including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. Growth factors were originally described as serum factors required to promote cell proliferation. Most growth factors are large, secreted polypeptides that act on cells in their local environment.
  • Growth factors bind to and activate specific cell surface receptors and initiate intracellular signal transduction cascades.
  • Many growth factor receptors are classified as receptor tyrosine kinases which undergo autophosphorylation upon ligand binding. Autophosphorylation enables the receptor to interact with signal transduction proteins characterized by the presence of SH2 or SH3 domains (Src homology regions 2 or 3). These proteins then modulate the activity state of small G-proteins, such as Ras, Rab, and Rho, along with GTPase activating proteins (GAPs), guanine nucleotide releasing proteins (GNRPs), and other guanine nucleotide exchange factors.
  • GAPs GTPase activating proteins
  • GNRPs guanine nucleotide releasing proteins
  • Small G proteins act as molecular switches that activate other downstream events, such as mitogen-activated protein kinase (MAP kinase) cascades. MAP kinases ultimately activate transcription of mitosis-
  • GPCR G-protein coupled receptor
  • TGF- ⁇ transforming growth factor beta
  • Some growth factors act on some cells to stimulate cell proliferation and on other cells to inhibit it. Growth factors may also stimulate a cell at one concentration and inhibit the same cell at another concentration. Most growth factors also have a multitude of other actions besides the regulation of cell growth and division: they can control the proliferation, survival, differentiation, migration, or function of cells depending on the circumstance.
  • the tumor necrosis factor/nerve growth factor (TNF/NGF) family can activate or inhibit cell death, as well as regulate proliferation and differentiation.
  • the cell response depends on the type of cell, its stage of differentiation and transformation status, which surface receptors are stimulated, and the types of stimuli acting on the cell (Smith, A. et al. (1994) Cell 76:959-962; and Nocentini, G. et al. (1997) Proc. Natl. Acad. Sci. USA 94:6216-6221).
  • Neighboring cells in a tissue compete for growth factors, and when provided with "unlimited” quantities in a perfused system will grow to even higher cell densities before reaching density- dependent inhibition of cell division.
  • Cells often demonstrate an anchorage dependence of cell division as well. This anchorage dependence may be associated with the formation of focal contacts linking the cytoskeleton with the extracellular matrix (ECM).
  • ECM components can be stimulated by growth factors. For example, TGF- ⁇ stimulates fibroblasts to produce a variety of ECM proteins, including fibronectin, collagen, and tenascin (Pearson, CA. et al. (1988) EMBO J.
  • ECM molecules such as laminin or fibronectin
  • Tenascin-C and -R expressed in developing and lesioned neural tissue, provide stimulatory/anti-adhesive or inhibitory properties, respectively, for axonal growth (Faissner,
  • Cancer represents a type of cell proliferative disorder that affects nearly every tissue in the body.
  • a wide variety of molecules, either aberrantly expressed or mutated, can be the cause of, or involved with, various cancers because tissue growth involves complex and ordered patterns of cell proliferation, cell differentiation, and apoptosis.
  • Cell proliferation must be regulated to maintain both the number of cells and their spatial organization. This regulation depends upon the appropriate expression of proteins which control cell cycle progression in response to extracellular signals such as growth factors and other mitogens, and intracellular cues such as DNA damage or nutrient starvation. Aberrant expression or mutations in any of these gene products can result in cell proliferative disorders such as cancer.
  • Oncogenes are genes generally derived from normal genes that, through abnormal expression or mutation, can effect the transformation of a normal cell to a malignant one (oncogenesis).
  • Oncoproteins encoded by oncogenes, can affect cell proliferation in a variety of ways and include growth factors, growth factor receptors, intracellular signal transducers, nuclear transcription factors, and cell-cycle control proteins. Molecules which directly or indirectly modulate cell cycle progression fall into several categories, including growth factors and their receptors, second messenger and signal transduction proteins, oncogene products, tumor-suppressor proteins, and mitosis-promoting factors. In contrast, tumor-suppressor genes are involved in inhibiting cell proliferation. Mutations which cause reduced function or loss of function in tumor-suppressor genes result in aberrant cell proliferation and cancer. Although many different genes and their products have been found to be associated with cell proliferative disorders such as cancer, many more may exist that are yet to be discovered.
  • oncoproteins are mutant isoforms of the normal protein, and other oncoproteins are abnormally expressed with respect to location or amount of expression.
  • Many oncogenes have been identified and characterized. These include sis, erbA, erbB, her-2, mutated G s , src, abl, ras, crk, jun, fos, myc, and mutated tumor-suppressor genes such as RB, p53, mdm2, Cipl, pl6, and cyclin D. Transformation of normal genes to oncogenes may also occur by chromosomal translocation.
  • the Philadelphia chromosome characteristic of chronic myeloid leukemia and a subset of acute lymphoblastic leukemias, results from a reciprocal translocation between chromosomes 9 and 22 that moves a truncated portion of the proto-oncogene c-abl to the breakpoint cluster region (bcr) on chromosome 22.
  • Viral oncogenes are integrated into the human genome after infection of human cells by certain viruses. Examples of viral oncogenes include v-src, v-abl, and v-fps.
  • Tumor-suppressor genes are involved in regulating cell proliferation. Mutations which cause reduced or loss of function in tumor-suppressor genes result in uncontrolled cell proliferation.
  • the retinoblastoma gene product RB
  • RB retinoblastoma gene product
  • Phosphorylation of RB causes it to dissociate from the genes, releasing the suppression, and allowing cell division to proceed.
  • Apoptosis is the genetically controlled process by which unneeded or defective cells undergo programmed cell death. Selective elimination of cells is as important for morphogenesis and tissue remodeling as is cell proliferation and differentiation.
  • Apoptosis may result in hyperplasia and other disorders associated with increased cell proliferation. Apoptosis is also a critical component of the immune response. Immune cells such as cytotoxic T-cells and natural killer cells prevent the spread of disease by inducing apoptosis in tumor cells and virus-infected cells. In addition, immune cells that fail to distinguish self molecules from foreign molecules must be eliminated by apoptosis to avoid an autoimmune response.
  • apoptosis includes cell shrinkage, nuclear and cytoplasmic condensation, and alterations in plasma membrane topology. Biochemically, apoptotic cells are characterized by increased intracellular calcium concentration, fragmentation of chromosomal DNA, and expression of novel cell surface components.
  • Apoptosis generally proceeds in response to a signal which is transduced intraceUularly and results in altered patterns of gene expression and protein activity.
  • Signaling molecules such as hormones and cytokines are known both to stimulate and to inhibit apoptosis through interactions with cell surface receptors. Transcription factors also play an important role in the onset of apoptosis.
  • a number of downstream effector molecules, particularly proteases such as the cysteine proteases called caspases have been implicated in the degradation of cellular components and the proteolytic activation of other apoptotic effectors. Aging and Senescence
  • Biochemical pathways are responsible for regulating metabolism, growth and development, protein secretion and trafficking, environmental responses, and ecological interactions including immune response and response to parasites.
  • DNA Deoxyribonucleic acid
  • DNA the genetic material
  • the bulk of human DNA is nuclear, in the form of linear chromosomes, while mitochondrial DNA is circular.
  • DNA replication begins at specific sites called origins of replication. Bidirectional synthesis occurs from the origin via two growing forks that move in opposite directions. Replication is semi-conservative, with each daughter duplex containing one old strand and its newly synthesized complementary partner. Proteins involved in DNA replication include DNA polymerases, DNA primase, telomerase, DNA helicase, topoisomerases, DNA ligases, replication factors, and DNA-binding proteins.
  • DNA Recombination and Repair include DNA polymerases, DNA primase, telomerase, DNA helicase, topoisomerases, DNA ligases, replication factors, and DNA-binding proteins.
  • Damage to DNA consists of any change that modifies the structure of the molecule. Changes to DNA can be divided into two general classes, single base changes and structural distortions. Any damage to DNA can produce a mutation, and the mutation may produce a disorder, such as cancer.
  • Repair systems can be divided into three general types, direct repair, excision repair, and retrieval systems. Proteins involved in DNA repair include DNA polymerase, excision repair proteins, excision and cross link repair proteins, recombination and repair proteins, RAD51 proteins, and BLN and WRN proteins that are homologs of RecQ helicase. When the repair systems are eliminated, cells become exceedingly sensitive to environmental mutagens, such as ultraviolet irradiation. Patients with disorders associated with a loss in DNA repair systems often exhibit a high sensitivity to environmental mutagens.
  • XP xeroderma pigmentosum
  • BS Bloom's syndrome
  • WS Werner's syndrome
  • Recombination is the process whereby new DNA sequences are generated by the movements of large pieces of DNA.
  • homologous recombination which occurs during meiosis and DNA repair, parent DNA duplexes align at regions of sequence similarity, and new DNA molecules form by the breakage and joining of homologous segments.
  • Proteins involved include RAD51 recombinase.
  • site-specific recombination two specific but not necessarily homologous DN sequences are exchanged, i the immune system this process generates a diverse collection of antibody and T cell receptor genes.
  • Proteins involved in site-specific recombination in the immune system include recombination activating genes 1 and 2 (RAG1 and RAG2).
  • mice 5 system site-specific recombination causes severe combined immunodeficiency disease in mice.
  • RNA Ribonucleic acid
  • ATP a linear single-stranded polymer of four nucleotides
  • CTP a linear single-stranded polymer of four nucleotides
  • RNA is transcribed as a copy of DNA, the genetic material of the organism.
  • DNA serves as the genetic material.
  • RNA copies of the 0 genetic material encode proteins or serve various structural, catalytic, or regulatory roles in organisms.
  • RNA is classified according to its cellular localization and function.
  • Messenger RNAs mRNAs
  • rRNAs Ribosomal RNAs
  • ribosomes which are cytoplasmic particles that translate mRNA into polypeptides.
  • Transfer RNAs are cytosolic adaptor molecules that function in mRNA translation by 5 recognizing both an mRNA codon and the amino acid that matches that codon.
  • Heterogeneous nuclear RNAs include mRNA precursors and other nuclear RNAs of various sizes.
  • Small nuclear RNAs are a part of the nuclear spliceosome complex that removes intervening, non-coding sequences (introns) and rejoins exons in pre-mRNAs.
  • RNA Transcription o The transcription process synthesizes an RNA copy of DNA. Proteins involved include multi-subunit RNA polymerases, transcription factors UA, UB, HD, HE, HF, HH, and HJ.
  • DNA-binding structural motifs which comprise either ⁇ -helices or ⁇ - sheets that bind to the major groove of DNA.
  • Four well-characterized structural motifs are helix- turn-helix, zinc finger, leucine zipper, and helix-loop-helix. 5 RNA Processing
  • RNAs RNAs
  • Pre- mRNA processing steps include capping at the 5' end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns.
  • the spliceosomal complex is composed of five small nuclear ribonucleoprotein particles (snRNPs) designated UI, U2, U4, U5, and U6.
  • snRNPs small nuclear ribonucleoprotein particles
  • U2 U4, U5, and U6 small nuclear ribonucleoprotein particles
  • Each snRNP contains a o single species of snRNA and about ten proteins.
  • the RNA components of some snRNPs recognize and base-pair with intron consensus sequences.
  • the protein components mediate spliceosome assembly and the splicing reaction.
  • snRNPs Heterogeneous nuclear ribonucleoproteins
  • Some examples of hnRNPs include the yeast proteins
  • Hrplp involved in cleavage and polyadenylation at the 3' end of the RNA
  • Cbp80p involved in capping the 5' end of the RNA
  • Npl3 ⁇ a homolog of mammalian hnRNP Al, involved in export of mRNA from the nucleus (Shen, E.C. et al. (1998) Genes Dev. 12:679-691).
  • HnRNPs have been shown to be important targets of the autoimmune response in rheumatic diseases (Biamonti, supra).
  • RNA recognition motif RRM
  • the RRM is about 80 amino acids in length and forms four ⁇ -strands and two ⁇ - helices arranged in an ⁇ / ⁇ sandwich.
  • the RRM contains a core RNP-1 octapeptide motif along with surrounding conserved sequences.
  • RNA helicases alter and regulate RNA conformation and secondary structure by using energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes.
  • the most well-characterized and ubiquitous family of RNA helicases is the DEAD-box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family.
  • DEAD-box helicases Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability. Some DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis. (Reviewed in Linder, P. et al. (1989) Nature 337: 121-122.)
  • DEAD-box 1 protein may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors.
  • DEAD-box helicases have been implicated either directly or indirectly in ultraviolet light-induced tumors, B cell lymphoma, and myeloid malignancies.
  • RNases Ribonucleases catalyze the hydrolysis of phosphodiester bonds in RNA chains, thus cleaving the RNA.
  • RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process.
  • RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and RNase H is an important enzyme in the retroviral replication cycle.
  • RNase H domains are often found as a domain associated with reverse transcriptases.
  • RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, CH. (1997) Nat. Biotechnol. 15:529-536). Regulation of RNase activity is being investigated as a means to control tumor angiogenesis, allergic reactions, viral infection and replication, and fungal infections.
  • the eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 80S ribosome.
  • the ribosome also contains more than fifty proteins.
  • the ribosomal prote ns ave a pre ix w ch denotes the subunit to which they belong, either L (large) or S (small).
  • Three important sites are identified on the ribosome.
  • the aminoacyl-tRNA site (A site) is where charged tRNAs (with the exception of the initiator-tRNA) bind on arrival at the ribosome.
  • the peptidyl-tRNA site (P site) is where new peptide bonds are formed, as well as where the initiator tRNA binds.
  • the exit site (E site) is where deacylated tRNAs bind prior to their release from the ribosome.
  • tRNA Charging Protein biosynthesis depends on each amino acid forming a linkage with the appropriate tRNA.
  • the aminoacyl-tRNA synthetases are responsible for the activation and correct attachment of an amino acid with its cognate tRNA.
  • the 20 aminoacyl-tRNA synthetase enzymes can be divided into two structural classes, Class I and Class 13. Autoantibodies against aminoacyl-tRNAs are generated by patients with dermatomyositis and polymyositis, and correlate strongly with complicating interstitial lung disease (ILD). These antibodies appear to be generated in response to viral infection, and coxsackie virus has been used to induce experimental viral myositis in animals.
  • Initiation of translation can be divided into three stages.
  • the first stage brings an initiator transfer RNA (Met-tRNA f ) together with the 40S ribosomal subunit to form the 43S preinitiation complex.
  • the second stage binds the 43S preinitiation complex to the mRNA, followed by migration of the complex to the correct AUG initiation codon.
  • the third stage brings the 60S ribosomal subunit to the 40S subunit to generate an 80S ribosome at the initiation codon.
  • Regulation of translation primarily involves the first and second stage in the initiation process (Pain, V.M. (1996) Eur. J. Biochem. 236:747-771).
  • eIF2 a guanine nucleotide binding protein, recruits the initiator tRNA to the 40S ribosomal subunit. Only when e_F2 is bound to GTP does it associate with the initiator tRNA.
  • eIF2B a guanine nucleotide exchange protein, is responsible for converting eIF2 from the GDP-bound inactive form to the GTP-bound active form.
  • eJJFlA and e_F3 bind and stabilize the 40S subunit by interacting with 18S ribosomal RNA and specific ribosomal structural proteins.
  • eIF3 is also involved in association of the 40S ribosomal subunit with mRNA.
  • the Met-tRNA f , elFIA, e_F3, and 40S ribosomal subunit together make up the 43S preinitiation complex (Pain, supra).
  • eIF4F is a complex consisting of three proteins: eIF4E, eIF4A, and eIF4G.
  • eIF4E recognizes and binds to the mRNA 5 -terminal m 7 GTP cap
  • e_F4A is a bidirectional RNA-dependent helicase
  • eIF4G is a scaffolding polypeptide.
  • eIF4G has three binding domains.
  • e_F4G acts as a bridge between the 40S ribosomal subunit and the mRNA (Hentze,
  • the ability of eIF4F to initiate binding of the 43S preinitiation complex is regulated by structural features of the mRNA.
  • the mRNA molecule has an untranslated region (UTR) between the 5' cap and the AUG start codon. In some mRNAs this region forms secondary structures that impede binding of the 43S preinitiation complex.
  • the helicase activity of eIF4A is thought to 0 function in removing this secondary structure to facilitate binding of the 43S preinitiation complex (Pain, supra).
  • Elongation is the process whereby additional amino acids are joined to the initiator methionine to form the complete polypeptide chain.
  • the elongation factors EFl ⁇ , EFl ⁇ ⁇ , and EF2 5 are involved in elongating the polypeptide chain following initiation.
  • EFl ⁇ is a GTP-binding protein. In EFl ⁇ 's GTP-bound form, it brings an aminoacyl-tRNA to the ribosome' s A site. The amino acid attached to the newly arrived aminoacyl-tRNA forms a peptide bond with the initiator methionine.
  • the GTP on EFl ⁇ is hydrolyzed to GDP, and EFl ⁇ -GDP dissociates from the ribosome.
  • EFl ⁇ ⁇ binds EFl ⁇ -GDP and induces the dissociation of GDP from EFl ⁇ , allowing EFl ⁇ to bind GTP and a o new cycle to begin.
  • EF-G another GTP-binding protein, catalyzes the translocation of tRNAs from the A site to the P site and finally to the E site of the ribosome. This allows the processivity of translation.
  • Translation Termination 5 The release factor eRF carries out termination of translation. eRF recognizes stop codons in the mRNA, leading to the release of the polypeptide chain from the ribosome.
  • Proteins may be modified after translation by the addition of phosphate, sugar, prenyl, fatty acid, and other chemical groups. These modifications are often required for proper protein activity. o Enzymes involved in post-translational modification include kinases, phosphatases, glycosyltransferases, and prenyltransf erases. The conformation of proteins may also be modified after translation by the introduction and rearrangement of disulfide bonds (rearrangement catalyzed by protein disulfide isomerase), the isomerization of proline sidechains by prolyl isomerase, and by interactions with molecular chaperone proteins. 5 Proteins may also be cleaved by proteases.
  • proteases include serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
  • Signal peptidase in the endoplasmic reticulum include serine proteases, cysteine proteases, aspartic proteases, and metalloproteases.
  • ER lumen cleaves the signal peptide from membrane or secretory proteins that are imported into the
  • Ubiquitin proteases are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria.
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression.
  • proteins targeted for degradation are conjugated to a ubiquitin, a small heat stable protein.
  • Proteins involved in the UCS include ubiquitin-activating enzyme, ubiquitin-conjugating enzymes, ubiquitin-ligases, and ubiquitin C-terminal hydrolases.
  • the ubiquitinated protein is then recognized and degraded by the proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease.
  • Lipids are water-insoluble, oily or greasy substances that are soluble in nonpolar solvents such as chloroform or ether.
  • Neutral fats triacylglycerols
  • Polar lipids such as phospholipids, sphingolipids, glycolipids, and cholesterol, are key structural components of cell membranes.
  • Lipid metabolism is involved in human diseases and disorders.
  • atherosclerosis fatty lesions form on the inside of the arterial wall. These lesions promote the loss of arterial flexibility and the formation of blood clots (Guyton, A.C Textbook of Medical Physiology (1991) W.B. Saunders Company, Philadelphia PA, pp.760-763).
  • Pn Tay-Sachs disease the GM 2 ganglioside (a sphingolipid) accumulates in lysosomes of the central nervous system due to a lack of the enzyme N-acetylhexosaminidase. Patients suffer nervous system degeneration leading to early death (Fauci, A.S. et al.
  • Niemann-Pick diseases are caused by defects in lipid metabolism.
  • Niemann-Pick diseases types A and B are caused by accumulation of sphingomyelin (a sphingolipid) and other lipids in the central nervous system due to a defect in the enzyme sphingomyelinase, leading to neurodegeneration and lung disease.
  • Niemann-Pick disease type C results from a defect in cholesterol transport, leading to the accumulation of sphingomyelin and cholesterol in lysosomes and a secondary reduction in sphingomyelinase activity.
  • Neurological symptoms such as grand mal seizures, ataxia, and loss of previously learned speech, manifest 1-2 years after birth.
  • a mutation in the NPC protein which contains a putative cholesterol-sensing domain, was found in a mouse model of Niemann-Pick disease type C (Fauci, supra, p. 2175; Loftus, S.K. et al. (1997) Science 277:232- 235).
  • Niemann-Pick disease type C Feuci, supra, p. 2175; Loftus, S.K. et al. (1997) Science 277:232- 235).
  • Lipid metabolism is reviewed in Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York NY; Lehninger, A.
  • Fatty Acid Synthesis Fatty acids are long-chain organic acids with a single carboxyl group and a long non-polar hydrocarbon tail. Long-chain fatty acids are essential components of glycolipids, phospholipids, and cholesterol, which are building blocks for biological membranes, and of triglycerides, which are biological fuel molecules. Long-chain fatty acids are also substrates for eicosanoid production, and
  • Fatty acid synthesis occurs in the cytoplasm.
  • acetyl-Coenzyme A (CoA) carboxylase (ACC) synthesizes malonyl-CoA from acetyl-CoA and bicarbonate.
  • the enzymes which catalyze the remaining reactions are covalently linked into a single polypeptide chain, referred to as 0 the multifunctional enzyme fatty acid synthase (FAS).
  • FAS catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA.
  • FAS contains acetyl transferase, malonyl transferase, ⁇ -ketoacetyl synthase, acyl carrier protein, ⁇ -ketoacyl reductase, dehydratase, enoyl reductase, and thioesterase activities.
  • the final product of the FAS reaction is the 16-carbon fatty acid palmitate. Further elongation, as well as unsaturation, of palmitate by accessory enzymes of the ER produces the variety 5 of long chain fatty acids required by the individual cell. These enzymes include a NADH-cytochrome b 5 reductase, cytochrome b 5 , and a desaturase.
  • Triacylglycerols also known as triglycerides and neutral fats, are major energy stores in animals. Triacylglycerols are esters of glycerol with three fatty acid chains. Glycerol-3-phosphate is o produced from dihydroxyacetone phosphate by the enzyme glycerol phosphate dehydrogenase or from glycerol by glycerol kinase. Fatty acid-CoA's are produced from fatty acids by fatty acyl-CoA synthetases. Glyercol-3-phosphate is acylated with two fatty acyl-CoA's by the enzyme glycerol phosphate acyltransferase to give phosphatidate.
  • Phosphatidate phosphatase converts phosphatidate to diacylglycerol, which is subsequently acylated to a triacylglyercol by the enzyme diglyceride 5 acyltransferase. Phosphatidate phosphatase and diglyceride acyltransferase form a triacylglyerol synthetase complex bound to the ER membrane.
  • a major class of phospholipids are the phosphoglycerides, which are composed of a glycerol backbone, two fatty acid chains, and a phosphorylated alcohol.
  • Phosphoglycerides are components of cell membranes. Principal phosphoglycerides are phosphatidyl choline, phosphatidyl ethanolamine, 0 phosphatidyl serine, phosphatidyl inositol, and diphosphatidyl glycerol. Many enzymes involved in phosphoglyceride synthesis are associated with membranes (Meyers, R.A. (1995) Molecular Biology and Biotechnology, VCH Publishers Inc., New York NY, pp. 494-501).
  • Phosphatidate is converted to CDP-diacylglycerol by the enzyme phosphatidate cytidylyltransferase (ExPASy ENZYME EC 2.7.7.41). Transfer of the diacylglycerol group from CDP-diacylglycerol to serine to yield 5 phosphatidyl serine, or to inositol to yield phosphatidyl inositol, is catalyzed by the enzymes CDP- diacylglycerol-serine O-phosphatidyltransferase and CDP-diacylglycerol-inositol 3- phosphatidyltransferase, respectively (ExPASy ENZYME EC 2.7.8.8; ExPASy ENZYME EC
  • the enzyme phosphatidyl serine decarboxylase catalyzes the conversion of phosphatidyl serine to phosphatidyl ethanolamine, using a pyruvate cofactor (Voelker, D.R. (1997) Biochim.
  • Phosphatidyl choline is formed using diet-derived choline by the
  • Cholesterol composed of four fused hydrocarbon rings with an alcohol at one end, moderates the fluidity of membranes in which it is incorporated, i addition, cholesterol is used in the synthesis 0 of steroid hormones such as cortisol, progesterone, estrogen, and testosterone. Bile salts derived from cholesterol facilitate the digestion of lipids. Cholesterol in the skin forms a barrier that prevents excess water evaporation from the body. Farnesyl and geranylgeranyl groups, which are derived from cholesterol biosynthesis intermediates, are post-translationally added to signal transduction proteins such as ras and protein-targeting proteins such as rab. These modifications are important for the 5 activities of these proteins (Guyton, supra; Stryer, supra, pp. 279-280, 691-702, 934).
  • Mammals obtain cholesterol derived from both de novo biosynthesis and the diet.
  • the liver is the major site of cholesterol biosynthesis in mammals.
  • Two acetyl-CoA molecules initially condense to form acetoacetyl-CoA, catalyzed by a thiolase.
  • Acetoacetyl-CoA condenses with a third acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA), catalyzed by HMG-CoA synthase.
  • HMG-CoA hydroxymethylglutaryl-CoA
  • Conversion of HMG-CoA to cholesterol is accomplished via a series of enzymatic steps known as the mevalonate pathway.
  • the rate-limiting step is the conversion of HMG-CoA to mevalonate by HMG- CoA reductase.
  • the drug lovastatin, a potent inhibitor of HMG-CoA reductase, is given to patients to reduce their serum cholesterol levels.
  • mevalonate pathway enzymes include mevalonate kinase, phosphomevalonate kinase, diphosphomevalonate decarboxylase, isopentenyldiphosphate 5 isomerase, dimethylallyl transferase, geranyl transferase, farnesyl-diphosphate farnesyltransferase, squalene monooxygenase, lanosterol synthase, lathosterol oxidase, and 7-dehydrocholesterol reductase.
  • Cholesterol is used in the synthesis of steroid hormones such as cortisol, progesterone, aldosterone, estrogen, and testosterone.
  • cholesterol is converted to pregnenolone by o cholesterol monooxygenases.
  • the other steroid hormones are synthesized from pregnenolone by a series of enzyme-catalyzed reactions including oxidations, isomerizations, hydroxylations, reductions, and demethylations. Examples of these enzymes include steroid ⁇ -isomerase, 3 ⁇ -hydroxy- ⁇ 5 -steroid dehydrogenase, steroid 21 -monooxygenase, steroid 19-hydroxylase, and 3 ⁇ -hydroxysteroid dehydrogenase.
  • Cholesterol is also the precursor to vitamin D. 5 Numerous compounds contain 5-carbon isoprene units derived from the mevalonate pathway intermediate isopentenyl pyrophosphate. Isoprenoid groups are found in vitamin K, ubiquinone, retinal, dolichol phosphate (a carrier of oligosaccharides needed for N- n ed glycosylation), and farnesyl and geranylgeranyl groups that modify proteins. Enzymes involved include farnesyl transferase, polyprenyl transferases, dolichyl phosphatase, and dolichyl kinase.
  • Sphingolipid Metabolism Sphingolipids are an important class of membrane lipids that contain sphingosine, a long chain amino alcohol. They are composed of one long-chain fatty acid, one polar head alcohol, and sphingosine or sphingosine derivative.
  • the three classes of sphingolipids are sphingomyelins, cerebrosides, and gangliosides. Sphingomyelins, which contain phosphocholine or phosphoethanolamine as their head group, are abundant in the myelin sheath surrounding nerve cells.
  • Galactocerebrosides which contain a glucose or galactose head group, are characteristic of the brain. Other cerebrosides are found in nonneural tissues. Gangliosides, whose head groups contain multiple sugar units, are abundant in the brain, but are also found in nonneural tissues.
  • Sphingolipids are built on a sphingosine backbone.
  • Sphingosine is acylated to ceramide by the enzyme sphingosine acetyltransferase.
  • Ceramide and phosphatidyl choline are converted to sphingomyelin by the enzyme ceramide choline phosphotransferase.
  • Cerebrosides are synthesized by the linkage of glucose or galactose to ceramide by a transferase. Sequential addition of sugar residues to ceramide by transferase enzymes yields gangliosides. Eicosanoid Metabolism
  • Eicosanoids including prostaglandins, prostacyclin, thromboxanes, and leukotrienes, are 20- carbon molecules derived from fatty acids. Eicosanoids are signaling molecules which have roles in pain, fever, and inflammation. The precursor of all eicosanoids is arachidonate, which is generated from phospholipids by phospholipase A 2 and from diacylglycerols by diacylglycerol lipase. Leukotrienes are produced from arachidonate by the action of lipoxygenases. Prostaglandin synthase, reductases, and isomerases are responsible for the synthesis of the prostaglandins.
  • Prostaglandins have roles in inflammation, blood flow, ion transport, synaptic transmission, and sleep.
  • Prostacyclin and the thromboxanes are derived from a precursor prostaglandin by the action of prostacyclin synthase and thromboxane synthases, respectively.
  • acetyl-CoA molecules derived from fatty acid oxidation in the liver can condense to form acetoacetyl-CoA, which subsequently forms acetoacetate, D-3-hydroxybutyrate, and acetone.
  • These three products are known as ketone bodies.
  • Enzymes involved in ketone body metabolism include HMG-CoA synthetase, HMG-CoA cleavage enzyme, D-3-hydroxybutyrate dehydrogenase, acetoacetate decarboxylase, and 3-ketoacyl-CoA transferase.
  • Ketone bodies are a normal fuel supply of the heart and renal cortex.
  • Acetoacetate produced by the liver is transported to cells where the acetoacetate is converted back to acetyl-CoA and enters the citric acid cycle, h times of starvation, ketone bodies produced from stored triacylglyerols become an important fuel source, especially for the brain. Abnormally high levels of ketone bodies are observed in diabetics. Diabetic coma can result if ketone body levels become too great.
  • Diazepam binding inhibitor also known as endozepine and acyl CoA-binding protein
  • GABA ⁇ -aminobutyric acid
  • DBI binds medium- and long-chain acyl-CoA esters with very high affinity and may function as an intracellular carrier of acyl-CoA esters (OMEVI * 125950 Diazepam Binding l o Inhibitor; DBI; PROSITE PDOC00686 Acyl-CoA-binding protein signature).
  • Fat stored in liver and adipose triglycerides may be released by hydrolysis and transported in the blood. Free fatty acids are transported in the blood by albumin. Triacylglycerols and cholesterol esters in the blood are transported in lipoprotein particles. The particles consist of a core of hydrophobic lipids surrounded by a shell of polar lipids and apolipoproteins. The protein components
  • Lipoproteins include chylomicrons, chylomicron remnants, very-low-density lipoproteins (VLDL), intermediate- density lipoproteins (DDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL).
  • VLDL very-low-density lipoproteins
  • DDL intermediate- density lipoproteins
  • LDL low-density lipoproteins
  • HDL high-density lipoproteins
  • Triacylglycerols in chylomicrons and VLDL are hydrolyzed by lipoprotein Upases that line blood vessels in muscle and other tissues that use fatty acids.
  • Cell surface LDL receptors bind LDL particles which are then internalized by endocytosis. Absence of the LDL receptor, the cause of the disease familial hypercholesterolemia, leads to increased plasma cholesterol levels and ultimately to atherosclerosis.
  • Plasma cholesteryl ester transfer protein mediates the transfer of cholesteryl esters
  • Cholesteryl ester transfer protein is important in the reverse cholesterol transport system and may play a role in atherosclerosis (Yamashita, S. et al. (1997) Curr. Opin. Lipidol. 8:101-110).
  • Macrophage scavenger receptors which bind and internalize modified lipoproteins, play a role in lipid transport and may contribute to atherosclerosis (Greaves, D.R. et al. (1998) Curr. Opin. Lipidol. 9:425-432).
  • the sterol regulatory element binding protein (SREBP) is a sterol- responsive transcription factor. Under normal cholesterol conditions, SREBP resides in the ER membrane. When cholesterol levels are low, a regulated cleavage of SREBP occurs which releases the extracellular domain of the protein. This cleaved domain is then transported to the nucleus where 35 it activates the transcription of the LDL receptor gene, and genes encoding enzymes of cholesterol synthesis, by binding the sterol regulatory element (SRE) upstream of the genes (Yang, J. et al. (1995) J. Biol. Chem. 270:12152-12161).
  • SRE sterol regulatory element
  • OSBP oxysterol-binding protein
  • Mitochondrial and peroxisomal beta-oxidation enzymes degrade saturated and unsaturated fatty acids by sequential removal of two-carbon units from CoA-activated fatty acids.
  • the main beta- oxidation pathway degrades both saturated and unsaturated fatty acids while the auxiliary pathway performs additional steps required for the degradation of unsaturated fatty acids.
  • the pathways of mitochondrial and peroxisomal beta-oxidation use similar enzymes, but have different substrate specificities and functions.
  • Mitochondria oxidize short-, medium-, and long- chain fatty acids to produce energy for cells.
  • Mitochondrial beta-oxidation is a major energy source for cardiac and skeletal muscle.
  • peroxisomes oxidize medium-, long-, and very-long-chain fatty acids, dicarboxylic fatty acids, branched fatty acids, prostaglandins, xenobiotics, and bile acid intermediates.
  • the chief roles of peroxisomal beta-oxidation are to shorten toxic lipophilic carboxylic acids to facilitate their excretion and to shorten very-long-chain fatty acids prior to mitochondrial beta-oxidation (Mannaerts, G.P. and P.P. van Veldhoven (1993) Biochimie 75: 147- 158).
  • Enzymes involved in beta-oxidation include acyl CoA synthetase, carnitine acyltransferase, acyl CoA dehydrogenases, enoyl CoA hydratases, L-3-hydroxyacyl CoA dehydrogenase, ⁇ - ketothiolase, 2,4-dienoyl CoA reductase, and isomerase.
  • Lipid Cleavage and Degradation Triglycerides are hydrolyzed to fatty acids and glycerol by lipases. Lysophospholipases
  • LPLs are widely distributed enzymes that metabolize intracellular lipids, and occur in numerous isoforms. Small isoforms, approximately 15-30 kD, function as hydrolases; large isoforms, those exceeding 60 kD, function both as hydrolases and transacylases.
  • a particular substrate for LPLs lysophosphatidylcholine, causes lysis of cell membranes when it is formed or imported into a cell.
  • LPLs are regulated by lipid factors including acylcamitine, arachidonic acid, and phosphatidic acid. These lipid factors are signaling molecules important in numerous pathways, including the inflammatory response. (Anderson, R. et al. (1994) Toxicol. Appl. Pharmacol. 125:176-183; Selle, H. et al. (1993); Eur. J. Biochem. 212:411-416.)
  • the secretory phospholipase A 2 (PLA2) superfamily comprises a number of heterogeneous enzymes whose common feature is to hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides. Hydrolysis of the glycerophospholipids releases free fatty acids and ysophospholipids. PLA2 activity generates precursors for the biosynthesis of biologically active lipids, hydroxy fatty acids, and platelet-activating factor. PLA2 hydrolysis of the sn-2 ester bond in phospholipids generates free fatty acids, such as arachidonic acid and lysophospholipids.
  • Carbohydrates including sugars or saccharides, starch, and cellulose, are aldehyde or ketone compounds with multiple hydroxyl groups. The importance of carbohydrate metabolism is demonstrated by the sensitive regulatory system in place for maintenance of blood glucose levels.
  • Carbohydrates have three important roles in mammalian cells. First, carbohydrates are used as energy stores, fuels, and metabolic intermediates. Carbohydrates are broken down to form energy in glycolysis and are stored as glycogen for later use. Second, the sugars deoxyribose and ribose form part of the structural support of DNA and RNA, respectively. Third, carbohydrate modifications are added to secreted and membrane proteins and lipids as they traverse the secretory pathway.
  • Cell surface carbohydrate- containing macromolecules including glycoproteins, glycolipids, and transmembrane proteoglycans, mediate adhesion with other cells and with components of the extracellular matrix.
  • the extracellular matrix is composed of diverse glycoproteins, glycosaminoglycans (GAGs), and carbohydrate-binding proteins which are secreted from the cell and assembled into an organized meshwork in close association with the cell surface.
  • GAGs glycosaminoglycans
  • carbohydrate-binding proteins which are secreted from the cell and assembled into an organized meshwork in close association with the cell surface.
  • the interaction of the cell with the surrounding matrix profoundly influences celhshape, strength, flexibility, motility, and adhesion.
  • Carbohydrate metabolism is altered in several disorders including diabetes mellitus, hyperglycemia, hypoglycemia, galactosemia, galactokinase deficiency, and UDP-galactose-4- epimerase deficiency (Fauci, A.S. et al. (1998) Harrison's Principles of Internal Medicine, McGraw- Hill, New York NY, pp. 2208-2209).
  • Altered carbohydrate metabolism is associated with cancer.
  • Reduced GAG and proteoglycan expression is associated with human lung carcinomas (Nackaerts, K. et al. (1997) Int. J. Cancer 74:335-345).
  • Enzymes of the glycolytic pathway convert the sugar glucose to pyruvate while simultaneously producing ATP.
  • the pathway also provides building blocks for the synthesis of cellular components such as long-chain fatty acids. After glycolysis, pyrvuate is converted to acetyl- Coenzyme A, which, in aerobic organisms, enters the citric acid cycle.
  • Glycolytic enzymes include hexokinase, phosphoglucose isomerase, phosphofructokinase, aldolase, triose phosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglyceromutase, enolase, and pyruvate kinase.
  • phosphofructokinase, hexokinase, and pyruvate kinase are important in regulating the rate of glycolysis.
  • Gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors such as lactate and amino acids.
  • the pathway which functions mainly in times of starvation and intense exercise, occurs mostly in the liver and kidney.
  • responsible enzymes include pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose-6-phosphatase.
  • Pentose phosphate pathway enzymes are responsible for generating the reducing agent NADPH, while at the same time oxidizing glucose-6-phosphate to ribose-5-phosphate. Ribose-5- phosphate and its derivatives become part of important biological molecules such as ATP, Coenzyme A, NAD + , FAD, RNA, and DNA.
  • the pentose phosphate pathway has both oxidative and non- oxidative branches. The oxidative branch steps, which are catalyzed by the enzymes glucose-6- phosphate dehydrogenase, lactonase, and 6- ⁇ hosphogluconate dehydrogenase, convert glucose-6- phosphate and NADP + to ribulose-6-phosphate and NADPH.
  • non-oxidative branch steps which are catalyzed by the enzymes phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase, allow the interconversion of three-, four-, five-, six-, and seven-carbon sugars.
  • Glucouronate Metabolism phosphopentose isomerase, phosphopentose epimerase, transketolase, and transaldolase
  • Glucuronate is a monosaccharide which, in the form of D-glucuronic acid, is found in the GAGs chondroitin and dermatan. D-glucuronic acid is also important in the detoxification and excretion of foreign organic compounds such as phenol. Enzymes involved in glucuronate metabolism include UDP-glucose dehydrogenase and glucuronate reductase. Disaccharide Metabolism
  • Disaccharides must be hydrolyzed to monosaccharides to be digested. Lactose, a disaccharide found in milk, is hydrolyzed to galactose and glucose by the enzyme lactase. Maltose is derived from plant starch and is hydrolyzed to glucose by the enzyme maltase. Sucrose is derived from plants and is hydrolyzed to glucose and fructose by the enzyme sucrase. Trehalose, a disaccharide found mainly in insects and mushrooms, is hydrolyzed to glucose by the enzyme trehalase (OMIM *275360 Trehalase; Ruf, J. et al. (1990) J. Biol. Chem. 265:15034-15039).
  • Lactase, maltase, sucrase, and trehalase are bound to mucosal cells lining the small intestine, where they participate in the digestion of dietary disaccharides.
  • lactose synthetase composed of the catalytic subunit galactosyltransferase and the modifier subunit ⁇ -lactalbumin, converts UDP- galactose and glucose to lactose in the mammary glands. Glycogen, Starch, and Chitin Metabolism
  • Glycogen is the storage form of carbohydrates in mammals. Mobilization of glycogen maintains glucose levels between meals and during muscular activity. Glycogen is stored mainly in the liver and in skeletal muscle in the form of cytoplasmic granules. These granules contain enzymes that catalyze the synthesis and degradation of glycogen, as well as enzymes that regulate these processes. Enzymes that catalyze the degradation of glycogen include glycogen phosphorylase, a transferase, ⁇ -l,6-glucosidase, and phosphoglucomutase.
  • Enzymes that catalyze the synthesis of glycogen include UDP-glucose pyrophosphorylase, glycogen synthetase, a branching enzyme, and nucleoside diphosphokinase.
  • the enzymes of glycogen synthesis and degradation are tightly regulated by the hormones insulin, glucagon, and epinephrine.
  • Starch a plant-derived polysaccharide, is hydrolyzed to maltose, maltotriose, and ⁇ -dextrin by ⁇ -amylase, an enzyme secreted by the salivary glands and pancreas.
  • Chitin is a polysaccharide found in insects and Crustacea.
  • GAGs are anionic linear unbranched polysaccharides composed of repetitive disaccharide units. These repetitive units contain a derivative of an amino sugar, either glucosamine or galactosamine. GAGs exist free or as part of proteoglycans, large molecules composed of a core protein attached to one or more GAGs.
  • GAGs are found on the cell surface, inside cells, and in the extracellular matrix. Changes in GAG levels are associated with several autoimmune diseases including autoimmune thyroid disease, autoimmune diabetes mellitus, and systemic lupus erythematosus (Hansen, C. et al. (1996) Clin. Exp. Rheum. 14 (Suppl. 15):S59-S67). GAGs include chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, dermatan sulfate, and hyaluronan.
  • HA GAG hyaluronan
  • GAG hyaluronan The GAG hyaluronan (HA) is found in the extracellular matrix of many cells, especially in soft connective tissues, and is abundant in synovial fluid (Pitsillides, A.A. et al. (1993) Int. J. Exp. Pathol. 74:27-34). HA seems to play important roles in cell regulation, development, and differentiation (Laurent, T.C and J.R. Fraser (1992) FASEB J. 6:2397-2404).
  • Hyaluronidase is an enzyme that degrades HA to oligosaccharides. Hyaluronidases may function in cell adhesion, infection, angiogenesis, signal transduction, reproduction, cancer, and inflammation.
  • Proteoglycans also known as peptidoglycans, are found in the extracellular matrix of connective tissues such as cartilage and are essential for distributing the load in weight-bearing joints.
  • Cell-surface-attached proteoglycans anchor cells to the extracellular matrix. Both extracellular and cell-surface proteoglycans bind growth factors, facilitating their binding to cell-surface receptors and subsequent triggering of signal transduction pathways.
  • Amino Acid and Nitrogen Metabolism NH 4 + is assimilated into amino acids by the actions of two enzymes, glutamate dehydrogenase and glutamine synthetase.
  • the carbon skeletons of amino acids come from the intermediates of glycolysis, the pentose phosphate pathway, or the citric acid cycle.
  • humans can synthesize only thirteen (nonessential amino acids). The remaining nine must come from the diet (essential amino acids).
  • Enzymes involved in nonessential amino acid biosynthesis include glutamate kinase dehydrogenase, pyrroline carboxylate reductase, asparagine synthetase, phenylalanine oxygenase, methionine adenosyltransferase, adenosylhomocysteinase, cystathionine ⁇ -synthase, cystathionine ⁇ -lyase, phosphoglycerate dehydrogenase, phosphoserine ttansaminase, phosphoserine phosphatase, serine hydroxylmethyltransferase, and glycine synthase.
  • Metabolism of amino acids takes place almost entirely in the liver, where the amino group is removed by aminotransferases (transaminases), for example, alanine aminotransferase.
  • the amino group is transferred to ⁇ -ketoglutarate to form glutamate.
  • Glutamate dehydrogenase converts glutamate to NH 4 + and ⁇ -ketoglutarate.
  • NH 4 + is converted to urea by the urea cycle which is catalyzed by the enzymes arginase, ornithine transcarbamoylase, arginosuccinate synthetase, and arginosuccinase.
  • Carbamoyl phosphate synthetase is also involved in urea formation.
  • Enzymes involved in the metabolism of the carbon skeleton of amino acids include serine dehydratase, asparaginase, glutaminase, propionyl CoA carboxylase, methylmalonyl CoA mutase, branched-chain ⁇ -keto dehydrogenase complex, isovaleryl CoA dehydrogenase, ⁇ -methylcrotonyl CoA carboxylase, phenylalanine hydroxylase, p-hydroxylphenylpyruvate hydroxylase, and homogentisate oxidase.
  • Polyamines which include spermidine, putrescine, and spermine, bind tightly to nucleic acids and are abundant in rapidly proliferating cells.
  • Enzymes involved in polyamine synthesis include omithine decarboxylase.
  • Cells derive energy from metabolism of ingested compounds that may be roughly categorized as carbohydrates, fats, or proteins. Energy is also stored in polymers such as triglycerides (fats) and glycogen (carbohydrates). Metabolism proceeds along separate reaction pathways connected by key intermediates such as acetyl coenzyme A (acetyl-CoA). Metabolic pathways feature anaerobic and aerobic degradation, coupled with the energy-requiring reactions such as phosphorylation of adenosine diphosphate (ADP) to the triphosphate (ATP) or analogous phosphorylations of guanosine
  • ADP adenosine diphosphate
  • ATP triphosphate
  • GDP/GTP GDP/GTP
  • UDP/UTP uridine
  • CDP/CTP cytidine
  • Digestive enzymes convert carbohydrates and sugars to glucose; fructose and galactose are converted in the liver to glucose. Enzymes involved in these conversions include galactose-1- phosphate uridyl transferase and UDP-galactose-4 epimerase. In the cytoplasm, glycolysis converts glucose to pyruvate in a series of reactions coupled to ATP synthesis.
  • Pyruvate is transported into the mitochondria and converted to acetyl-CoA for oxidation via the citric acid cycle, involving pyruvate dehydrogenase components, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase.
  • Enzymes involved in the citric acid cycle include: citrate synthetase, aconitases, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex including transsuccinylases, succinyl CoA synthetase, succinate dehydrogenase, fumarases, and malate dehydrogenase.
  • Acetyl CoA is oxidized to C0 2 with concomitant formation of NADH, FADH 2 , and GTP.
  • oxidative phosphorylation the transport of electrons from NADH and FADH 2 to oxygen by dehydrogenases is coupled to the synthesis of ATP from ADP and P; by the F ⁇
  • Enzyme complexes responsible for electron transport and ATP synthesis include the F Q F J ATPase complex, ubiquinone(CoQ)-cytocl ⁇ rome c reductase, ubiquinone reductase, cytochrome b, cytochrome C ] , FeS protein, and cytochrome c oxidase.
  • Triglycerides are hydrolyzed to fatty acids and glycerol by Upases. Glycerol is then phosphorylated to gIycerol-3 -phosphate by glycerol kinase and glycerol phosphate dehydrogenase, and degraded by the glycolysis. Fatty acids are transported into the mitochondria as fatty acylcamitine esters and undergo oxidative degradation.
  • Cofactors are small molecular weight inorganic or organic compounds that are required for the action of an enzyme. Many cofactors contain vitamins as a component. Cofactors include thiamine pyrophosphate, flavin adenine dinucleotide, flavin mononucleotide, nicotinamide adenine dinucleotide, pyridoxal phosphate, coenzyme A, tetrahydrofolate, lipoamide, and heme. The vitamins biotin and cobalamin are associated with
  • Heme a prosthetic group found in myoglobin and hemoglobin, consists of protoporphyrin group bound to iron. Porphyrin groups contain four substituted pyrroles covalently joined in a ring, often with a bound metal atom. Enzymes involved in porphyrin synthesis include ⁇ - aminolevulinate synthase, ⁇ -aminolevulinate dehydrase, porphobilinogen deaminase, and cosynthase.
  • heme formation causes porphyrias. Heme is broken down as a part of erythrocyte 0 turnover. Enzymes involved in heme degradation include heme oxygenase and biliverdin reductase. Iron is a required cofactor for many enzymes. Besides the heme-containing enzymes, iron is found in iron-sulfur clusters in proteins including aconitase, succinate dehydrogenase, and NADH-Q reductase. Iron is transported in the blood by the protein transferrin. Binding of transferrin to the transferrin receptor on cell surfaces allows uptake by receptor mediated endocytosis. Cytosolic iron is 5 bound to ferritin protein.
  • a molybdenum-containing cofactor (molybdopterin) is found in enzymes including sulfite oxidase, xanthine dehydrogenase, and aldehyde oxidase. Molybdopterin biosynthesis is performed by two molybdenum cofactor synthesizing enzymes. Deficiencies in these enzymes cause mental retardation and lens dislocation. Other diseases caused by defects in cofactor metabolism include o pernicious anemia and methylmalonic aciduria. Secretion and Trafficking
  • Eukaryotic cells are bound by a lipid bilayer membrane and subdivided into functionally distinct, membrane bound compartments.
  • the membranes maintain the essential differences between the cytosol, the extracellular environment, and the lumenal space of each intracellular organelle.
  • As 5 lipid membranes are highly impermeable to most polar molecules, transport of essential nutrients, metabolic waste products, cell signaling molecules, macromolecules and proteins across lipid membranes and between organelles must be mediated by a variety of transport-associated molecules. Protein Trafficking
  • ER-bound ribosomes In eukaryotes, some proteins are synthesized on ER-bound ribosomes, co-translationally o imported into the ER, delivered from the ER to the Golgi complex for post-translational processing and sorting, and transported from the Golgi to specific intracellular and extracellular destinations. All cells possess a constitutive transport process which maintains homeostasis between the cell and its environment. In many differentiated cell types, the basic machinery is modified to carry out specific transport functions. For example, in endocrine glands, hormones and other secreted proteins 5 are packaged into secretory granules for regulated exocytosis to the cell exterior.
  • ER-bound ribosomes Synthesis of most integral membrane proteins, secreted proteins, and proteins destined for the lumen of a particular organelle occurs on ER-bound ribosomes. These proteins are co-translationally imported into the ER. The proteins leave the ER via membrane-bound vesicles which bud off the ER at specific sites and fuse with each other (homotypic fusion) to form the ER-Golgi Intermediate Compartment (ERGIC). The ERGIC matures progressively through the cis, medial, and trans asternal stacks of the Golgi, modifying the enzyme composition by retrograde transport of specific Golgi enzymes. In this way, proteins moving through the Golgi undergo post-translational modification, such as glycosylation.
  • post-translational modification such as glycosylation.
  • the final Golgi compartment is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination.
  • TGN Trans-Golgi Network
  • secretory vesicle which contains proteins destined for the plasma membrane, such as receptors, adhesion molecules, and ion channels, and secretory proteins, such as hormones, neurotransmitters, and digestive enzymes. Secretory vesicles eventually fuse with the plasma membrane (Glick, B.S. and V. Malhotra (1998) Cell 95:883-889).
  • the secretory process can be constitutive or regulated. Most cells have a constitutive pathway for secretion, whereby vesicles derived from maturation of the TGN require no specific signal to fuse with the plasma membrane. In many cells, such as endocrine cells, digestive cells, and neurons, vesicle pools derived from the TGN collect in the cytoplasm and do not fuse with the plasma membrane until they are directed to by a specific signal. Endocytosis
  • Endocytosis wherein cells internalize material from the extracellular environment, is essential for transmission of neuronal, metabolic, and proliferative signals; uptake of many essential nutrients; and defense against invading organisms. Most cells exhibit two forms of endocytosis. The first, phagocytosis, is an actin-driven process exemplified in macrophage and neutrophils. Material to be endocytosed contacts numerous cell surface receptors which stimulate the plasma membrane to extend and surround the particle, enclosing it in a membrane-bound phagosome. In the mammalian immune system, IgG-coated particles bind Fc receptors on the surface of phagocytic leukocytes.
  • Activation of the Fc receptors initiates a signal cascade involving src-family cytosolic kinases and the monomeric GTP-binding (G) protein Rho.
  • G GTP-binding
  • the resulting actin reorganization leads to phagocytosis of the particle.
  • This process is an important component of the humoral immune response, allowing the processing and presentation of bacterial-derived peptides to antigen-specific T-lymphocytes.
  • the second form of endocytosis, pinocytosis is a more generalized uptake of material from the external milieu. Like phagocytosis, pinocytosis is activated by ligand binding to cell surface receptors.
  • Activation of individual receptors stimulates an internal response that includes coalescence of the receptor-ligand complexes and formation of clathrin-coated pits. Invagination of the plasma membrane at clathrin-coated pits produces an endocytic vesicle within the cell cytoplasm.
  • vesicles undergo homotypic fusion to form an early endosomal (EE) compartment.
  • EE early endosomal
  • the tubulovesicular EE serves as a sorting site for incoming material. ATP-driven proton pumps in the
  • EE membrane lowers the pH of the EE lumen (pH 6.3-6.8).
  • the acidic environment causes many ligands to dissociate from their receptors.
  • the receptors, along with membrane and other integral membrane proteins, are recycled back to the plasma membrane by budding off the, tubular extensions of the EE in recycling vesicles (RV).
  • RV recycling vesicles
  • This selective removal of recycled components produces a carrier vesicle containing ligand and other material from the external environment.
  • the carrier vesicle fuses with TGN-derived vesicles which contain hydrolytic enzymes.
  • the acidic environment of the resulting late endosome (LE) activates the hydrolytic enzymes which degrade the ligands and other material.
  • vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane (PM), and tubular extensions of the endosomes.
  • tER transitional endoplasmic reticulum
  • TGN Trans-Golgi Network
  • PM plasma membrane
  • tubular extensions of the endosomes The process begins with the budding of a vesicle out of the donor membrane.
  • the membrane-bound vesicle contains proteins to be transported and is surrounded by a protective coat made up of protein subunits recruited from the cytosol.
  • the initial budding and coating processes are controlled by a cytosolic ras-like GTP-binding protein, ADP- ribosylating factor (Arf), and adapter proteins (AP). Different isoforms of both Arf and AP are involved at different sites of budding.
  • Another small G-protein, dynamin forms a ring complex around the neck of the forming vesicle and may provide the mechanochemical force to accomplish the final step of the budding process.
  • the coated vesicle complex is then transported through the cytosol. During the transport process, Arf-bound GTP is hydrolyzed to GDP and the coat dissociates from the transport vesicle (West, M.A. et al. (1997) J. Cell Biol.
  • coat protein Two different classes have also been identified. Clathrin coats form on the TGN and PM surfaces, whereas coatomer or COP coats form on the ER and Golgi. COP coats can further be distinguished as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra).
  • the COP coat consists of two major components, a G-protein (Arf or Sar) and coat protomer (coatomer).
  • Coatomer is an equimolar complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP. (Harter, C and F.T. Wieland (1998) Proc. Natl. Acad. Sci. USA 95: 11649-11654.)
  • Transport vesicles undergo homotypic or heterotypic fusion in the secretory and endocytotic pathways.
  • Molecules required for appropriate targeting and fusion of vesicles with their target membrane include proteins incorporated in the vesicle membrane, the target membrane, and proteins recruited from the cytosol.
  • VAMP vesicle-associated membrane protein
  • a cytosolic prenylated GTP-binding protein, Rab is inserted into the vesicle membrane.
  • GTP-bound Rab proteins are directed into nascent transport vesicles where they interact with VAMP. Following vesicle transport, GTPase activating proteins (GAPs) in the target membrane convert Rab proteins to the GDP-bound form. A cytosolic protein, guanine-nucleotide dissociation inhibitor (GDI) helps return GDP-bound Rab proteins to their membrane of origin.
  • GAPs GTPase activating proteins
  • GDI guanine-nucleotide dissociation inhibitor
  • Rab proteins appear to play a role in mediating the function of a viral gene, Rev, which is essential for replication of FflV-l, the virus responsible for AIDS (Flavell, R.A. et al. (1996) Proc. Natl. Acad. Sci. USA 93:4421-4424).
  • v-SNARE vesicle SNAP receptor
  • t- target membrane
  • cytosolic proteins a complex between the vesicle SNAP receptor (v-SNARE), target membrane (t-) SNAREs, and certain other membrane and cytosolic proteins. Many of these other proteins have been identified although their exact functions in the docking complex remain uncertain (Tellam, J.T. et al. (1995) J. Biol. Chem. 270:5857-5863; and Hata, Y. and T.C. Sudhof (1995) J. Biol. Chem. 270: 13022-13028).
  • N-ethylmaleimide sensitive factor (NSF) and soluble NSF-attachment protein ( ⁇ -SNAP and ⁇ -SNAP) are two such proteins that are conserved from yeast to man and function in most intracellular membrane fusion reactions.
  • Seel represents a family of yeast proteins that function at many different stages in the secretory pathway including membrane fusion. Recently, mammalian homologs of Seel, called Munc-18 proteins, have been identified (Katagiri, H. et al. (1995) J. Biol. Chem. 270:4963-4966; Hata et al. supra).
  • the SNARE complex involves three SNARE molecules, one in the vesicular membrane and two in the target membrane.
  • Synaptotagmin is an integral membrane protein in the synaptic vesicle which associates with the t-SNARE syntaxin in the docking complex.
  • Synaptotagmin binds calcium in a complex with negatively charged phosphoUpids, which allows the cytosolic SNAP protein to displace synaptotagmin from syntaxin and fusion to occur.
  • synaptotagmin is a negative regulator of fusion in the neuron (Littleton, J.T. et al. (1993) Cell 74: 1125-1134).
  • the most abundant membrane protein of synaptic vesicles appears to be the glycoprotein synaptophysin, a 38 kDa protein with four transmembrane domains.
  • v-SNARE v-SNARE
  • t-SNAREs t-SNAREs
  • associated proteins v-SNARE
  • Different isoforms of SNAREs and Rabs show distinct cellular and subcellular distributions.
  • VAMP-1/synaptobrevin, membrane-anchored synaptosome-associated protein of 25 kDa (SNAP-25), syntaxin-1, Rab3A, Rabl5, and Rab23 are predominantly expressed in the brain and nervous system.
  • syntaxin, VAMP, and Rab proteins are associated with distinct subcellular compartments and their vesicular carriers.
  • NPCs nuclear pore complexes
  • All nuclear proteins are imported from the cytoplasm, their site of synthesis.
  • tRNA and mRNA are exported from the nucleus, their site of synthesis, to the cytoplasm, their site of function.
  • Processing of small nuclear RNAs involves export into the cytoplasm, assembly with proteins and modifications such as hypermethylation to produce small nuclear ribonuclear proteins (snRNPs), and subsequent import of the snRNPs back into the nucleus.
  • snRNPs small nuclear ribonuclear proteins
  • ribosomes require the initial import of ribosomal proteins from the cytoplasm, their incorporation with RNA into ribosomal subunits, and export back to the cytoplasm. (G ⁇ rlich, D. and I.W. Mattaj (1996) Science 271:1513-1518.)
  • NLS nuclear localization signals
  • NLS nuclear localization signals
  • NTF2 homodimeric protein nuclear transport factor 2
  • abnormal hormonal secretion is linked to disorders such as diabetes insipidus (vasopressin), hyper- and hypoglycemia (insulin, glucagon), Grave's disease and goiter (thyroid hormone), and Cushing's and Addison's diseases (adrenocorticotropic hormone, ACTH).
  • cancer cells secrete excessive amounts of hormones or other biologically active peptides.
  • Disorders related to excessive secretion of biologically active peptides by tumor cells include fasting hypoglycemia due to increased insulin secretion from insulinoma-islet cell tumors; hypertension due to increased epinephrine and norepinephrine secreted from pheochromocytomas of the adrenal medulla and sympathetic paraganglia; and carcinoid syndrome, which is characterized by abdominal cramps, diarrhea, and valvular heart disease caused by excessive amounts of vasoactive substances such as serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones, secreted from intestinal tumors.
  • vasoactive substances such as serotonin, bradykinin, histamine, prostaglandins, and polypeptide hormones, secreted from intestinal tumors.
  • Biologically active peptides that are ectopically synthesized in and secreted from tumor cells include ACTH and vasopressin (lung and pancreatic cancers); parathyroid hormone (lung and bladder cancers); calcitonin (lung and breast cancers); and thyroid-stimulating hormone (medullary thyroid carcinoma).
  • Such peptides may be useful as diagnostic markers for tumorigenesis (Schwartz, M.Z. (1997) Semin. Pediatr. Surg. 3:141-146; and Said, S.I. and G.R. Faloona (1975) N. Engl. J. Med. 293:155-160).
  • Defective nuclear transport may play a role in cancer.
  • the BRCA1 protein contains three potential NLSs which interact with importin alpha, and is transported into the nucleus by the importin/NPC pathway.
  • the BRCA1 protein is aberrantly localized in the cytoplasm.
  • the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the NPC nuclear import pathway (Chen, CF. et al. (1996) J. Biol. Chem. 271:32863-32868). It has been suggested that in some breast cancers, the tumor-suppressing activity of p53 is inactivated by the sequestration of the protein in the cytoplasm, away from its site of action in the cell nucleus. Cytoplasmic wild-type p53 was also found in human cervical carcinoma cell lines.
  • Organisms respond to the environment by a number of pathways.
  • Heat shock proteins including hsp70, hsp60, hsp90, and hsp40, assist organisms in coping with heat damage to cellular proteins.
  • Aquaporins are channels that transport water and, in some cases, nonionic small solutes such as urea and glycerol. Water movement is important for a number of physiological processes including renal fluid filtration, aqueous humor generation in the eye, cerebrospinal fluid production in the brain, and appropriate hydration of the lung. Aquaporins are members of the major intrinsic protein (MEP) family of membrane transporters (King, L.S. and P. Agre (1996) Annu. Rev. Physiol. 58:619-648; Ishibashi, K. et al. (1997) J. Biol. Chem. 272:20782-20786).
  • MEP major intrinsic protein
  • OMIM *154050 Major Intrinsic Protein of Lens Fiber MIP
  • the metallothioneins MTs
  • cysteine-rich proteins that bind heavy metals such as cadmium, zinc, mercury, lead, and copper and are thought to play a role in metal detoxification or the metabolism and homeostasis of metals.
  • Arsenite-resistance proteins have been identified in hamsters that are resistant to toxic levels of arsenite (Rossman, T.G. et al. (1997)
  • Proteins involved in light perception include rhodopsin, transducin, and cGMP phosphodiesterase.
  • Proteins involved in odor perception include multiple olfactory receptors. Other proteins are important in human Orcadian rhythms and responses to wounds.
  • the cellular components of the humoral immune system include six different types of leukocytes: monocytes, lymphocytes, polymorphonuclear granulocytes (consisting of neutrophils, eosinophils, and basophils) and plasma cells. Additionally, fragments of megakaryocytes, a seventh type of white blood cell in the bone marrow, occur in large numbers in the blood as platelets.
  • Leukocytes are formed from two stem cell lineages in bone marrow.
  • the myeloid stem cell line produces granulocytes and monocytes and, the lymphoid stem cell produces lymphocytes.
  • Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes. Leukocytes are responsible for defending the body against invading pathogens.
  • monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis.
  • Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes specific foreign molecules and organisms and inactivates them, as well as signals other cells to attack the invaders.
  • Granulocytes and monocytes are formed and stored in the bone marrow until needed.
  • Megakaryocytes are produced in bone marrow, where they fragment into platelets and are released into the bloodstream.
  • the main function of platelets is to activate the blood clotting mechanism.
  • Lymphocytes and plasma cells are produced in various lymphogenous organs, including the lymph nodes, spleen, thymus, and tonsils. Both neutrop ls and macrophages exhibit chemotaxis towar s s tes of inflammation.
  • Tissue inflammation in response to pathogen invasion results in production of chemo-attractants for leukocytes, such as endotoxins or other bacterial products, prostaglandins, and products of leukocytes or platelets.
  • Basophils participate in the release of the chemicals involved in the inflammatory process.
  • basophils The main function of basophils is secretion of these chemicals to such a degree that they have been referred to as "unicellular endocrine glands."
  • basophils A distinct aspect of basophilic secretion is that the contents of granules go directly into the extracellular environment, not into vacuoles as occurs with neutrophils, eosinophils and monocytes.
  • Basophils have receptors for the Fc fragment of immunoglobulin E (IgE) that are not present on other leukocytes. Crosslinking of membrane IgE with anti-IgE or other ligands triggers degranulation.
  • IgE immunoglobulin E
  • Eosinophils are bi- or multi-nucleated white blood cells which contain eosinophilic granules. Their plasma membrane is characterized by Ig receptors, particularly IgG and IgE. GeneraUy, eosinophils are stored in the bone marrow until recruited for use at a site of inflammation or invasion. They have specific functions in parasitic infections and allergic reactions, and are thought to detoxify some of the substances released by mast cells and basophils which cause inflammation. Additionally, they phagocytize antigen-antibody complexes and further help prevent spread of the inflammation.
  • Macrophages are monocytes that have left the blood stream to settle in tissue. Once monocytes have migrated into tissues, they do not re-enter the bloodstream.
  • the mononuclear phagocyte system is composed of precursor cells in the bone marrow, monocytes in circulation, and macrophages in tissues. The system is capable of very fast and extensive phagocytosis. A macrophage may phagocytize over 100 bacteria, digest them and extrude residues, and then survive for many more months.
  • Macrophages are also capable of ingesting large particles, including red blood cells and malarial parasites. They increase several-fold in size and transform into macrophages that are characteristic of the tissue they have entered, surviving in tissues for several months.
  • Mononuclear phagocytes are essential in defending the body against invasion by foreign pathogens, particularly intracellular microorganisms such as M. tuberculosis, listeria, leishmania and toxoplasma. Macrophages can also control the growth of tumorous cells, via both phagocytosis and secretion of hydrolytic enzymes. Another important function of macrophages is that of processing antigen and presenting them in a biochemically modified form to lymphocytes.
  • the immune system responds to invading microorganisms in two major ways: antibody production and cell mediated responses.
  • Antibodies are immunoglobulin proteins produced by B-lymphocytes which bind to specific antigens and cause inactivation or promote destruction of the antigen by other cells.
  • Cell-mediated immune responses involve T-lymphocytes (T cells) that react with foreign antigen on the surface of infected host cells. Depending on the type of T cell, the infected cell is either killed or signals are secreted which activate macrophages and other cells to destroy the infected cell (Paul, supra).
  • T-lymphocytes originate in the bone marrow or liver in fetuses. Precursor cells migrate via the blood to the thymus, where they are processed to mature into T-lymphocytes. This processing is crucial because of positive and negative selection of T cells that will react with foreign antigen and not with self molecules. After processing, T cells continuously circulate in the blood and secondary lymphoid tissues, such as lymph nodes, spleen, certain epithelium-associated tissues in the gastrointestinal tract, respiratory tract and skin. When T-lymphocytes are presented with the complementary antigen, they are stimulated to proliferate and release large numbers of activated T cells into the lymph system and the blood system. These activated T cells can survive and circulate for several days.
  • T memory cells are created, which remain in the lymphoid tissue for months or years. Upon subsequent exposure to that specific antigen, these memory cells will respond more rapidly and with a stronger response than induced by the original antigen. This creates an "immunological memory” that can provide immunity for years.
  • T cells There are two major types of T cells: cytotoxic T cells destroy infected host cells, and helper T cells activate other white blood cells via chemical signals.
  • helper T cells activate other white blood cells via chemical signals.
  • T H 1 activates macrophages to destroy ingested microorganisms, while another, T H 2, stimulates the production of antibodies by B cells.
  • Cytotoxic T cells directly attack the infected target cell.
  • peptides derived from viral proteins are generated by the proteasome. These peptides are transported into the ER by the transporter associated with antigen processing (TAP) (Pamer, E. and P. Cresswell (1998) Annu. Rev. Immunol. 16:323-358).
  • TAP antigen processing
  • the peptides bind MHC I chains, and the peptide/MHC I complex is transported to the cell surface.
  • Receptors on the surface of T cells bind to antigen presented on cell surface MHC molecules.
  • T cells Once activated by binding to antigen, T cells secrete ⁇ -interferon, a signal molecule that induces the expression of genes necessary for presenting viral (or other) antigens to cytotoxic T cells. Cytotoxic T cells kill the infected cell by stimulating programmed cell death.
  • Helper T cells constitute up to 75% of the total T cell population. They regulate the immune functions by producing a variety of lymphokines that act on other cells in the immune system and on bone marrow. Among these lymphokines are: interleukins-2,3,4,5,6; granulocyte-monocyte colony stimulating factor, and ⁇ -interferon.
  • Helper T cells are required for most B cells to respond to antigen.
  • an activated helper cell contacts a B cell, its centrosome and Golgi apparatus become oriented toward the B cell, aiding the directing of signal molecules, such as transmembrane-bound protein called CD40 ligand, onto the B cell surface to interact with the CD40 transmembrane protein.
  • Secreted signals also help B cells to proliferate and mature and, in some cases, to switch the class of antibody being produced.
  • B-lymphocytes produce antibodies which react with specific antigenic proteins presented by pathogens. Once activated, B cells become filled with extensive rough endoplasmic reticulum and are known as plasma cells. As with T cells, interaction of B cells with antigen stimulates proliferation of only those B cells which produce antibody specific to that antigen.
  • immunoglobulins There are five classes of antibodies, known as immunoglobulins, which together comprise about 20% of total plasma protein. Each class mediates a characteristic biological response after antigen binding.
  • the immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens.
  • antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site- specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.
  • Ig immunoglobulin
  • MHC major histocompatibility
  • MHC proteins are cell surface markers that bind to and present foreign antigens to T cells. MHC molecules are classified as either class I or class II. Class I MHC molecules (MHC I) are expressed on the surface of almost all cells and are involved in the presentation of antigen to cytotoxic T cells. For example, a cell infected with virus will degrade intracellular viral proteins and express the protein fragments bound to MHC I molecules on the cell surface. The MHC I/antigen complex is recognized by cytotoxic T-cells which destroy the infected cell and the virus within. Class II MHC molecules are expressed primarily on specialized antigen-presenting cells of the immune system, such as B-cells and macrophages. These cells ingest foreign proteins from the extracellular fluid and express MHC Il/antigen complex on the cell surface. This complex activates helper T-ce ls, w ich then secrete cytokines and other factors that stimulate the immune response.
  • MHC I Class I MHC molecules
  • MHC molecules also play an important role in organ rejection following transplantation. Rejection occurs when the recipient's T-cells respond to foreign MHC molecules on the transplanted organ in the same way as to self MHC molecules bound to foreign antigen. (Reviewed in Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing, New York NY, pp. 1229-1246.)
  • Antibodies or immunoglobulins (Ig), are the founding members of the Ig superfamily and the central components of the humoral immune response. Antibodies are either expressed on the surface of B cells or secreted by B cells into the circulation. Antibodies bind and neutralize blood- borne foreign antigens.
  • the prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition.
  • the five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the , ⁇ , e, ⁇ , and ⁇ .
  • H-chain types There are two types of L- chains, K and ⁇ , either of which may associate as a pair with any H-chain pair.
  • IgG the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure.
  • H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region.
  • the constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains.
  • the amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class.
  • the variable region consists of about 110 amino acids in both H- and L-chains.
  • Both H-chains and L-chains contain repeated Ig domains. For example, a typical H- chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site.
  • a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
  • H chains such as ⁇ have been shown to associate with other polypeptides during differentiation of the B cell.
  • the amino acid sequence of the variable region differs among H- or L-chains of a particular class.
  • Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site. (Reviewed in Alberts, supra, pp. 1206-1213 and 1216- 1217.)
  • Antibodies can be described in terms of their two main functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region of the antibody, while effector functions are mediated by the Fc (crystallizable fragment) region. Binding of antibody to an antigen, such as a bacterium, triggers the destruction of the antigen by phagocytic white blood cells such as macrophages and neutrophils. These cells express surface receptors that specifically bind to the antibody Fc region and allow the phagocytic cells to engulf, ingest, and degrade the antibody-bound antigen. The Fc receptors expressed by phagocytic cells are single-pass transmembrane glycoproteins of about 300 to 400 amino acids (Sears, D.W. et al. (1990) J. Immunol. 144:371-378).
  • the extracellular portion of the Fc receptor typically contains two or three Ig domains. Diseases which cause over- or under-abundance of any one type of leukocyte usually result in the entire immune defense system becoming involved.
  • a well-known autoimmune disease is AIDS
  • acquired Immunodeficiency Syndrome where the number of helper T cells is depleted, leaving the patient susceptible to infection by microorganisms and parasites.
  • Another widespread medical condition attributable to the immune system is that of allergic reactions to certain antigens. Allergic reactions include: hay fever, asthma, anaphylaxis, and urticaria (hives).
  • Leukemias are an excess production of white blood cells, to the point where a major portion of the body's metabolic resources are directed solely at proliferation of white blood cells, leaving other tissues to starve.
  • Leukopenia or agranulocytosis occurs when the bone marrow stops producing white blood cells. This leaves the body unprotected against foreign microorganisms, including those which normally inhabit skin, mucous membranes, and gastrointestinal tract. If all white blood cell production stops completely, infection will occur within two days and death may follow only 1 to 4 days later.
  • Impaired phagocytosis occurs in several diseases, including monocytic leukemia, systemic lupus, and granulomatous disease. In such a situation, macrophages can phagocytize normally, but the enveloped organism is not killed. A defect in the plasma membrane enzyme which converts oxygen to lethally reactive forms results in abscess formation in liver, lungs, spleen, lymph nodes, and beneath the skin. Eosinophilia is an excess of eosinophils commonly observed in patients with allergies (hay fever, asthma), allergic reactions to drugs, rheumatoid arthritis, and cancers (Hodgkin's disease, lung, and liver cancer) (Isselbacher, K.J. et al.
  • the complement system serves as an effector system and is involved in infectious agent recognition. It can function as an independent immune network or in conjunction with other humoral immune responses.
  • the complement system is composed of numerous plasma and membrane proteins that act in a cascade of reaction sequences whereby one component activates the next. The result is a rapid and amplified response to infection through either an inflammatory response or increased phagocytosis.
  • the complement system has more than 30 protein components which can be divided into functional groupings including modified serine proteases, membrane-binding proteins and regulators of complement activation. Activation occurs through two different pathways the classical and the alternative. Both pathways serve to destroy infectious agents through distinct triggering mechanisms that eventually merge with the involvement of the component C3.
  • the classical pathway requires antibody binding to infectious agent antigens.
  • the antibodies serve to define the target and initiate the complement system cascade, culminating in the destruction of the infectious agent.
  • the complement can be seen as an effector arm of the humoral immune system.
  • the alternative pathway of the complement system does not require the presence of pre- 5 existing antibodies for targeting infectious agent destruction. Rather, this pathway, through low levels of an activated component, remains constantly primed and provides surveillance in the non- immune host to enable targeting and destruction of infectious agents. In this case foreign material triggers the cascade, thereby facilitating phagocytosis or lysis (Paul, supra, pp.918-919).
  • Inflammatory 0 responses are divided into four categories on the basis of pathology and include allergic inflammation, cytotoxic antibody mediated inflammation, immune complex mediated inflammation and monocyte mediated inflammation. Inflammation manifests as a combination of each of these forms with one predominating.
  • Allergic acute inflammation is observed in individuals wherein specific antigens stimulate 5 IgE antibody production.
  • Mast cells and basophils are subsequently activated by the attachment of antigen-IgE complexes, resulting in the release of cytoplasmic granule contents such as histamine.
  • the products of activated mast cells can increase vascular permeability and constrict the smooth muscle of breathing passages, resulting in anaphylaxis or asthma.
  • Acute inflammation is also mediated by cytotoxic antibodies and can result in the destruction of tissue through the binding of o complement-fixing antibodies to cells.
  • the responsible antibodies are of the IgG or IgM types. Resultant clinical disorders include autoimmune hemolytic anemia and thrombocytopenia as associated with systemic lupus erythematosis.
  • Immune complex mediated acute inflammation involves the IgG or IgM antibody types which combine with antigen to activate the complement cascade.
  • immune complexes 5 bind to neutrophils and macrophages they activate the respiratory burst to form protein- and vessel- damaging agents such as hydrogen peroxide, hydroxyl radical, hypochlorous acid, and chloramines.
  • Clinical manifestations include rheumatoid arthritis and systemic lupus erythematosus.
  • MPs Matrix proteins
  • the expression and balance of MPs may be perturbed by biochemical changes that result from congenital, epigenetic, or infectious diseases, hi addition, MPs affect leukocyte migration, proliferation, differentiation, and activation in the immune response.
  • MPs are frequently characterized by the presence of one or more domains which may include collagen-like domains, EGF-like domains, immunoglobulin-like domains, and f ⁇ bronectin-like domains.
  • MPs may be heavily glycosylated and may contain an Arginine-Glycine-Aspartate (RGD) tripeptide motif which may play a role in adhesive interactions.
  • MPs include extracellular proteins such as fibronectin, collagen, galectin, vitronectin and its proteolytic derivative somatomedin B; and cell adhesion receptors such as cell adhesion molecules (CAMs), cadherins, and integrins.
  • Growth and differentiation factors are secreted proteins which function in intercellular communication. Some factors require oligomerization or association with MPs for activity. Complex interactions among these factors and their receptors trigger intracellular signal transduction pathways that stimulate or inhibit cell division, cell differentiation, cell signaling, and cell motility. Most growth and differentiation factors act on cells in their local environment (paracrine signaling).
  • the first class includes the large polypeptide growth factors such as epidermal growth factor, fibroblast growth factor, transforming growth factor, insulin-like growth factor, and platelet-derived growth factor.
  • the second class includes the hematopoietic growth factors such as the colony stimulating factors (CSFs).
  • CSFs colony stimulating factors
  • Hematopoietic growth factors stimulate the proliferation and differentiation of blood cells such as B- lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, macrophages, and their stem cell precursors.
  • the third class includes small peptide factors such as bombesin, vasopressin, oxytocin, endothelin, transferrin, angiotensin ⁇ , vasoactive intestinal peptide, and bradykinin which function as hormones to regulate cellular functions other than proliferation.
  • Growth and differentiation factors play critical roles in neoplastic transformation of cells in vitro and in tumor progression in vivo.
  • Inappropriate expression of growth factors by tumor cells may contribute to vascularization and metastasis of tumors.
  • growth factor misregulation can result in anemias, leukemias, and lymphomas.
  • Certain growth factors such as interferon are cytotoxic to tumor cells both in vivo and in vitro.
  • some growth factors and growth factor receptors are related both structurally and functionally to oncoproteins.
  • growth factors affect transcriptional regulation of both proto-oncogenes and oncosuppressor genes. (Reviewed in Pimentel, E. (1994) Handbook of Growth Factors, CRC Press, Ann Arbor MI, pp. 1-9.) Extracellular Information Transmission Molecules
  • Intercellular communication is essential for the growth and survival of multicellular organisms, and in particular, for the function of the endocrine, nervous, and immune systems.
  • intercellular communication is critical for developmental processes such as tissue construction and organogenesis, in which cell proliferation, cell differentiation, and morphogenesis must be spatially and temporally regulated in a precise and coordinated manner.
  • Cells communicate with one another through the secretion and uptake of diverse types of signaling molecules such as hormones, growth factors, neuropeptides, and cytokines.
  • Hormones include hormones, growth factors, neuropeptides, and cytokines.
  • Hormones are secreted molecules that travel through the circulation and bind to specific receptors on the surface of, or within, target cells. Although they have diverse biochemical compositions and mechanisms of action, hormones can be grouped into two categories.
  • One category includes small lipophilic hormones that diffuse through the plasma membrane of target cells, bind to cytosolic or nuclear receptors, and form a complex that alters gene expression. Examples of these molecules include retinoic acid, thyroxine, and the cholesterol-derived steroid hormones such as progesterone, estrogen, testosterone, cortisol, and aldosterone.
  • the second category includes hydrophilic hormones that function by binding to cell surface receptors that transduce signals across the plasma membrane.
  • hormones include amino acid derivatives such as catecholamines and peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin.
  • catecholamines amino acid derivatives
  • peptide hormones such as glucagon, insulin, gastrin, secretin, cholecystokinin, adrenocorticotropic hormone, follicle stimulating hormone, luteinizing hormone, thyroid stimulating hormone, and vasopressin.
  • Hormones are signaling molecules that coordinately regulate basic physiological processes from embryogenesis throughout adulthood. These processes include metabolism, respiration, reproduction, excretion, fetal tissue differentiation and organogenesis, growth and development, homeostasis, and the stress response. Hormonal secretions and the nervous system are tightly integrated and interdependent. Hormones are secreted by endocrine glands, primarily the hypothalamus and pituitary, the thyroid and parathyroid, the pancreas, the adrenal glands, and the ovaries and testes.
  • Hormones are often secreted in diumal, pulsatile, and cyclic patterns. Hormone secretion is regulated by perturbations in blood biochemistry, by other upstream-acting hormones, by neural impulses, and by negative feedback loops. Blood hormone concentrations are constantly monitored and adjusted to maintain optimal, steady-state levels. Once secreted, hormones act only on those target cells that express specific receptors.
  • hyposecretion often occurs when a hormone's gland of origin is damaged or otherwise impaired. Hypersecretion often results from the proliferation of tumors derived from hormone- secreting cells. Inappropriate hormone levels may also be caused by defects in regulatory feedback loops or in the processing of hormone precursors. Endocrine malfunction may also occur when the target cell fails to respond to the hormone.
  • Hormones can be classified biochemically as polypeptides, steroids, eicosanoids, or amines.
  • Polypeptides which include diverse hormones such as insulin and growth hormone, vary in size and function and are often synthesized as inactive precursors that are processed intraceUularly into
  • Amines which include epinephrine and dopamine, are amino acid derivatives that function in neuroendocrine signaling.
  • Steroids which include the cholesterol-derived hormones estrogen and testosterone, function in sexual development and reproduction.
  • Eicosanoids which include prostaglandins and prostacyclins, are fatty acid derivatives that function in a variety of processes.
  • Most polypeptides and some amines are soluble in the circulation where they are highly 0 susceptible to proteolytic degradation within seconds after their secretion. Steroids and lipids are insoluble and must be transported in the circulation by carrier proteins. The following discussion will focus primarily on polypeptide hormones.
  • Hypothalamic hormones include thyrotropin-releasing hormone, gonadotropin- releasing hormone, somatostatin, growth-hormone releasing factor, corticotropin-releasing hormone, substance P, dopamine, and prolactin-releasing hormone. These hormones directly regulate the secretion of hormones from the anterior lobe of the pituitary.
  • Hormones secreted by the anterior pituitary include adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone, o somatotropic hormones such as growth hormone and prolactin, glycoprotein hormones such as thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), ⁇ - lipotropin, and ⁇ -endorphins.
  • ACTH adrenocorticotropic hormone
  • melanocyte-stimulating hormone such as growth hormone and prolactin
  • glycoprotein hormones such as thyroid-stimulating hormone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH), ⁇ - lipotropin, and ⁇ -endorphins.
  • FSH follicle-stimulating hormone
  • ⁇ -endorphins ⁇ -endorphins.
  • disorders of the hypothalamus and pituitary often result from lesions such as primary brain tumors, adenomas, infarction associated with pregnancy, hypophysectomy, aneurysms, vascular malformations, thrombosis, infections, immunological disorders, and complications due to head trauma. Such disorders have profound effects on the function of other endocrine glands.
  • disorders o associated with hypopituitarism include hypogonadism, Sheehan syndrome, diabetes insipidus,
  • Kallman's disease Hand-Schuller-Christian disease, Letterer-Siwe disease, sarcoidosis, empty sella syndrome, and dwarfism.
  • Disorders associated with hyperpituitarism include acromegaly, giantism, and syndrome of inappropriate ADH secretion (SIADH), often caused by benign adenomas.
  • SIADH inappropriate ADH secretion
  • Thyroid hormones secreted by the thyroid and parathyroid primarily control metabolic rates and the 5 regulation of serum calcium levels, respectively.
  • Thyroid hormones include calcitonin, somatostatin, and thyroid hormone.
  • the parathyroid secretes parathyroid hormone.
  • Disorders associated with hypothyroidism include goiter, myxedema, acute thyroiditis associated with bacterial infection, subacute thyroiditis associated with viral infection, autoimmune thyroiditis (Hashimoto's disease), and cretinism.
  • Disorders associated with hyperthyroidism include thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and
  • pancreatic hormones include insulin, glucagon, amylin, ⁇ - aminobutyric acid, gastrin, somatostatin, and pancreatic polypeptide.
  • the principal disorder 0 associated with pancreatic dysfunction is diabetes mellitus caused by insufficient insulin activity. Diabetes mellitus is generally classified as either Type I (insulin-dependent, juvenile diabetes) or Type II (non-insulin-dependent, adult diabetes). The treatment of both forms by insulin replacement therapy is well known.
  • Diabetes mellitus often leads to acute complications such as hypoglycemia (insulin shock), coma, diabetic ketoacidosis, lactic acidosis, and chronic complications leading to 5 disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection.
  • hypoglycemia insulin shock
  • coma coma
  • diabetic ketoacidosis lactic acidosis
  • chronic complications leading to 5 disorders of the eye, kidney, skin, bone, joint, cardiovascular system, nervous system, and to decreased resistance to infection.
  • Growth factors are secreted proteins that mediate intercellular communication. Unlike hormones, which travel great distances via the circulatory system, most growth factors are primarily local mediators that act on neighboring cells. Most growth factors contain a hydrophobic N-terminal 5 signal peptide sequence which directs the growth factor into the secretory pathway. Most growth factors also undergo post-translational modifications within the secretory pathway. These modifications can include proteolysis, glycosylation, phosphorylation, and intramolecular disulfide bond formation. Once secreted, growth factors bind to specific receptors on the surfaces of neighboring target cells, and the bound receptors trigger intracellular signal transduction pathways. o These signal transduction pathways elicit specific cellular responses in the target cells. These responses can include the modulation of gene expression and the stimulation or inhibition of cell division, cell differentiation, and cell motility.
  • Growth factors fall into at least two broad and overlapping classes.
  • the broadest class includes the large polypeptide growth factors, which are wide-ranging in their effects. These factors 5 include epidermal growth factor (EGF), fibroblast growth factor (FGF), transforming growth factor- ⁇ (TGF- ⁇ ), insulin-like growth factor (IGF), nerve growth factor (NGF), and platelet-derived growth factor (PDGF), eac efining a family of numerous related factors.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • TGF- ⁇ transforming growth factor- ⁇
  • IGF insulin-like growth factor
  • NGF nerve growth factor
  • PDGF platelet-derived growth factor
  • eac efining a family of numerous related factors.
  • the arge polypept e growth factors act as mitogens on diverse cell types to stimulate wound healing, bone synthesis and remodeling, extracellular matrix synthesis, and proliferation of epithelial, epidermal, and connective tissues.
  • NGF functions specifically as a neurotrophic factor, promoting neuronal growth and differentiation.
  • Another class of growth factors includes the hematopoietic growth factors, which are narrow in their target specificity. These factors stimulate the proliferation and differentiation of blood cells such as B-lymphocytes, T-lymphocytes, erythrocytes, platelets, eosinophils, basophils, neutrophils, 0 macrophages, and their stem cell precursors. These factors include the colony-stimulating factors (G-CSF, M-CSF, GM-CSF, and CSF1-3), erythropoietin, and the cytokines. The cytokines are specialized hematopoietic factors secreted by cells of the immune system and are discussed in detail below.
  • Growth factors play critical roles in neoplastic transformation of cells in vitro and in tumor 5 progression in vivo. Overexpression of the large polypeptide growth factors promotes the proliferation and transformation of cells in culture. Inappropriate expression of these growth factors by tumor cells in vivo may contribute to tumor vascularization and metastasis. Inappropriate activity of hematopoietic growth factors can result in anemias, leukemias, and lymphomas. Moreover, growth factors are both structurally and functionally related to oncoproteins, the potentially cancer- o causing products of proto-oncogenes.
  • FGF and PDGF family members are themselves homologous to oncoproteins, whereas receptors for some members of the EGF, NGF, and FGF families are encoded by proto-oncogenes. Growth factors also affect the transcriptional regulation of both proto-oncogenes and oncosuppressor genes (Pimentel, E. (1994) Handbook of Growth Factors. CRC Press, Ann Arbor MI; McKay, I. and I. Leigh, eds. (1993) Growth Factors: A Practical 5 Approach, Oxford University Press, New York NY; Habenicht, A., ed. (1990) Growth Factors, Differentiation Factors, and Cytokines. Springer- Verlag, New York NY).
  • Neuropeptides and vasomediators comprise a family of small peptide factors, typically of 20 amino acids or less. These factors generally function in neuronal excitation and 5 inhibition of vasoconstriction/vasodilation, muscle contraction, and hormonal secretions from the brain and other endocrine tissues.
  • neuropeptides and neuropeptide hormones such as bombesin, neuropeptide Y, neurotens n, neuromedin N, melanocortins, opioi s, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-bome signaling molecules such as angiotensin, complement, calcitonin, endothelins, formyl-methionyl peptides,
  • neuropeptide hormones such as bombesin, neuropeptide Y, neurotens n, neuromedin N, melanocortins, opioi s, galanin, somatostatin, tachykinins, urotensin II and related peptides involved in smooth muscle stimulation, vasopressin, vasoactive intestinal peptide, and circulatory system-bome signal
  • NP/VMs can transduce signals directly, modulate the activity or release of other neurotransmitters and hormones, and act as catalytic enzymes in signaling cascades.
  • the effects of NP/VMs range from extremely brief to long-lasting.
  • Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as B- and T-lymphocytes, monocytes, macrophages, 5 and granulocytes. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which alter gene expression patterns. There is considerable potential for the use of cytokines in the treatment of inflammation and immune system disorders.
  • Cytokines are secreted by hematopoietic cells in response to injury or infection. Interleukins, 0 neurotrophins, growth factors, interferons, and chemokines all define cytokine families that work in conjunction with cellular receptors to regulate cell proliferation and differentiation. In addition, cytokines effect activities such as leukocyte migration and function, hematopoietic cell proliferation, temperature regulation, acute response to infection, tissue remodeling, and apoptosis.
  • Cytokine structure and function have been extensively characterized in vitro. Most cytokines 5 are small polypeptides of about 30 kilodaltons or less. Over 50 cytokines have been identified from human and rodent sources. Examples of cytokine subfamilies include the interferons (IFN- ⁇ , - ⁇ , and - ⁇ ), the interleukins (BL1-IL13), the tumor necrosis factors (TNF- ⁇ and - ⁇ ), and the chemokines. Many cytokines have been produced using recombinant DNA techniques, and the activities of individual cytokines have been determined in vitro. These activities include regulation of leukocyte o proliferation, differentiation, and motility.
  • cytokine activity may not reflect the full scope of that cytokine' s activity in vivo.
  • Cytokines are not expressed individually in vivo but are instead expressed in combination with a multitude of other cytokines when the organism is challenged with a stimulus. Together, these cytokines collectively modulate the immune response in a manner appropriate for that 5 particular stimulus. Therefore, the physiological activity of a cytokine is determined by the stimulus itself and by complex interactive networks among co-expressed cytokines which may demonstrate both synergistic and antagonistic relationships.
  • Chemokines comprise a cytokine subfamily with over 30 members. (Reviewed in Wells, T.
  • Chemokines were initially identified as chemotactic proteins that recruit monocytes and macrophages to sites of inflammation. Chemokines
  • Chemokines are small chemoattractant cytokines involved in inflammation, leukocyte proliferation and migration, angiogenesis and angiostasis, regulation of hematopoiesis, HTV infectivity, and stimulation of cytokine secretion.
  • Chemokines generally contain 70-100 amino acids and are subdivided into four subfamilies based on the presence of conserved cysteine-based motifs. (Callard, R. and Gearing, A.
  • Chemokines may also play key roles in hematopoiesis and HTV-1 infection. Chemokines are small proteins which range from about 6-15 kilodaltons in molecular weight. Chemokines are further classified as C, CC, CXC, or CX 3 C based on the number and position of critical cysteine residues.
  • the CC chemokines for example, each contain a conserved motif consisting of two consecutive cysteines followed by two additional cysteines which occur 5 downstream at 24- and 16-residue intervals, respectively (ExPASy PROSITE database, documents PS00472 and PDOC00434).
  • the presence and spacing of these four cysteine residues are highly conserved, whereas the intervening residues diverge significantly.
  • a conserved tyrosine located about 15 residues downstream of the cysteine doublet seems to be important for chemotactic activity.
  • Most of the human genes encoding CC chemokines are clustered on chromosome 17, o although there are a few examples of CC chemokine genes that map elsewhere.
  • chemokines include lymphotactin (C chemokine); macrophage chemotactic and activating factor (MCAF/MCP-1; CC chemokine); platelet factor 4 and IL-8 (CXC chemokines); and fractalkine and neurotractin (CX 3 C chemokines).
  • C chemokine lymphotactin
  • MCAF/MCP-1 macrophage chemotactic and activating factor
  • CXC chemokines platelet factor 4 and IL-8
  • CX 3 C chemokines fractalkine and neurotractin
  • Receptor Molecules 5 The term receptor describes proteins that specifically recognize other molecules. The category is broad and includes proteins with a variety of functions. The bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis, and immune response.
  • receptors facilitate the selective transport of proteins out of the endoplasmic reticulum and localize enzymes to particular locations in o the cell.
  • the term may also be applied to proteins which act as receptors for ligands with known or unknown chemical composition and which interact with other cellular components.
  • the steroid hormone receptors bind to and regulate transcription of DNA.
  • Regulatory proteins such as growth factors coordinately control these cellular 5 processes and act as mediators in cell-cell signaling pathways.
  • Growth factors are secreted proteins that bind to specific cell-surface receptors on target cells. The bound receptors trigger intracellular signal transduction pathways which activate various downstream effectors that regulate gene expression, cell division, cell differentiation, cell motility, and other cellular processes.
  • Cell surface receptors are typically integral plasma membrane proteins. These receptors recognize hormones such as catecholamines; peptide hormones; growth and differentiation factors; small peptide factors such as thyrotropin-releasing hormone; galanin, somatostatin, and tachykinins; and circulatory system-bome signaling molecules.
  • Cell surface receptors on immune system cells recognize antigens, antibodies, and major histocompatibility complex (MHC)-bound peptides. Other cell surface receptors bind ligands to be internalized by the cell.
  • MHC major histocompatibility complex
  • LDL low density lipoproteins
  • transferrin glucose- or mannose-terminal glycoproteins, galactose-terminal glycoproteins, immunoglobulins, phosphovitellogenins, fibrin, proteinase-inhibitor complexes, plasminogen activators, and thrombospondin
  • growth factor receptors including receptors for epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, as well as the growth modulator ⁇ -thrombin, contain intrinsic protein kinase activities. When growth factor binds to the receptor, it triggers the autophosphorylation of a serine, threonine, or tyrosine residue on the receptor. These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins. These proteins participate in signaling pathways that eventually link the initial receptor activation at the cell surface to the activation of a specific intracellular target molecule.
  • SH2 domains and SH3 domains are found in phospholipase C- ⁇ , PI-3-K p85 regulatory subunit, Ras-GTPase activating protein, and pp60 c"src (Lowenstein, E.J. et al. (1992) Cell 70:431-442).
  • the cytokine family of receptors share a different common binding domain and include transmembrane receptors for growth hormone (GH), interleukins, erythropoietin, and prolactin.
  • receptors and second messenger-binding proteins have intrinsic serine/threonine protein kinase activity. These include activin/TGF- ⁇ /BMP-superfamily receptors, calcium- and diacylglycerol-activated/phospholipid-dependant protein kinase (PK-C), and RNA-dependant protein kinase (PK-R).
  • PKI calcium- and diacylglycerol-activated/phospholipid-dependant protein kinase
  • PK-R RNA-dependant protein kinase
  • serine/threonine protein kinases including nematode Twitchin, have fibronectin-like, immunoglobulin C2-like domains.
  • GPCR G-protein coupled receptors
  • N-terminus is of variable length and often glycosylated; the carboxy-terminus is cytoplasmic and generally phosphorylated.
  • GPCRs Three extracellular loops alternate with three intracellular loops to link the seven transmembrane regions. Cysteine disulfide bridges connect the second and third extracellular loops.
  • the most conserved regions of GPCRs are the transmembrane regions and the first two cytoplasmic loops. A conserved, acidic-Arg-aromatic residue triplet present in the second cytoplasmic loop may interact with G proteins.
  • a GPCR consensus pattern is characteristic of most proteins belonging to this superfamily (ExPASy PROSITE document PS00237; and Watson, S. and S. Arkinstall (1994) The G-protein Linked Receptor Facts Book, Academic Press, San Diego CA, pp. 2-6).
  • the transmembrane domains account for structural and functional features of the receptor.
  • the extracellular N-terminal segment or one or more of the three extracellular loops may also participate in ligand binding.
  • Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
  • the activated receptor interacts with an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or interactions with ion channel proteins (Baldwin, J.M. (1994) Curr. Opin. Cell Biol. 6: 180-190).
  • G guanine nucleotide binding
  • GPCRs include receptors for biogenic amines such as dopamine, epinephrine, histamine, glutamate (metabotropic-type), acetylcholine
  • lipid mediators of inflammation such as prostaglandins, platelet activating factor, and leukotrienes
  • peptide hormones such as calcitonin, C5a anaphylatoxin, follicle stimulating hormone, gonadotropin releasing hormone, neurokinin, oxytocin, and thrombin
  • sensory signal mediators such as retinal photopigments and olfactory stimulatory molecules.
  • the N-terminus interacts with ligands, the disulfide bridges interact with agonists and antagonists, and the large third intracellular loop interacts with G proteins to activate second messengers such as cyclic AMP, phospholipase C, inositol triphosphate, or ion channels.
  • second messengers such as cyclic AMP, phospholipase C, inositol triphosphate, or ion channels.
  • GPCRs include those for acetylcholine, adenosine, epinephrine and norepinephrine, bombesin, bradykinin, chemokines, dopamine, endothelin, ⁇ -aminobutyric acid (GABA), follicle- stimulating hormone (FSH), glutamate, gonadotropin-releasing hormone (GnRH), hepatocyte growth factor, histamine, leukotrienes, melanocortins, neuropeptide Y, opioid peptides, opsins, prostanoids, serotonin, somatostatin, tachykinins, thrombin, thyrotropin-releasing hormone (TRH), vasoactive intestinal polypeptide family, vasopressin and oxytocin, and orphan receptors.
  • GABA ⁇ -aminobutyric acid
  • FSH follicle- stimulating hormone
  • GnRH gonadotropin-releasing
  • GPCR mutations which may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin, supra). Mutations and changes in transcriptional activation of GPCR-encoding genes have been associated with neurological disorders such as schizophrenia, Parkinson's disease, Alzheimer's disease, drug addiction, and feeding disorders. For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene.
  • Rhodopsin is the retinal photoreceptor which is located within the discs of the eye rod cell.
  • Parma, J. et al. (1993, Nature 365:649-651) report that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as protooncogenes.
  • Nuclear receptors bind small molecules such as hormones or second messengers, leading to increased receptor-binding affinity to specific chromosomal DNA elements. In addition the affinity for other nuclear proteins may also be altered. Such binding and protein-protein interactions may regulate and modulate gene expression. Examples of such receptors include the steroid hormone receptors family, the retinoic acid receptors family, and the thyroid hormone receptors family. Ligand-Gated Receptor Ion Channels
  • Ligand-gated receptor ion channels fall into two categories.
  • the first category extracellular ligand-gated receptor ion channels (ELGs), rapidly transduce neurotransmitter-binding events into electrical signals, such as fast synaptic neurotransmission. ELG function is regulated by post- translational modification.
  • the second category intracellular ligand-gated receptor ion channels (ILGs), are activated by many intracellular second messengers and do not require post-translational modification(s) to effect a channel-opening response.
  • ELGs depolarize excitable cells to the threshold of action potential generation, h non- excitable cells, ELGs permit a limited calcium ion-influx during the presence of agonist.
  • ELGs include channels directly gated by neurotransmitters such as acetylcholine, L-glutamate, glycine, ATP, serotonin, GABA, and histamine.
  • ELG genes encode proteins having strong structural and functional similarities. ILGs are encoded by distinct and unrelated gene families and include receptors for cAMP, cGMP, calcium ions, ATP, and metabolites of arachidonic acid.
  • Macrophage Scavenger Receptors are integral membrane proteins with broad ligand specificity may participate in the binding of low density lipoproteins (LDL) and foreign antigens.
  • Scavenger receptors types I and II are trimeric membrane proteins with each subunit containing a small N- terminal intracellular domain, a transmembrane domain, a large extracellular domain, and a C- terminal cysteine-rich domain.
  • the extracellular domain contains a short spacer domain, an ⁇ -helical coiled-coil domain, and a triple helical coUagenous domain.
  • receptors have been shown to bind a spectrum of ligands, including chemically modified lipoproteins and albumin, polyribonucleotides, polysaccharides, phospholipids, and asbestos (Matsumoto, A. et al. (1990) Proc.
  • T-Cell Receptors are thought to play a key role in atherogenesis by mediating uptake of modified LDL in arterial walls, and in host defense by binding bacterial endotoxins, bacteria, and protozoa.
  • T-cell receptors are both structurally and functionally related to antibodies.
  • T-cell receptors are cell surface proteins that bind foreign antigens and mediate diverse aspects of the immune response.
  • a typical T-cell receptor is a heterodimer composed of two disulfide-linked polypeptide chains called ⁇ and ⁇ . Each chain is about 280 amino acids in length and contains one variable region and one constant region. Each variable or constant region folds into an Ig domain. The variable regions from the ⁇ and ⁇ chains come together in the heterodimer to form the antigen recognition site.
  • T-cell receptor diversity is generated by somatic rearrangement of gene segments encoding the ⁇ and ⁇ chains.
  • T-cell receptors recognize small peptide antigens that are expressed on the surface of antigen-presenting cells and pathogen-infected cells. These peptide antigens are presented on the cell surface in association with major histocompatibility proteins which provide the proper context for antigen recognition. / T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition with the transmission of signals that induce cell death in infected cells and stimulate proliferation of other immune cells.
  • TCR T cell receptor
  • MHC major histocompatibility molecule
  • Intracellular signaling is the general process by which cells respond to extracellular signals (hormones, neurotransmitters, growth and differentiation factors, etc.) through a cascade of biochemical reactions that begins with the binding of a signaling molecule to a cell membrane receptor and ends with the activation of an intracellular target molecule. Intermediate steps in the process involve the activation of various cytoplasmic proteins by phosphorylation via protein kinases, and their deactivation by protein phosphatases, and the eventual translocation of some of these activated proteins to the cell nucleus where the transcription of specific genes is triggered.
  • the intracellular signaling process regulates all types of cell functions including cell proliferation, cell differentiation, and gene transcription, and involves a diversity of molecules including protein kinases and phosphatases, and second messenger molecules, such as cyclic nucleotides, calcium-
  • Protein kinases and phosphatases play a key role in the intracellular signaling process by controlling the phosphorylation and activation of various signaling proteins.
  • the high energy phosphate for this reaction is generally transferred from the adenosine triphosphate molecule (ATP) 0 to a particular protein by a protein kinase and removed from that protein by a protein phosphatase.
  • ATP adenosine triphosphate molecule
  • Protein kinases are roughly divided into two groups: those that phosphorylate tyrosine residues (protein tyrosine kinases, PTK) and those that phosphorylate serine or threonine residues (serine/threonine kinases, STK).
  • a few protein kinases have dual specificity for serine/threonine and tyrosine residues. Almost all kinases contain a conserved 250-300 amino acid catalytic domain 5 containing specific residues and sequence motifs characteristic of the kinase family (Hardie, G. and S. Hanks (1995) The Protein Kinase Facts Books, Vol 1:7-20, Academic Press, San Diego CA).
  • STKs include the second messenger dependent protein kinases such as the cyclic-AMP dependent protein kinases (PKA), involved in mediating hormone-induced cellular responses; calcium-calmodulin (CaM) dependent protein kinases, involved in regulation of smooth muscle o contraction, glycogen breakdown, and neurotransmission; and the mitogen-activated protein kinases
  • PKA cyclic-AMP dependent protein kinases
  • CaM calcium-calmodulin dependent protein kinases
  • MAP MAP which mediate signal transduction from the cell surface to the nucleus via phosphorylation cascades.
  • Altered PKA expression is implicated in a variety of disorders and diseases including cancer, thyroid disorders, diabetes, atherosclerosis, and cardiovascular disease (Isselbacher, K.J. et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, New York NY, pp. 416-431, 1887). 5 PTKs are divided into transmembrane, receptor PTKs and nontransmembrane, non-receptor
  • Transmembrane PTKs are receptors for most growth factors.
  • Non-receptor PTKs lack transmembrane regions and, instead, form complexes with the intracellular regions of cell surface receptors.
  • Receptors that function through non-receptor PTKs include those for cytokines and hormones (growth hormone and prolactin) and antigen-specific receptors on T and B lymphocytes. o Many of these PTKs were first identified as the products of mutant oncogenes in cancer cells in which their activation was no longer subject to normal cellular controls.
  • HPK histidine protein kinase family
  • a histidine residue in the N-terminal half of the molecule (region I) is an autophosphorylation site.
  • Three additional motifs located in the C-terminal half of the molecule include an invariant asparagine residue in region II and two glycine-rich loops characteristic of
  • the two principal categories of protein phosphatases are the protein (serine/threonine) phosphatases (PPs) and the protein tyrosine 0 phosphatases (PTPs).
  • PPs dephosphorylate phosphoserine/threonine residues and are important regulators of many cAMP-mediated hormone responses (Cohen, P. (1989) Annu. Rev. Biochem. 58:453-508).
  • PTPs reverse the effects of protein tyrosine kinases and play a significant role in cell cycle and cell signaling processes (Charbonneau, supra).
  • PTPs may prevent or reverse cell transformation and the growth of various cancers by controlling the levels of tyrosine phosphorylation in cells. This hypothesis is supported by studies showing that overexpression of PTPs can suppress transformation in cells, and that specific inhibition of PTPs can enhance cell transformation (Charbonneau, supra).
  • Phospholipid and Inositol-Phosphate Signaling o Inositol phospholipids (phosphoinositides) are involved in an intracellular signaling pathway that begins with binding of a signaling molecule to a G-protein linked receptor in the plasma membrane.
  • lP 3 diffuses through the plasma membrane to induce calcium release from the endoplasmic reticulum (ER), while diacylglycerol remains in the membrane and helps activate protein kinase C, an STK that phosphorylates selected proteins in the target cell.
  • the calcium o response initiated by IP 3 is terminated by the dephosphorylation of IP 3 by specific inositol phosphatases.
  • Cellular responses that are mediated by this pathway are glycogen breakdown in the liver in response to vasopressin, smooth muscle contraction in response to acetylcholine, and thrombin-induced platelet aggregation.
  • cyclic- AMP dependent protein kinases (PKA) are thought to account or a l o t e effects of cAMP in most mammalian cells, including various hormone-induced cellular responses.
  • Visual excitation and the phototransmission of light signals in the eye is controlled by cyclic-GMP regulated, Ca 2+ -specific channels. Because of the importance of cellular levels of cyclic nucleotides
  • adenylyl cyclase which synthesizes cAMP from AMP, is activated to increase cAMP levels in muscle by binding of adrenaline to ⁇ -andrenergic receptors, while activation of guanylate cyclase and increased cGMP levels in photoreceptors leads to reopening of the
  • PDEs cyclic 0 nucleotides
  • cAMP and cGMP-specific phosphodiesterases produce the opposite of these and other effects mediated by increased cyclic nucleotide levels.
  • PDEs appear to be particularly important in the regulation of cyclic nucleotides, considering the diversity found in this family of proteins.
  • PDE1-7 At least seven families of mammalian PDEs (PDE1-7) have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo, 5 J.A. (1995) Physiological Reviews 75:725-748).
  • PDE inhibitors have been found to be particularly useful in treating various clinical disorders.
  • Rolipram a specific inhibitor of PDE4
  • Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases (Banner, K.H. and CP. Page (1995) Eur. Respir. J. 8:996-1000).
  • G-proteins are critical mediators of signal transduction between a particular class of extracellular receptors, the G-protein coupled receptors (GPCR), and intracellular second messengers such as cAMP and Ca 2+ .
  • G-proteins are linked to the cytosolic side of a GPCR such that activation of the GPCR by ligand binding stimulates binding of the G-protein to 5 GTP, inducing an "active" state in the G-protein. In the active state, the G-protein acts as a signal to trigger other events in the cell such as the increase of cAMP levels or the release of Ca 2+ into the cytosol from the ER, which, in turn, regulate phosphorylation and activation of other intracellular proteins.
  • the three polypeptide subunits of heterotrimeric G-proteins are the ⁇ , ⁇ , and ⁇ subunits.
  • the ⁇ subunit binds and hydrolyzes GTP.
  • the ⁇ and ⁇ subunits form a tight complex that anchors the 5 protein to the inner side of the plasma membrane.
  • the ⁇ subunits also known as G- ⁇ proteins or ⁇ transducins, contain seven tandem repeats of the WD-repeat sequence motif, a motif found in many proteins with regulatory functions. Mutations and variant expression of ⁇ transducin proteins are linked with various disorders (Neer, E.J. et al. (1994) Nature 371:297-300; Margottin, F. et al. (1998)
  • LMW GTP-proteins are GTPases which regulate cell growth, cell cycle control, protein secretion, and intracellular vesicle interaction. They consist of single polypeptides which, like the subunit of the heterotrimeric G-proteins, are able to bind and hydrolyze GTP, thus cycling between an inactive and an active state. At least sixty members of the LMW G-protein superfamily have been identified and are currently grouped into the six subfamilies of ras, rho, arf, sari, ran, and rab.
  • ras genes were initially found in human cancers, and subsequent studies confirmed that ras function is critical in determining whether cells continue to grow or become differentiated.
  • Other members of the LMW G-protein superfamily have roles in signal transduction that vary with the function of the activated genes and the locations of the G-proteins.
  • Guanine nucleotide exchange factors regulate the activities of LMW G-proteins by determining whether GTP or GDP is bound.
  • GTPase-activating protein GAP
  • GTP-ras GTPase-activating protein
  • GDP-ras guanine nucleotide releasing protein
  • RGS G-protein signaling
  • cytokine interleukin
  • Ca +2 is another second messenger molecule that is even more widely used as an intracellular mediator than cAMP.
  • Ca 2+ directly activates regulatory enzymes, such as protein kinase C, which trigger signal transduction pathways.
  • Ca 2+ also binds to specific Ca 2+ -binding proteins (CBPs) such as calmodulin (CaM) which then activate multiple target proteins in the cell including enzymes, membrane transport pumps, and ion channels.
  • CBPs Ca 2+ -binding proteins
  • CaM interactions are involved in a multitude of cellular processes including, but not limited to, gene regulation, DNA synthesis, cell cycle progression, mitosis, cytokinesis, cytoskeletal organization, muscle contraction, signal transduction, ion homeostasis, exocytosis, and metabolic regulation (Celio, M.R. et al. (1996) Guidebook to
  • Calsequestrin is one such CBP that is expressed in isoforms specific to cardiac muscle and skeletal muscle. It is suggested that calsequestrin binds Ca 2+ in a rapidly exchangeable state that is released during Ca 2+ -signaling conditions (Celio, M.R. et al.
  • Cell division is the fundamental process by which all living things grow and reproduce. In most organisms, the cell cycle consists of three principle steps; interphase, mitosis, and cytokinesis.
  • Interphase involves preparations for cell division, replication of the DNA and production of essential 0 proteins.
  • mitosis the nuclear material is divided and separates to opposite sides of the cell.
  • Cytokinesis is the final division and fission of the cell cytoplasm to produce the daughter cells.
  • Cyclins act by binding to and activating a group of cyclin-dependent protein kinases (Cdks) which then phosphorylate and activate selected proteins 5 involved in the mitotic process.
  • Cdks cyclin-dependent protein kinases
  • cyclin B which controls entry of the cell into mitosis
  • Gl cyclin which controls events that drive the cell out of mitosis.
  • Ceretain proteins in intracellular signaling pathways serve to link or cluster other proteins o involved in the signaling cascade.
  • a conserved protein domain called the PDZ domain has been identified in various membrane-associated signaling proteins. This domain has been implicated in receptor and ion channel clustering and in the targeting of multiprotein signaling complexes to specialized functional regions of the cytosolic face of the plasma membrane. (For a review of PDZ domain-containing proteins, see Ponting, CP. et al.
  • PDZ domains are found in die eukaryotic MAGUK (membrane-associated guanylate kinase) protein family, members of which bind to the intracellular domains of receptors and channels.
  • MAGUK membrane-associated guanylate kinase
  • PDZ domains are also found in diverse membrane-localized proteins such as protein tyrosine phosphatases, serine/threonine kinases, G-protein cofactors, and synapse-associated proteins such as syntrophins and neuronal nitric oxide synthase (nNOS).
  • nNOS neuronal nitric oxide synthase
  • the plasma membrane acts as a barrier to most molecules. Transport between the cytoplasm and the extracellular environment, and between the cytoplasm and lumenal spaces of cellular 5 organelles requires specific transport proteins. Each transport protein carries a particular class of molecule, such as ions, sugars, or amino acids, and often is specific to a certain molecular species of t e class. A variety o uman in erite diseases are caused by a mutat on in a transport protein. For example, cystinuria is an inherited disease that results from the inability to transport cystine, the disulfide-linked dimer of cysteine, from the urine into the blood. Accumulation of cystine in the urine leads to the formation of cystine stones in the kidneys. Transport proteins are multi-pass transmembrane proteins, which either actively transport molecules across the membrane or passively allow them to cross. Active transport involves directional pumping of a solute across the membrane, usually against an electrochemical gradient.
  • Active transport is tightly coupled to a source of metabolic energy, such as ATP hydrolysis or an electrochemically favorable ion gradient.
  • Passive transport involves the movement of a solute down its electrochemical gradient.
  • Transport proteins can be further classified as either carrier proteins or channel proteins.
  • Carrier proteins which can function in active or passive transport, bind to a specific solute to be transported and undergo a conformational change which transfers the bound solute across the membrane.
  • Channel proteins which only function in passive transport, form hydrophilic pores across the membrane. When the pores open, specific solutes, such as inorganic ions, pass through the membrane and down the electrochemical gradient of the solute.
  • Carrier proteins which transport a single solute from one side of the membrane to the other are called uniporters.
  • coupled transporters link the transfer of one solute with simultaneous or sequential transfer of a second solute, either in the same direction (symport) or in the opposite direction (antiport).
  • intestinal and kidney epithelium contains a variety of symporter systems driven by the sodium gradient that exists across the plasma membrane. Sodium moves into the cell down its electrochemical gradient and brings the solute into the cell with it. The sodium gradient that provides the driving force for solute uptake is maintained by the ubiquitous Na + /K + ATPase.
  • Sodium-coupled transporters include the mammalian glucose transporter (SGLT1), iodide transporter (NIS), and multivitamin transporter (SMVT). All three transporters have twelve putative transmembrane segments, extracellular glycosylation sites, and cytoplasmically-oriented N- and C-termini. NIS plays a crucial role in the evaluation, diagnosis, and treatment of various thyroid pathologies because it is the molecular basis for radioiodide thyroid-imaging techniques and for specific targeting of radioisotopes to the thyroid gland (Levy, O. et al. (1997) Proc. Natl. Acad. Sci. USA 94:5568-5573).
  • SMVT is expressed in the intestinal mucosa, kidney, and placenta, and is implicated in the transport of the water-soluble vitamins, e.g., biotin and pantothenate (Prasad, P.D. et al. (1998) J. Biol. Chem. 273:7501-7506).
  • Transporters play a major role in the regulation of pH, excretion of drugs, and the cellular K7Na + balance.
  • Monocarboxylate anion transporters are proton-coupled symporters with a broad substrate specificity that includes L-lactate, pyruvate, and the ketone bodies acetate, acetoacetate, and beta-hydroxybutyrate.
  • At least seven isoforms have been identified to date. The isoforms are predicted to have twelve transmembrane (TM) helical domains with a large intracellular loop between TM6 and TM7, and play a critical role in maintaining intracellular pH by removing t e protons t at are produced stoichiometrically with lactate during glycolysis. The best characterized
  • H(+)-monocarboxylate transporter is that of the erythrocyte membrane, which transports L-lactate and a wide range of other aliphatic monocarboxylates.
  • Other cells possess H(+)-linked monocarboxylate transporters with differing substrate and inhibitor selectivities.
  • cardiac muscle and tumor cells have transporters that differ in their K m values for certain substrates, including stereoselectivity for L- over D-lactate, and in their sensitivity to inhibitors.
  • Na(+)-monocarboxylate cotransporters on the luminal surface of intestinal and kidney epithelia, which allow the uptake of lactate, pyruvate, and ketone bodies in these tissues.
  • organic anion transporters are selective for hydrophobic, charged molecules with electron-attracting side groups.
  • Organic cation transporters such as the ammonium transporter, mediate the secretion of a variety of drugs and endogenous metabolites, and contribute to the maintenance of intercellular pH.
  • ABC transporters are also called the "traffic ATPases” comprising a superfamily of membrane proteins that mediate transport and channel functions in prokaryotes and eukaryotes (Higgins, CF. (1992) Annu. Rev. Cell Biol. 8:67-113). ABC proteins share a similar overall structure and significant sequence homology. AU ABC proteins contain a conserved domain of approximately two hundred amino acid residues which includes one or more nucleotide binding domains.
  • ABC proteins consist of four modules: two nucleotide-binding domains (NBD), which hydrolyze ATP to supply the energy required for transport, and two membrane-spanning domains (MSD), each containing six putative transmembrane segments.
  • NBD nucleotide-binding domains
  • MSD membrane-spanning domains
  • These four modules may be encoded by a single gene, as is the case for the cystic fibrosis transmembrane regulator (CFTR), or by separate genes. When encoded by separate genes, each gene product contains a single NBD and MSD.
  • CFTR cystic fibrosis transmembrane regulator
  • each gene product contains a single NBD and MSD.
  • These "half-molecules” form homo- and heterodimers, such as Tapl and Tap2, the endoplasmic reticulum- based major l ⁇ stocompatibility (MHC) peptide transport system.
  • MHC major l ⁇ stocompatibility
  • ABC transporters can transport substances that differ markedly in chemical structure and size, ranging from small molecules such as ions, sugars, amino acids, peptides, and phospholipids, to lipopeptides, large proteins, and complex hydrophobic drugs. Mutations in ABC transporter genes are associated with various disorders, such as hyperbilirubinemia II/Dubin- Johnson syndrome, recessive Stargardt's disease, and celiac disease.
  • cystic fibrosis an ion channel
  • X-linked adrenoleukodystrophy adrenoleukodystrophy protein, ALDP
  • Zellweger syndrome peroxisomal membrane protein-70, PMP70
  • ABC transporter in human cancer cells makes the cells resistant to a variety of cytotoxic drags used in chemotherapy (Taglight, D. and S. Michaelis (1998) Meth. Enzymol. 292:131-163). Transport of fatty acids across the plasma membrane can occur by diffusion, a high capacity, low affinity process. However, under normal physiological conditions a significant fraction of fatty acid transport appears to occur via a high affinity, low capacity protein-mediated transport process.
  • Fatty acid transport protein an integral membrane protein with four transmembrane segments, is expressed in tissues exhibiting high levels of plasma membrane fatty acid flux, such as muscle, heart, and adipose.
  • FATP Fatty acid transport protein
  • Expression of FATP is upregulated in 3T3-L1 cells during adipose conversion, and expression in COS7 fibroblasts elevates uptake of long-chain fatty acids (Hui, T.Y. et al. (1998) J. Biol. Chem. 273:27420-27429).
  • the proteins in this family contain a highly conserved, large transmembrane domain composed of 12 ⁇ -helices, and several weakly conserved, cytoplasmic and exoplasmic domains. (Pessin, J.E. and Bell, G.I. (1992) Annu. Rev. Physiol. 54:911-930.)
  • Amino acid transport is mediated by Na + dependent amino acid transporters. These transporters are involved in gastrointestinal and renal uptake of dietary and cellular amino acids and in neuronal reuptake of neurotransmitters. Transport of cationic amino acids is mediated by the system y+ family and the cationic amino acid transporter (CAT) family. Members of the CAT family share a high degree of sequence homology, and each contains 12-14 putative transmembrane domains. (Ito, K. and Groudine, M. (1997) J. Biol. Chem. 272:26780-26786.) Ion Channels The electrical potential of a cell is generated and maintained by controlling the movement of ions across the plasma membrane.
  • Ion transporters utilize the energy obtained from ATP hydrolysis to actively transport an ion against the ion's concentration gradient.
  • Gated ion channels allow passive flow of an ion down the ion' s electrochemical gradient under restricted conditions. Together, these types of ion channels generate, maintain, and utilize an electrochemical gradient that is used in 1) electrical impulse conduction down the axon of a nerve cell, 2) transport of molecules into cells against concentration gradients, 3) initiation of muscle contraction, and 4) endocrine cell secretion.
  • Ion transporters generate and maintain the resting electrical potential of a cell. Utilizing the energy derived from ATP hydrolysis, they transport ions against the ion' s concentration gradient. These transmembrane ATPases are divided into three families.
  • the phosphorylated (P) class ion transporters, nc u ng a - ase, a - ase, an - T ase, are act vate y a phosphorylation event.
  • P-class ion transporters are responsible for maintaining resting potential distributions such that cytosolic concentrations of Na + and Ca 2+ are low and cytosolic concentration of K + is high.
  • the vacuolar (V) class of ion transporters includes H + pumps on intracellular
  • V-class ion transporters are responsible for generating the low pH within the lumen of these organelles that is required for function.
  • the coupling factor (F) class consists of H + pumps in the mitochondria. F-class ion transporters utilize a proton gradient to generate ATP from ADP and inorganic phosphate (P ; ).
  • the resting potential of the cell is utilized in many processes involving carrier proteins and 0 gated ion channels.
  • Carrier proteins utilize the resting potential to transport molecules into and out of the cell.
  • Amino acid and glucose transport into many cells is linked to sodium ion co-transport (symport) so that the movement of Na + down an electrochemical gradient drives transport of the other molecule up a concentration gradient.
  • cardiac muscle links transfer of Ca 2+ out of the cell with transport of Na + into the cell (antiport).
  • 5 Ion channels share common structural and mechanistic themes.
  • the channel consists of four or five subunits or protein monomers that are arranged like a barrel in the plasma membrane.
  • Each subunit typically consists of six potential transmembrane segments (SI, S2, S3, S4, S5, and S6).
  • the center of the barrel forms a pore lined by ⁇ -helices or ⁇ -strands.
  • the side chains of the amino acid residues comprising the ⁇ -helices or ⁇ -strands establish the charge (cation or anion) selectivity of the o channel.
  • the degree of selectivity, or what specific ions are allowed to pass through the channel depends on the diameter of the narrowest part of the pore.
  • Gated ion channels control ion flow by regulating the opening and closing of pores. These channels are categorized according to the manner of regulating the gating function. Mechanically- gated channels open pores in response to mechanical stress, voltage-gated channels open pores in 5 response to changes in membrane potential, and ligand-gated channels open pores in the presence of a specific ion, nucleotide, or neurotransmitter.
  • Voltage-gated Na + and K + channels are necessary for the function of electrically excitable cells, such as nerve and muscle cells. Action potentials, which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeability of the membrane to Na + 0 and K + ions. Depolarization of the membrane beyond the threshold level opens voltage-gated Na + channels. Sodium ions flow into the cell, further depolarizing the membrane and opening more voltage-gated Na + channels, which propagates the depolarization down the length of the cell. Depolarization also opens voltage-gated potassium channels. Consequently, potassium ions flow outward, which leads to repolarization of the membrane.
  • Voltage-gated channels utilize charged 5 residues in the fourth transmembrane segment (S4) to sense voltage change.
  • the open state lasts only about 1 millisecond, at which time the channel spontaneously converts into an inactive state that cannot be opened irrespective of the membrane potential, hiactivation is mediated by the channel's
  • N-terminus which acts as a plug that closes the pore.
  • the transition from an inactive to a closed state requires a return to resting potential.
  • Voltage-gated Na + channels are heterotrimeric complexes composed of a 260 kDa pore
  • the ⁇ 2 subunit is an integral membrane glycoprotein that contains an extracellular Ig domain, and its association with ⁇ and ⁇ l subunits correlates with increased functional expression of the channel, a change in its gating properties, and an increase in whole cell capacitance due to an increase in membrane surface area.
  • o Voltage-gated Ca 2+ channels are involved in presynaptic neurotransmitter release, and heart and skeletal muscle contraction.
  • the voltage-gated Ca 2+ channels from skeletal muscle (L-type) and brain (N-type) have been purified, and though their functions differ dramatically, they have similar subunit compositions.
  • the channels are composed of three subunits.
  • the ⁇ j subunit forms the membrane pore and voltage sensor, while the ⁇ 2 ⁇ and ⁇ subunits modulate the voltage-dependence, 5 gating properties, and the current amplitude of the channel.
  • subunits are encoded by at least six ⁇ 1; one ⁇ 2 ⁇ , and four ⁇ genes.
  • a fourth subunit, ⁇ has been identified in skeletal muscle. (Walker, D. et al. (1998) J. Biol. Chem. 273:2361-2367; and Jay, S.D. et al. (1990) Science 248:490- 492.)
  • Chloride channels are necessary in endocrine secretion and in regulation of cytosolic and o organelle pH.
  • Cl " enters the cell across a basolateral membrane tlirough an Na + , K7CI " cotransporter, accumulating in the cell above its electrochemical equilibrium concentration.
  • the cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel encoded by the gene for cystic fibrosis, a common fatal 5 genetic disorder in humans.
  • Loss of CFTR function decreases transepithelial water secretion and, as a result, the layers of mucus that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated and difficult to clear. The resulting blockage of these sites leads to pancreatic insufficiency, "meconium ileus", and devastating "chronic obstructive pulmonary disease” (Al- Awqati, Q. et al. (1992) J. Exp. Biol. 172:245-266).
  • Many intracellular organelles contain H + -ATPase pumps that generate transmembrane pH and electrochemical differences by moving protons from the cytosol to the organelle lumen.
  • Functions that require a low vacuolar pH include uptake of small molecules such as biogenic am nes in chromaffin granules, processing of vacuolar const tuents such as pro-hormones by proteolytic enzymes, and protein degradation in lysosomes (Al-Awqati, supra).
  • Ligand-gated channels open their pores when an extracellular or intracellular mediator binds
  • Neurotransmitter-gated channels are channels that open when a neurotransmitter binds to their extracellular domain. These channels exist in the postsynaptic membrane of nerve or muscle cells. There are two types of neurotransmitter-gated channels. Sodium channels open in response to excitatory neurotransmitters, such as acetylcholine, glutamate, and serotonin. This opening causes an influx of Na + and produces the initial localized depolarization that activates the 0 voltage-gated channels and starts the action potential. Chloride channels open in response to inhibitory neurotransmitters, such as ⁇ -aminobutyric acid (GABA) and glycine, leading to hyperpolarization of the membrane and the subsequent generation of an action potential.
  • GABA ⁇ -aminobutyric acid
  • Ligand-gated channels can be regulated by intracellular second messengers.
  • Calcium- activated K + channels are gated by internal calcium ions. In nerve cells, an influx of calcium during 5 depolarization opens K + channels to modulate the magnitude of the action potential (Ishi, T.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656).
  • Cyclic nucleotide-gated (CNG) channels are gated by cytosolic cyclic nucleotides. The best examples of these are the cAMP-gated Na + channels involved in off action and the cGMP-gated cation channels involved in vision. Both systems involve ligand-mediated activation of a G-protein coupled receptor which then alters the level of cyclic o nucleotide within the cell.
  • Ion channels are expressed in a number of tissues where they are implicated in a variety of processes. CNG channels, while abundantly expressed in photoreceptor and olfactory sensory cells, are also found in kidney, lung, pineal, retinal ganglion cells, testis, aorta, and brain. Calcium- activated K + channels may be responsible for the vasodilatory effects of bradykinin in the kidney and 5 for shunting excess K + from brain capillary endothelial cells into the blood. They are also implicated in repolarizing granulocytes after agonist-stimulated depolarization (Ishi, supra). Ion channels have been the target for many drug therapies.
  • Neurotransmitter-gated channels have been targeted in therapies for treatment of insomnia, anxiety, depression, and schizophrenia.
  • Voltage-gated channels have been targeted in therapies for arrhythmia, ischemic stroke, head trauma, and neurodegenerative o disease (Taylor, CP. and L.S. Narasimhan (1997) Adv. Pharmacol. 39:47-98).
  • Protein Modification and Maintenance Molecules The cellular processes regulating modification and maintenance of protein molecules coordinate their conformation, stabilization, and degradation. Each of these processes is mediated by key enzymes or proteins such as proteases, protease inhibitors, transferases, isomerases, and molecular chaperones.
  • proteases protease inhibitors
  • transferases transferases
  • isomerases and molecular chaperones.
  • Proteases proteave proteins and peptides at the peptide bond that forms the backbone of the peptide and protein chain. Proteolytic processing is essential to cell growth, differentiation, remodeling, and homeostasis as well as inflammation and immune response.
  • Typical protein half- lives range from hours to a few days, so that within all living cells, precursor proteins are being cleaved to then active form, signal sequences proteolytically removed from targeted proteins, and aged or defective proteins degraded by proteolysis.
  • Proteases function in bacterial, parasitic, and viral invasion and replication within a host.
  • Four principal categories of mammalian proteases have been identified based on active site structure, mechanism of action, and overall three-dimensional structure. (Beynon, R.J. and J.S. Bond (1994) Proteolytic Enzymes: A Practical Approach, Oxford University Press, New York NY, pp. 1-5).
  • SPs serine proteases
  • the serine proteases have a serine residue, usually within a conserved sequence, in an active site composed of the serine, an aspartate, and a histidine residue.
  • SPs include the digestive enzymes trypsin and chymotrypsin, components of the complement cascade and the blood-clotting cascade, and enzymes that control extracellular protein degradation.
  • the main SP sub-families are trypases, which cleave after arginine or lysine; aspartases, which cleave after aspartate; chymases, which cleave after phenylalanine or leucine; metases, which cleavage after methionine; and serases which cleave after serine.
  • Enterokinase the initiator of intestinal digestion, is a serine protease found in the intestinal brush border, where it cleaves the acidic propeptide from trypsinogen to yield active trypsin (Kitamoto, Y. et al. (1994) Proc. Natl. Acad. Sci. USA 91:7588-7592).
  • Prolylcarboxypeptidase a lysosomal serine peptidase that cleaves peptides such as angiotensin II and UI and [des-Arg9] bradykinin, shares sequence homology with members of both the serine carboxypeptidase and prolylendopeptidase families (Tan, F. et al. (1993) J. Biol. Chem. 268:16631- 16638).
  • Cysteine proteases have a cysteine as the major catalytic residue at an active site where catalysis proceeds via an intermediate thiol ester and is facilitated by adjacent histidine and aspartic acid residues.
  • CPs are involved in diverse ceUular processes ranging from the processing of precursor proteins to intracellular degradation. Mammalian CPs include lysosomal cathepsins and cytosolic calcium activated proteases, calpains.
  • CPs are produced by monocytes, macrophages and other cells of the immune system which migrate to sites of inflammation and secrete molecules involved in tissue repair. Overabundance of these repair molecules plays a role in certain disorders.
  • autoimmune diseases such as rheumatoid arthritis
  • secretion of the cysteine peptidase cathepsin C degrades collagen, laminin, elastin and other structural proteins found in the extracellular matrix of bones.
  • Aspartic proteases are members of the cathepsin family of lysosomal proteases and include pepsin A, gastricsin, chymosin, renin, and cathepsins D and E. Aspartic proteases have a pair of aspartic acid residues in the active site, and are most active in the pH 2 - 3 range, in which one of the aspartate residues is ionized, the other un-ionized. Aspartic proteases include bacterial penicillopepsin, mammalian pepsin, renin, chymosin, and certain fungal proteases. Abnormal regulation and expression of cathepsins is evident in various inflammatory disease states.
  • the mRNA for stromelysin, cytokines, TIMP-1, cathepsin, gelatinase, and other molecules is preferentially expressed.
  • Expression of cathepsins L and D is elevated in synovial tissues from patients with rheumatoid arthritis and osteoarthritis.
  • Cathepsin L expression may also contribute to the influx of mononuclear cells which exacerbates the destruction of the rheumatoid synovium. (Keyszer, G.M. (1995) Arthritis Rheum.
  • Metalloproteases have active sites that include two glutamic acid residues and one histidine residue that serve as binding sites for zinc.
  • Carboxypeptidases A and B are the principal mammalian metalloproteases. Both are exoproteases of similar structure and active sites.
  • Carboxypeptidase A like chymotrypsin, prefers C-terminal aromatic and aliphatic side chains of hydrophobic nature, whereas carboxypeptidase B is directed toward basic arginine and lysine residues.
  • Glycoprotease (GCP), or O-sialoglycoprotein endopeptidase is a metallopeptidase which specifically cleaves O-sialoglycoproteins such as glycophorin A.
  • P-LAP placental leucine aminopeptidase
  • Ubiquitin proteases are associated with the ubiquitin conjugation system (UCS), a major pathway for the degradation of cellular proteins in eukaryotic cells and some bacteria.
  • the UCS mediates the elimination of abnormal proteins and regulates the half-lives of important regulatory proteins that control cellular processes such as gene transcription and cell cycle progression.
  • proteins targeted for degradation are conjugated to a ubiquitin, a small heat stable protein.
  • the ubiquitinated protein is then recognized and degraded by proteasome, a large, multisubunit proteolytic enzyme complex, and ubiquitin is released for reutilization by ubiquitin protease.
  • the UCS is implicated in the degradation of mitotic cyclic kinases, oncoproteins, tumor suppressor genes such as p53, viral proteins, cell surface receptors associated with signal transduction, transcriptional regulators, and mutated or damaged proteins (Ciechanover, A. (1994)
  • a murine proto-oncogene, Unp encodes a nuclear ubiquitin protease whose overexpression leads to oncogenic transformation of NIH3T3 cells, and the human homolog of this gene is consistently elevated in small cell tumors and adenocarcinomas of the lung (Gray, D.A.
  • the mechanism for the translocation process into the endoplasmic reticulum (ER) involves the recognition of an N-terminal signal peptide on the elongating protein.
  • the signal peptide directs the protein and attached ribosome to a receptor on the ER membrane.
  • the polypeptide chain passes through a pore in the ER membrane into the lumen while the N-terminal signal peptide remains attached at the membrane surface.
  • the process is completed when signal peptidase located inside the 5 ER cleaves the signal peptide from the protein and releases the protein into the lumen.
  • Protease inhibitors and other regulators of protease activity control the activity and effects of proteases.
  • Protease inhibitors have been shown to control pathogenesis in animal models of proteolytic disorders (Murphy, G. (1991) Agents Actions Suppl. 35:69-76).
  • Low levels of the 0 cystatins, low molecular weight inhibitors of the cysteine proteases correlate with malignant progression of tumors. (Calkins, C. et al (1995) Biol. Biochem. Hoppe Seyler 376:71-80).
  • Serpins are inhibitors of mammalian plasma serine proteases. Many serpins serve to regulate the blood clotting cascade and/or the complement cascade in mammals.
  • Sp32 is a positive regulator of the mammalian acrosomal protease, acrosin, that binds the proenzyme, proacrosin , and thereby aides in 5 packaging the enzyme into the acrosomal matrix (Baba, T. et al. (1994) J. Biol. Chem. 269:10133- 10140).
  • the Kunitz family of serine protease inhibitors are characterized by one or more "Kunitz domains" containing a series of cysteine residues that are regularly spaced over approximately 50 amino acid residues and form three intrachain disulfide bonds.
  • TFPI-1 and TFPI-2 tissue factor pathway inhibitor
  • inter- ⁇ -trypsin inhibitor inter- ⁇ -trypsin inhibitor
  • bikunin bikunin.
  • TFPI-1 and TFPI-2 tissue factor pathway inhibitor
  • TFPI-2 tissue factor pathway inhibitor
  • bikunin bikunin.
  • o Marlor, C.W. et al. (1997) J. Biol. Chem. 272: 12202-12208.
  • Aprotinin has clinical utility in reduction of perioperative blood loss.
  • Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl-prolyl isomerase (PPI).
  • PDI protein disulfide isomerase
  • PPI peptidyl-prolyl isomerase
  • PDI catalyzes the oxidation of free sulfhydryl groups in cysteine residues to form intramolecular disulfide bonds in proteins.
  • PPI an enzyme that catalyzes the isomerization of certain proline irnidic bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation.
  • cyclophilins represent a major class of PPI that was originally identified as the major receptor for the immunosuppressive drug cyclosporin A (Handschumacher, R.E. et al. (1984) Science 226: 544-547). Protein Glycosylation
  • O-linked glycosylation of proteins also occurs in the ER by the addition of N-acetylgalactosamine to the hydroxyl group of a serine or threonine residue followed by the sequential addition of other sugar residues to the first. This process is catalysed by a series of glycosyltransferases each specific for a particular donor sugar nucleotide and acceptor molecule (Lodish, H. et al. (1995) Molecular Cell Biology, W.H. Freeman and Co., New York NY, pp.700-708). In many cases, both N- and O-linked oligosaccharides appear to be required for the secretion of proteins or the movement of plasma membrane glycoproteins to the cell surface.
  • An additional glycosylation mechanism operates in the ER specifically to target lysosomal enzymes to lysosomes and prevent their secretion.
  • Lysosomal enzymes in the ER receive an N- linked oligosaccharide, like plasma membrane and secreted proteins, but are then phosphorylated on one or two mannose residues.
  • the phosphorylation of mannose residues occurs in two steps, the first step being the addition of an N-acetylglucosamine phosphate residue by N-acetylglucosamine phosphotransferase, and the second the removal of the N-acetylglucosamine group by phosphodiesterase.
  • Chaperones Molecular chaperones are proteins that aid in the proper folding of immature proteins and refolding of improperly folded ones, the assembly of protein subunits, and in the transport of unfolded proteins across membranes. Chaperones are also called heat-shock proteins (hsp) because of their tendency to be expressed in dramatically increased amounts following brief exposure of cells to elevated temperatures. This latter property most likely reflects their need in the refolding of proteins that have become denatured by the high temperatures.
  • hsp heat-shock proteins
  • Chaperones may be divided into several classes according to their location, function, and molecular weight, and include hsp60, TCPl, hsp70, hsp40 (also called DnaJ), and hsp90.
  • hsp90 binds to steroid hormone receptors, represses transcription in the absence of the ligand, and provides proper folding of the ligand-binding domain of the receptor in the presence of the hormone (Burston, S.G. and A.R. Clarke (1995) Essays
  • Hsp60 and hsp70 chaperones aid in the transport and folding of newly synthesized proteins.
  • Hsp70 acts early in protein folding, binding a newly synthesized protein before it leaves the ribosome and transporting the protein to the mitochondria or ER before releasing the folded protein.
  • Hsp60, along with hsp 10 binds misfolded proteins and gives them the opportunity to refold correctly.
  • AU chaperones share an affinity for hydrophobic patches on incompletely folded proteins and the ability to hydrolyze ATP. The energy of ATP hydrolysis is used to release the hsp- bound protein in its properly folded state (Alberts, supra, pp 214, 571-572).
  • DNA and RNA replication are critical processes for cell replication and function.
  • DNA and RNA replication are mediated by the enzymes DNA and RNA polymerase, respectively, by a "templating" process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
  • templating the process in which the nucleotide sequence of a DNA or RNA strand is copied by complementary base-pairing into a complementary nucleic acid sequence of either DNA or RNA.
  • DNA polymerase catalyzes the stepwise addition of a deoxyribonucleotide to the 3' -OH end of a polynucleotide strand (the primer strand) that is paired to a second (template) strand.
  • the new DNA strand therefore grows in the 5' to 3' direction (Alberts, B. et al. (1994) The Molecular Biology of the Cell, Garland Publishing Inc., New York NY, pp. 251-254).
  • the substrates for the polymerization reaction are the corresponding deoxynucleotide triphosphates which must base-pair with the correct nucleotide on the template strand in order to be recognized by the polymerase.
  • DNA polymerase is also involved in the repair of damaged DNA as discussed below under “Ligases.”
  • RNA polymerase uses a DNA template strand to "transcribe"
  • RNA polymerization proceeds in a 5' to 3' direction by addition of a ribonucleoside monophosphate to the
  • RNA polymerase 3' -OH end of a growing RNA chain. Transcription of DNA into RNA takes place in the nucleus and is catalyzed by RNA polymerases. DNA transcription generates messenger RNAs (mRNA) that carry information for protein synthesis, as well as the transfer, ribosomal, and other RNAs that have structural or catalytic functions.
  • mRNA messenger RNAs
  • RNA polymerase I makes the large ribosomal RNAs
  • RNA polymerase II makes the mRNAs that will be translated into proteins
  • RNA polymerase III makes a variety of small, stable RNAs, including
  • RNA synthesis is initiated by binding of the RNA polymerase to a promoter region on the DNA and synthesis begins at a start site within the promoter. Synthesis is completed at a broad, general stop or termination region in the
  • RNA polymerase II The primary transcript of RNA polymerase II is called heterogenous nuclear RNA (hnRNA), and must be further processed by splicing to remove non-coding sequences called introns. RNA splicing is mediated by small nuclear ribonucleoprotein complexes, or snRNPs, producing mature messenger RNA (mRNA) which is then transported out of the nucleus for translation into proteins.
  • DNA repair is the process by which accidental base changes, such as those produced by oxidative damage, hydrolytic attack, or uncontrolled methylation of DNA are corrected before replication or transcription of the DNA can occur. Because of the efficiency of the DNA repair process, fewer than one in one thousand accidental base changes causes a mutation (Alberts, supra, pp. 245-249).
  • the three steps common to most types of DNA repair are (1) excision of the damaged or altered base or nucleotide by DNA nucleases, leaving a gap; (2) insertion of the correct nucleotide in this gap by DNA polymerase using the complementary strand as the template; and (3) sealing the break left between the inserted nucleotide(s) and the existing DNA strand by DNA ligase.
  • DNA ligase uses the energy from ATP hydrolysis to activate the 5' end of the broken phosphodiester bond before forming the new bond with the 3'-OH of the DNA strand.
  • Bloom's syndrome an inherited human disease, individuals are partially deficient in DNA ligation and consequently have an increased incidence of cancer (Alberts, supra, p. 247).
  • Nucleases comprise both enzymes that hydrolyze DNA (DNase) and RNA (RNase). They serve different purposes in nucleic acid metabolism. Nucleases hydrolyze the phosphodiester bonds between adjacent nucleotides either at internal positions (endonucleases) or at the terminal 3' or 5' nucleotide positions (exonucleases).
  • a DNA exonuclease activity in DNA polymerase serves to remove improperly paired nucleotides attached to the 3'-OH end of the growing DNA strand by the polymerase and thereby serves a "proofreading" function.
  • DNA endonuclease activity is involved in the excision step of the DNA repair process.
  • RNases also serve a variety of functions.
  • RNase P is a ribonucleoprotein enzyme which cleaves the 5' end of pre-tRNAs as part of their maturation process.
  • RNase H digests the RNA strand of an RNA/DNA hybrid. Such hybrids occur in cells invaded by retroviruses, and
  • RNase H is an important enzyme in the retroviral replication cycle. Pancreatic RNase secreted by the pancreas into the intestine hydrolyzes RNA present in ingested foods. RNase activity in serum and cell extracts is elevated in a variety of cancers and infectious diseases (Schein, CH. (1997) Nat.
  • Methylases Methylation of specific nucleotides occurs in both DNA and RNA, and serves different functions in the two macromolecules. Methylation of cytosine residues to form 5-methyl cytosine in DNA occurs specifically at CG sequences which are base-paired with one another in the DNA double-helix. This pattern of methylation is passed from generation to generation during DNA replication by an enzyme called "maintenance methylase" that acts preferentially on those CG sequences that are base-paired with a CG sequence that is already methylated. Such methylation appears to distinguish active from inactive genes by preventing the binding of regulatory proteins that "turn on” the gene, but permit the binding of proteins that inactivate the gene (Alberts, supra, pp.
  • tRNA methylase produces one of several nucleotide modifications in tRNA that affect the conformation and base-pairing of the molecule and facilitate the recognition of the appropriate mRNA codons by specific tRNAs.
  • the primary methylation pattern is the dimethylation of guanine residues to form N,N-dimethyI guanine.
  • Helicases are enzymes that destabilize and unwind double helix structures in both DNA and RNA. Since DNA replication occurs more or less simultaneously on both strands, the two strands must first separate to generate a replication "fork" for DNA polymerase to act on. Two types of replication proteins contribute to this process, DNA helicases and single-stranded binding proteins. DNA helicases hydrolyze ATP and use the energy of hydrolysis to separate the DNA strands. Single- stranded binding proteins (SSBs) then bind to the exposed DNA strands without covering the bases, thereby temporarily stabilizing them for templating by the DNA polymerase (Alberts, supra, pp. 255- 256).
  • SSBs Single- stranded binding proteins
  • RNA helicases also alter and regulate RNA conformation and secondary structure. Like the DNA helicases, RNA helicases utilize energy derived from ATP hydrolysis to destabilize and unwind RNA duplexes.
  • the most well-characterized and ubiquitous family of RNA helicases is the DEAD- box family, so named for the conserved B-type ATP-binding motif which is diagnostic of proteins in this family.
  • DEAD-box helicases Over 40 DEAD-box helicases have been identified in organisms as diverse as bacteria, insects, yeast, amphibians, mammals, and plants. DEAD-box helicases function in diverse processes such as translation initiation, splicing, ribosome assembly, and RNA editing, transport, and stability.
  • DEAD-box helicases play tissue- and stage-specific roles in spermatogenesis and embryogenesis.
  • Overexpression of the DEAD-box 1 protein (DDXl) may play a role in the progression of neuroblastoma (Nb) and retinoblastoma (Rb) tumors (Godbout, R. et al. (1998) J. Biol. Chem. 273:21161-21168). These observations suggest that DDXl may promote or enhance tumor progression by altering the normal secondary structure and expression levels of RNA in cancer cells.
  • DDX6 is located at a chromosomal breakpoint associated with B-cell lymphoma.
  • a chimeric protein composed of DDX10 and NUP98, a nucleoporin protein may be involved in the pathogenesis of certain myeloid malignancies.
  • DNA topoisomerases are enzymes that affect the topological state of DNA. For example, defects in topoisomerases or their regulation can affect normal physiology. DNA topoisomerase effectively acts as a reversible nuclease that hydrolyzes a phosphodiesterase bond in a DNA strand, permitting the two strands to rotate freely about one another to remove the strain of the helix, and then rejoins the original phosphodiester bond between the two strands.
  • Two types of DNA topoisomerase exist, types I and II.
  • DNA Topoisomerase I causes a single-strand break in a DNA helix to allow the rotation of the two strands of the helix about the remaining phosphodiester bond in the opposite strand.
  • DNA topoisomerase II causes a transient break in both strands of a DNA helix where two double helices cross over one another. This type of topoisomerase can efficiently separate two interlocked DNA circles (Alberts, supra, pp.260-262).
  • Type II topoisomerases are largely confined to proliferating cells in eukaryotes, such as cancer cells. For this reason they are targets for anticancer drugs.
  • Topoisomerase II has been implicated in multi- drug resistance (MDR) as it appears to aid in the repair of DNA damage inflicted by DNA binding agents such as doxorubicin and vincristine. Reduced levels of topoisomerase II have been correlated with some of the DNA processing defects associated with the disorder ataxia-telangiectasia (Singh, S.P. et al. (1988) Nucleic Acids Res. 16:3919-3929). Recombinases
  • Genetic recombination is the process of rearranging DNA sequences within an organism's genome to provide genetic variation for the organism in response to changes in the environment.
  • DNA recombination allows variation in the particular combination of genes present in an individual's genome, as well as the timing and level of expression of these genes (see Alberts, supra, pp. 263- 273).
  • Two broad classes of genetic recombination are commonly recognized, general recombination and site-specific recombination.
  • General recombination involves genetic exchange between any homologous pair of DNA sequences usually located on two copies of the same chromosome.
  • recombinases that "nick" one strand of a DNA duplex more or less randomly and permit exchange with the complementary strand of another duplex.
  • the process does not normally change the arrangement of genes on a chromosome.
  • the recombinase recognizes specific nucleotide sequences present in one or both of the recombining molecules. Base-pairing is not involved in this form of recombination and therefore does not require DNA homology between the recombining molecules.
  • this form of recombination can alter the relative positions of nucleotide sequences in chromosomes.
  • RNA processing steps include capping at the 5' end with methylguanosine, polyadenylating the 3' end, and splicing to remove introns.
  • the primary RNA transcript from DNA is a faithful copy of the gene containing both exon and intron sequences, and the latter sequences must be cut out of the RNA transcript to produce an mRNA that codes for a protein.
  • This "splicing" of the mRNA sequence takes place in the nucleus with the aid of a large, multicomponent ribonucleoprotein complex known as a spliceosome.
  • the spliceosomal complex is composed of five small nuclear ribonucleoprotein particles (snRNPs) designated UI, U2, U4, U5, and U6, and a number of additional proteins.
  • snRNP small nuclear ribonucleoprotein particles
  • Each snRNP contains a single species of snRNA and about ten proteins.
  • the RNA components of some snRNPs recognize and base pair with intron consensus sequences.
  • the protein components mediate spliceosome assembly and the splicing reaction.
  • Autoantibodies to snRNP proteins are found in the blood of patients with systemic lupus erythematosus (Stryer, L. (1995) Biochemistry, W.H. Freeman and Company, New York NY, p. 863).
  • Adhesion Molecules The surface of a cell is rich in transmembrane proteoglycans, glycoproteins, glycolipids, and receptors. These macromolecules mediate adhesion with other cells and with components of the extracellular matrix (ECM). The interaction of the cell with its surroundings profoundly influences cell shape, strength, flexibility, motility, and adhesion. These dynamic properties are intimately associated with signal transduction pathways controlling cell proliferation and differentiation, tissue construction, and embryonic development. Cadherins
  • Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. These proteins share multiple repeats of a cadherin-specific motif, and the repeats form the folding units of the cadherin extracellular domain. Cadherin molecules cooperate to form focal contacts, or adhesion plaques, between adjacent epithelial cells.
  • the cadherin family includes the classical cadherins and pro oca er ns. assica ca er ns inc u e e -ca e n, -ca erm, an -ca e n su am es.
  • E-cadherin is present on many types of epithelial cells and is especially important for embryonic development.
  • N-cadherin is present on nerve, muscle, and lens cells and is also critical for embryonic development.
  • P-cadherin is present on cells of the placenta and epidermis. Recent studies report that protocadherins are involved in a variety of cell-cell interactions (Suzuki, S.T. (1996) J.
  • cadherins The intracellular anchorage of cadherins is regulated by their dynamic association with catenins, a family of cytoplasmic signal transduction proteins associated with the actin cytoskeleton.
  • the anchorage of cadherins to the actin cytoskeleton appears to be regulated by protein tyrosine phosphorylation, and the cadherins are the target of phosphorylation-induced junctional disassembly (Aberle, H. et al. (1996) J. Cell. Biochem. 61:514-523). Integrins
  • Integrins are ubiquitous transmembrane adhesion molecules that link the ECM to the internal cytoskeleton. Integrins are composed of two noncovalently associated transmembrane glycoprotein subunits called ⁇ and ⁇ . Integrins function as receptors that play a role in signal transduction. For example, binding of integrin to its extracellular ligand may stimulate changes in intracellular calcium levels or protein kinase activity (Sjaastad, M.D. and W.J. Nelson (1997) BioEssays 19:47-55). At least ten cell surface receptors of the integrin family recognize the ECM component fibronectin, which is involved in many different biological processes including cell migration and embryogenesis (Johansson, S. et al. (1997) Front. Biosci. 2:D126-D146). Lectins
  • Lectins comprise a ubiquitous family of extracellular glycoproteins which bind cell surface carbohydrates specifically and reversibly, resulting in the agglutination of cells (reviewed in Drickamer, K. and M.E. Taylor (1993) Annu. Rev. Cell Biol. 9:237-264). This function is particularly important for activation of the immune response. Lectins mediate the agglutination and mitogenic stimulation of lymphocytes at sites of inflammation (Lasky, L.A. (1991) J. Cell. Biochem. 45:139-146; Paietta, E. et al. (1989) J. Immunol. 143:2850-2857).
  • Lectins are further classified into subfamilies based on carbohydrate-binding specificity and other criteria.
  • the galectin subfamily includes lectins that bind ⁇ -galactoside carbohydrate moieties in a thiol-dependent manner (reviewed in Hadari, Y.R. et al. (1998) J. Biol. Chem. 270:3447-3453). Galectins are widely expressed and developmentally regulated. Because aU galectins lack an N-terminal signal peptide, it is suggested that galectins are externalized through an atypical secretory mechanism. Two classes of galectins have been defined based on molecular weight and oligomerization properties.
  • Galectins form homodimers and are about 14 to 16 kilodaltons in mass, while large galectins are monomeric and about 29-37 kilodaltons.
  • Galectins contain a characteristic carbohydrate recognition domain (CRD).
  • the CRD is about 140 amino acids and contains several stretches of about 1 - 10 amino acids which are highly conserve among a ga ect ns. part cu ar -am no ac mot w t n t e conta ns conserve tryptophan and arginine residues which are critical for carbohydrate binding.
  • the CRD of some galectins also contains cysteine residues which may be important for disulfide bond formation.
  • Galectins play a number of roles in diseases and conditions associated with cell-cell and cell- matrix interactions. For example, certain galectins associate with sites of inflammation and bind to cell surface immunoglobulin E molecules. In addition, galectins may play an important role in cancer metastasis. Galectin overexpression is correlated with the metastatic potential of cancers in humans and mice. Moreover, anti-galectin antibodies inhibit processes associated with cell transformation, such as cell aggregation and anchorage-independent growth (See, for example, Su, Z.-Z. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7252-7257). Selectins
  • Selectins comprise a specialized lectin subfamily involved primarily in inflammation and leukocyte adhesion (Reviewed in Lasky, supra). Selectins mediate the recruitment of leukocytes from the circulation to sites of acute inflammation and are expressed on the surface of vascular endothelial cells in response to cytokine signaling. Selectins bind to specific ligands on the leukocyte cell membrane and enable the leukocyte to adhere to and migrate along the endothelial surface. Binding of selectin to its ligand leads to polarized rearrangement of the actin cytoskeleton and stimulates signal transduction within the leukocyte (Brenner, B. et al. (1997) Biochem. Biophys. Res. Commun.
  • selectins include lymphocyte adhesion molecule-1 (Lam-1 or L-selectin), endothelial leukocyte adhesion molecule-1 (ELAM-1 or E-selectin), and granule membrane protein-140 (GMP-140 or P-selectin) (Johnston, G.I. et al. (1989) Cell 56:1033-1044).
  • Lam-1 or L-selectin lymphocyte adhesion molecule-1
  • ELAM-1 or E-selectin endothelial leukocyte adhesion molecule-1
  • GMP-140 or P-selectin granule membrane protein-140
  • Protein transport and secretion are essential for cellular function. Protein transport and secretion are mediated by a signal peptide located at the amino terminus of the protein to be secreted.
  • the signal peptide is composed of about ten to twenty hydrophobic amino acids wliich target the nascent protein from the ribosome to the endoplasmic reticulum (ER). Proteins targeted to the ER may either proceed through the secretory pathway or remain in any of the secretory organelles such as the ER, Golgi apparatus, or lysosomes. Proteins that transit through the secretory pathway are either secreted into the extracellular space or retained in the plasma membrane.
  • Proteins that are retained in the plasma membrane contain one or more transmembrane domains, each comprised of about 20 hydrophobic amino acid residues.
  • Secreted proteins are often synthesized as inactive precursors that are activate by post-translational processing events during transit through the secretory pathway.
  • Such events include glycosylation, proteolysis, and removal of the signal peptide by a signal peptidase.
  • Other events that may occur during protein transport include chaperone-dependent unfolding and folding of the nascent protein and interaction of the protein with a receptor or pore complex.
  • Examples of secreted proteins with amino terminal signal peptides include receptors, extracellular matrix molecules, cytokines, hormones, growth and differentiation factors, neuropeptides, vasomediators, ion channels, transporters/pumps, and proteases.
  • the extracellular matrix is a complex network of glycoproteins, polysaccharides, proteoglycans, and other macromolecules that are secreted from the cell into the extracellular space.
  • the ECM remains in close association with the cell surface and provides a supportive meshwork that profoundly influences cell shape, motility, strength, flexibility, and adhesion. In fact, adhesion of a cell to its surrounding matrix is required for cell survival except in the case of metastatic tumor cells, which have overcome the need for cell-ECM anchorage. This phenomenon suggests that the ECM plays a critical role in the molecular mechanisms of growth control and metastasis. (Reviewed in Ruoslahti, E. (1996) Sci. Am.
  • the ECM determines the structure and physical properties of connective tissue and is particularly important for morphogenesis and other processes associated with embryonic development and pattern formation.
  • the collagens comprise a family of ECM proteins that provide structure to bone, teeth, skin, ligaments, tendons, cartilage, blood vessels, and basement membranes. Multiple collagen proteins have been identified. Three collagen molecules fold together in a triple helix stabilized by interchain disulfide bonds. Bundles of these triple helices then associate to form fibrils.
  • Collagen primary structure consists of hundreds of (Gly-X-Y) repeats where about a third of the X and Y residues are Pro.
  • Glycines are crucial to helix formation as the bulkier amino acid sidechains cannot fold into the triple helical conformation. Because of these strict sequence requirements, mutations in collagen genes have severe consequences. Osteogenesis imperfecta patients have brittle bones that fracture easily; in severe cases patients die in utero or at birth. Ehlers-Danlos syndrome patients have hyperelastic skin, hypermobile joints, and susceptibility to aortic and intestinal rupture. Chondrodysplasia patients have short stature and ocular disorders. Alport syndrome patients have hematuria, sensorineural deafness, and eye lens deformation. (Isselbacher, K.J. et al.
  • Elastin and related proteins confer elasticity to tissues such as skin, blood vessels, and lungs.
  • Elastin is a highly hydrophobic protein of about 750 amino acids that is rich in proline and glycine residues. Elastin molecules are highly cross-linked, forming an extensive extracellular network of fibers and sheets. Elastin fibers are surrounded by a sheath of microfibrils which are composed of a number of glycoproteins, including fibrillin. Mutations in the gene encoding fibrillin are responsible for Marian's syndrome, a genetic disorder characterized by defects in connective tissue. In severe
  • Fibronectin is a large ECM glycoprotein found in all vertebrates. Fibronectin exists as a di er of two subunits, each containing about 2,500 amino acids. Each subunit folds into a rod-like structure containing multiple domains. The domains each contain multiple repeated modules, the 0 most common of which is the type HI fibronectin repeat.
  • the type HI fibronectin repeat is about 90 amino acids in length and is also found in other ECM proteins and in some plasma membrane and cytoplasmic proteins.
  • some type HI fibronectin repeats contain a characteristic tripeptide consisting of Arginine-Glycine-Aspartic acid (RGD).
  • the RGD sequence is recognized by the integrin family of cell surface receptors and is also found in other ECM proteins. Disruption of 5 both copies of the gene encoding fibronectin causes early embryonic lethality in mice. The mutant embryos display extensive morphological defects, including defects in the formation of the notochord, somites, heart, blood vessels, neural tube, and extraembryonic structures. (Reviewed in Alberts, supra, pp. 986-987.)
  • Laminin is a major glycoprotein component of the basal lamina which underlies and supports o epithelial cell sheets.
  • Laminin is one of the first ECM proteins synthesized in the developing embryo.
  • Laminin is an 850 kilodalton protein composed of three polypeptide chains joined in the shape of a cross by disulfide bonds.
  • Laminin is especially important for angiogenesis and in particular, for guiding the formation of capillaries. (Reviewed in Alberts, supra, pp. 990-991.)
  • proteoglycans are composed of unbranched polysaccharide chains (glycosaminoglycans) attached to protein cores. Common proteoglycans include aggrecan, betaglycan, decorin, perlecan, serglycin, and syndecan-1. Some of these molecules not only provide mechanical support, but also bind to extracellular signaling molecules, such as fibroblast growth factor and transforming growth factor ⁇ , suggesting a role for proteoglycans in cell-cell 0 communication and cell growth. (Reviewed in Alberts, supra, pp.
  • the cytoskeleton is a cytoplasmic network of protein fibers that mediate cell shape, structure, and movement.
  • the cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol.
  • the cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements.
  • Major cytoskeletal fibers include the microtubules, the microfilaments, and the intermediate filaments.
  • 5 dynamin drives the formation of membrane vesicles.
  • Accessory or associated proteins modify the structure or activity of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane.
  • Microtubules cytoskeletal fibers with a diameter of about 24 nm, have multiple roles in the 0 cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported in the cell along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi- 5 directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. Microtubules are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
  • Microtubules are polymers of GTP-binding tubulin protein subunits. Each subunit is a o heterodimer of ⁇ - and ⁇ - tubulin, multiple isoforms of which exist.
  • the hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule.
  • the subunits interact head to tail to form protofilaments; the protofilaments interact side to side to form a microtubule.
  • a microtubule is polarized, one end ringed with ⁇ -tubulin and the other with ⁇ -tubulin, and the two ends differ in their rates of assembly.
  • each microtubule is composed of 13 protofilaments although 11 or 15 5 protofilament-microtubules are sometimes found.
  • Cilia and flagella contain doublet microtubules.
  • Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules.
  • the basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole.
  • Gamma tubulin present in the MTOC is important for nucleating the o polymerization of - and ⁇ - tubulin heterodimers but does not polymerize into microtubules.
  • MAPs Microtubule-associated proteins
  • assembly MAPs can be identified in neurons as well as non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. 5 These MAPs are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules. Based on sequence analysis, assembly MAPs can be further grouped into two types: Type I and Type H. Type I MAPs, which include MAPIA and MAPIB, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testes. Type I
  • MAPIA and MAPIB are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain.
  • LC3 Another light chain, is a 16.4 kDa molecule that binds MAPIA, MAPIB, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAPIA or 0 MAPIB transcripts, and that the expression of LC3 may be important in regulating the microtubule binding activity of MAPIA and MAPIB during cell proliferation (Mann, S.S. et al. (1994) J. Biol.
  • Type H MAPs which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain.
  • MAP2a, 5 MAP2b, and MAP2c are found only in dendrites
  • MAP4 is found in non-neuronal cells
  • Tau is found in axons and dendrites of nerve cells.
  • Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein.
  • Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17.
  • the o altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates (Spillantini, M.G. and M. Goedert (1998) Trends Neurosci. 21:428-
  • the protein pericentrin is found in the MTOC and has a role in microtubule assembly.
  • Microfilaments cytoskeletal filaments with a diameter of about 7-9 nm, are vital to cell locomotion, cell shape, cell adhesion, cell division, and muscle contraction. Assembly and disassembly of the microfilaments allow cells to change their morphology. Microfilaments are the polymerized form of actin, the most abundant intracellular protein in the eukaryotic cell. Human cells contain six isoforms of actin. The three ⁇ -actins are found in different kinds of muscle, o nonmuscle ⁇ -actin and nonmuscle ⁇ -actin are found in nonmuscle cells, and another ⁇ -actin is found in intestinal smooth muscle cells.
  • G-actin the monomeric form of actin, polymerizes into polarized, helical F-actin filaments, accompanied by the hydrolysis of ATP to ADP.
  • Actin filaments associate to form bundles and networks, providing a framework to support the plasma membrane and determine cell shape. These bundles and networks are connected to the cell membrane.
  • thin filaments containing actin slide past thick filaments containing the motor protein myosin during contraction.
  • a family of actin-related proteins exist that are not part of the actin cytoskeleton, but rather associate with microtubules and dynein.
  • Actin-associated proteins have roles in cross-linking, severing, and stabilization of actin filaments and in sequestering actin monomers. Several of the actin-associated proteins have multiple functions. Bundles and networks of actin filaments are held together by actin cross-linking proteins.
  • Group I cross-linking proteins have unique actin-binding domains and include the 30 kD protein, EF-la, fascin, and scruin.
  • Group II cross-linking proteins have a 7,000- MW actin-binding domain and include villin and dematin.
  • Group III cross-linking proteins have pairs of a 26,000-MW actin-binding domain and include fimbrin, spectrin, dystrophin, ABP 120, and filamin.
  • Severing proteins regulate the length of actin filaments by breaking them into short pieces or by blocking their ends.
  • Severing proteins include gCAP39, severin (fragmin), gelsolin, and villin.
  • Capping proteins can cap the ends of actin filaments, but cannot break filaments.
  • Capping proteins include CapZ and tropomodulin.
  • the proteins thymosin and profilin sequester actin monomers in the cytosol, allowing a pool of unpolymerized actin to exist.
  • Intermediate filaments are cytoskeletal fibers with a diameter of about 10 nm, intermediate between that of microfilaments and microtubules. IFs serve structural roles in the cell, reinforcing cells and organizing cells into tissues. IFs are particularly abundant in epidermal cells and in neurons. IFs are extremely stable, and, in contrast to microfilaments and microtubules, do not function in cell motility .
  • Type I and Type H proteins are the acidic and basic keratins, respectively. Heterodimers of the acidic and basic keratins are the building blocks of keratin IFs. Keratins are abundant in soft epithelia such as skin and cornea, hard epithelia such as nails and hair, and in epithelia that line internal body cavities.
  • keratin genes lead to epithelial diseases including epidermolysis bullosa simplex, bullous congenital ichthyosiform erythroderma (epidermolytic hyperkeratosis), non-epidermolytic and epidermolytic palmoplantar keratoderma, ichthyosis bullosa of Siemens, pachyonychia congenita, and white sponge nevus.
  • Some of these diseases result in severe skin blistering.
  • Type HI IF proteins include desmin, glial fibrillary acidic protein, vimentin, and peripherin.
  • Glial fibrillary acidic protein filaments are found in the glial cells that surround neurons and astrocytes. Vimentin filaments are found in blood vessel endothelial cells, some epithelial cells, and mesenchymal cells such as fibroblasts, and are commonly associated with microtubules. Vimentin filaments may have roles in keeping the nucleus and other organelles in place in the cell.
  • Type IV IFs include the neurofilaments and nestin. Neurofilaments, composed of three polypeptides NF-L, NF-M, and NF-H, are frequently associated with microtubules in axons.
  • Neurofilaments are responsible for the radial growth and diameter of an axon, and ultimately for the speed of nerve impulse transmission. Changes in phosphorylation and metabolism of neurofilaments are observed in neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease (Julien, J.P. and W.E. Mushynski (1998) Prog. Nucleic Acid Res. Mol. Biol.
  • Type V IFs the lamins, are found in the nucleus where they support the nuclear membrane.
  • IFs have a central ⁇ -helical rod region interrupted by short nonhelical linker segments.
  • the rod region is bracketed, in most cases, by non-helical head and tail domains.
  • the rod regions of intermediate filament proteins associate to form a coiled-coil dimer.
  • a highly ordered assembly process leads from the dimers to the IFs. Neither ATP nor GTP is needed for IF assembly, unlike that of microfilaments and microtubules.
  • IF-associated proteins mediate the interactions of IFs with one another and with other cell structures.
  • IFAPs cross-link IFs into a bundle, into a network, or to the plasma membrane, and may cross-link IFs to the microfilament and microtubule cytoskeleton.
  • Microtubules and IFs are in particular closely associated.
  • IFAPs include BPAGl, plakoglobin, desmoplakin I, desmoplakin H, plectin, ankyrin, filaggrin, and lamin B receptor. Cytoskeletal-Membrane Anchors
  • Cytoskeletal fibers are attached to the plasma membrane by specific proteins. These attachments are important for maintaining cell shape and for muscle contraction.
  • the spectrin-actin cytoskeleton is attached to cell membrane by three proteins, band 4.1, ankyrin, and adducin. Defects in this attachment result in abnormally shaped cells which are more rapidly degraded by the spleen, leading to anemia.
  • the spectrin-actin cytoskeleton is also linked to the membrane by ankyrin; a second actin network is anchored to the membrane by filamin.
  • the protein dystrophin links actin filaments to the plasma membrane; mutations in the dystrophin gene lead to Duchenne muscular dystrophy.
  • peripheral membrane proteins ⁇ -actinin and vinculin attach actin filaments to the cell membrane.
  • IFs are also attached to membranes by cytoskeletal-membrane anchors.
  • the nuclear lamina is attached to the inner surface of the nuclear membrane by the lamin B receptor.
  • Vimentin IFs are attached to the plasma membrane by ankyrin and plectin.
  • Desmosome and hemidesmosome membrane junctions hold together epithelial cells of organs and skin. These membrane junctions allow shear forces to be distributed across the entire epithelial cell layer, thus providing strength and rigi ity to the epithelium.
  • IFs in epithelial ce s are attached to the desmosome y plakoglobin and desmoplakins.
  • the proteins that link IFs to hemidesmosomes are not known.
  • Desmin IFs surround the sarcomere in muscle and are linked to the plasma membrane by paranernin, synemin, and ankyrin.
  • Myosins are actin-activated ATPases, found in eukaryotic cells, that couple hydrolysis of
  • Myosin provides the motor function for muscle contraction and intracellular movements such as phagocytosis and rearrangement of cell contents during mitotic cell division
  • the contractile unit of skeletal muscle termed the sarcomere
  • the contractile unit of skeletal muscle termed the sarcomere
  • sarcomere consists of highly ordered arrays of thin actin-containing filaments and thick myosin-containing filaments.
  • l o Crossbridges form between the thick and thin filaments, and the ATP-dependent movement of myosin heads within the thick filaments pulls the thin filaments, shortening the sarcomere and thus the muscle fiber.
  • Myosins are composed of one or two heavy chains and associated light chains.
  • Myosin heavy chains contain an amino-terminal motor or head domain, a neck that is the site of light-chain
  • the tail domains may associate to form an ⁇ -helical coiled coil.
  • Conventional myosins such as those found in muscle tissue, are composed of two myosin heavy-chain subunits, each associated with two light-chain subunits that bind at the neck region and play a regulatory role.
  • Unconventional myosins believed to function in intracellular motion, may contain either one or two heavy chains and associated light chains.
  • Dyneins are (-) end-directed motor proteins which act on microtubules. Two classes of dyneins, cytosolic and axonemal, have been identified. Cytosolic dyneins are responsible for translocation of materials along cytoplasmic microtubules, for example, transport from the nerve
  • Cytoplasmic dyneins are also reported to play a role in mitosis.
  • Axonemal dyneins are responsible for the beating of flagella and cilia.
  • Dynein on one microtubule doublet walks along the adjacent microtubule doublet. This sliding force produces bending forces that cause the flagellum or cilium to beat.
  • Dyneins have a native mass between 1000 and 2000 kDa and contain either two or three force-producing heads driven
  • Kinesins are (+) end-directed motor proteins which act on microtubules.
  • the prototypical kinesin molecule is involved in the transport of membrane-bound vesicles and organelles. This
  • Kinesin is also important in all cell types for the transport of vesicles from the Golgi complex to the endoplasmic reticulum. This role is cri ica or main aining e i en i y an unc iona y o ese secre ory organe es.
  • Kinesins define a ubiquitous, conserved family of over 50 proteins that can be classified into at least 8 subfamilies based on primary amino acid sequence, domain structure, velocity of movement, and cellular function. (Reviewed in Moore, J.D. and S.A. Endow (1996) Bioessays 18:207-219; and
  • the prototypical kinesin molecule is a heterotetramer composed of two heavy polypeptide chains (KHCs) and two light polypeptide chains
  • KLCs The KHC subunits are typically referred to as "kinesin.”
  • KHC is about 1000 amino acids in length
  • KLC is about 550 amino acids in length.
  • Two KHCs dimerize to form a rod-shaped molecule with three distinct regions of secondary structure.
  • At one end of the molecule is a globular 0 motor domain that functions in ATP hydrolysis and microtubule binding.
  • Kinesin motor domains are highly conserved and share over 70% identity. Beyond the motor domain is an ⁇ -helical coiled-coil region wliich mediates dimerization.
  • a fan-shaped tail that associates with molecular cargo. The tail is formed by the interaction of the KHC C-termini with the two KLCs. 5
  • kinesin-related proteins are called kinesin-related proteins
  • KRPs KRPs
  • Hoyt eukaryotes
  • Dynamin is a large GTPase motor protein that functions as a "molecular pinchase," 5 generating a mechanochemical force used to sever membranes. This activity is important in forming clathrin-coated vesicles from coated pits in endocytosis and in the biogenesis of synaptic vesicles in neurons. Binding of dynamin to a membrane leads to dynamin' s self-assembly into spirals that may act to constrict a flat membrane surface into a tubule. GTP hydrolysis induces a change in conformation of the dynamin polymer that pinches the membrane tubule, leading to severing of the o .
  • dynamin disassembly. Following disassembly the dynamin may either dissociate from the membrane or remain associated to the vesicle and be transported to another region of the cell.
  • Three homologous dynamin genes have been discovered, in addition to several dynamin-related proteins.
  • conserveed dynamin regions are the N-terminal GTP-binding domain, a central pleckstrin homology 5 domain that binds membranes, a central coiled-coil region that may activate dynamin' s GTPase activity, and a C-terminal proline-rich domain that contains several motifs that bind SH3 domains on o pro eins, me - pro o no n ai e proline-rich domain. (See McNiven, M.A. (1998) Cell 94:151-154; Scaife, R.M. and R.L. Margolis
  • the cytoskeleton is reviewed in Lodish, H. et al. (1995) Molecular Cell Biology. Scientific American Books, New York NY.
  • Ribosomal RNAs are assembled, along with ribosomal proteins, into ribosomes, which are cytoplasmic particles that translate messenger RNA into polypeptides.
  • the eukaryotic ribosome is composed of a 60S (large) subunit and a 40S (small) subunit, which together form the 0 80S ribosome.
  • the ribosome also contains more than fifty proteins.
  • the ribosomal proteins have a prefix which denotes the subunit to which they belong, either L (large) or S (small).
  • Ribosomal protein activities include binding rRNA and organizing the conformation of the junctions between rRNA helices (Woodson, S.A. and N.B. Leontis (1998) Curr. Opin. Struct. Biol. 8:294-300; Ramakrishnan, V. and S.W. White (1998) Trends 5 Biochem. Sci. 23:208-212.)
  • Three important sites are identified on the ribosome.
  • the aminoacyl- tRNA site (A site) is where charged tRNAs (with the exception of the initiator-tRNA) bind on arrival at the ribosome.
  • the peptidyl-tRNA site (P site) is where new peptide bonds are formed, as well as where the initiator tRNA binds.
  • the exit site (E site) is where deacylated tRNAs bind prior to their release from the ribosome. (The ribosome is reviewed in Stryer, L. (1995) Biochemistry W.H. o Freeman and Company, New York NY, pp. 888-908; and Lodish, H. et al. (1995) Molecular Cell
  • chromatin The nuclear DNA of eukaryotes is organized into chromatin. Two types of chromatin are observed: euchromatin, some of which may be transcribed, and heterochromatin so densely packed 5 that much of it is inaccessible to transcription. Chromatin packing thus serves to regulate protein expression in eukaryotes. Bacteria lack chromatin and the chromatin-packing level of gene regulation.
  • the fundamental unit of chromatin is the nucleosome of 200 DNA base pairs associated with two copies each of histones H2A, H2B, H3, and H4. Adjascent nucleosomes are linked by another o class of histones, HI .
  • Low molecular weight non-histone proteins called the high mobility group
  • HMG HMG
  • Chromodomain proteins function in compaction of chromatin into its transcriptionally silent heterochromatin form.
  • the inactive state of this chromosome is inherited, so that adult females are mosaics of clusters of paternal-X and maternal-X clonal cell groups.
  • the condensed X chromosome is reactivated in meiosis.
  • Chromatin is associated with disorders of protein expression such as thalassemia, a genetic anemia resulting from the removal of the locus control region (LCR) required for decondensation of the globin gene locus.
  • Electron carriers such as cytochromes accept electrons from NADH or FADH 2 and donate them to other electron carriers.
  • Adrenodoxin for example, is an FeS protein that forms a complex with NADPH: adrenodoxin o reductase and cytochrome p450.
  • Cytochromes contain a heme prosthetic group, a porphyrin ring containing a tightly bound iron atom. Electron transfer reactions play a crucial role in cellular energy production.
  • Glucose is initially converted to pyruvate in the cytoplasm.
  • Fatty acids and pyruvate are transported to the mitochondria for 5 complete oxidation to C0 2 coupled by enzymes to the transport of electrons from NADH and FADH 2 to oxygen and to the synthesis of ATP (oxidative phosphorylation) from ADP and P
  • ATP synthesis requires membrane transport enzymes including the phosphate transporter and the ATP- ADP antiport protein.
  • the ATP-binding casette (ABC) superfamily has also been suggested as belonging to the mitochondrial transport group (Hogue, D.L. et al. (1999) J. Mol. Biol. 285:379- 0 389). Brown fat uncoupling protein dissipates oxidative energy as heat, and may be involved the fever response to infection and trauma (Cannon, B. et al. (1998) Ann. NY Acad. Sci. 856:171-187).
  • Mitochondria are oval-shaped organelles comprising an outer membrane, a tightly folded inner membrane, an intermembrane space between the outer and inner membranes, and a matrix inside the inner membrane.
  • the outer membrane contains many porin molecules that allow ions and 5 charged molecules to enter the intermembrane space, while the inner membrane contains a variety of transport proteins that transfer only selected molecules.
  • Mitochondria are the primary sites of energy production in cells.
  • Mitochondria contain a small amount of DNA.
  • Human mitochondrial DNA encodes 13 proteins, 22 tRNAs, and 2 rRNAs.
  • Mitochondrial-DNA encoded proteins include NADH-Q reductase, a cytochrome reductase subunit, cytochrome oxidase subunits, and ATP synthase subunits.
  • Cytochrome b5 is a central electron donor for various reductive reactions occurring on the cytoplasmic surface of liver endoplasmic reticulum. Cytochrome b5 has been found in Golgi, plasma, endoplasmic reticulum (ER), and microbody membranes. 0 For a review of mitochondrial metabolism and regulation, see Lodish, H. et al. (1995)
  • mitochondrial proteins The majority of mitochondrial proteins are encoded by nuclear genes, are synthesized on cytosolic ribosomes, and are imported into the mitochondria. Nuclear-encoded proteins which are 5 destined for the mitochondrial matrix typically contain positively-charged amino terminal signal sequences. Import of these preproteins from the cytoplasm requires a multisubunit protein complex in the outer membrane known as the translocase of outer mitochondrial membrane (TOM; previously designated MOM; Pfanner, N. et al. (1996) Trends Biochem. Sci. 21:51-52) and at least three inner membrane proteins which comprise the translocase of inner mitochondrial membrane (TIM; o previously designated MTM; Pfanner, supra). An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import.
  • TOM translocase of outer mitochondrial membrane
  • TIM inner membrane proteins which comprise the translocase of inner mitochondrial membrane
  • An inside-negative membrane potential across the inner mitochondrial membrane is also required for preprotein import.
  • Preproteins are recognized by surface receptor components of the TOM complex and are translocated through a proteinaceous pore formed by other TOM components. Proteins targeted to the matrix are then recognized by the import machinery of the TIM complex.
  • the import systems of the outer and inner membranes can function 5 independently (Segui-Real, B. et al. (1993) EMBO J. 12:2211-2218).
  • leader peptide is cleaved by a signal peptidase to generate the mature protein.
  • Most leader peptides are removed in a one step process by a protease termed mitochondrial processing peptidase (MPP) (Paces, V. et al. (1993) Proc. Natl. Acad. Sci. USA 90:5355-5358).
  • MPP mitochondrial processing peptidase
  • a two-step process occurs in which MPP generates an o intermediate precursor form which is cleaved by a second enzyme, mitochondrial intermediate peptidase, to generate the mature protein.
  • Mitochondrial dysfunction leads to impaired calcium buffering, generation of free radicals that may participate in deleterious intracellular and extracellular processes, changes in mitochondrial permeability and oxidative damage which is observed in several neurodegenerative diseases.
  • 5 Neurodegenerative diseases linked to mitochondrial dysfunction include some forms of Alzheimer's disease, Friedreich's ataxia, familial amyotrophic lateral sclerosis, and Huntington's disease (Beal, M.F. (1998) Biochim. Biophys. Acta 1366:211-213).
  • the myocardium is heavily dependent on oxidative metabolism, so mitochondrial dysfunction often leads to heart disease (DiMauro, S. and M.
  • Mitochondria are implicated in disorders of cell proliferation, since they play an important role in a cell's decision to proliferate or self-destruct
  • the oncoprotein Bcl-2 promotes cell proliferation by stabilizing mitochondrial membranes so that apoptosis signals are not released (Susin, S.A. (1998) Biochim.
  • Multicellular organisms are composed of diverse cell types that differ dramatically both in 0 structure and function.
  • the identity of a cell is determined by its characteristic pattern of gene expression, and different cell types express overlapping but distinctive sets of genes throughout development. Spatial and temporal regulation of gene expression is critical for the control of cell proliferation, cell differentiation, apoptosis, and other processes that contribute to organismal development.
  • gene expression is regulated in response to extracellular signals that 5 mediate cell-cell communication and coordinate the activities of different cell types. Appropriate gene regulation also ensures that cells function efficiently by expressing only those genes whose functions are required at a given time.
  • Transcriptional regulatory proteins are essential for the control of gene expression. Some of these proteins function as transcription factors that initiate, activate, repress, or terminate gene o transcription. Transcription factors generally bind to the promoter, enhancer, and upstream regulatory regions of a gene in a sequence-specific manner, although some factors bind regulatory elements within or downstream of a gene's coding region. Transcription factors may bind to a specific region of DNA singly or as a complex with other accessory factors. (Reviewed in Lewin, B. (1990) Genes IV. Oxford University Press, New York NY, and Cell Press, Cambridge MA, pp. 5 554-570.) Many transcription factors incorporate DNA-binding structural motifs which comprise either ⁇ helices or ⁇ sheets that bind to the major groove of DNA.
  • helix-turn-helix helix-turn-helix
  • zinc finger helix-turn-helix
  • leucine zipper helix-loop-helix
  • Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA.
  • the double helix structure and repeated sequences of DNA create topological and chemical features which can be recognized by transcription factors. These features are hydrogen bond donor and acceptor groups, hydrophobic patches, major and minor grooves, and regular, repeated stretches of sequence which induce distinct bends in the helix.
  • transcription factors recognize specific DNA sequence motifs of about 20 nucleotides in length. Multiple, adjacent transcription 5 factor-binding motifs may be required for gene regulation.
  • DNA-binding structural motifs which comprise either helices or ⁇ sheets that bind to the major groove of DNA.
  • structural motifs are helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix. Proteins containing these motifs may act alone as monomers, or they may form homo- or heterodimers that interact with DNA.
  • the helix-turn-helix motif consists of two helices connected at a fixed angle by a short chain of amino acids. One of the helices binds to the major groove. Helix-turn-helix motifs are exemplified by the homeobox motif which is present in homeodomain proteins. These proteins are critical for specifying the anterior-posterior body axis during development and are conserved throughout the animal kingdom.
  • the Antennapedia and Ultrabithorax proteins of Drosophila melanogaster are prototypical homeodomain proteins (Pabo, CO. and R.T. Sauer (1992) Annu. Rev. Biochem. 61:1053-1095).
  • the zinc finger motif which binds zinc ions, generally contains tandem repeats of about 30 amino acids consisting of periodically spaced cysteine and histidine residues. Examples of this sequence pattern, designated C2H2 and C3HC4 ("RING" finger), have been described (Lewin, supra). Zinc finger proteins each contain an ⁇ helix and an antiparallel ⁇ sheet whose proximity and conformation are maintained by the zinc ion. Contact with DNA is made by the arginine prece ding the ⁇ helix and by the second, tliird, and sixth residues of the ⁇ helix. Variants of the zinc finger motif include poorly defined cysteine-rich motifs which bind zinc or other metal ions. These motifs may not contain histidine residues and are generally nonrepetitive.
  • the leucine zipper motif comprises a stretch of amino acids rich in leucine which can form an amphipathic helix. This structure provides the basis for dimerization of two leucine zipper proteins.
  • the region adjacent to the leucine zipper is usually basic, and upon protein dimerization, is optimally positioned for binding to the major groove. Proteins containing such motifs are generally referred to as bZIP transcription factors.
  • the helix-loop-helix motif (HLH) consists of a short ⁇ helix connected by a loop to a longer ⁇ helix.
  • the loop is flexible and allows the two helices to fold back against each other and to bind to DNA.
  • the transcription factor Myc contains a prototypical HLH motif.
  • Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-104). Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy.
  • the immune system responds to infection or trauma by activating a cascade ot events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process.
  • hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Isselbacher, K.J. et al. (1996) Harrison's Principles of Internal Medicine, 13/e,
  • Cell Membrane Molecules Eukaryotic cells are surrounded by plasma membranes which enclose the cell and maintain an environment inside the cell that is distinct from its surroundings. In addition, eukaryotic organisms are distinct from prokaryotes in possessing many intracellular organelle and vesicle structures. Many of the metabolic reactions which distinguish eukaryotic biochemistry from prokaryotic biochemistry take place within these structures.
  • the plasma membrane and the membranes surrounding organelles and vesicles are composed of phosphoglycerides, fatty acids, cholesterol, phospholipids, glycolipids, proteoglycans, and proteins. These components confer identity and functionality to the membranes with which they associate. Integral Membrane Proteins
  • TM proteins transmembrane proteins
  • TM domains are typically composed of 15 to 25 hydrophobic amino acids which are predicted to adopt an ⁇ -helical conformation.
  • TM proteins are classified as bitopic (Types I and'II) and polytopic (Types III and IV) (Singer, S.J. (1990) Annu. Rev. Cell Biol. 6:247-296).
  • Bitopic proteins span the membrane once while polytopic proteins contain multiple membrane-spanning segments.
  • TM proteins function as cell-surface receptors, receptor-interacting proteins, transporters of ions or metabolites, ion channels, cell anchoring proteins, and cell type-specific surface antigens.
  • Many membrane proteins contain amino acid sequence motifs that target these proteins to specific subcellular sites. Examples of these motifs include PDZ domains, KDEL, RGD, NGR, and
  • NGR NGR
  • GSL motif-containing peptides have been used as drag delivery agents in targeted cancer
  • membrane proteins may also contain amino acid sequence motifs, such as the carbohydrate recognition domain (CRD), that mediate interactions with extracellular or intracellular molecules.
  • CCD carbohydrate recognition domain
  • the transmembrane 4 superfamily (TM4SF) or tetraspan family is a multigene family 0 encoding type in integral membrane proteins (Wright, M.D. and M.G. Tomlinson (1994) Immunol. Today 15:588-594).
  • the TM4SF is composed of membrane proteins which traverse the cell membrane four times.
  • Members of the TM4SF include platelet and endothelial cell membrane proteins, melanoma-associated antigens, leukocyte surface glycoproteins, colonal carcinoma antigens, tumor-associated antigens, and surface proteins of the schistosome parasites (Jankowski, 5 S.A. (1994) Oncogene 9:1205-1211).
  • Members of the TM4SF share about 25-30% amino acid sequence identity with one another.
  • TM4SF members have been implicated in signal transduction, control of cell adhesion, regulation of cell growth and proliferation, including development and oncogenesis, and cell motility, including tumor cell metastasis.
  • Expression of TM4SF proteins is associated with a o variety of tumors and the level of expression may be altered when ceUs are growing or activated.
  • Tumor antigens are cell surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens distinguish tumor cells immunologically from normal cells and provide diagnostic and therapeutic targets for human cancers (Takagi, S. et al. (1995) Int. J. 5 Cancer 61:706-715; Liu, E. et al. (1992) Oncogene 7: 1027-1032).
  • Leukocyte Antigens are cell surface molecules that are differentially expressed in tumor cells relative to normal cells. Tumor antigens.
  • cell surface antigens include those identified on leukocytic cells of the immune system. These antigens have been identified using systematic, monoclonal antibody (mAb)- based "shot gun” techniques. These techniques have resulted in the production of hundreds of mAbs o directed against unknown cell surface leukocytic antigens. These antigens have been grouped into
  • CD antigens in a given cluster are presumed to identify a single cell surface protein and are assigned a "cluster of differentiation” or "CD” designation.
  • Some of the genes encoding proteins identified by CD antigens have been cloned and 5 verified by standard molecular biology techniques.
  • CD antigens have been characterized as both transmembrane proteins and cell surface proteins anchored to the plasma membrane via covalent attachment to fatty acid-containing glycolipids such as glycosylphosphatidylinositol (GPl).
  • GPl glycosylphosphatidylinositol
  • Ion channels are found in the plasma membranes of virtually every cell in the body.
  • chloride channels mediate a variety of cellular functions including regulation of membrane potentials and absorption and secretion of ions across epithelial membranes.
  • Chloride channels also regulate the pH of organelles such as the Golgi apparatus and endosomes (see, e.g., Greger, R. (1988)
  • Electrophysiological and pharmacological properties of chloride 0 channels including ion conductance, current-voltage relationships, and sensitivity to modulators, suggest that different chloride channels exist in muscles, neurons, fibroblasts, epithelial cells, and lymphocytes.
  • Proton channels have sites for phosphorylation by one or more protein kinases including protein kinase A, protein kinase C, tyrosine kinase, and casein kinase II, all of which regulate ion 5 channel activity in cells. Inappropriate phosphorylation of proteins in cells has been linked to changes in cell cycle progression and cell differentiation. Changes in the cell cycle have been linked to induction of apoptosis or cancer. Changes in cell differentiation have been linked to diseases and disorders of the reproductive system, immune system, skeletal muscle, and other organ systems.
  • Proton Pumps o Proton ATPases comprise a large class of membrane proteins that use the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane.
  • the resultant gradient may be used to transport other ions across the membrane (Na + , K + , or Cl " ) or to maintain organelle pH.
  • Proton ATPases are further subdivided into the mitochondrial F-ATPases, the plasma membrane ATPases, and the vacuolar ATPases.
  • the vacuolar ATPases establish and maintain an acidic pH 5 within various organelles involved in the processes of endocytosis and exocytosis (Mellman, I. et al. (1986) Annu. Rev. Biochem. 55:663-700).
  • Proton-coupled, 12 membrane-spanning domain transporters such as PEPT 1 and PEPT 2 are responsible for gastrointestinal absorption and for renal reabsorption of peptides using an electrochemical H + gradient as the driving force.
  • Another type of peptide transporter, the TAP o transporter is a heterodimer consisting of TAP 1 and TAP 2 and is associated with antigen processing. Peptide antigens are transported across the membrane of the endoplasmic reticulum by TAP so they can be expressed on the cell surface in association with MHC molecules.
  • Each TAP protein consists of multiple hydrophobic membrane spanning segments and a highly conserved ATP-binding cassette (Boll, M. et al. (1996) Proc. Natl. Acad. Sci.
  • Pathogenic 5 microorganisms such as herpes simplex virus, may encode inhibitors of TAP-mediated peptide transport in order to evade immune surveillance (Marusina, K. and JJ Manaco (1996) Curr. Opin. emato . : - .
  • membrane proteins are not membrane-spanning but are attached to the plasma membrane via membrane anchors or interactions with integral membrane proteins.
  • Membrane anchors are covalently joined to a protein post-translationally and include such moieties as prenyl, myristyl, and glycosylphosphatidyl inositol groups.
  • Membrane localization of peripheral and anchored proteins is important for their function in processes such as receptor-mediated signal transduction. For example, prenylation of Ras is required for its localization to the plasma membrane and for its normal and oncogenic functions in signal transduction.
  • Intercellular communication is essential for the development and survival of multicellular organisms.
  • Cells communicate with one another through the secretion and uptake of protein signaling molecules.
  • the uptake of proteins into the cell is achieved by the endocytic pathway, in which the interaction of extracellular signaling molecules with plasma membrane receptors results in the formation of plasma membrane-derived vesicles that enclose and transport the molecules into the cytosol. These transport vesicles fuse with and mature into endosomal and lysosomal (digestive) compartments.
  • the secretion of proteins from the cell is achieved by exocytosis, in which molecules inside of the cell proceed through the secretory pathway.
  • vesicles form at the transitional endoplasmic reticulum (tER), the rim of Golgi cisternae, the face of the Trans-Golgi Network (TGN), the plasma membrane (PM), and tubular extensions of the endosomes. Vesicle formation occurs when a region of membrane buds off from the donor organelle.
  • the membrane-bound vesicle contains proteins to be transported and is surrounded by a proteinaceous coat, the components of which are recruited from the cytosol.
  • a proteinaceous coat the components of which are recruited from the cytosol.
  • Two different classes of coat protein have been identified. Clathrin coats form on vesicles derived from the TGN and PM, whereas coatomer (COP) coats form on vesicles derived from the ER and Golgi.
  • COP coats can be further classified as COPI, involved in retrograde traffic through the Golgi and from the Golgi to the ER, and COPII, involved in anterograde traffic from the ER to the Golgi (Mellman, supra).
  • adapter proteins bring vesicle cargo and coat proteins together at the surface of the budding membrane.
  • Adapter protein- 1 and -2 select cargo from the TGN and plasma membrane, respectively, based on molecular information encoded on the cytoplasmic tail of integral membrane cargo proteins.
  • Adapter proteins also recruit clathrin to the bud site.
  • Clathrin is a protein complex consisting of three large and three small polypeptide chains arranged in a three-legged structure called a triskelion. Multiple triskelions and other coat proteins appear to se -assem e on t e mem rane to orm a coate p t.
  • s assem y process may serve to deform the membrane into a budding vesicle.
  • GTP-bound ADP-ribosylation factor (Arf) is also incorporated into the coated assembly.
  • Another small G-protein, dynamin forms a ring complex around the neck of the forming vesicle and may provide the mechanochemical force to seal the bud,
  • the coated vesicle complex is then transported through the cytosol.
  • the coat protein is assembled from cytosolic 0 precursor molecules at specific budding regions on the organelle.
  • the COP coat consists of two major components, a G-protein (Arf or Sar) and coat protomer (coatomer).
  • Coatomer is an equimolar complex of seven proteins, termed alpha-, beta-, beta'-, gamma-, delta-, epsilon- and zeta-COP.
  • the coatomer complex binds to dilysine motifs contained on the cytoplasmic tails of integral membrane proteins.
  • Eukaryotic cells are organized into various cellular organelles which has the effect of o separating specific molecules and their functions from one another and from the cytosol. Within the cell, various membrane structures surround and define these organelles while allowing them to interact with one another and the cell environment through both active and passive transport processes. Important cell organelles include the nucleus, the Golgi apparatus, the endoplasmic reticulum, mitochondria, peroxisomes, lysosomes, endosomes, and secretory vesicles. 5 Nucleus
  • the cell nucleus contains all of the genetic information of the cell in the form of DNA, and the components and machinery necessary for replication of DNA and for transcription of DNA into RNA. (See Alberts, B. et al. (1994) Molecular Biology of the Cell, Garland Publishing Inc., New York NY, pp. 335-399.) DNA is organized into compact structures in the nucleus by interactions o with various DNA-binding proteins such as histones and non-histone chromosomal proteins.
  • DNA-specific nucleases DNAses
  • DNAses partially degrade these compacted structures prior to DNA replication or transcription.
  • DNA replication takes place with the aid of DNA helicases which unwind the double-stranded DNA helix, and DNA polymerases that duplicate the separated DNA strands. 5
  • DNA helicases which unwind the double-stranded DNA helix
  • DNA polymerases that duplicate the separated DNA strands. 5
  • Many neoplastic disorders in humans can be attributed to inappropriate gene expression.
  • Malignant cell growth may result from either excessive expression of tumor promoting genes or insufficient expression of tumor suppressor genes (Cleary, M.L. (1992) Cancer Surv. 15:89-10 ).
  • Chromosomal translocations may also produce chimeric loci which fuse the coding sequence of one gene with the regulatory regions of a second unrelated gene. Such an arrangement likely results in inappropriate gene transcription, potentially contributing to malignancy.
  • the immune system responds to infection or trauma by activating a cascade of events that coordinate the progressive selection, amplification, and mobilization of cellular defense mechanisms. A complex and balanced program of gene activation and repression is involved in this process.
  • hyperactivity of the immune system as a result of improper or insufficient regulation of gene expression may result in considerable tissue or organ damage. This damage is well documented in immunological responses associated with arthritis, allergens, heart attack, stroke, and infections (Isselbacher, K.J. et al. (1996) Harrison's Principles of Internal Medicine, 13/e, McGraw Hill, Inc. and Teton Data Systems Software).
  • the nucleolus is a highly organized subcompartment in the nucleus that contains high concentrations of RNA and proteins and functions mainly in ribosomal RNA synthesis and assembly (Alberts, et al. supra, pp. 379-382).
  • Ribosomal RNA is a structural RNA that is complexed with proteins to form ribonucleoprotein structures called ribosomes. Ribosomes provide the platform on which protein synthesis takes place.
  • Ribosomes are assembled in the nucleolus initially from a large, 45S combined with a variety of proteins imported from the cytoplasm, as well as smaller, 5S rRNAs. Later processing of the immature ribosome results in formation of smaller ribosomal subunits which are transported from the nucleolus to the cytoplasm where they are assembled into functional ribosomes. Endoplasmic Reticulum
  • proteins are synthesized within the endoplasmic reticulum (ER), delivered from the ER to the Golgi apparatus for post-translational processing and sorting, and transported from the Golgi to specific intracellular and extracellular destinations. Synthesis of integral membrane proteins, secreted proteins, and proteins destined for the lumen of a particular organelle occurs on the rough endoplasmic reticulum (ER).
  • the rough ER is so named because of the rough appearance in electron micrographs imparted by the attached ribosomes on which protein synthesis proceeds.
  • Protein destined for the ER actually begins in the cytosol with the synthesis of a specific signal peptide which directs the growing polypeptide and its attached ribosome to the ER membrane where the signal peptide is removed and protein synthesis is completed.
  • Soluble proteins destined for the ER lumen, for secretion, or for transport to the lumen of other organelles pass completely into the ER lumen.
  • Transmembrane proteins destined for the ER or for other cell membranes are translocated across the ER membrane but remain anchored in the lipid bilayer of the membrane by one or more membrane-spanning ⁇ -helical regions.
  • Translocated polypeptide chains destined for other organelles or for secretion also told and assemble in the ER lumen with the aid of certain "resident" ER proteins.
  • Protein folding in the ER is aided by two principal types of protein isomerases, protein disulfide isomerase (PDI), and peptidyl- prolyl isomerase (PPI).
  • PDI protein disulfide isomerase
  • PPI peptidyl- prolyl isomerase
  • PPI peptidyl- prolyl isomerase
  • PPI an enzyme that catalyzes the isomerization of certain proline imide bonds in oligopeptides and proteins, is considered to govern one of the rate limiting steps in the folding of many proteins to their final functional conformation.
  • the cyclophilins represent a major class of PPI that was originally identified as the major receptor for the immunosuppressive drag cyclosporin A (Handschumacher, R.E. et al. (1984) Science 226:544-547).
  • Molecular "chaperones” such as BiP (binding protein) in the ER recognize incorrectly folded proteins as well as proteins not yet folded into their final form and bind to them, both to prevent improper aggregation between them, and to promote proper folding.
  • the Golgi apparatus is a complex structure that lies adjacent to the ER in eukaryotic cells and serves primarily as a sorting and dispatching station for products of the ER (Alberts, et al. supra, pp. 600-610). Additional posttranslational processing, principally additional glycosylation, also occurs in the Golgi including "O-linked" glycosylation of proteins. Indeed, the Golgi is a major site of carbohydrate synthesis, including most of the glycosaminoglycans of the extracellular matrix. N- linked oligosaccharides, added to proteins in the ER, are also further modified in the Golgi by the addition of more sugar residues to form complex N-linked oligosaccharides.
  • the terminal compartment of the Golgi is the Trans-Golgi Network (TGN), where both membrane and lumenal proteins are sorted for their final destination.
  • Other transport vesicles bud off containing proteins destined for the plasma membrane, such as receptors, adhesion molecules, and ion channels, and secretory proteins, such as hormones, neurotransmitters, and digestive enzymes.
  • the vacuole system is a collection of membrane bound compartments in eukaryotic cells that functions in the processes of endocytosis and exocytosis. They include phagosomes, lysosomes, endosomes, and secretory vesicles.
  • Endocytosis is the process in cells of internalizing nutrients, solutes or small particles (pinocytosis) or large particles such as internalized receptors, viruses, bacteria, or bacterial toxins (phagocytosis).
  • Exocytosis is the process of transporting molecules to the cell surface. It facilitates placement or localization of membrane-bound receptors or other membrane _ proteins ano secretion o ormones, neuro ransmitters, igestive enzymes, wastes, etc.
  • a common property of all of these vacuoles is an acidic pH environment ranging from approximately pH 4.5-5.0. This acidity is maintained by the presence of a proton ATPase that uses the energy of ATP hydrolysis to generate an electrochemical proton gradient across a membrane (Mellman, I. et al. (1986) Annu. Rev. Biochem. 55:663-700). Eukaryotic vacuolar proton ATPase
  • vp-ATPase is a multimeric enzyme composed of 3-10 different subunits.
  • One of these subunits is a highly hydrophobic polypeptide of approximately 16 kDa that is similar to the proteolipid component of vp-ATPases from eubacteria, fungi, and plant vacuoles (Mandel, M. et al. (1988) Proc. Natl. Acad.
  • the 16 kDa proteolipid component is the major subunit of the membrane portion of vp-ATPase and functions in the transport of protons across the membrane. Lysosomes
  • Lysosomes are membranous vesicles containing various hydrolytic enzymes used for the controlled intracellular digestion of macromolecules. Lysosomes contain some 40 types of enzymes including proteases, nucleases, glycosidases, Upases, phospholipases, phosphatases, and sulfatases, all of which are acid hydrolases that function at a pH of about 5. Lysosomes are surrounded by a unique membrane containing transport proteins that allow the final products of macromolecule degradation, such as sugars, amino acids, and nucleotides, to be transported to the cytosol where they may be either excreted or reutilized by the cell. A vp-ATPase, such as that described above, maintains the acidic environment necessary for hydrolytic activity (Alberts, supra, pp. 610-611). Endosomes
  • Endosomes are another type of acidic vacuole that is used to transport substances from the cell surface to the interior of the cell in the process of endocytosis. Like lysosomes, endosomes have an acidic environment provided by a vp-ATPase (Alberts et al. supra, pp. 610-618). Two types of endosomes are apparent based on tracer uptake studies that distinguish their time of formation in the cell and their cellular location. Early endosomes are found near the plasma membrane and appear to function primarily in the recycling of internalized receptors back to the cell surface.
  • Late endosomes appear later in the endocytic process close to the Golgi apparatus and the nucleus, and appear to be associated with delivery of endocytosed material to lysosomes or to the TGN where they may be recycled.
  • Specific proteins are associated with particular transport vesicles and their target compartments that may provide selectivity in targeting vesicles to their proper compartments.
  • a cytosolic prenylated GTP-binding protein, Rab is one such protein. Rabs 4, 5, and 11 are associated with the early endosome, whereas Rabs 7 and 9 associate with the late endosome.
  • Mitochondria are oval-shaped organelles comprising an outer membrane, a tightly folded inner membrane, an intermembrane space between the outer and inner membranes, and a matrix inside the inner membrane.
  • the outer membrane contains many porin molecules that allow ions and charged molecules to enter the intermembrane space, while the inner mem rane contains a variety of transport proteins that transfer only selected molecules. Mitochondria are the primary sites of energy production in cells.
  • Peroxisomes like mitochondria, are a major site of oxygen utilization. They contain one or 0 more enzymes, such as catalase and urate oxidase, that use molecular oxygen to remove hydrogen atoms from specific organic substrates in an oxidative reaction that produces hydrogen peroxide
  • Catalase oxidizes a variety of substrates including phenols, formic acid, formaldehyde, and alcohol and is important in peroxisomes of liver and kidney cells for detoxifying various toxic molecules that enter the bloodstream.
  • Another major function of oxidative 5 reactions in peroxisomes is the breakdown of fatty acids in a process called ⁇ oxidation, ⁇ oxidation results in shortening of the alkyl chain of fatty acids by blocks of two carbon atoms that are converted to acetyl CoA and exported to the cytosol for reuse in biosynthetic reactions.
  • peroxisomes import their proteins from the cytosol using a specific signal sequence located near the C-terminus of the protein.
  • the importance of this import process is o evident in the inherited human disease Zellweger syndrome, in which a defect in importing proteins into perixosomes leads to a perixosomal deficiency resulting in severe abnormalities in the brain, liver, and kidneys, and death soon after birth.
  • One form of this disease has been shown to be due to a mutation in the gene encoding a perixosomal integral membrane protein called peroxisome assembly factor- 1.
  • DNA-based arrays can provide a simple way to explore the expression of a single polymorphic gene or a large number of genes. When the expression of a single gene is explored, DNA-based arrays are employed to detect the expression of specific gene variants. For example, a p53 tumor suppressor gene array is used to determine whether individuals are carrying mutations that o predispose them to cancer. A cytochrome p450 gene array is useful to determine whether individuals have one of a number of specific mutations that could result in increased drag metabolism, drug resistance or drug toxicity.
  • DNA-based array technology is especially relevant for the rapid screening of expression of a large number of genes.
  • gene expression is affected in a global 5 fashion.
  • a genetic predisposition, disease or therapeutic treatment may affect, directly or indirectly, the expression of a large number of genes.
  • the interactions may be expected, such as when the genes are part of the same signaling pathway. In other cases, such as when the genes participate in separate signaling pathways, the interactions may be totally unexpected. Therefore,
  • DNA-based arrays can be used to investigate how genetic predisposition, disease, or therapeutic treatment affects the expression of a large number of genes.
  • the present invention relates to nucleic acid sequences comprising human diagnostic and therapeutic polynucleotides (dithp) as presented in the Sequence Listing.
  • the dithp uniquely identify genes encoding human structural, functional, and regulatory molecules.
  • the invention provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID 5 NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:l- 2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
  • the polynucleotide comprises a polynucleotide sequence selected from the group o consisting of SEQ ID NO: 1-2722.
  • the polynucleotide comprises at least 30 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of 5 SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a)
  • the polynucleotide comprises at least 60 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a o naturally occurring polynucleotide comprising a polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b); and e) an RNA equivalent of a) through d).
  • the invention further provides a composition for the detection of expression of human diagnostic and therapeutic polynucleotides 5 comprising at least one isolated polynucleotide comprising a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b) ; and e) an RNA equivalent of a) through d); and a detectable label.
  • a composition for the detection of expression of human diagnostic and therapeutic polynucleotides 5 compris
  • the invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a polynucleotide sequence of a polyneucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence of a polynucleotide selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b) ; and e) an RNA equivalent of a) through d).
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof, and, optionally, if present, the amount thereof.
  • the invention also provides a method for detecting a target polynucleotide in a sample, said target polynucleotide having a polynucleotide sequence of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b) ; and e) an RNA equivalent of a) through d).
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide, and b) detecting the presence or absence of said hybridization complex, and, optionally, if present, the amount thereof.
  • the invention provides a composition comprising a target polynucleotide of the method, wherein said probe comprises at least 30 contiguous nucleotides.
  • the invention provides a composition comprising a target polynucleotide of the method, wherein said probe comprises at least 60 contiguous nucleotides.
  • the invention further provides a recombinant polynucleotide comprising a promoter sequence operably Unked to an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1
  • a polynucleotide comprising a naturally occurring polynucleotide sequence at least ent ca to a po ynuc eot e sequence se ecte om t e group cons st ng o : -
  • the invention provides a cell transformed with the recombinant polynucleotide.
  • the invention provides a transgenic organism comprising the recombinant polynucleotide.
  • the invention also provides a method for producing a human diagnostic and therapeutic polypeptide, the method comprising a) culturing a cell under conditions suitable for expression of the human diagnostic and therapeutic polypeptide, wherein said cell is transformed with a recombinant polynucleotide, said recombinant polynucleotide comprising an isolated polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; iii) a polynucleotide complementary to the polynucleotide of i); iv) a polynucleotide complementary to the polynucleotide of ii
  • the invention also provides an isolated human diagnostic and therapeutic polypeptide (DITHP) encoded by at least one polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722.
  • DITHP diagnostic and therapeutic polypeptide
  • the invention further provides a method of screening for a test compound that specifically binds to the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the method comprises a) combining the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723- 5444 with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723- 5444 to the test compound, thereby identifying a compound that specifically binds to the polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the invention further provides a microarray wherein at least one element of the microarray is an isolated polynucleotide comprising at least 30 contiguous nucleotides of a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b) ; and e) an RNA equivalent of a) through d).
  • the invention also provides a method for generating a transcript image of a sample which contains polynucleotides.
  • the method comprises a) labeling the polynucleotides of the sample, b) contacting the elements of the microarray with the labeled polynucleotides of the sample under conditions suitable for the formation of a hybridization complex, and c) quantifying the expression of the polynucleotides in the sample.
  • the invention provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence 0 selected from the group consisting of SEQ ID NO: 1-2722; c) a polynucleotide complementary to the polynucleotide of a); d) a polynucleotide complementary to the polynucleotide of b) ; and e) an RNA equivalent of a) through d).
  • a target polynucleotide comprises a polynucleotide selected from the group consisting
  • the method comprises a) exposing a sample comprising the target polynucleotide to a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the 5 compound and in the absence of the compound.
  • the invention further provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide o comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; iii) a polynucleotide complementary to the polynucleotide of i); iv) a polynucleotide complementary to the polynucleotide of ii); and v) an RNA equivalent
  • Hybridization occurs under 5 conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1-2722; ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical to a o polynucleotide sequence selected from the group consisting of SEQ H NO: 1-2722; iii) a polynucleotide complementary to the polynucleotide of i); iv) a polynucleotide complementary to the polynucleotide of ii); and v) an RNA equivalent of i) through iv), and alternatively, the target
  • the invention further provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1;
  • the invention provides an isolated polypeptide comprising an amino acid sequence 0 selected from the group consisting of SEQ ID NO:2723-5444.
  • the invention further provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 5 SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the polynucleotide encodes a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ
  • the invention provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a 5 naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723- 5444, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the invention further provides a composition comprising a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence 5 selected from the group consisting of SEQ ID NO:2723-5444, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723- 5444, and a pharmaceutically acceptable excipient.
  • the composition comprises a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-
  • the invention additionally provides a method of treating a disease or condition associated with decreased expression of functional DITHP, comprising administering to a patient in need of such treatment the composition.
  • the invention also provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting agonist activity in the sample.
  • the invention provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • the invention provides a method of treating a disease or condition associated with decreased expression of functional DITHP, comprising administering to a patient in need of such treatment the composition.
  • the invention provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444.
  • the method comprises a) exposing a sample comprising the polypeptide to a compound, and b) detecting antagonist activity in the sample, fn one alternative, the invention provides a composition comprising an antagonist compound identified by the method and a pharmaceutically acceptable excipient. hi another alternative, the invention provides a method of treating a disease or condition associated with overexpression of functional DITHP, comprising administering to a patient in need of such treatment the composition.
  • the invention further provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2723-5444, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity of the polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity of the polypeptide in the presence of the test compound with the activity of the polypeptide in the absence of the test compound, wherein a change in the activity of the polypeptide in the presence of the test compound is indicative of a compound that modulates the activity of the polypeptide.
  • CD-R 1 is identified by: PN-0100 PCT, Copy 1- SEQUENCE LISTING PART, recorded on 09/11/03 and contains the Sequence Listing formatted in plain ASCII text. The file for the Sequence Listing is entitled PN-0100.seq.listing.txt, was recorded on 09/11/03 and is 22,079 KB in size.
  • CD-R 5 2 is an exact copy of CD-R-1.
  • CD-R 2 is identified by PN-0100 PCT, Copy 2 - SEQUENCE
  • CD-R 3 is an exact copy of CD-R-1.
  • CD-R 3 is identified by PN-0100 PCT, Copy 3 - SEQUENCE LISTING PART, recorded on 09/11/03.
  • CD-R 4 is an exact copy of CD-R-1.
  • CD-R 4 is identified by PN-0100 PCT, CRF, recorded on 09/11/03.
  • CD-R 5 is identified by: PN-0100 PCT, Copy 1- TABLES PART, recorded on 09/12/03 and contains: Tables 1, 2, 3, 4, and 5 formatted in plain ASCII text (tab delimited).
  • the file for Table 1 is entitled pn0100tl.txt, was recorded on 09/12/03 and is 112 KB in size.
  • the file for Table 2 is entitled pn0100t2.txt, was recorded on 09/12/03 and is 183 KB in size.
  • the file for Table 3 is entitled 5 pn0100t3.txt, was recorded on 09/12/03 and is 732 KB in size.
  • the file for Table 4 is entitled pn0100t4.txt, was recorded on 09/12/03 and is 565 KB in size.
  • the file for Table 5 is entitled pn0100t5.txt, was recorded on 09/12/03 and is 173 KB in size.
  • CD-R 6 is an exact copy of CD-R 5.
  • CD-R 6 is identified by: PN-0100 PCT, Copy 2 - TABLES PART, recorded on 09/12/03.
  • CD-R 7 is an exact copy of CD-R 5.
  • CD-R 7 is identified o by: PN-0100 PCT, Copy 3 - TABLES PART, recorded on 09/12/03.
  • Table 1 shows the sequence identification numbers (SEQ ID NO:s) and Incyte identification numbers (Incyte ID No.) corresponding to the polynucleotides of the present invention, along with the sequence ent cat on num ers :s an open rea ng rame i ent fication numbers (O
  • Table 2 shows the sequence identification numbers (SEQ ID NO:s) and and Incyte
  • Table 3 shows the sequence identification numbers (SEQ ID NO:s) and open reading frame identification numbers (ORF IDs) corresponding to polypeptides encoded by the Incyte ID numbers, 0 along with the annotations corresponding to the BioKnowledge hits.
  • Table 4 shows the sequence identification numbers (SEQ ID NO:s) and open reading frame identification numbers (ORF IDs) corresponding to the polypeptides of the present invention, along with polypeptide segments of each sequence as defined by the indicated “start” and “stop” polypeptide positions.
  • the polypeptide regions constitute either signal peptide (SP) or 5 transmembrane (TM) domains, as indicated.
  • SP signal peptide
  • TM transmembrane domains
  • the membrane topology of the polypeptide sequence is indicated as being transmembrane or on the cytosolic or non-cytosolic side of the cell membrane or organelle.
  • Table 5 shows the tissue distribution profiles for the polynucleotides of the invention.
  • Table 6 summarizes the bioinformatics tools which are useful for analysis of the o polynucleotides of the present invention.
  • the first column of Table 6 lists analytical tools, programs, and algorithms, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the 5 homology between two sequences).
  • dithp refers to a nucleic acid sequence
  • DITHP refers to an amino acid sequence encoded by dithp.
  • a "full-length” dithp refers to a nucleic acid sequence containing the entire coding region of a gene endogenously expressed in human tissue.
  • Adjuvants are materials such as Freund's adjuvant, mineral gels (aluminum hydroxide), and surface active substances (lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole 0 limpet hemocyanin, and dinitrophenol) which may be administered to increase a host's immunological response.
  • mineral gels aluminum hydroxide
  • surface active substances lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole 0 limpet hemocyanin, and dinitrophenol
  • Alleles refers to an alternative form of a nucleic acid sequence. Alleles result from a “mutation,” a change or an alternative reading of the genetic code. Any given gene may have none, one, or many allelic forms. Mutations which give rise to alleles include deletions, additions, or 5 substitutions of nucleotides. Each of these changes may occur alone, or in combination with the others, one or more times in a given nucleic acid sequence.
  • the present invention encompasses allelic dithp.
  • allelic variant is an alternative form of the gene encoding DITHP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in o polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding DITHP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as DITHP or a polypeptide with at least one functional characteristic of DITHP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding DITHP, and improper or unexpected hybridization to allelic variants, o with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent DITHP.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the 5 residues, as long as the biological or immunological activity of DITHP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged ammo acids may include lysine and arginine.
  • Ammo acids with uncharged polar side cnams navmg similar hydrophilicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having similar hydrophilicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • Amino acid sequence refers to a peptide, a polypeptide, or a protein of either natural or synthetic origin. The amino acid sequence is not limited to the complete, endogenous amino acid sequence and may be a fragment, epitope, variant, or derivative of a protein expressed by a nucleic acid sequence.
  • Amplification refers to the production of additional copies of a sequence and is carried out using polymerase chain reaction (PCR) technologies well known in the art.
  • PCR polymerase chain reaction
  • Antibody refers to intact molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding the epitopic determinant.
  • Antibodies that bind DITHP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen.
  • the polypeptide or peptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • RNA e.g., a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance of the aptamer from the circulatory system.
  • Aptamers may be specifically cross-linked to their cognate ligands, e.g., by photo-activation of a cross-linker. (See, e.g., Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13.)
  • introduction refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturally occurring enzymes, which normally act on substrates containing right-handed nucleotides.
  • Antisense sequence refers to a sequence capable of specifically hybridizing to a target sequence.
  • the antisense sequence may include DNA, RNA, or any nucleic acid mimic or analog such as peptide nucleic acid (PNA); oligonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, or benzylphosphonates; oligonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or oligonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyuracil, or 7-deaza-2'-deoxyguanosine.
  • PNA peptide nucleic acid
  • Antisense technology refers to any technology which relies on the specific hybridization of an antisense sequence to a target sequence.
  • a “bin” is a portion of computer memory space used by a computer program for storage of data, and bounded in such a manner that data stored in a bin may be retrieved by the program.
  • Bioly active refers to an amino acid sequence having a structural, regulatory, or biochemical function of a naturally occurring amino acid sequence.
  • Canonical splice site refers to the polynucleotide GTAG located on the positive strand of DNA.
  • “Clone joining” is a process for combining gene bins based upon the bins' containing sequence information from the same clone.
  • the sequences may assemble into a primary gene transcript as well as one or more splice variants.
  • “Complementary” describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing (5'-A-G-T-3' pairs with its complement 3'-T-C-A-5').
  • a “component sequence” is a nucleic acid sequence selected by a computer program such as PHRED and used to assemble a consensus or template sequence from one or more component sequences.
  • a "consensus sequence” or “template sequence” is a nucleic acid sequence which has been assembled from overlapping sequences, using a computer program for fragment assembly such as the
  • GEL VIEW fragment assembly system (Genetics Computer Group (GCG), Madison WI) or using a relational database management system (RDMS).
  • GCG Genetics Computer Group
  • RDMS relational database management system
  • Constant amino acid substitutions are those substitutions that, when made, least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative substitutions.
  • Trp Phe Tyr Tyr His, Phe, Trp Val Ue, Leu, Thr
  • Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • Genomic contig refers to contiguous genomic sequence obtained from a group of overlapping clones or sequences. Contigs derived from the NCBI may include draft and finished sequences and may contain sequence gaps (within a clone) or gaps between clones when the gap is spanned by another clone which is not sequenced. "Coverage start position” refers to the position of a nucleotide basepair (bp) on the genomic sequence where the first bp of a cDNA-to-genomic sequence alignment is located.
  • Crossage stop position refers to the position of a nucleotide bp on the genomic sequence where the last bp of a cDNA-to-genomic sequence alignment is located.
  • “Deletion” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or amino acid residue, respectively, is absent.
  • Derivative refers to the chemical modification of a nucleic acid sequence, such as by replacement of hydrogen by an alkyl, acyl, amino, hydroxyl, or other group.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • array element refers to a polynucleotide, polypeptide, or other chemical compound having a unique and defined position on a microarray.
  • modulate refers to a change in the activity of DITHP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of DITHP.
  • E-value refers to the statistical probability that a match between two sequences occurred by chance.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a structural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus allowing acceleration of the evolution of new protein functions.
  • a “fragment” is a unique portion of dithp or DITHP which can be identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue. For example, a fragment may comprise from 10 to 1000 contiguous amino acid residues or nucleotides.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50,
  • Fragments may be preferentially selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing and the figures, may be encompassed by the present embodiments.
  • a fragment of dithp comprises a region of unique polynucleotide sequence that specificaUy identifies dithp, for example, as distinct from any other sequence in the same genome.
  • a fragment of dithp is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish dithp from related polynucleotide sequences.
  • the precise length of a fragment of dithp and the region of dithp to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of DITHP is encoded by a fragment of dithp.
  • a fragment of DITHP comprises a region of unique amino acid sequence that specifically identifies DITHP. For example, a fragment of
  • DITHP is useful as an immunogenic peptide for the development of antibodies that specifically recognize DITHP.
  • the precise length of a fragment of DITHP and the region of DITHP to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a "full length" nucleotide sequence is one containing at least a start site for translation to a protein sequence, followed by an open reading frame and a stop site, and encoding a "full length" polypeptide.
  • “Hit” refers to a sequence whose annotation will be used to describe a given template. Criteria for selecting the top hit are as follows: if the template has one or more exact nucleic acid matches, the top hit is the exact match with highest percent identity. If the template has no exact matches but has significant protein hits, the top hit is the protein hit with the lowest E-value. If the template has no significant protein hits, but does have significant non-exact nucleotide hits, the top hit is the nucleotide hit with the lowest E-value.
  • Homology refers to sequence similarity either between a reference nucleic acid sequence and at least a fragment of a dithp or between a reference amino acid sequence and a fragment of a
  • Hybridization refers to the process by which a strand of nucleotides anneals with a complementary strand through base pairing. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under defined annealing conditions, and remain hybridized after the "washing" step.
  • the defined hybridization 0 conditions include the annealing conditions and the washing step(s), the latter of which is particularly important in determining the stringency of the hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid probes that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable and may be consistent among hybridization experiments, whereas wash conditions may 5 be varied among experiments to achieve the desired stringency.
  • T m thermal melting point
  • High stringency conditions for hybridization between polynucleotides of the present 5 invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1% SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, or 55°C may be used. SSC concentration may be varied from about 0.2 to 2 x SSC, with SDS being present at about 0.1%.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, denatured salmon sperm DNA at about 100-200 ⁇ g/ml. Useful variations on o these conditions will be readily apparent to those skilled in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their resultant proteins. Other parameters, such as temperature, salt concentration, and detergent concentration may be varied to achieve the desired stringency. Denaturants, such as formamide at a concentration of 5 about 35-50% v/v, may also be used under particular circumstances, such as RNA:DNA hybridizations. Appropriate hybridization conditions are routinely determinable by one of ordinary skill in the art.
  • Immunologically active or “immunogenic” describes the potential for a natural, recombinant, or synthetic peptide, epitope, polypeptide, or protein to induce antibody production in appropriate animals, cells, or cell lines.
  • Immunogenic response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, which may affect cellular and systemic defense systems.
  • An "immunogenic fragment” is a polypeptide or oligopeptide fragment of DITHP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • the term “immunogenic fragment” also includes any polypeptide or oligopeptide fragment of DITHP which can be useful in any of the antibody production methods disclosed herein or known in the art.
  • “Insertion” or “addition” refers to a change in either a nucleic or amino acid sequence in which at least one nucleotide or residue, respectively, is added to the sequence.
  • Labeling refers to the covalent or noncovalent joining of a polynucleotide, polypeptide, or antibody with a reporter molecule capable of producing a detectable or measurable signal.
  • “Microarray” is any arrangement of nucleic acids, amino acids, antibodies, etc., on a substrate.
  • the substrate may be a solid support such as beads, glass, paper, nitrocellulose, nylon, or an appropriate membrane.
  • Linkers are short stretches of nucleotide sequence which may be added to a vector or a dithp to create restriction endonuclease sites to facilitate cloning.
  • Polylinkers are engineered to incorporate multiple restriction enzyme sites and to provide for the use of enzymes which leave 5' or 3' overhangs (e.g., BamHI, EcoRI, and HindHI) and those which provide blunt ends (e.g., EcoRV,
  • Naturally occurring refers to an endogenous polynucleotide or polypeptide that may be isolated from viruses or prokaryotic or eukaryotic cells.
  • Nucleic acid sequence refers to the specific order of nucleotides joined by phosphodiester bonds in a linear, polymeric arrangement. Depending on the number of nucleotides, the nucleic acid sequence can be considered an oligomer, oligonucleotide, or polynucleotide. The nucleic acid can be
  • DNA, RNA, or any nucleic acid analog, such as PNA may be of genomic or synthetic origin, may be either double-stranded or single-stranded, and can represent either the sense or antisense
  • Oligomer refers to a nucleic acid sequence of at least about 6 nucleotides and as many as about 60 nucleotides, preferably about 15 to 40 nucleotides, and most preferably between about 20 and 30 nucleotides, that may be used in hybridization or amplification technologies. Oligomers may be used as, e.g., primers for PCR, and are usually chemically synthesized.
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNAs refers to a DNA mimic in which nucleotide bases are attached to a pseudopeptide backbone to increase stability. PNAs, also designated antigene agents, can prevent gene expression by targeting complementary messenger RNA.
  • percent identity and % identity refer to the percentage of residue matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence alignment program. This program is part of the LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5:151-153 and in Higgins, D.G. et al. (1992)
  • CABIOS 8 189-191.
  • the "weighted” residue weight table is selected as the default.
  • Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequence pairs.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • BLAST 215:403-410 which is available from several sources, including the NCBI, Bethesda, MD, and on the Internet at ncbi.nlm.nih.gov/BLAST/.
  • the BLAST software suite includes various sequence analysis programs including "BLASTN,” that is used to determine alignment between a known polynucleotide sequence and other sequences on a variety of databases. Also available is a tool called “BLAST 2 Sequences” that is used for direct pairwise comparison of two nucleotide sequences. "BLAST 2 Sequences" can be accessed and used interactively at ncbi.nlm.nih.gov/gorf/bl2/.
  • BLAST 2 Sequences can be used for both BLASTN and BLASTP (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use BLASTN with the "BLAST 2 Sequences” tool Version 2.0.9 (May-07-1999) set at default parameters. Such default parameters may be, for example:
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • percent identity and % identity refer to the percentage of residue matches between at least two polypeptide sequences aligned using a standardized algorithm.
  • Methods of polypeptide sequence alignment are well-known. Some alignment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detail above, generally preserve the hydrophobicity and acidity of the substituted residue, thus preserving the structure (and therefore function) of the folded polypeptide.
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in figures or Sequence Listings, may be used to describe a length over which percentage identity may be measured.
  • Probe refers to dithp or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences. Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • Primers are short nucleic acids, usually DNA oligonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme.
  • Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence, hi order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the figures and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) allows the user to input a "mispriming library," in which 5 sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, o thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to 5 identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • “Purified” refers to molecules, either polynucleotides or polypeptides that are isolated or separated from then natural environment and are at least 60% free, preferably at least 75% free, and most preferably at least 90% free from other compounds with which they are naturally associated.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook, supra.
  • recombinant includes nucleic acids that have 5 been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence.
  • Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is 5 expressed, inducing a protective immunological response in the mammal.
  • regulatory element refers to a nucleic acid sequence from nontranslated regions of a gene, and includes enhancers, promoters, introns, and 3' untranslated regions, which interact with host proteins to carry out or regulate transcription or translation.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, 0 an amino acid, or an antibody. They include radionuclides; enzymes; fluorescent, chemiluminescent, or chromogenic agents; substrates; cofactors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA sequence, is composed of the same linear sequence of nucleotides as the reference DNA sequence with the exception that all occurrences of the 5 nitrogenous base thymine are replaced with uracil, and the sugar backbone is composed of ribose instead of deoxyribose.
  • Samples may contain nucleic or amino acids, antibodies, or other materials, and may be derived from any source (e.g., bodily fluids including, but not limited to, saliva, blood, and urine; chromosome(s), organelles, or membranes isolated from a o cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; and cleared cells or tissues or blots or imprints from such cells or tissues).
  • source e.g., bodily fluids including, but not limited to, saliva, blood, and urine; chromosome(s), organelles, or membranes isolated from a o cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; and cleared cells or tissues or blots or imprints from such cells or tissues).
  • Specific binding or “specifically binding” refers to the interaction between a protein or peptide and its agonist, antibody, antagonist, or other binding partner. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, 5 recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide containing epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substitution refers to the replacement of at least one nucleotide or amino acid by a different 0 nucleotide or amino acid.
  • Substrate refers to any suitable rigid or semi-rigid support including, e.g., membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles or capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the collective pattern of gene expression by a particular cell type or tissue under given conditions at a given time.
  • Transformation refers to a process by which exogenous DNA enters a recipient ceU.
  • Transformation may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed.
  • Transformants include stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as cells which transiently express inserted DNA or RNA.
  • a "ttansgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by ttansgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the ttansgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, and plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA of the present invention into such organisms are widely known and provided in references such as Sambrook et al. (1989), supra.
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 25% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using BLASTN with the "BLAST 2 Sequences" tool Version 2.0.9 (May- 07-1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • the variant may result in "conservative" amino acid changes which do not affect structural and/or chemical properties.
  • a variant may be described as, for example, an "allelic” (as defined above), “splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species. Polymorphic variants also may encompass
  • SNPs single nucleotide polymorphisms
  • SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • variants of the polynucleotides of the present invention may be generated through recombinant methods.
  • One possible method is a DNA shuffling technique such as
  • MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No. 5,837,458;
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties. These preferred variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening. Thus, genetic diversity is created through "artificial" breeding and rapid molecular evolution.
  • fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized.
  • fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occurring genes in a directed and controllable manner.
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using BLASTP with the "BLAST 2 Sequences" tool Version 2.0.9 (May- 07-1999) set at default parameters.
  • Such a pah of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater identity over a certain defined length of one of the polypeptides.
  • cDNA sequences derived from human tissues and cell lines were aligned based on nucleotide sequence identity and assembled into "consensus" or "template” sequences which are designated by the Incyte identification numbers (Incyte ID No.) in column 2 of Table 2.
  • the sequence identification numbers (SEQ ED NO:s) corresponding to the Incyte IDs are shown in column 1.
  • the template sequences have similarity to GenBank sequences, or "hits,” as designated by the Gl Numbers in column 3.
  • the statistical probability of each GenBank hit is indicated by a probability score in column 4, and the functional annotation corresponding to each
  • GenBank hit is listed in column 5.
  • An alternative embodiment utilizes cDNA sequences disclosed in publically available DNA sequence databases (for example, GenBank, National Center for Biotechnology Information (NCBI), Bethesda, MD) as well as cDNA sequences derived from human tissues and cell lines which have been aligned to genomic contigs obtained from NCBI to identify cDNA transcripts.
  • the cDNA transcript template sequences are identified by the Incyte identification numbers (Incyte IDs) in column 2 of Table 2.
  • the sequence identification numbers (SEQ ID NO:s) corresponding to the cDNA transcript template IDs are shown in column 1.
  • the cDNA transcript template sequences have similarity to GenBank sequences, or "hits," as designated by the Gl Numbers in column 3.
  • the statistical probability of each GenBank hit is indicated by a probability score in column 4, and the functional annotation corresponding to each GenBank hit is listed in column 5.
  • the invention incorporates the nucleic acid sequences of these templates as disclosed in the Sequence Listing and the use of these sequences in the diagnosis and treatment of disease states characterized by defects in human molecules.
  • the invention further utilizes these sequences in hybridization and amplification technologies, and in particular, in technologies which assess gene expression patterns correlated with specific cells or tissues and their responses in vivo or in vitro to pharmaceutical agents, toxins, and other treatments.
  • the sequences of the present invention are used to develop a transcript image for a particular cell or tissue.
  • Derivation of Nucleic Acid Sequences cDNA was isolated from libraries constructed using RNA derived from normal and diseased human tissues and cell lines.
  • the human tissues and cell lines used for cDNA library construction were selected from a broad range of sources to provide a diverse population of cDNAs representative of gene transcription throughout the human body. Descriptions of the human tissues and cell lines used for cDNA library construction are provided in the LIFESEQ database (Incyte Corporation).
  • Human tissues were broadly selected from, for example, cardiovascular, dermatologic, endocrine, gastrointestinal, hematopoietic/immune system, musculoskeletal, neural, reproductive, and urologic sources.
  • Cell lines used for cDNA library construction were derived from, for example, leukemic cells, teratocarcinomas, neuroepitheliomas, cervical carcinoma, lung fibroblasts, and endothelial cells. Such cell lines include, for example, THP-1, Jurkat, HUVEC, hNT2, WI38, HeLa, and other cell lines commonly used and available from public depositories (American Type Culture Collection, Manassas VA).
  • cell lines Prior to mRNA isolation, cell lines were untreated, treated with a pharmaceutical agent such as 5'-aza-2'-deoxycytidine, tteated with an activating agent such as lipopolysaccharide in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress.
  • a pharmaceutical agent such as 5'-aza-2'-deoxycytidine
  • an activating agent such as lipopolysaccharide in the case of leukocytic cell lines, or, in the case of endothelial cell lines, subjected to shear stress.
  • Chain termination reaction products may be electrophoresed on urea- polyacrylamide gels and detected either by autoradiography (for radioisotope-labeled nucleotides) or by fluorescence (for fluorophore-labeled nucleotides).
  • Automated methods for mechanized reaction preparation, sequencing, and analysis using fluorescence detection methods have been developed.
  • Machines used to prepare cDNAs for sequencing can include the MICROLAB 2200 liquid transfer system (Hamilton Company (Hamilton), Reno NV), Peltier thermal cycler (PTC200; MJ Research, Inc. (MJ Research), Watertown MA), and ABI CATALYST 800, thermal cycler (Applied
  • Sequencing can be carried out using, for example, the ABI 373 or 377 (Applied Biosystems) or MEGABACE 1000 (Molecular Dynamics, Inc. (Molecular Dynamics), Sunnyvale CA) DNA sequencing systems, or other automated and manual sequencing systems well known in the art.
  • the clones are pooled into 'shot-gun' libraries.
  • the cDNA inserts from these pools are amplified by PCR, mechanically sheared into smaller pieces and cloned into plasmid vectors for vector primer sequencing. Assembly of the nucleic acid sequences of the small pieces into their respective parent fuU-length insert can then be accomplished using sequence assembly programs such as PHRAP phrap.org/phrap.docs/phrap.html).
  • DNA sequencing primers derived from cloning vectors generate only nucleic acid sequence coverage of the cDNA insert ends of a clone
  • the complete sequence of the internal regions of cDNA inserts can be achieved by performing DNA sequencing using gene specific primers and "primer-walking" methods (Shyamala, V. and G.F. Ames (1989) Gene 84:1-8, hereby incorporated by reference in its entkety).
  • Primer-walking is carried out in iterative cycles until the primer-walk sequences can be assembled into a non-gapped, contiguous consensus sequence.
  • nucleotide sequences of the Sequence Listing have been prepared by current, state-of- the-art, automated methods and, as such, may contain occasional sequencing errors or unidentified nucleotides. Such unidentified nucleotides are designated by an N. These infrequent unidentified bases do not represent a hindrance to practicing the invention for those skilled in the art.
  • Several methods employing standard recombinant techniques may be used to correct errors and complete the missing sequence information. (See, e.g., those described in Ausubel, F.M. et al. (1997) S ort
  • IRISS Incyte Research Informatics Sequence Search
  • Comparable pairings can be achieved using publically available alignment algorithms such as MEGABLAST (Zhang, Z. et al. (2000) J. Comput. Biol. 7:203-214). A pairing occurred if 50% of the length of the 5 cDNA sequence was aligned.
  • the cDNA/ genomic pairings identified by IRISS were analyzed using the SEVI4 alignment algorithm (version May 2000 with optimization for high throughput and strand assignment confidence, Florea et al., (1998) Genome Res. 8:967-974). For cDNAs which hit multiple genomic locations, only the SIM4 alignment providing the highest percent identity was retained. The SIM4 results were then analyzed by determining alignment quality, strand assignment o and polyA location.
  • the alignment quality was assessed first by examining the terminal exons of cDNAs. Terminal exons were cleaved if the exon was less than 9 bp in length or the intron length exceeded 40 Kb and a certain exon length and percent identity threshold was not met (as determined by the intron length). Terminal exons were also cleaved if the exons appeared to be derived from poly A tails. Any cDNA sequence meeting any of the following criteria was determined to be a false 5 result: i) a gap of more than 5 bp present within the aligned cDNA/genomic pairing; ii) the global identity or coverage of the alignment was below 95%; or iii) the global coverage length of the alignment was less than 50 bp.
  • ESTScan (Iseli, C et al. (1999) Roc. Int. Conf. Intel! Syst. Mol. Biol. AAAI, 1999:138-148) if such predictions were available.
  • strand assignment was either based on overlap with an assigned cDNA, or the cDNA was assigned by default to the positive strand. 5 Determination of the polyA site was performed by examining an area of the cDNA 35 bp upstream to 120 bp downstream of the 3' end and searching for the presence of the eleven known variants of the poly-adenylation signal (Beaudoing, E. et al., (2000) Genome Res. 10:1001-1010).
  • the clustering is performed by sorting all clones on the genomic contig, left to right (smaller to larger genomic start coordinate). The contig was then examined left to right. The first clone encountered was placed into a gene bound. AU subsequent clones found to "link" to that initial clone via the above criteria were placed into that gene bound. This linking process was carried out for all of the clones which linked to the initial clone, and then performed for all of the clones that linked to a clone that linked to the initial clone, in a reiterative process. When no further clones were found which could be linked and placed into the gene bound, the examination of the genomic contig continued 3' to 5' until a new clone was found. A new gene bound was created and the linking/clustering process was repeated anew. This process was performed until the end of the genomic contig was reached. This resulted in determining all of the allowed gene bounds within that particular genomic contig.
  • cDNA transcripts were generated within gene bounds by a multi-step process that first modified the start and stop coordinates of cDNAs in order to generate transcripts which were devoid of hnRNA (heteronuclear RNA) contamination and potentially faulty misalignments due to SIM4.
  • hnRNA was identified as start/stop coordinates of one cDNA transcript appearing within the intron of another cDNA transcript.
  • Correction involved moving the coverage start or coverage stop locations of the cDNA transcript to the next nearest splice start or stop, or moving to a second more distant splice start and stop if both cDNA transcripts in question shared the same splice start/stop next to the coverage start/stop in question. 5 cDNAs were grouped together for misalignment analysis if they had the same intron size (+/-
  • Introns were identified as misaligned if the splice site window identity average was greater than 98% (window size 10 bp on each side of splice site) and the intron had a total depth of greater than or equal to 5% (of cDNAs in the cluster). The depth was calculated from the number of cDNAs used as evidence for o this splicing event over all cDNAs used in the analysis and whether a canonical splice site was present, or the splice site window identity of any cDNA was 100%, regardless of depth and score. If none of these criteria were met, the splice sites of the cDNAs were adjusted so that then intron was the same as the splice site represented by the majority of cDNAs.
  • the transcripts are generated from the cDNAs in the following manner. 5
  • the cDNAs are represented as nodes in a directed acyclic graph (DAG). Each node is linked in a directed manner to other nodes if the cDNA of the original node can be extended by the cDNAs represented by the other nodes. Extension is defined by "extending a cDNA in the 3' direction” if the extending cDNA has a subset of exons which in turn are a subset of the original cDNA exons. This process is called "mating". Whenever a cDNA can be mated to another, a directed link is created in o the DAG. After all such links are created, transcript generation can proceed.
  • DAG directed acyclic graph
  • a transcript is generated by starting at a node that has no incoming links, (current cDNA cannot be extended 5' by another cDNA), following an outgoing link (current cDNA can be extended 3 'by another cDNA) to the next node, and repeating that process until finally a node is reached which has no outgoing links (current cDNA cannot be extended 3' by another possible cDNA).
  • This is 5 called “traversing" the DAG. All the distinct exons encountered by such a traversal in going from node to node (i,e., from cDNA to cDNA) are then assembled into the exons of a transcript. All distinct traversals of the DAG (distinct sets of exons) lead to the generation of all distinct transcripts for that gene bound.
  • Human polynucleotide sequences may be assembled using programs or algorithms well known in the art. Sequences to be assembled are related, wholly or in part, and may be derived from a single or many different transcripts. Assembly of the sequences can be performed using such programs as PHRAP (Phils Revised Assembly Program) and the GELVIEW fragment assembly system (GCG), or other methods known in the art.
  • PHRAP Phils Revised Assembly Program
  • GCG GELVIEW fragment assembly system
  • cDNA sequences are used as "component" sequences that are assembled into “template” or “consensus” sequences as follows. Sequence chromatograms are processed, verified, and quality scores are obtained using PHRED. Raw sequences are edited using an editing pathway known as Block 1 (See, e.g., the LIFESEQ Assembled User Guide, Incyte). A series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's", or masked, to prevent spurious matches. Mitochondrial and ribosomal RNA sequences are also removed.
  • Block 1 See, e.g., the LIFESEQ Assembled User Guide, Incyte.
  • a series of BLAST comparisons is performed and low-information segments and repetitive elements (e.g., dinucleotide repeats, Alu repeats, etc.) are replaced by "n's
  • the processed sequences are then loaded into a relational database management system (RDMS) which assigns edited sequences to existing templates, if available.
  • RDMS relational database management system
  • a process is initiated which modifies existing templates or creates new templates from works in progress (i.e., nonfinal assembled sequences) containing queued sequences or the sequences themselves.
  • the templates can be merged into bins. If multiple templates exist in one bin, the bin can be split and the templates reannotated.
  • bins are "clone joined" based upon clone information. Clone joining occurs when the 5' sequence of one clone is present in one bin and the 3' sequence from the same clone is present in a different bin, indicating that the two bins should be merged into a single bin. Only bins which share at least two different clones are merged.
  • a resultant transcprit template sequence may contain either a partial or a full length open reading frame, or all or part of a genetic regulatory element. This variation is due in part to the fact that the full length cDNAs of many genes are several hundred, and sometimes several thousand, bases in length. With current technology, cDNAs comprising the coding regions of large genes cannot be cloned because of vector limitations, incomplete reverse transcription of the mRNA, or incomplete "second strand" synthesis. Template sequences may be extended to include additional contiguous sequences derived from the parent RNA transcript using a variety of methods known to those of skill in the art. Extension may thus be used to achieve the full length coding sequence of a gene.
  • cDNA sequences are analyzed using a variety of programs and algorithms which are well known in the art. (See, e.g., Ausubel, 1997, supra. Chapter 7.7; Meyers, R.A. (Ed.) (1995) Molecular
  • 25:3389-3402 is especially useful in determining exact and gapped matches and by comparing two sequence fragments of arbitrary but equal lengths, whose alignment is locally maximal and for which the alignment score 5 meets or exceeds a threshold or cutoff score set by the user (Karlin, S. et al. (1988) Proc. Natl. Acad. Sci. USA 85:841-845).
  • an appropriate search tool e.g., BLAST or HMM
  • GenBank, SwissProt, PFAM, Protein Data Bank (PDB) and other databases may be searched for sequences containing regions of homology to a query dithp or DITHP of the present invention.
  • the cDNA transcript template sequences were further analyzed by translating each template using BLASTX against either the SwissProt or GenPept (version 130) databases, saving those hits with an E-value less than or equal to le-45.
  • Transcripts having a predicted protein were evaluated by 5 both a global alignment-based translation method and a maximal size ORF (Open Reading Frame)- based ttanslation method, hi the global alignment method, the transcript was realigned to its top BLASTX hit to either SwissProt or GenPept (version 130) using a global, end-gaps-free, frameshift- tolerant alignment algorithm (GCG FRAMEALIGN, Accelrys Inc. San Diego, CA).
  • GCG FRAMEALIGN Accelrys Inc. San Diego, CA
  • Evaluation of the predicted protein translation by the ORF-based translation method used all ORFs of at least 60 amino acids in length which were identified using the translation generation program available in LIFESEQ (Incyte).
  • the ORFs which met the following two criteria were considered further.
  • the reading frame of the ORF was identical to that of the top BLASTX HSP 5 (local alternative alignment) from either SwissProt or GenPept (version 130) with an E-value less than or equal to le-45 and second, the ORF overlapped the nucleotide alignment coordinates of the top BLASTX HSP. The longest such ORF was used as the predicted protein.
  • DITHP The identities of the DITHP encoded by the dithp of the present invention were obtained by analysis of the assembled cDNA and template sequences. Human molecules encoding DITHP are 0 classified by then GenBank annotation into a hierarchical classification system. Table 1, column 5 indicates the identities of DITHP which correspond to the following Protein Functional Hierarchy (PFH) classification:
  • PSH Protein Functional Hierarchy
  • the dithp of the present invention may be used for a variety of diagnostic and therapeutic purposes.
  • a dithp may be used to diagnose a particular condition, disease, or disorder associated with human molecules.
  • Such conditions, diseases, and disorders include, but are not limited to, a cell proliferative disorder, such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, a cancer of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, gan
  • MCTD mixed connective tissue disease
  • multiple sclerosis myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, polycythemia vera, polymyositis,
  • hypothalamic hormone a disorder associated with hyperthyroidism including thyrotoxicosis and its various forms, Grave's disease, pretibial myxedema, toxic multinodular goiter, thyroid carcinoma, and Plummer's disease; a disorder associated with hyperparathyroidism including
  • Conn disease chronic hypercalemia
  • a pancreatic disorder such as Type I or Type H diabetes mellitus and associated complications
  • a disorder associated with the adrenals such as hyperplasia, carcinoma, or adenoma of the adrenal cortex, hypertension associated with alkalosis, amyloidosis, hypokalemia, Cushing's disease, Liddle's syndrome, and Arnold-Healy-Gordon syndrome, pheochromocytoma tumors, and Addison's disease
  • a disorder associated with gonadal steroid hormones such as: in women, abnormal prolactin production, infertility, endometriosis, perturbation of the menstrual cycle, polycystic ovarian disease, hyperprolactinemia, isolated gonadotropin deficiency, amenorrhea, galactorrhea, hermaphroditism, hirsutism and virilization, breast cancer, and, in post-menopausal women, osteop
  • Tay-Sachs disease Sandhoff s disease, hyperlipidemia, hyperlipemia, lipid myopathies, and obesity; and disorders of copper metabolism such as Menke's disease, Wilson's disease, and Ehlers-Danlos syndrome type IX; a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Huntington's disease, dementia, Parkinson's 0 disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease, prion diseases including kuru, Creutzfeldt
  • ttansport e.g., neurofibromatosis, postherpetic neuralgia, trigeminal neuropathy, sarcoidosis, sickle cell anemia, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, Grave's disease, goiter, glucose-galactose malabsorption syndrome, hypercholesterolemia, Cushing's disease, and Addison's disease; and a connective tissue disorder 0 such as osteogenesis imperfecta, Ehlers-Danlos syndrome, chondrodysplasias, Marfan syndrome, Alport syndrome, familial aortic aneurysm, achondroplasia, mucopolysaccharidoses, osteoporosis, osteopetrosis, Paget's disease, rickets, osteomalacia, hyper
  • the dithp can be used to detect the presence of, or to quantify the amount of, a dithp-related polynucleotide in a sample. This information is then compared to information 5 obtained from appropriate reference samples, and a diagnosis is established.
  • a polynucleotide complementary to a given dithp can inhibit or inactivate a therapeutically relevant gene related to the dithp.
  • the expression of dithp may be routinely assessed by hybridization-based methods to 0 determine, for example, the tissue-specificity, disease-specificity, or developmental stage-specificity of dithp expression.
  • the level of expression of dithp may be compared among different cell types or tissues, among diseased and normal cell types or tissues, among cell types or tissues at different developmental stages, or among cell types or tissues undergoing various treatments. This type of analysis is useful, for example, to assess the relative levels of dithp expression in fully or 5 partially differentiated cells or tissues, to determine if changes in dithp expression levels are correlated with the development or progression of specific disease states, and to assess the response of a cell or tissue to a specific therapy, for example, in pharmacological or toxicologica stu ies.
  • Methods for the analysis of dithp expression are based on hybridization and amplification technologies and include membrane-based procedures such as northern blot analysis, high-throughput procedures that utilize, for example, microarrays, and PCR-based procedures.
  • the dithp, their fragments, or complementary sequences may be used to identify the presence of and/or to determine the degree of similarity between two (or more) nucleic acid sequences.
  • the dithp may be hybridized to naturally occurring or recombinant nucleic acid sequences under appropriately selected temperatures and salt concentrations. Hybridization with a probe based on the l o nucleic acid sequence of at least one of the dithp allows for the detection of nucleic acid sequences, including genomic sequences, which are identical or related to the dithp of the Sequence Listing. Probes may be selected from non-conserved or unique regions of at least one of the polynucleotides of SEQ ID NO: 1-2722 and tested for their ability to identify or amplify the target nucleic acid sequence using standard protocols.
  • SEQ ID NO: 1-2722 and fragments thereof can be identified using various conditions of stringency.
  • stringency See, e.g., Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511.
  • Hybridization conditions are discussed in "Definitions.”
  • a probe for use in Southern or northern hybridization may be derived from a fragment of a
  • probes 20 dithp sequence, or its complement, that is up to several hundred nucleotides in length and is either single-sttanded or double-stranded.
  • probes may be hybridized in solution to biological materials such as plasmids, bacterial, yeast, or human artificial chromosomes, cleared or sectioned tissues, or to artificial substrates containing dithp.
  • Microarrays are particularly suitable for identifying the presence of and detecting the level of expression for multiple genes of interest by examining gene
  • An array analogous to a dot or slot blot may be used to arrange and link polynucleotides to the surface of a substrate using one or more of the, following: mechanical (vacuum), chemical, thermal, or UV bonding procedures.
  • Such an array may contain any number of dithp and may be produced by hand or by using available devices, materials, and machines.
  • Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g.,
  • Probes may be labeled by either PCR or enzymatic techniques using a variety of commercially available reporter molecules.
  • commercial kits are available for radioactive and chemiluminescent labeling (Amersham Pharmacia Biotech) and for alkaline phosphatase labeling (Life Technologies).
  • dithp may be cloned into commercially available vectors for the production of RNA probes.
  • Such probes may be transcribed in the presence of at least one labeled nucleotide (e.g., 32 P-ATP, Amersham Pharmacia Biotech).
  • polynucleotides of SEQ ID NO: 1-2722 or suitable fragments thereof can be used to isolate full length cDNA sequences utilizing hybridization and/or amplification procedures well known in the art, e.g., cDNA library screening, PCR amplification, etc.
  • the molecular cloning of such full length cDNA sequences may employ the method of cDNA library screening with probes using the hybridization, stringency, washing, and probing strategies described above and in Ausubel, 0 supra. Chapters 3, 5, and 6. These procedures may also be employed with genomic libraries to isolate genomic sequences of dithp in order to analyze, e.g., regulatory elements. Genetic Mapping
  • Gene identification and mapping are important in the investigation and treatment of almost all conditions, diseases, and disorders. Cancer, cardiovascular disease, Alzheimer's disease, arthritis, 5 diabetes, and mental illnesses are of particular interest. Each of these conditions is more complex than the single gene defects of sickle cell anemia or cystic fibrosis, with select groups of genes being predictive of predisposition for a particular condition, disease, or disorder.
  • cardiovascular disease may result from malfunctioning receptor molecules that fail to clear cholesterol from the bloodstream
  • diabetes may result when a particular individual' s immune o system is activated by an infection and attacks the insulin-producing cells of the pancreas, hi some studies, Alzheimer's disease has been linked to a gene on chromosome 21; other studies predict a different gene and location.
  • Mapping of disease genes is a complex and reiterative process and generally proceeds from genetic linkage analysis to physical mapping.
  • a genetic linkage map traces parts of 5 chromosomes that are inherited in the same pattern as the condition.
  • Statistics link the inheritance of particular conditions to particular regions of chromosomes, as defined by RFLP or other markers.
  • RFLP radio frequency domain
  • markers and their locations are known from previous studies. More often, however, the markers are simply stretches of DNA that differ among individuals. Examples of o genetic linkage maps can be found in various scientific journals or at the Online Mendelian
  • dithp sequences may be used to generate hybridization probes useful in chromosomal mapping of naturally occurring genomic sequences. Either coding or noncoding sequences of dithp may be used, and in some instances, noncoding 5 sequences may be preferable over coding sequences. For example, conservation of a dithp coding sequence among members of a multi-gene family may potentially cause undesired cross hybridization ur ng c romosoma mapp ng.
  • e sequences may e mappe to a part cu ar c romosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial PI constructions
  • chromosome cDNA libraries See, e.g., Harrington, JJ.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Meyers, supra, pp. 965-968.) Correlation between the location of dithp on a physical chromosomal map and a specific disorder, or a i o predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the dithp sequences may also be used to detect polymorphisms that are genetically linked to the inheritance of a particular condition, disease, or disorder.
  • In situ hybridization of chromosomal preparations and genetic mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending existing genetic
  • nucleotide sequences of the subject invention may also be used to detect differences in chromosomal architecture due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • a disease-associated gene is mapped to a chromosomal region, the gene must be cloned in order to identify mutations or other alterations (e.g., translocations or inversions) that may be correlated with disease.
  • This process requires a physical map of the chromosomal region containing the disease-gene of interest along with associated markers. A physical map is necessary for determining the nucleotide sequence of and order of marker genes on a particular chromosomal
  • Physical mapping techniques are well known in the art and require the generation of overlapping sets of cloned DNA fragments from a particular organelle, chromosome, or genome. These clones are analyzed to reconstruct and catalog their order. Once the position of a marker is determined, the DNA from that region is obtained by consulting the catalog and selecting clones from that region. The gene of interest is located through positional cloning techniques using hybridization
  • the dithp of the present invention may be used to design probes useful in diagnostic assays.
  • Such assays may be used to detect or confirm conditions, disorders, or diseases associated with abnormal levels of dithp expression.
  • Labeled probes developed from dithp sequences are added to a sample under hybridizing conditions of desired stringency.
  • dithp, or fragments or oligonucleotides derived from dithp may be used as primers in amplification steps prior to hybridization.
  • the amount of hybridization complex formed is quantified and compared with standards for that cell or tissue. If dithp expression varies significantly from the standard, the assay indicates the presence of the condition, disorder, or disease.
  • Qualitative or quantitative diagnostic methods may include northern, dot blot, or other membrane or dip-stick based technologies or multiple-sample format technologies such as PCR, enzyme-linked immunosorbent assay (ELISA)-like, pin, or chip-based assays.
  • PCR enzyme-linked immunosorbent assay
  • the probes described above may also be used to monitor the progress of conditions, disorders, or diseases associated with abnormal levels of dithp expression, or to evaluate the efficacy of a particular therapeutic treatment.
  • the candidate probe may be identified from the dithp that are specific to a given human tissue and have not been observed in GenBank or other genome databases.
  • Such a probe may be used in animal studies, preclinical tests, clinical trials, or in monitoring the treatment of an individual patient, hi a typical process, standard expression is established by methods well known in the art for use as a basis of comparison, samples from patients affected by the disorder or disease are combined with the probe to evaluate any deviation from the standard profile, and a therapeutic agent is administered and effects are monitored to generate a treatment profile.
  • Efficacy is evaluated by determining whether the expression progresses toward or returns to the standard normal pattern. Treatment profiles may be generated over a period of several days or several months. Statistical methods well known to those skilled in the art may be use to determine the significance of such therapeutic agents.
  • the polynucleotides are also useful for identifying individuals from minute biological samples, for example, by matching the RFLP pattern of a sample's DNA to that of an individual's DNA.
  • the polynucleotides of the present invention can also be used to determine the actual base-by-base DNA sequence of selected portions of an individual's genome. These sequences can be used to prepare PCR primers for amplifying and isolating such selected DNA, which can then be sequenced. Using this technique, an individual can be identified through a unique set of DNA sequences. Once a unique ID database is established for an individual, positive identification of that individual can be made from extremely small tissue samples.
  • oligonucleotide primers derived from the dithp of the invention may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • oligonucleotide primers derived from dit p are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodily fluids, and the like.
  • sequence database analysis methods termed in silico SNP (isSNP) are capable of identifying polymorphisms by comparing the sequences of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • DNA-based identification techniques are critical in forensic technology. DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, semen, etc., can be amplified using, e.g., PCR, to identify individuals. (See, e.g., Erlich, H. (1992) PCR Technology, Freeman and Co., New York, NY).
  • polynucleotides of the present invention can be used as polymorphic markers. There is also a need for reagents capable of identifying the source of a particular tissue.
  • reagents can comprise, for example, DNA probes or primers prepared from the sequences of the present invention that are specific for particular tissues. Panels of such reagents can identify tissue by species and/or by organ type. In a similar fashion, these reagents can be used to screen tissue cultures for contamination.
  • the polynucleotides of the present invention can also be used as molecular weight markers on nucleic acid gels or Southern blots, as diagnostic probes for the presence of a specific mRNA in a particular cell type, in the creation of subtracted cDNA libraries which aid in the discovery of novel polynucleotides, in selection and synthesis of oligomers for attachment to an array or other support, and as an antigen to elicit an immune response.
  • the dithp of the invention or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) cells.
  • ES embryonic stem
  • Such techniques are well known in the art and are useful for the generation of animal models of human disease.
  • mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown in culture.
  • the ES cells are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the , . . _, - . integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des polynucléotides humains purifiés utilisés à des fins diagnostiques et thérapeutiques (dithp) ainsi que des polypeptides (DITHP) codés par ces dithp. L'invention concerne également l'utilisation de ces dithp, ou de leurs compléments, oligonucléotides ou fragments dans des analyses diagnostiques. L'invention concerne des vecteurs et des cellules hôtes contenant ces dithp et permettant l'expression de DITHP. L'invention concerne également l'utilisation de DITHP isolés et purifiés pour induire des anticorps et passer au crible des banques de composés ainsi que l'utilisation d'anticorps anti-DITHP dans des analyses diagnostiques. L'invention concerne également des microéchantillons contenant ces dithp et leurs procédés d'utilisation.
PCT/US2003/028227 2002-09-12 2003-09-12 Molecules utilisees a des fins diagnostiques et therapeutiques WO2004023973A2 (fr)

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