WO2004016641A2 - Proteines intervenant dans la regulation de l'homeostase energetique - Google Patents

Proteines intervenant dans la regulation de l'homeostase energetique Download PDF

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WO2004016641A2
WO2004016641A2 PCT/EP2003/008826 EP0308826W WO2004016641A2 WO 2004016641 A2 WO2004016641 A2 WO 2004016641A2 EP 0308826 W EP0308826 W EP 0308826W WO 2004016641 A2 WO2004016641 A2 WO 2004016641A2
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nucleic acid
polypeptide
protein
acid molecule
homologous
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WO2004016641A3 (fr
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Karsten Eulenberg
Günter BRÖNNER
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DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
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    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the use of nucleic acid sequences encoding foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri homologous proteins, to the use of polynucleotides encoding these, and to the use of modulators/effectors of the proteins and polynucleotides in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation, for example, but not limited to, metabolic diseases or dysfunctions such as obesity, metabolic syndrome, diabetes mellitus, eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • metabolic diseases or dysfunctions such as obesity, metabolic syndrome, diabetes mellitus, eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease,
  • Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes more and more relevant for western society. Obesity is defined as a body weight more than 20% in excess of the ideal body weight, frequently resulting in a significant impairment of health. Obesity may be measured by body mass index, an indicator of adiposity or fatness. Further parameters for defining obesity are waist circumferences, skinfold thickness and bioimpedance. Obesity is associated with an increased risk for cardiovascular disease, hypertension, diabetes, hyperlipidaemia and an increased mortality rate.
  • Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors, and can be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate bound energy and low energy expenditure. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Since obesity is not to be considered as a single disorder but as a heterogeneous group of conditions with (potential) multiple causes, it is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Kolterman O.G., (1980) Clin. Invest 65: 1272-1284). A clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman P.G., (2000) Nature 404: 635-643).
  • the technical problem underlying the present invention was to provide for means and methods for modulating/effecting (pathological) metabolic conditions influencing body-weight regulation and/or energy homeostatic circuits.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to novel functions of proteins and nucleic acids encoding these in body-weight regulation, energy homeostasis, metabolism, and obesity.
  • the proteins disclosed herein and polynucleotides encoding these are thus suitable to investigate metabolic diseases and disorders.
  • Further new compositions are provided that are useful in diagnosis, treatment, and prognosis of metabolic diseases and disorders as described.
  • the present invention discloses that foraging (GadFly Accession Number CG10033), CG5535 (GadFly Accession Number), CG14217 (GadFly Accession Number), Klp67A (GadFly Accession Number CG 10923), CG10133 (GadFly Accession Number), CG3967 (GadFly Accession Number), CG32048 (GadFly Accession Number), CG13625 (GadFly Accession Number), Baldspot (GadFly Accession Number CG3971), CG31605 (GadFly Accession Number), fusilli (GadFly Accession Number CG8205), or schnurri (GadFly Accession Number CG7734) homologous proteins (herein referred to as "proteins of the invention” or "a protein of the invention”) are regulating the energy homeostasis and fat metabolism, especially the metabolism and storage of triglycerides, and polynu
  • the invention also relates to vectors, host cells, and recombinant methods for producing the polypeptides and polynucleotides of the invention.
  • the invention also relates to the use of these compounds and effectors/modulators thereof, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynulceotides or polypeptides, in the diagnosis, study, prevention, and treatment of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • Cyclic GMP-dependent protein kinase (PKG) 1 and 2 play a role in several physiological processes including intestinal fluid secretion, ossification, cell adhesion and smooth muscle function (see, Pfeifer A. et al., (1996) Science 274(5295):2082-2086; Talts J. F. et al., (1998) Ann N Y Acad Sci. 857:74-85).
  • PKG1 is a specific mediator of the NO/cGMP effects in murine smooth muscle. Loss of PKG1 abolishes nitric oxide (NO)/cGMP-dependent relaxation of smooth muscle, resulting in severe vascular and intestinal dysfunctions (see, for example, Pfeifer A.
  • NO nitric oxide
  • CAT cationic amino acid transporters
  • BAT broad-scope transport proteins
  • the mCAT-1 gene encodes a basic amino acid transporter that also acts as the receptor for murine ecotropic leukemia viruses.
  • Targeted mutagenesis in embryonic stem cells has been used to introduce a germ-line null mutation into this gene. This mutation removes a domain critical for virus binding and inactivates amino acid transport activity.
  • Homozygous mutant pups generated from these cells were approximately 25% smaller than normal littermates, very anemic, and died on the day of birth.
  • Peripheral blood from homozygotes contained 50% fewer red blood cells, reduced hemoglobin levels, and showed a pronounced normoblastosis. Histological analyses of bone marrow, spleen, and liver showed a decrease in both erythroid progenitors and mature red blood cells.
  • mCAT-1 not only appears to be the sole receptor for a group of murine ecotropic retroviruses associated with hematological disease but also plays a critical role in both hematopoiesis and growth control during mouse development (Perkins C. P. et al., (1997) Genes Dev 1 1 (7):914-925).
  • IGF-II insulin-like growth factor II
  • CAT1 Na + -independent cationic amino acid transporter protein 1
  • TAO1 The thousand and one-amino acid protein kinase 1 (TAO1 ) is highly expressed in brain.
  • TAO1 selectively activates mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) 3, 4, and vitro.
  • MAP mitogen-activated protein
  • ERK extracellular signal-regulated kinase
  • MEKs extracellular signal-regulated kinase
  • TAO1 lie in stress-sensitive kinase cascades and define a mechanism by which these kinases may organize downstream targets.
  • Prostate-derived STE20-like kinase (PSK), a member of the sterile 20 (STE20) family of serine-threonine kinases activates the c-Jun N-terminal kinase mitogen-activated protein kinase pathway and regulates the actin cytoskeleton organization (Moore T.M. et al., (2000) J Biol Chem 275(6):431 1-4322).
  • KIFs kinesin superfamily proteins
  • the members of the kinesin superfamily are involved in microtubule-based movement of very different proteins, e.g. mannose-6-phosphate receptor, or even organelles, e.g. mitochondria, and vesicles (see, for example, Nakagawa T. et al., (2000) Cell 103(4):569-581 ; Tanaka Y. et al., (1998) Cell 93(7):1 147-1 158; Yonekawa Y. et al., (1998) J Cell Biol 1998 Apr 20; 141 (2) :431 -41 ).
  • Some members are also essential for axonal transport and endocytosis, e.g. clathrin-mediated (see, for example, Nakamura Y. et al., (1994) Neurosci Lett 180(1):25-28).
  • PLA2 enzymes are found in all living species and form a diverse family of enzymes. PLA2 enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor (PAF) from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation, reproduction and neurotoxicity (Bonventre J. V. et al., (1997) Nature 390(6660):622-625). PLA2 are involved in endocytosis in different organs and cell types (e.g.
  • phagocytosis e.g. prostaglandins
  • Cytosolic PLA2 are found in small amounts within the cell and play a key role in the biosynthetic pathway leading to the formation of the platelet activating factors and the eicosanoids (Sharp J. D. et al., (1991 ) J Biol Chem 266(23): 14850-14853). Cytosolic PLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia, and plays a non-redundant role in allergic responses and reproductive physiology (see, Bonventre J. V. et al., supra; Uozumi N. et al., (1997) Nature 390(6660):618-622).
  • phospholipase A2 group IV genes in man and mouse
  • PLA2G4A group IV genes in man and mouse
  • PLA2G4B group IV genes in man and mouse
  • PLA2G4C group IV genes in man and mouse
  • Cytosolic PLA2 preferentially releases arachidonic acid from phospholipids
  • PLA2G4A is regulated by changes in intracellular calcium concentration and by phosphorylation by mitogen-activated protein kinases (MAP kinases) (Clark J. D. et al., (1995) J Lipid Mediat Cell Signal 12(2-3):83-1 17).
  • MAP kinases mitogen-activated protein kinases
  • Acyl-CoA N-acyltransferases are modifying enzymes that transfer fatty acids on specific substrates like histones, aminoglycoside, proteins (e.g. serotonin), cholesterol, other sterols, glucosamine-phosphate, diaglycerol, glycerol-3-phosphate (see, for example, Farese R. V., (1998) Curr Opin Lipidol 9(2):1 19-123; Chen H. C. and Farese R. V., (2000) Trends Cardiovasc Med 10(5): 188-192; Dircks L. K. and Sul H. S., (1997) Biochim Biophys Acta 1348(1 -2): 17-26).
  • the Drosophila gene CG3967 (GadFly Accession Number), encodes for a protein that contains an acyl-CoA N-acyltransferase domain, which is most homologous to the splice variants of the human hypothetical protein FLJ13158 (GenBank Accession Numbers XP_042375.2 and NP_079185.1 for the proteins, XM_042375 and NM_024909 for the cDNAs). No functional data are available for these proteins.
  • Nitric oxide (NO) produced by neuronal nitric oxide synthase (nNOS) is important for N-methyl-D-aspartate (NMDA) receptor-dependent neurotransmitter release, neurotoxicity, and cyclic GMP elevations.
  • NMDA N-methyl-D-aspartate
  • the coupling of NMDA receptor-mediated calcium influx and nNOS activation is postulated to be due to a physical coupling of the receptor and the enzyme by an intermediary adaptor protein, PSD95, through a unique PDZ-PDZ domain interaction between PSD95 and nNOS.
  • PSD95 physical coupling of the receptor and the enzyme
  • PSD95 intermediary adaptor protein
  • the nNOS adaptor protein CAPON interacts with the nNOS PDZ domain through its C terminus.
  • CAPON competes with PSD95 for interaction with nNOS, and overexpression of CAPON results in a loss of PSD95/nNOS complexes in transfected cells.
  • CAPON may influence nNOS by regulating its ability to associate with PSD95/NMDA receptor complexes (Jaffrey S. R. et al., (1998) Neuron 20(1 ):1 15-124).
  • CAPON might be involved in worm muscle degeneration (Gieseler K. et al., (2000) Curr Biol 10(18):1092-1097).
  • nNOS adaptor protein CAPON interacts selectively with Dexrasl , a brain-enriched member of the Ras family of small monomeric G proteins, and with synapsins I, II, and III (see, for example, Fang M. et al., (2000) Neuron 28(1 ):183-193; Jaffrey S. R. et al., (2002) Proc Natl Acad Sci U S A 99(5):3199-3204).
  • Diabetic gastropathy in mice reflects an insulin-sensitive reversible loss of nNOS. nNOS expression and pyloric function are restored to normal levels by insulin treatment.
  • Basigin is a component of the blood-brain barier (see, for example, Schlosshauer B., (1 993) Bioessays 1 5(5):341 -346; Igakura T. et al., (1 996) Biochem Biophys Res Commun 224:33 36). Basigin, also named extracellular matrix metalloproteinase inducer (EMMPRIN), stimulates the production of interstitial collagenase, gelatinase A, and stromelysin-1 by fibroblasts (DeCastro R. et al., (1 996) J Invest Dermatol 106(6): 1 260-1 265). Basigin is an important cell-surface molecule involved in early embryogenesis and reproduction (Igakura T.
  • Basigin which is closely colocalized with MCT1 on acinar cell membranes, was absent from islet cell membranes (Zhao C. et al., (2001 ) Diabetes 50(2):361 -366). Basigin is a cell-surface receptor for CyPA and CyPB that are essential components of cyclophilin-initiated signaling cascades that culminates in ERK activation and chemotaxis (Yurchenko V. et al., (2001 ) Biochem Biophys Res Commun 288(4):786-788).
  • the Stromal cell-derived factor-1 may be implicated in the aggressiveness of the autoimmune process leading to type 1 diabetes (Dubois-Laforgue D. et al., (2001) Diabetes 50 ⁇ 5):121 1 -1213).
  • Fatty acids are synthesized de novo from acetyl-CoA and malonyl-CoA through a series of reactions mediated by acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS).
  • ACC acetyl-CoA carboxylase
  • FAS fatty acid synthase
  • the principal fatty acid produced by FAS is palmitic acid (16:0).
  • the fatty acids that accumulate in livers of sterol regulatory element-binding protein (SREBP) transgenic mice are 18 carbons rather than 16 carbons in length, suggesting that the enzymes required for the elongation of palmitic to stearic acid may be induced.
  • SREBP sterol regulatory element-binding protein
  • LCE long chain fatty acyl elongase
  • Cig30 Cold inducible glycoprotein of 30 kDa; a synonym for Elovl3, Elongation of very long chain fatty acids protein 3 is implicated in the thermogenic function of brown adipose tissue of mice. Cig30 is expressed in brown fat and liver. Animals exposed to cold stress showed a selective elevation of Cig30 expression in brown fat. Similar increases were brought about in two other conditions of brown fat recruitment, namely during perinatal development and after cafeteria diet. The Cig30 protein is involved in a pathway connected with brown fat hyperplasia (Tvrdik P. et al., (1997) J Biol Chem 272(50):31738-31746).
  • Cig30 reverted the phenotype of the Flap endonuclease/Elongation of fatty acids protein 2 (fenl /elo2) mutant that has reduced levels of fatty acids in the C(20)-C(24) range.
  • the dramatic induction of Cig30 expression during brown fat recruitment coincided with elevated elongation activity (Tvrdik P. et al., (2000) J Cell Biol 149(3):707-71 8).
  • the Drosophila gene with GadFly Accession Number CG 1 3625 encodes for a protein which is most homologous to human unnamed protein product (GenBank Accession Number BAB71 593.1 for the protein, AK057832 for the cDNA) and to human hypothetical protein MGC13125 (GenBank Accession Number NP_1 1 61 14.1 for the protein, NM_032725 for the cDNA). No functional data are available for these proteins.
  • Drosophila fusilli encodes a RNA binding protein involved in the EGF receptor signalling pathway. Fusilli interacts genetically with cactus, a putative transcription factor, which is a component of the the EGF receptor signalling pathway, too (Wakabayashi-lto N. et al., (2001 ) Dev Biol 229(1 ):44-54).
  • the Drosophila gene fusilli (GadFly Accession Number CG8205) encodes for a protein which is most homologous to human protein similar to fusilli; Enhancer of cactus (GenBank Accession Number XP 007770.7 for the protein, XM 007770 for the cDNA) and to human hypothetical protein FLJ21 91 8 (GenBank Accession Number NP_07921 5.1 for the protein, NM D24939 for the cDNA). No functional data are available for these proteins.
  • the zinc finger transcription factor kappa recognition component KRC (HIVEP-3) is a hitherto unrecognized participant in the signal transduction pathway leading from the TNF receptor to gene activation and may play a critical role in inflammatory and apoptotic responses (Oukka M. et al., (2002) Mol Cell 9(1 ): 1 21 -1 31 ).
  • TNF-alpha is a powerful autocrine and paracrine regulator of adipose tissue, too (Coppack S. W., (2001 ) Proc Nutr Soc 60(3) :349-356).
  • the Smad-Shn-2 (Smad-Hivep-2) complex is involved in the thymic selection of T cells (Takagi T.
  • TGF-beta is a key factor in experimental models of diabetic kidney disease as well as in patients with diabetic nephropathy. TGF-beta may also be involved in mediating the vascular dysfunction of diabetic kidney disease via its effects on the key intracellular calcium channel, the inositol trisphosphate receptor (IP(3)R) (Sharma K. and McGowan T.
  • IP(3)R inositol trisphosphate receptor
  • CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG 13625, Baldspot, CG31605, fusilli, or schnurri homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • Particularly preferred are homologous nucleic acids, particularly nucleic acids encoding a human protein as described in Table 1.
  • the invention particularly relates to a nucleic acid molecule encoding a polypeptide contributing to regulating the energy homeostasis and the metabolism of triglycerides, wherein said nucleic acid molecule comprises
  • nucleotide sequence encoding foraging CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri or homologous nucleic acids, particularly nucleic acids encoding a human protein as described in Table 1 , and/or a sequence complementary thereto,
  • a nucleotide sequence which hybridizes at 50°C in a solution containing 1 x SSC and 0.1 % SDS to a sequence of (a), (c) a sequence corresponding to the sequences of (a) or (b) within the degeneration of the genetic code, (d) a sequence which encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the amino acid sequences of foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri protein, preferably of the human homologous proteins, particularly a human protein as described in Table 1 ,
  • (f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 15-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.
  • the invention is based on the finding that foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri and/or homologous proteins and the polynucleotides encoding these, are involved in the regulation of triglyceride storage and therefore energy homeostasis.
  • the invention describes the use of these compositions for the diagnosis, study, prevention, or treatment of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, liver fibrosis, or gallstones.
  • the present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity, functional fragments of said genes, polypeptides encoded by said genes or functional fragments thereof, and modulators/effectors thereof, e.g. antibodies, biologically active nucleic acids, such as antisense molecules.
  • RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • model organisms such as the fly Drosophila melanogaster
  • Identification of novel gene functions in model organisms can directly contribute to the elucidation of correlative pathways in mammals (humans) and of methods of modulating them.
  • a correlation between a pathology model (such as changes in triglyceride levels as indication for metabolic syndrome including obesity) and the modified expression of a fly gene can identify the association of the human ortholog with the particular human disease.
  • a forward genetic screen is performed in fly displaying a mutant phenotype due to misexpression of a known gene (see, St Johnston D., (2002) Nat Rev Genet 3: 176-188; Rorth P., (1996) Proc Natl Acad Sci U S A 93: 12418-12422).
  • this invention we have used a genetic screen to identify mutations that cause changes in the body weight, which are reflected by a significant change of triglyceride levels.
  • Triglycerides are the most efficient storage for energy in cells.
  • genes with a function in energy homeostasis several thousand proprietary and publicly available EP-lines were tested for their triglyceride content after a prolonged feeding period (see Examples and Figures for more detail). Lines with significantly changed triglyceride content were selected as positive candidates for further analysis.
  • the increase or decrease of triglyceride content due to the loss or gain of a gene function suggests gene activities in energy homeostasis in a dose dependent manner that controls the amount of energy stored as triglycerides.
  • the content of triglycerides of a pool of flies with the same genotype was analyzed after prolonged feeding using a triglyceride assay.
  • Male flies homozygous, hemizygous, or heterozygous for the integration of vectors for Drosophila EP-lines were analyzed in an assay measuring the triglyceride contents of these flies, illustrated in more detail in the Examples section.
  • the results of the triglyceride content analysis are shown in Figures 1 , 6, 10, 14, 17, 20, 22, 24, 27, 31 , 33, and 35.
  • Genomic DNA sequences were isolated that are localized adjacent to the EP vector integration. Using those isolated genomic sequences public databases like Berkeley Drosophila Genome Project (GadFly; see also FlyBase (1999) Nucleic Acids Research 27: 85-88) were screened thereby identifying the integration site of the vectors, and the corresponding genes, described in more detail in the Examples section. The molecular organization of the genes is shown in Figures 2, 7, 1 1 , 15, 18, 21 , 23, 25, 28, 32, 34, and 36.
  • An additional screen using Drosophila mutants with modifications of the eye phenotype identified a modification of UCP activity by CG32048 and schnurri, thereby leading to an altered mitochondrial activity.
  • An additional genetic screen uses a Drosophila line with ectopic expression of adp in the developing eye thereby leading to a visible change of the eye phenotype.
  • Modifications of the eye phenotype by coexpression of schnurri or CG32048 identified an interaction of schnurri and CG32048 with adp, a protein regulating, causing or contributing to obesity.
  • Another genetic screen uses a Drosophila line with ectopic expression of adipose (adp) mainly in the fatbody thereby leading to lethality.
  • adp adipose
  • Modifications of the lethality phenotype by coexpression of CG3967, CG32048, or schnurri identified an interaction of CG3967, CG32048, and schnurri with adp.
  • Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride metabolism were further analysed in publicly available sequence databases (see Examples for more detail) and mammalian homologs were identified.
  • mice carrying gene knockouts in the leptin pathway for example, ob (leptin) or db (leptin receptor) mice
  • mice developing typical symptoms of diabetes show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • Microarrays are analytical tools routinely used in bioanalysis.
  • a microarray has molecules distributed over, and stably associated with, the surface of a solid support.
  • the term "microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as monitoring gene expression, drug discovery, gene sequencing, gene mapping, bacterial identification, and combinatorial chemistry.
  • One area in particular in which microarrays find use is in gene expression analysis (see Example 7).
  • array technology can be used to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (see for example, Brennan T.M., (1995) U.S. Patent No. US5474796; Schena M. et al., (1996) Proc. Natl. Acad. Sci. USA 93:
  • Oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques, which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents, which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • PRKG2 , BSG, ELOVL6, and HIVEP1 are strong candidates for the manufacture of a pharmaceutical composition and a medicament for the treatment of conditions related to human metabolism, such as obesity, diabetes, and/or metabolic syndrome.
  • the invention also encompasses polynucleotides that encode a protein of the invention or a homologous protein. Accordingly, any nucleic acid sequence, which encodes the amino acid sequences of a protein of the invention or a homologous protein, can be used to generate recombinant molecules that express a protein of the invention or a homologous protein.
  • the invention encompasses a nucleic acid encoding Drosophila foraging, CG5535, CG 1421 7, Klp67A, CG101 33, CG3967, CG32048, CG13625, Baldspot, CG31 605, fusilli, or schnurri, or human foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31 605, fusilli, or schnurri homologs, preferably a human homologous protein as described in Table 1 ; referred to as the proteins of the invention.
  • nucleotide sequences encoding the proteins may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that can be made by selecting combinations based on possible codon choices.
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those of the polynucleotide encoding foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri, or a homologous protein, preferably a human homologous protein as described in Table 1 , under various conditions of stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Wahl G.M.
  • Tm melting temperature
  • hybridization under stringent conditions means that after washing for 1 h with 1 x SSC and 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, particularly for 1 h in 0.2 x SSC and 0.1 % SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, a positive hybridization signal is observed.
  • Altered nucleic acid sequences encoding the proteins which are encompassed by the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent protein.
  • the encoded proteins may also contain deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in functionally equivalent proteins. 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 residues as long as the biological activity of the protein is retained.
  • the invention relates to peptide fragments of the proteins or derivatives thereof such as cyclic peptides, retro-inverso peptides or peptide mimetics having a length of at least 4, preferably at least 6 and up to 50 amino acids.
  • an 'allele' or 'allelic sequence' is an alternative form of the gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structures or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, 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.
  • nucleic acid sequences encoding a protein of the invention or a homologous protein may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method which may be employed, 'restriction-site' PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar G. et al., (1993) PCR Methods Applic. 2: 318-322).
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia T. et al., (1988) Nucleic Acids Res. 16: 8186).
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom M. et al., (1991 ) PCR Methods Applic. 1 : 1 1 1-1 19). Another method which may be used to retrieve unknown sequences is that of Parker J.D. et al., (1991 ) Nucleic Acids Res. 19: 3055-3060. Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • nucleotide sequences encoding the proteins or functional equivalents may be inserted into appropriate expression vectors, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vectors i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art, may be used to construct expression vectors containing sequences encoding the proteins and the appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
  • natural, modified or recombinant nucleic acid sequences encoding a protein of the invention or a homologous protein may be ligated to a heterologous sequence to encode a fusion protein.
  • Heterologous sequences are preferably located at the N-and/or C-terminus of the fusion protein.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding the proteins or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or PBR322 plasmids); or animal cell systems.
  • micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus
  • polynucleotide sequences encoding a protein of the invention or a homologous protein in a sample can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of said polynucleotides.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences specific for the gene to detect transformants containing DNA or RNA encoding the corresponding protein.
  • 'oligonucleotides' or 'oligomers' refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
  • Means for producing labeled hybridization or PCR probes for detecting polynucleotide sequences encoding a protein of the invention or a homologous protein include oligo-labeling, nick translation, end-labeling of labeled RNA probes, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).
  • Suitable reporter molecules or labels which may be used for nucleic acid and protein assays, include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a protein of the invention may be cultured under conditions suitable for the expression and recovery of said protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides, which encode the protein may be designed to contain signal sequences, which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may be used to join sequences encoding the protein to nucleotide sequence encoding a polypeptide domain, which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAG extension/affinity purification system (Immunex Corp., Seattle, Wash.)
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAG extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the desired protein may be used to facilitate purification.
  • nucleic acids and proteins of the invention and effector/modulator molecules thereof are useful in diagnostic and therapeutic applications implicated, for example but not limited to, metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • diagnostic and therapeutic uses for the nucleic acids and proteins of the invention are, for example but not limited to, the following: (i) protein therapy, (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi) research tools, and (vii) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues).
  • nucleic acids and proteins of the invention and modulators/effectors thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below.
  • cDNAs encoding a protein of the invention and particularly their human homologues may be useful in gene therapy, and the proteins of the invention and particularly their human homologues may be useful when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from, for example, but not limited to, in metabolic disorders as described above.
  • nucleic acids encoding a protein of the invention, a homologous protein, or a functional fragment thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acids or the proteins are to be assessed. These materials are further useful in the generation of antibodies that bind immunospecifically to the novel substances of the invention may be used in therapeutic or diagnostic methods.
  • antibodies which are specific for a protein of the invention or a homologous protein, may be used directly as a modulator/effector, e.g. an antagonist or an agonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein.
  • the antibodies may be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric single chain, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralising antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the protein or any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response. It is preferred that the peptides, fragments or oligopeptides used to induce antibodies to the protein have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.
  • Monoclonal antibodies to the proteins may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (K ⁇ hler G. and Milstein C. (1975) Nature 256: 495-497; Kozbor D. et al. (1985) J. Immunol. Methods 81 : 31 -42; Cote R.J. et al., (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole S.P. et al., (1984) Mol Cell Biochem. 62: 109-120).
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Kang A.S. et al., (1991 ) Proc. Natl. Acad. Sci. 88: 1 1 120-1 1 123). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi R. et al., (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter G. and Milstein C, (1991 ) Nature 349: 293-299).
  • Antibody fragments which contain specific binding sites for the proteins may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by Pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al., (1989) Science 246: 1275-1281 ).
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding and immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reacive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox D.E. et al., (1983) J. Exp. Med. 158: 121 1 -1216).
  • the polynucleotides of the invention or fragments thereof or nucleic acid modulator/effector molecules such as aptamers, antisense molecules, RNAi molecules or ribozymes may be used for therapeutic purposes.
  • nucleic acid modulator/effector molecules such as aptamers, antisense molecules, RNAi molecules or ribozymes
  • aptamers i.e. nucleic acid molecules, which are capable of binding to a protein of the invention and modulating its activity, may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries.
  • antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to polynucleotides encoding a protein of the invention or a homologous protein.
  • antisense molecules may be used to modulate/effect protein activity or to achieve regulation of gene function.
  • sense or antisense oligomers or larger fragments can be designed from various locations along the coding or control regions of sequences encoding the proteins.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding a protein of the invention or a homologous protein. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
  • Genes encoding a protein of the invention or a homologous protein can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode a protein of the invention, or a homologous protein, or a functional fragment thereof.
  • Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing antisense molecules, e.g.
  • DNA, RNA or PNA to the control regions of the genes encoding a protein of the invention or a homologous protein, i.e., the promoters, enhancers, and introns.
  • Oligonucleotides derived from the transcription initiation site e.g., between positions -10 and + 10 from the start site, are preferred.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee J.E. et al. (1994) Gene 149: 109-1 14.; Huber B.E.
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples, which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding a protein of the invention or a homologous protein.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Nucleic acid effector molecules, e.g. antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding a protein of the invention or a homologous protein. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient.
  • Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • compositions may consist of a nucleic acid sequence or protein of the invention or a homologous nucleic acid sequence or protein, antibodies to a protein of the invention or a homologous protein, mimetics, agonists, antagonists or inhibitors of a nucleic acid sequence or protein of the invention or a homologous nucleic acid sequence or protein.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids. After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of proteins, such labeling would include amount, frequency, and method of administration.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of preadipocyte cell lines or in animal models, usually mice, rabbits, dogs or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example a protein of the invention or a homologous protein or nucleic acid sequence or a functional fragment thereof or an antibody, which is sufficient for treating a specific condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • antibodies which specifically bind to a protein of the invention may be used for the diagnosis of conditions or diseases characterized by or associated with over- or underexpression of a protein of the invention or a homologous protein or in assays to monitor patients being treated with a protein of the invention or a homologous protein, or modulators/effectors thereof, e.g. agonists, antagonists, or inhibitors.
  • Diagnostic assays include methods which utilize the antibody and a label to detect the protein in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
  • a wide variety of reporter molecules which are known in the art may be used several of which are described above.
  • a variety of protocols including ELISA, RIA, and FACS for measuring proteins are known in the art and provide a basis for diagnosing altered or abnormal levels of gene expression.
  • Normal or standard values for gene expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibodies to the protein under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of protein expressed in control and disease, samples e.g. from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides specific for a protein of the invention or a homologous protein may be used for diagnostic purposes.
  • the polynucleotides, which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which gene expression may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess gene expression, and to monitor regulation of protein levels during therapeutic intervention.
  • hybridization with probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding a protein of the invention or a homologous protein or closely related molecules may be used to identify nucleic acid sequences which encode the respective protein.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of the polynucleotide encoding a protein of the invention or a homologous protein or from a genomic sequence including promoter, enhancer elements, and introns of the naturally occurring gene.
  • Means for producing specific hybridization probes for DNAs encoding a protein of the invention or a homologous protein include the cloning of nucleic acid sequences specific for a protein of the invention or a homologous protein into vectors for the production of mRNA probes.
  • Such vectors are known in the art, commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • reporter groups for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences specific for a protein of the invention or homologous nucleic acids may be used for the diagnosis of conditions or diseases, which are associated with the expression of the protein.
  • Examples of such conditions or diseases include, but are not limited to, metabolic diseases and disorders, including obesity or diabetes.
  • Polynucleotide sequences specific for a protein of the invention or a homologous protein may also be used to monitor the progress of patients receiving treatment for metabolic diseases and disorders, including obesity or diabetes.
  • the polynucleotide sequences may be used qualitative or quantitative assays, e.g. in Southern or Northern analysis, dot blot or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered gene expression. Such qualitative or quantitative methods are well known in the art.
  • nucleotide sequences specific for a protein of the invention or homologous nucleic acids may be useful in assays that detect activation or induction of various metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • the nucleotide sequences may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • nucleotide sequences encoding a protein of the invention or a homologous protein in the sample indicates the presence of the associated disease.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence or a fragment thereof, which is specific for the nucleic acids encoding a protein of the invention or homologous nucleic acids, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease.
  • Deviation between standard and subject values is used to establish the presence of disease. Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that, which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • oligonucleotides designed from the sequences encoding a protein of the invention or a homologous protein may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically or produced from a recombinant source.
  • Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'.fwdarw.3') and another with antisense (3'.rarw.5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of a protein of the invention or a homologous protein include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby P.C. et al., (1993) J. Immunol. Methods, 159: 235-244; Duplaa C. et al., (1993) Anal. Biochem. 212: 229-236).
  • the speed of quantification of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantification.
  • the nucleic acid sequences which are specific for a protein of the invention or homologous nucleic acids may also be used to generate hybridization probes, which are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques.
  • Such techniques include FISH, FACS or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price CM., (1993) Blood Rev. 7: 127-134, and Trask B.J., (1991 ) Trends Genet. 7: 149-154.
  • FISH Fluorescence In situ hybridization
  • Verma R.S. and Babu A. (1989) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y..
  • the results may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265:1981 f). Correlation between the location of the gene encoding a protein of the invention on a physical chromosomal map and a specific disease or predisposition to a specific disease, may help to delimit the region of DNA associated with that genetic disease.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. For example, an analysis of polymorphisms, e.g. single nucleotide polymorphisms may be carried out. Further, in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms or parts thereof, by physical mapping.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequences of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.
  • the proteins of the invention can be used for screening libraries of compounds, e.g. peptides or low molecular weight organic compounds, in any of a variety of drug screening techniques.
  • modulators/effectors e.g. receptors, enzymes, proteins, ligands, or substrates that bind to, modulate or mimic the action of one or more of the proteins of the invention.
  • the protein or fragment thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between a protein of the invention and the agent tested, may be measured. Agents could also, either directly or indirectly, influence the activity of a protein of the invention.
  • the enzymatic kinase activity of the unmodified polypeptides of foraging or CG14217 homologous kinase towards a substrate can be measured.
  • Activation of the kinases may be induced in the natural context by extracellular or intracellular stimuli, such as signaling molecules or environmental influences.
  • One may generate a system containing foraging or CG14217 homologous kinase may it be an organism, a tissue, a culture of cells or cell-free environment, by exogenously applying this stimulus or by mimicking this stimulus by a variety of the techniques, some of them described further below.
  • a system containing activated foraging or CG14217 homologous kinase may be produced (i) for the purpose of diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases, (ii) for the purpose of identifying or validating therapeutic candidate agents, pharmaceuticals or drugs that influence the genes of the invention or their encoded polypeptides, (iii) for the purpose of generating cell lysates containing activated polypeptides encoded by the genes of the invention, (iv) for the purpose of isolating from this source activated polypeptides encoded by the genes of the invention.
  • an agent that mimics the natural stimulus such as activators of the foraging or CG14217 homologous kinase, phorbol ester, anisomycin, constitu
  • CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri homologous proteins against their physiological substrate(s) or derivatives thereof could be measured in cell-based or cell-free assays.
  • Agents may also interfere with posttranslational modifications of a protein of the invention, such as phosphorylation and dephosphorylation, farnesylation, palmitoylation, acetylation, alkylation, ubiquitination, proteolytic processing, subcellular localization or degradation.
  • agents could influence the dimerization or oligomerization of a protein of the invention or, in a heterologous manner, of a protein of the invention with other proteins, for example, but not exclusively, docking proteins, enzymes, receptors, ion channels, uncoupling proteins, or translation factors. Agents could also act on the physical interaction of the proteins of this invention with other proteins, which are required for protein function, for example, but not exclusively, their downstream signaling.
  • binding of a fluorescently labeled peptide derived from a protein of the invention to the interacting protein could be detected by a change in polarisation.
  • binding partners which can be either the full length proteins as well as one binding partner as the full length protein and the other just represented as a peptide are fluorescently labeled
  • binding could be detected by fluorescence energy transfer (FRET) from one fluorophore to the other.
  • FRET fluorescence energy transfer
  • the two or more binding partners can be different proteins with one being the protein of the invention, or in case of dimerization and/or oligomerization the protein of the invention itself.
  • Proteins of the invention, for which one target mechanism of interest, but not the only one, would be such protein/protein interactions are foraging, CG5535, CG1421 7, Klp67A, CG 101 33, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri homologous proteins.
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of one or more of the proteins of the invention.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the screening assay is a binding assay
  • one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal.
  • kinase substrate such as a protein, a peptide, a lipid, or an organic compound, which may or may not include modifications as further described below, or others are phosphorylated by the proteins or protein fragments of the invention.
  • the kinase can be a protein of the invention (e.g. foraging or CG14217 homologous kinase) or a kinase, which is influenced in its activity by a protein of the invention.
  • a therapeutic candidate agent may be identified by its ability to increase or decrease the enzymatic activity of a protein of the invention.
  • the kinase activity may be detected by change of the chemical, physical or immunological properties of the substrate due to phosphorylation.
  • One example could be the transfer of radioisotopically labelled phosphate groups from an appropriate donor molecule to the kinase substrate catalyzed by the polypeptides of the invention.
  • the phosphorylation of the substrate may be followed by detection of the substrates autoradiography with techniques well known in the art.
  • the change of mass of the substrate due to its phosphorylation may be detected by mass spectrometry techniques.
  • Such an analyte may act by having different affinities for the phosphorylated and unphosphorylated forms of the substrate or by having specific affinity for phosphate groups.
  • Such an analyte could be, but is not limited to, an antibody or antibody derivative, a recombinant antibody-like structure, a protein, a nucleic acid, a molecule containing a complexed metal ion, an anion exchange chromatography matrix, an affinity chromatography matrix or any other molecule with phosphorylation dependend selectivity towards the substrate.
  • analyte could be employed to detect the kinase substrate, which is immobilized on a solid support during or after an enzymatic reaction. If the analyte is an antibody, its binding to the substrate could be detected by a variety of techniques as they are described in Harlow E. and Lane D., 1988, Antibodies: A Laboratory Manual, CSH Lab Press, NY. If the analyte molecule is not an antibody, it may be detected by virtue of its chemical, physical or immunological properties, being endogenously associated with it or engineered to it.
  • the kinase substrate may have features, designed or endogenous, to facilitate its binding or detection in order to generate a signal that is suitable for the analysis of the substrates phosphorylation status.
  • These features may be, but are not limited to, a biotin molecule or derivative thereof, a glutathione-S-transferase moiety, a moiety of six or more consecutive histidine residues, an amino acid sequence or hapten to function as an epitope tag, a fluorochrome, an enzyme or enzyme fragment.
  • the kinase substrate may be linked to these or other features with a molecular spacer arm to avoid steric hindrance.
  • the kinase substrate may be labelled with a fluorochrome.
  • the binding of the analyte to the labelled substrate in solution may be followed by the technique of fluorescence polarization as it is described in the literature (see, for example, Deshpande S. et al., (1999) Prog. Biomed. Optics (SPIE) 3603: 261 ; Parker G.J. et al., (2000) J. Biomol. Screen. 5: 77-88; Wu P. et al., (1997) Anal. Biochem. 249: 29-36).
  • a fluorescent tracer molecule may compete with the substrate for the analyte to detect kinase activity by a technique which is known to those skilled in the art as indirect fluorescence polarization.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • this method as applied to the proteins of the invention large numbers of different small test compounds are synthesized or provided on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with a protein of the invention or fragments thereof, and washed. Bound proteins are then detected by methods well known in the art. Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the antibodies can be used to detect the presence of any peptide, which shares one or more antigenic determinants with foraging, CG5535, CG14217, Klp67A, CG10133, CG3967, CG32048, CG13625, Baldspot, CG31605, fusilli, or schnurri homologous protein.
  • the nucleic acids encoding a protein of the invention can be used to generate transgenic animals or site-specific gene modifications in cell lines. These transgenic non-human animals are useful in the study of the function and regulation of said protein in vivo. Transgenic animals, particularly mammalian transgenic animals, can serve as a model system for the investigation of many developmental and cellular processes common to humans. A variety of non-human models of metabolic disorders can be used to test modulators/effectors of the proteins of the invention. Misexpression (for example, overexpression or lack of expression) of a protein of the invention, particular feeding conditions, and/or administration of biologically active compounds can create models of metablic disorders.
  • such assays use mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice).
  • leptin pathway for example, ob (leptin) or db (leptin receptor) mice.
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • Susceptible wild type mice for example C57BI/6) show similiar symptoms if fed a high fat diet.
  • mice could be used to test whether administration of a candidate modulator/effector alters for example lipid accumulation in the liver, in plasma, or adipose tissues using standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • Transgenic animals may be made through homologous recombination in non-human embryonic stem cells, where the normal locus of the gene encoding a protein of the invention is altered.
  • a nucleic acid construct encoding a protein of the invention is injected into oocytes and is randomly integrated into the genome.
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, yeast artificial chromosomes (YACs), and the like. The modified cells or animal are useful in the study of the function and regulation of the proteins of the invention.
  • a series of small deletions and/or substitutions may be made in the gene that encodes a protein of the invention to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.
  • variants of the genes of the invention like specific constructs of interest include anti-sense molecules, which will block the expression of the proteins of the invention, or expression of dominant negative mutations.
  • a detectable marker such as for example lac-Z or luciferase may be introduced in the locus of a gene of the invention, where up regulation of expression of the genes of the invention will result in an easily detected change in phenotype.
  • genes of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development.
  • proteins of the invention in cells in which they are not normally produced, one can induce changes in cell behavior.
  • DNA constructs for homologous recombination will comprise at least portions of the genes of the invention with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration do not need to contain regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. DNA constructs for random integration will consist of the nucleic acids encoding the proteins of the invention, a regulatory element (promoter), an intron and a poly-adenylation signal. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For non-human embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer and are grown in the presence of leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • non-human ES or embryonic cells or somatic pluripotent stem cells When non-human ES or embryonic cells or somatic pluripotent stem cells have been transfected, they may be used to produce transgenic animals. After transfection, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be selected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo transfection and morula aggregation. Briefly, morulae are obtained from 4 to 6 week old superovulated females, the Zona Pellucida is removed and the morulae are put into small depressions of a tissue culture dish.
  • the ES cells are trypsinized, and the modified cells are placed into the depression closely to the morulae.
  • the aggregates are transfered into the uterine horns of pseudopregnant females.
  • Females are then allowed to go to term.
  • Chimeric offsprings can be readily detected by a change in coat color and are subsequently screened for the transmission of the mutation into the next generation (F1 -generation).
  • Offspring of the F1-generation are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogenic or congenic grafts or transplants, or in vitro culture.
  • the transgenic animals may be any non-human mammal, such as laboratory animal, domestic animals, etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and others.
  • the transgenic animals may be used in functional studies, drug screening, and other applications and are useful in the study of the function and regulation of the proteins of the invention in vivo.
  • the invention also relates to a kit comprising at least one of
  • a host cell comprising the nucleic acid of (a) or the vector of (c); (e) a polypeptide encoded by the nucleic acid of (a);
  • the kit may be used for diagnostic or therapeutic purposes or for screening applications as described above.
  • the kit may further contain user instructions.
  • Figure 1 shows the triglyceride content of a Drosophila foraging (GadFly Accession Number CG10033) mutant. Shown is the change of triglyceride content of HD-EP(2)21996, HD-EP(2)22008, HD-EP(2)26800 flies caused by homozygous viable integration of the P-vector into the promoter/enhancer or into the cDNA of the foraging gene (referred to as
  • 'EP-control' column 1
  • 'HD-EP20702/CyO' column 6
  • CyO balancer chromosome (referred to as 'EP-control CyO', column 5).
  • Figure 2 shows the molecular organization of the mutated foraging gene locus.
  • Figure 3 shows the BLASTP search result for the foraging gene product (Query) with the two best human homologous matches (Sbjct).
  • Figure 4 shows the expression of the foraging homologs in mammalian
  • Figure 4A shows the real-time PCR analysis of protein kinase, cGMP-dependent, type I (Prkgl ) expression in wild-type mouse tissues.
  • Figure 4B shows the real-time PCR analysis of Prkg 1 expression in different mouse models.
  • Figure 4C shows the real-time PCR analysis of Prkgl expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 4D shows the real-time PCR analysis of Prkgl expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 4E shows the real-time PCR analysis of protein kinase, cGMP-dependent, type II (Prkg2) expression in wild-type mouse tissues.
  • Figure 4F shows the real-time PCR analysis of Prkg2 expression in different mouse models.
  • Figure 4G shows the real-time PCR analysis of Prkg2 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 4H shows the real-time PCR analysis of Prkg2 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 5 shows the expression of a foraging homolog in mammalian (human) tissue. Shown is the quantitative analysis of protein kinase, cGMP-dependent, type II (PRKG2) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • protein kinase cGMP-dependent, type II (PRKG2) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 6 shows the triglyceride content of a Drosophila cationic amino acid transporter (GadFly Accession Number CG5535) mutant. Shown is the change of triglyceride content of HD-EP(3)37469 flies caused by integration of the P-vector 5' of the cDNA (referred to as 'HD-EP37469', column 2), or by ectopic expression of the gene mainly in the fat body (referred to as 'HD-EP37469/FB', column 3) or mainly in the neurons (referred to as 'HD-EP37469/elav', column 4) of these flies in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 7 shows the molecular organization of the mutated CG5535 (Gadfly Accession Number) gene locus.
  • Figure 8 shows the BLASTP search results for the CG5535 (Gadfly Accession Number) gene product (Query) with the three best human homologous matches (Sbjct).
  • Figure 9 shows the expression of the CG5535 homologs in mammalian (mouse) tissues.
  • Figure 9A shows the real-time PCR analysis of solute carrier family 7, member 1 (Slc7a1 ) expression in wild-type mouse tissues.
  • Figure 9B shows the real-time PCR analysis of Slc7a1 expression in different mouse models.
  • Figure 9C shows the real-time PCR analysis of Slc7a1 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 9D shows the real-time PCR analysis of Slc7a1 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 9E shows the real-time PCR analysis of solute carrier family 7, member 2 (Slc7a2) expression in wild-type mouse tissues.
  • Figure 9F shows the real-time PCR analysis of Slc7a2 expression in different mouse models.
  • Figure 9G shows the real-time PCR analysis of Slc7a2 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 9H shows the real-time PCR analysis of Slc7a2 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 91 shows the real-time PCR analysis of solute carrier family 7, member 3 (Slc7a3) expression in wild-type mouse tissues.
  • Figure 9J shows the real-time PCR analysis of Slc7a3 expression in different mouse models.
  • Figure 9K shows the real-time PCR analysis of Slc7a3 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 10 shows the triglyceride content of a Drosophila CG1421 7 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(X) 10632 flies caused by integration of the P-vector into the annotated cDNA (referred to as 'HD-EP10632', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 1 1 shows the molecular organization of the mutated CG 1421 7 (Gadfly Accession Number) gene locus.
  • Figure 1 2 shows the BLASTP search results for the CG 1421 7 gene product (Query) with the four best human homologous matches (Sbjct).
  • Figure 1 3 shows the expression of CG 1421 7 homologs in mammalian (mouse) tissues.
  • Figure 1 3A shows the real-time PCR analysis of RIKEN cDNA 1 1 10033K02 gene (1 1 10033K02Rik) expression in wild-type mouse tissues.
  • Figure 1 3B shows the real-time PCR analysis of 1 1 10033K02Rik expression in different mouse models.
  • Figure 1 3C shows the real-time PCR analysis of 1 1 10033K02Rik expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 1 3D shows the real-time PCR analysis of 1 1 10033K02Rik expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 13E shows the real-time PCR analysis of protein similar to thousand and one amino acid protein kinase (LOC192767) expression in wild-type mouse tissues.
  • Figure 13F shows the real-time PCR analysis of LOC192767 expression in different mouse models.
  • Figure 13G shows the real-time PCR analysis of LOC192767 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 13H shows the real-time PCR analysis of LOC192767 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 14 shows the triglyceride content of a Drosophila Kinesin-like protein at 67A (Klp67A; GadFly Accession Number CG 10923) mutant. Shown is the change of triglyceride content of HD-EP(3)31624 flies caused by integration of the P-vector into the cDNA (referred to as 'HD-EP31624/TM3,Ser', column 2), or by ectopic expression of the Klp67A gene mainly in the fat body (referred to as 'HD-EP31624/FB', column 3) or mainly in the neurons (referred to as 'HD-EP31624/elav', column 4) of these flies in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 15 shows the molecular organization of the mutated Klp67A gene locus.
  • Figure 16 shows the expression of the Klp67A homolog in mammalian
  • Figure 16A shows the real-time PCR analysis of kinesin family member
  • Figure 16B shows the real-time PCR analysis of Kif18a expression in different mouse models.
  • Figure 16C shows the real-time PCR analysis of Kif18a expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 16D shows the real-time PCR analysis of Kif1 8a expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 17 shows the triglyceride content of a Drosophila phospholipase A2 (GadFly Accession Number CG 10133) mutant. Shown is the change of triglyceride content of HD-EP(3)31 769 flies caused by integration of the P-vector into the cDNA (referred to as 'HD-EP31 769', column 2), or by ectopic expression of the CG 10133 gene mainly in the fat body (referred to as 'HD-EP31769/FB', column 3) or mainly in the neurons (referred to as 'HD-EP31 769/elav', column 4) of these flies in comparison to controls ('EP-control', column 1 ).
  • Figure 18 shows the molecular organization of the mutated CG10133 (Gadfly Accession Number) gene locus.
  • Figure 1 9 shows the expression of a CG101 33 homolog in mammalian
  • Figure 1 9A shows the real-time PCR analysis of protein similar to phospholipase A2, group IVB (cytosolic) (LOC21 1429) expression in wild-type mouse tissues.
  • Figure 19B shows the real-time PCR analysis of LOC21 1429 expression in different mouse models.
  • Figure 19C shows the real-time PCR analysis of LOC21 1429 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 20 shows triglyceride content of a Drosophila CG3967 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(3)37458 flies (referred to as 'HD-EP37458', column 2) caused by integration of the P-vector into the promoter of CG3967 (in comparison to controls (referred to as 'EP-control', column 1 ) .
  • Figure 21 shows the molecular organization of the mutated CG3967 (Gadfly Accession Number) gene locus.
  • Figure 22 shows the triglyceride content of Drosophila CG32048 (GadFly Accession Number) mutants.
  • Figure 22A shows the change of triglyceride content of HD-EP(3)36547, HD-EP(3)37389, and HD-EP(3)36941 flies caused by integration of the P-vector into the promoter/enhancer region (referred to as 'HD-EP36547', column 2, 'HD-EP37389', column 3, 'HD-EP36941 ', column 4) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 22B shows the change of triglyceride content of HD-EP(3)30418 flies caused by integration of the P-vector into the promoter/enhancer region (referred to as 'HD-EP30418', column 2) in comparison to controls containing all flies of the EP collection (referred to as 'EP-control', column 1 ), and by ectopic expression of the CG32048 gene mainly in the fat body of these flies (referred to as 'HD-EP30418/FB', column 4) in comparison to controls containing all flies with the FB-Gal4 insertion (referred to as 'random EP/FB', column 3).
  • Figure 22C shows the change of triglyceride content of HD-EP(3)36364 flies caused by integration of the P-vector into the promoter/enhancer region (referred to as 'HD-EP36364', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 22D shows the change of triglyceride content of HD-EP(3)31 171 flies caused by integration of the P-vector into the promoter/enhancer region (referred to as 'HD-EP31171 ', column 2), or by ectopic expression of the CG32048 gene mainly in the fat body (referred to as 'HD-EP31 171 /FB', column 3), or mainly in the neurons (referred to as 'HD-EP31 171 /elav', column 4) of these flies, in comparison to controls (referred to as 'EP-control', column 1).
  • Figure 23 shows the molecular organization of the mutated CG32048 (Gadfly Accession Number) gene locus.
  • Figure 24 shows the triglyceride content of a Drosophila CG31605 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(2)26044 flies caused by integration of the P-vector 9 base pairs 5' of the cDNA/ into the cDNA (referred to as 'HD-EP26044', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 25 shows the molecular organization of the mutated CG31605 (Gadfly Accession Number) gene locus.
  • Figure 26 shows the expression of the CG31605 homologs in mammalian (human) tissue.
  • Figure 26A shows the quantitative analysis of stromal cell derived factor receptor 1 (SDFR1 ) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 26B shows the quantitative analysis of basigin (BSG) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • SDFR1 stromal cell derived factor receptor 1
  • Figure 27 shows the triglyceride content of a Drosophila Baldspot (GadFly Accession Number CG3971 ) mutant. Shown is the change of triglyceride content of HD-EP(3)37185 flies caused by integration of the P-vector into the promoter/enhancer of the cDNA (referred to as 'HD-EP37185', column 2), or by ectopic expression of the Baldspot gene mainly in the fat body (referred to as 'HD-EP37185/FB', column 3) or mainly in the neurons (referred to as 'HD-EP37185/elav', column 4) of these flies in comparison to controls ('EP-control', column 1 ).
  • Figure 28 shows the molecular organization of the mutated Baldspot gene locus.
  • Figure 29 shows the expression of the Baldspot homologs in mammalian (mouse) tissues.
  • Figure 29A shows the real-time PCR analysis of ELOVL family member 6, elongation of long chain fatty acids (yeast) (Elovl ⁇ ) expression in wild-type mouse tissues.
  • Figure 29B shows the real-time PCR analysis of Elovl ⁇ expression in different mouse models.
  • Figure 29C shows the real-time PCR analysis of Elovl6 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 29D shows the real-time PCR analysis of Elovl6 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 29E shows the real-time PCR analysis of elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3 (Elovl3) expression in wild-type mouse tissues.
  • Figure 29F shows the real-time PCR analysis of Elovl3 expression in different mouse models.
  • Figure 29G shows the real-time PCR analysis of Elovl3 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 29H shows the real-time PCR analysis of Elovl3 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 30 shows the expression of a Baldspot homolog in mammalian (human) tissue. Shown is the quantitative analysis of ELOVL family member 6, elongation of long chain fatty acids (ELOVL6) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • ELOVL6 long chain fatty acids
  • Figure 31 shows the triglyceride content of a Drosophila CG13625 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(3)37410 flies caused by integration of the P-vector 5' of the cDNA (referred to as 'HD-EP37410', column 2), or by ectopic expression of the CG 13625 gene mainly in the fat body (referred to as 'HD-EP37410/FB', column 3) or mainly in the neurons (referred to as 'HD-EP37410/elav', column 4) of these flies in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 32 shows the molecular organization of the mutated CG 13625 (Gadfly Accession Number) gene locus.
  • Figure 33 shows the triglyceride content of a Drosophila fusilli (GadFly Accession Number CG8205) mutant. Shown is the change of triglyceride content of HD-EP(2)25801 flies caused by integration of the P-vector 5' of/into the cDNA (referred to as 'HD-EP25801 ', column 2), or by ectopic expression of the fusilli gene mainly in the fat body (referred to as 'HD-EP25801 /FB', column 3) or mainly in the neurons of these flies (referred to as 'HD-EP25801 /elav', column 4) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 34 shows the molecular organization of the mutated fusilli gene locus.
  • Figure 35 shows the triglyceride content of a Drosophila schnurri (shn; GadFly Accession Number CG7734) mutant. Shown is the change of triglyceride content of HD-EP(2)21 919, HD-EP(2)22018, HD-EP(2)26878, and HD-EP(2)26808 flies caused by integration of the P-vector 5' of the cDNA /into an intron of schnurri (referred to as 'HD-EP21 91 9', column 2, 'HD-EP22018', column 3, 'HD-EP26878/CyO', column 4, and 'HD-EP26808', column 5) in comparison to controls ('EP-control', column 1 ).
  • Figure 36 shows the molecular organization of the mutated schnurri gene locus.
  • Figure 37 shows the expression of a schnurri homolog in mammalian (human) tissue. Shown is the quantitative analysis of human immunodeficiency virus type I enhancer binding protein 1 (HIVEP1 ) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • HIVEP1 human immunodeficiency virus type I enhancer binding protein 1
  • Example 1 Measurement of triglyceride content in Drosophila
  • Mutant flies are obtained from proprietary or publicly available fly mutation stock collections. The flies are grown under standard conditions known to those skilled in the art. In the course of the experiment, additional feedings with bakers yeast (Saccharomyces cerevisiae) are provided. The average change of triglyceride content of Drosophila containing the EP-vectors in homozygous viable, hemizygous viable, or heterozygous viable/homozygous lethal integration, was investigated in comparison to control flies (see Figures 1 , 6, 10, 14, 17, 20, 22, 24, 27, 31 , 33, and 35).
  • the offspring of males containing the EP-vectors that are crossed to elav-Gal4 virgins carries a copy of the EP-vector and a copy of the elav-Gal4 vector, leading to ectopic expression of adjacent genomic DNA sequences 3' of the EP-vector integration locus, mainly in neurons of these flies.
  • flies were incubated for 5 min at 90°C in an aqueous buffer using a waterbath, followed by hot extraction.
  • the triglyceride content of the flies extract was determined using Sigma Triglyceride (INT 336-10 or -20) assay by measuring changes in the optical density according to the manufacturer's protocol.
  • the protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol. The assays were repeated several times.
  • the average triglyceride level of all flies of the EP collection (referred to as 'EP-control') is shown as 100% in the first columns in Figures 1 , 6, 10, 14, 17, 20, 22 A-D, 24, 27, 31 , 33, and 35.
  • the average triglyceride level of all heterozygous flies of the EP collection containing a CyO balancer chromosome (referred to as 'EP-control CyO') is shown as 100% in column 5 in Figure 1 .
  • the average triglyceride level of all flies containing the FB-Gal4 vector (referred to as 'random EP/FB') is shown as 100% in column 3 in Figure 22B. Standard deviations of the measurements are shown as thin bars.
  • HD-EP(2)21996 homozygous flies (column 2 in Figure 1 , 'HD-EP21996') and HD-EP(2)22008 homozygous flies (column 3 in Figure 1 , 'HD-EP22008') show constantly a lower triglyceride content
  • HD-EP(2)26800 homozygous flies (column 4 in Figure 1 , 'HD-EP26800') show constantly a higher triglyceride content than the controls.
  • HD-EP(2)20702 heterozygous flies (column 6 in Figure 1 , 'HD-EP20702/CyO') show constantly a higher triglyceride content than the heterozygous controls.
  • HD-EP(3)37469 homozygous flies (column 2 in Figure 6, 'HD-EP37469') show a slightly higher triglyceride content than the controls.
  • the offspring of HD-EP(3)37469 males that are crossed to FB-Gal4 virgins (column 3 in
  • Figure 6 shows constantly a slightly lower triglyceride content than the controls. Both values are in the normal range compared to controls and therfore represent no significant change of triglyceride content.
  • HD-EP(X) 10632 hemizygous flies show constantly a higher triglyceride content than the controls (column 2 in Figure 10, 'HD-EP10632').
  • HD-EP(3)31624 heterozygous flies (column 2 in Figure 14, 'HD-EP31624/TM3,Ser') show a slightly lower triglyceride content than the controls.
  • the offspring of HD-EP(3)31624 males that are crossed to FB-Gal4 virgins (column 3 in Figure 14, 'HD-EP31624/FB') shows constantly a slightly higher triglyceride content than the controls. Both values are in the normal range compared to controls and therfore represent no significant change of triglyceride content.
  • the offspring of HD-EP(3)31624 males that are crossed to elav-Gal4 virgins (column 4 in Figure 14, 'HD-EP31624/elav') shows constantly a higher triglyceride content than the controls.
  • HD-EP(3)31769 homozygous flies (column 2 in Figure 17, 'HD-EP31769') show constantly a slightly lower triglyceride content than the controls.
  • the offspring of HD-EP(3)31769 males that are crossed to FB-Gal4 virgins (column 3 in Figure 17, 'HD-EP31769/FB') shows constantly a slightly higher triglyceride content than the controls. Both values are in the normal range compared to controls and therfore represent no significant change of triglyceride content.
  • HD-EP(3)37458 homozygous flies show constantly a higher triglyceride content than the controls (column 2 in Figure 20, 'HD-EP37458').
  • HD-EP(3)36547 homozygous flies (column 2 in Figure 22A, 'HD-EP36547') and HD-EP(3)37389 homozygous flies (column 3 in Figure 22A, 'HD-EP37389') show constantly a lower triglyceride content than the controls.
  • HD-EP(3)36941 homozygous flies (column 4 in Figure 22A, 'HD-EP36941 ') show constantly a higher triglyceride content than the controls.
  • HD-EP(3)30418 homozygous flies (column 2 in Figure 22B, 'HD-EP3041 8') show constantly a higher triglyceride content than the controls.
  • the offspring of HD-EP(3)3041 8 males that are crossed to FB-Gal4 virgins (column 4 in Figure 22B, 'HD-EP3041 8/FB') shows constantly a higher triglyceride content than the controls.
  • HD-EP(3)36364 homozygous flies (column 2 in Figure 22C, 'HD-EP36547') show constantly a higher triglyceride content than the controls.
  • HD-EP(3)31 1 71 homozygous flies (column 2 in Figure 22D, 'HD-EP31 1 71 ') show constantly a slightly lower triglyceride content than the controls. This value is in the normal range compared to controls and therfore represents no significant change of triglyceride content.
  • the offspring of HD-EP(3)31 1 71 males that are crossed to FB-Gal4 virgins (column 3 in Figure 22D, 'HD-EP31 171 /FB')shows constantly a higher triglyceride content than the controls.
  • HD-EP(2)26044 homozygous flies (column 2 in Figure 24, 'HD-EP26044') show constantly a lower triglyceride content than the controls.
  • HD-EP(3)371 85 homozygous flies (column 2 in Figure 27, 'HD-EP371 85') show constantly a lower triglyceride content than the controls.
  • the offspring of HD-EP(3)37185 males that are crossed to elav-Gal4 virgins (column 4 in Figure 27, 'HD-EP37185/elav') shows constantly a higher triglyceride content than the controls.
  • Example 2 Identification of Drosophila genes associated with triglyceride metabolism
  • Nucleic acids encoding the proteins of the present invention were identified using a plasmid-rescue technique. Genomic DNA sequences were isolated that are localized adjacent to the EP vector (herein HD-EP(2)21996, HD-EP(2)22008, HD-EP(2)26800, HD-EP(2)20702, HD-EP(3)37469, HD-EP(X) 10632, HD-EP(3)31624, HD-EP(3)31769, HD-EP(3)37458, HD-EP(3)31600, HD-EP(3)36681 , HD-EP(3)36956, HD-EP(3)36547, HD-EP(3)37389, HD-EP(3)36941 , HD-EP(3)30418, HD-EP(3)36364, HD-EP(3)31 171 , HD-EP(2)26044, HD-EP(3)37185, HD-EP(3)37410, HD-EP(2)25801 , HD-EP(2)21919, HD-EP(2)22018, HD-EP(2)26878, or HD-EP(2)26808) integration.
  • the EP vector herein HD
  • genomic DNA sequence is represented by the assembly as a black scaled double-headed arrow in the middle that includes the integration sites of the vectors for lines HD-EP(2)21996, HD-EP(2)22008, HD-EP(2)26800, HD-EP(2)20702, HD-EP(3)37458, HD-EP(3)31600, HD-EP(3)36681 , HD-EP(3)36956, HD-EP(3)36547, HD-EP(3)37389, HD-EP(3)36941 , HD-EP(3)30418, HD-EP(3)36364, HD-EP(3)31 171 , HD-EP(2)26044, HD-EP(2)25801 , HD-EP(2)21919, HD-EP(2)22018, HD-EP(2)26878, or HD-EP(2)26808.
  • Ticks represent the coordinates of the genomic DNA (1000 base pairs ( Figure 21 ) or 10000 base pairs ( Figures 2, 23, 25, 34, and 36) per tick).
  • the grey arrows in the upper part of the figures represent BAG clones, the black arrows in the topmost part of the figures represent the sections of the chromosomes.
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles and are labeled.
  • the cDNA sequences of the predicted genes are shown as dark grey bars (exons), linked by dark grey lines (introns), and are labeled (see also key at the bottom of the figures).
  • genomic DNA sequence is represented by the assembly as a dotted black line in the middle that includes the integration sites of the vectors for lines HD-EP(3)37469, HD-EP(X) 10632, HD-EP(3)31624, HD-EP(3)31769, HD-EP(3)37458, HD-EP(3)31600, HD-EP(3)36681 , HD-EP(3)36956, HD-EP(3)36547, HD-EP(3)37389, HD-EP(3)36941 , HD-EP(3)30418, HD-EP(3)36364, HD-EP(3)31 171 , HD-EP(2)26044, HD-EP(3)37185, HD-EP(3)37410, HD-EP(2)25801 , HD-EP(2)21919, HD-EP(2)22018, HD-EP(2)26878, or HD-EP(2)26808.
  • Numbers represent the coordinates of the genomic DNA.
  • the upper parts of the figures represent the sense strand " + ", the lower parts represent the antisense strand "-".
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles or boxes in the "P-elements + “ and/or "P-elements -” lines.
  • Transcribed DNA sequences ESTs are shown as grey bars in the "EST + “, "EST -”, “IPI + “, and/or the "IPI -” lines, and predicted cDNAs are shown as bars in the "cDNA + " and/ or "cDNA -” lines.
  • Predicted exons of the cDNAs are shown as dark grey bars and predicted introns are shown as light grey bars.
  • the HD-EP(2)21996, HD-EP(2)22008, and HD-EP ⁇ 2)26800 vectors are homozygous viable integrated 80 base pairs 5' / into an intron of the cDNA of a Drosophila gene
  • the HD-EP(2)20702 vector is homozygous lethal/heterozygous viable integrated 6243 base pairs 5' / into an intron of the cDNA of a Drosophila gene in sense orientation, identified as foraging (for, GadFly Accession Number CG 10033).
  • the chromosomal localization site of integrations of the vectors of HD-EP(2)21996, HD-EP(2)22008, HD-EP(2)26800, and HD-EP(2)20702 are at gene locus 2L, 24A2-4.
  • the HD-EP(3)37469 vector is homozygous viable integrated about 4500 base pairs 5' of a Drosophila gene in sense orientation, identified as CG5535 (GadFly Accession Number; GenBank Accession Number AY1 18300 for the cDNA, AAM48329, AAF49291 , and AAF49292 for the protein).
  • the chromosomal localization site of integration of the vector of HD-EP(3)37469 is at gene locus 3L, 75A4. In Figure 7, the coordinates of the genomic DNA start at position 17748000 on chromosome 3L, ending at position 17759500.
  • the insertion site of the P-element in Drosophila HD-EP(3)37469 line is shown in the "P Elements -" line and is labeled.
  • the predicted cDNA of the CG5535 gene shown in the "cDNA -" line is labeled, the corresponding ESTs are shown in the "EST -” and the "IPI -” lines.
  • the HD-EP(X) 10632 vector is hemizygous viable integrated into the cDNA (base pair 188) of a Drosophila gene in antisense orientation, identified as CG14217 (GadFly Accession Number).
  • the chromosomal localization site of integration of the vector of HD-EP(X) 10632 is at gene locus X, 18D3.
  • Figure 1 1 the coordinates of the genomic DNA start at position 19307388 on chromosome X, ending at position 19319888.
  • the insertion site of the P-element in Drosophila HD-EP(X) 10632 line is shown in the "P Elements + " line and is labeled.
  • the gene CG14217 shown in the "cDNA -" line is labeled, the corresponding ESTs are shown in the "EST -" and the "IPI -" lines.
  • the HD-EP(3)31624 vector is homozygous lethal/heterozygous viable integrated into the cDNA (base pair 25; 5' of the translation start) of a Drosophila gene in sense orientation, identified as Klp67A (GadFly Accession Number CG 10923, GenBank Accession Number NM 079268 for the cDNA, NP 523992 for the protein).
  • the chromosomal localization site of integration of the vector of HD-EP(3)31624 is at gene locus 3L, 67A9-B1. In Figure 15, the coordinates of the genomic DNA start at position 9282080 on chromosome 3L, ending at position 9288330.
  • the insertion site of the P-element in Drosophila HD-EP(3)31624 line is shown in the "P Elements -" line and is labeled.
  • the gene Klp67A shown in the "cDNA -” line is labeled, the corresponding ESTs are shown in the "EST -” and the "IPI -” lines.
  • the HD-EP(3)31769 vector is homozygous viable integrated into the cDNA (base pair 91 ; 5' of the translation start) of a Drosophila gene in sense orientation, identified as CG10133 (GadFly Accession Number, GenBank Accession Numbers AY1221 13 for the cDNA, AAM52625, and AAF49813 for the protein).
  • the chromosomal localization site of the integration of the vectors of HD-EP(3)31769 is at gene locus 70B1 on chromosome 3L. In Figure 18, the coordinates of the genomic DNA start at position 13363718 on chromosome 3L, ending at position 13365281 .
  • the insertion site of the P-element in Drosophila HD-EP(3)31769 line is shown in the "P Elements -” line and is labeled.
  • the gene CG10133 is shown in the "cDNA -” line, the corresponding ESTs are shown in the “EST -” and the “IPI -” lines and are labeled.
  • the HD-EP(3)37458 vector is homozygous viable integrated into the promoter (5' of EST clone HL01728) of a Drosophila gene in sense orientation, identified as CG3967 (GadFly Accession Number) .
  • the chromosomal localization site of integration of the vector of HD-EP(3)37458 is at gene locus 3L, 67B2-3.
  • the coordinates of the genomic DNA start at position 9331000 on chromosome 3L, ending at position 9349000.
  • the insertion site of the P-element in Drosophila HD-EP(3)37458 line is shown in the "P Elements -" line and is labeled.
  • the gene CG3967 is shown in the "cDNA -" line and is labeled, the corresponding ESTs are shown in the "EST -” line and are labeled.
  • the splice variants of CG3967 show different 5' ends of the gene resulting in an integration site of the HD-EP(3)37458 1 base pair 5' of the cDNA of three CG3967 splice variants. Additionally the insertion sites of the P-elements in Drosophila HD-EP(3)31 600, HD-EP(3)36681 and HD-EP(3)36959 lines are are labeled.
  • the HD-EP(3)36941 and HD-EP(3)31 171 vectors are homozygous viable integrated in the promoter/ enhancer region of a Drosophila gene in sense oriantation, and the HD-EP(3)36547, HD-EP(3)37389, HD-EP(3)30418, and HD-EP(3)36364 vectors are homozygous viable integrated into the promoter/enhancer region of a Drosophila gene in antisense orientation, identified as CG32048 (GadFly Accession Number).
  • the chromosomal localization site of integration of the vectors is at gene locus 3L, 67B1 2-C1 .
  • the coordinates of the genomic DNA start at position 9460000 on chromosome 3L, ending at position 951 7000.
  • the insertion site of the P-elements in Drosophila HD-EP(3)36941 , and HD-EP(3)31 171 lines is shown in the "P Elements + " line and is labeled, and the insertion site of the P-elements in Drosophila HD-EP(3)36547, HD-EP(3)37389, HD-EP(3)30418, and HD-EP(3)36364 lines is shown in the "P Elements -" line and is labeled.
  • the gene CG32048 is shown in the "cDNA + " line, the corresponding ESTs are shown in the "EST + " and the "IPI + " lines and are labeled.
  • the molecular organization of the CG32048 gene locus as annotated by GadFly (release 3), is shown.
  • the insertion sites of the P-elements in the Drosophila lines are labeled.
  • the splice variants show different 5' ends of the CG32048 gene.
  • the HD-EP(2)26044 vector is homozygous viable integrated into the promoter of a Drosophila gene in sense orientation, identified as CG31605 (GadFly Accession Number; also referred to as 'dmel2L_DKmel2L_DK12').
  • the chromosomal localization site of integration of the vector of HD-EP(2)26044 is at gene locus 2L, 28E3-5. In the upper part of Figure 25, the coordinates of the genomic DNA start at position 7984000 on chromosome 2L, ending at position 8019000.
  • the insertion site of the P-element in Drosophila HD-EP(2)26044 line is shown in the "P Elements + " line and is labeled.
  • the gene CG31605 (referred to as 'dmel2L_DKdmel2L_DK12') shown in the "cDNA + " line is labeled, the corresponding ESTs are shown in the "EST + " line.
  • the molecular organization of the CG31605 gene as annotated by GadFly (release 3), is shown.
  • the insertion site of the P-element in Drosophila HD-EP(2)26044 line is labeled.
  • the splice variants show different 5' ends of the gene resulting in an integration site of the vector of HD-EP(2)26044 into the promoter (9 base pairs 5') of four CG31605 splice variants) and into the first intron of four other CG31605 splice variants.
  • the HD-EP(3)37185 vector is homozygous viable integrated 14 base pairs 5' of a Drosophila gene in sense orientation, identified as Baldspot (GadFly
  • the chromosomal localization site of the integration of the vector of HD-EP(3)37185 is at gene locus 73B4-5 on chromosome 3L.
  • the coordinates of the genomic DNA start at position 16547000 on chromosome 3L, ending at position 16562000.
  • the insertion site of the P-element in Drosophila HD- HD-EP(3)37185 line is shown in the "P Elements -" line and is labeled. Magpie).
  • the gene Baldspot is shown in the "cDNA -" line, the corresponding ESTs are shown in the "EST -" and "IPI -" lines and are labeled.
  • the HD-EP(3)37410 vector is homozygous viable integrated about 3500 base pairs 5' of a Drosophila gene in sense orientation, identified as CG13625 (GadFly Accession Number; GenBank Accession Numbers NM 143015 for the cDNA, NP 651272 for the protein).
  • the chromosomal localization site of the integration of the vector of HD-EP(3)37410 is at gene locus 3R, 96A9.
  • the coordinates of the genomic DNA start at position 20314000 on chromosome 3R, ending at position 20324000.
  • the insertion site of the P-element in Drosophila HD-EP(3)37410 line is shown in the "P Elements -" line and is labeled.
  • the gene CG 13625 is shown in the "cDNA -" line, the corresponding ESTs are shown in the "EST -" and "IPI -” lines and are labeled.
  • the HD-EP(2)25801 vector is homozygous viable integrated into the cDNA (base pair 21 ; 5' of the translation start) of a Drosophila gene in sense orientation, identified as fusilli (fus; GadFly Accession Number CG8205; GenBank Accession Number NM 079952 for the cDNA, NP 524691 for the protein).
  • the chromosomal localization site of integration of the vector of HD-EP(2)25801 is at gene locus 2R, 52C3-4. In the upper part of Figure 34, the coordinates of the genomic DNA start at position 10618000 on chromosome 2R, ending at position 10648000.
  • the insertion site of the P-element in Drosophila HD-EP ⁇ 2)25801 line is shown in the "P Elements -" line and is labeled.
  • the gene fusilli is shown in the "cDNA -” line, the corresponding ESTs are shown in the "EST -” and "IPI -” lines and are labeled.
  • the molecular organization of the fusilli gene (referred to as 'fus'), as annotated by GadFly (release 3), is shown.
  • the splice variants show different 5' ends of the gene resulting in an integration site of the HD-EP(2)25801 into the cDNA CG8205-RE but very likely affecting also three other fusilli splice variants.
  • the HD-EP(2)21919 and HD-EP(2)26808 vectors are homozygous viable integrated into the promoter, the HD-EP(2)22018 vector is homozygous viable integrated into an of a Drosophila gene in sense orientation, and the HD-EP(2)26878 vector is homozygous lethal/heterozygous viable integrated into the promoter or an intron of a Drosophila gene in antisense orientation, identified as schnurri (shn; GadFly Accession Number CG7734).
  • the chromosomal localization sites of integration of the vectors of HD-EP(2)21919, HD-EP(2)22018, HD-EP(2)26878, and HD-EP(2)26808 are at gene locus 2R, 47E1 .
  • Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride metabolism were further analysed using the BLAST algorithm searching in publicly available sequence databases and mammalian homologs were identified (see Table 1 and Figures 3, 8, and 12).
  • polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number.
  • GenBank Accession number relates to NCBI GenBank database entries (Ref.: Benson et al., Nucleic Acids Res. 28 (2000) 15-18).
  • nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • Particularly preferred are nucleic acids as described in Table 1.
  • gene product of Drosophila foraging (GadFly Accession Number CG10033, GenBank Accession Number NM_058139 for the cDNA, NP_477487 for the protein) is 74% homologous to human protein kinase, cGMP-dependent, regulatory, type 1 , beta isozym (GenBank Accession Number NP 006249.1 for the protein, NM_006258 for the cDNA) and 63% homologous to human protein kinase, cGMP-dependent, type II (GenBank Accession Number NP 006250.1 for the protein, NM 006259 for the cDNA).
  • gene product of Drosophila foraging is 74% homologous to human cGMP-dependent protein kinase 1 , alpha isozym (GenBank Accession Number Q13976 for the protein), 81 %/38% homologous to human protein kinase, cGMP-dependent, type 1 (GenBank Accession Number XP 1 65548.1 for the protein, XM_1 65548 for the cDNA), 70%/55% homologous to human protein kinase, cGMP-dependent, type II (GenBank Accession Number XP_1 1 3447.1 for the protein, XM 1 1 3447 for the cDNA), and 63% homologous to human cGMP-dependant protein kinase (GenBank Accession Number CAA76073.1 for the protein, Y1 6106 for the cDNA) .
  • Drosophila foraging also shows homology on protein level to mouse protein kinase, cGMP-dependent, type 1 (GenBank Accession Number NM_01 1 1 60 for the cDNA), and type II (GenBank Accession Number NM_008926 for the cDNA).
  • gene product of Drosophila CG5535 (GadFly Accession Number; GenBank Accession Number AY1 1 8300 for the cDNA, AAM48329, AAF49291 , and AAF49292 for the protein) is 64% homologous to human solute carrier family 7 (cationic amino acid transporter, V + system), member 2 (GenBank Accession Number NP 003037.1 for the protein, NM_003046 for the cDNA), 64% homologous to human solute carrier familiy 7 (cationic amino acid transporter, y + system), member 1 (GenBank Accession Number NP_003036.1 for the protein, NM_003045 for the cDNA), and 64% homologous to human solute carrier family 7 (cationic amino acid transporter, y + system), member 3 (GenBank Accession Number NP_1 1 61 92.2 for the protein, NM 032803 for the cDNA).
  • Drosophila CG5535 also shows homology on protein level to mouse solute carrier familiy 7 (cationic amino acid transporter, y + system), members 1 , 2, and 3 (GenBank Accession Numbers NM_00751 3, NM 007514, and NM 00751 5, respectively) .
  • gene product of Drosophila CG1421 7 (GadFly Accession Number, GenBank Accession Number NM_1 34475 for the cDNA, NP_60831 9 and AAF48973 for the protein) is 84%/56%/43/41 % homologous to human prostate derived STE20-like kinase PSK (GenBank Accession Number NP_057235 for the protein, NM 016151 for the cDNA), 73%/66% homologous to human KIAA1361 protein (GenBank Accession Number BAA92599.1 for the protein, AB037782 for the cDNA), 71 %/65% homologous to human STE20-like kinase (GenBank Accession Number NP 057365.2 for the protein, NM_016281 for the cDNA), and 84% homologous to human thousand and one amino acid protein kinase (GenBank Accession Number NP 004774.1 for the protein, NM 004783 for
  • gene product of Drosophila CG14217 is 73%/66% homologous to human serin/threonin kinase TAO1 (GenBank Accession Number AAL12217.1 for the protein, AY049015 for the cDNA), 72%/66% homologous to human STE20-like kinase (GenBank Accession Number AAG38502.1 for the protein, AF263312 for the cDNA), 70%/65% homologous to human STE20-like kinase (GenBank Accession Number AAF14559.1 for the protein, AF179867 for the cDNA), 71 %/65% homologous to human protein similar to serine kinase (JIK; GenBank Accession Number XP_045006.1 for the protein, XM_045006 for the cDNA), 71 %/65% homologous to human STE20-like kinase (GenBank Accession Number AAG38501 .1 for the protein, AF26331 1
  • Drosophila Klp67A The gene product of Drosophila Klp67A (Kinesin-like protein at 67A; GadFly Accession Number CG10923, GenBank Accession Number NM_079268 for the cDNA, NP_523992 for the protein) is 61 % homologous to human hypothetical protein DKFZp434G2226 (GenBank Accession Number NP_1 12494.2 for the protein, NM_031217 for the cDNA). Drosophila Klp67A also shows homology on protein level to mouse kinesin superfamily protein 18A (GenBank Accession Number NM_139303 for the cDNA).
  • Drosophila CG10133 The gene product of Drosophila CG10133 (GadFly Accession Number, GenBank Accession Numbers AY1221 13 for the cDNA, AAM52625, and AAF49813 for the protein) is 64% homologous to human unknown protein (protein for MGC:39228; GenBank Accession Number AAH25290.1 for the protein, BC025290 for the cDNA), 61 % homologous to human unnamed protein product (GenBank Accession Number BAA91385.1 for the protein, AK000814 for the cDNA), and 59% homologous to human phospholipase A2, group IVB (GenBank Accession Number NP_005081 for the protein, NM_005090 for the cDNA). Drosophila CG10133 also shows homology on protein level to mouse protein similar to phospholipase A2, group IVB (GenBank Accession Number XM 130446 for the cDNA).
  • Drosophila CG3967 The gene product of Drosophila CG3967 (GadFly Accession Number) is 61 % homologous to human hypothetical protein FLJ13158 (GenBank Accession Number XP 042375.2 for the protein, XM_042375 for the cDNA), and 61 % homologous to human hypothetical protein FLJ13158 (GenBank Accession Number NP_079185.1 for the protein, NMJD24909 for the cDNA). Drosophila CG3967 also shows homology on protein level to mouse RIKEN cDNA 31 10080J08 and RIKEN cDNA 31 10080J08 (GenBank Accession Numbers NM_028476 and XM 28622 for the cDNAs, respectively).
  • Drosophila CG32048 (GadFly Accession Number CG32048-PA) is 58% homologous to human protein similar to C-terminal PDZ domain ligand of neuronal nitric oxide synthase (GenBank Accession Number XP_034002.1 for the protein, XM_034002 for the cDNA), and 58% homologous to human KIAA0464 protein (GenBank Accession Number BAA32309.2 for the protein, AB007933 for the cDNA) .
  • Drosophila CG32048 also shows homology on protein level to mouse protein (GenBank Accession Number XM 129577 for the cDNA).
  • Drosophila CG31605 (GadFly Accession Number CG31605-PG) is 45% homologous to human basigin; collagenase stimulatory factor; M6 antigen; extracellular matrix metalloproteinase inducer (GenBank Accession Number NP 001719.1 for the protein, NM_001728 for the cDNA), 46% homologous to human emmprin (GenBank Accession Number BAB88938.1 for the protein, AB072923 for the cDNA), 42% homologous to human stromal cell derived factor receptor 1 isoform b (GenBank Accession Number NP_036560.1 for the protein, NM 012428 for the cDNA), and 42% homologous to human stromal cell derived factor receptor 1 isoform a (GenBank Accession Number NP 059429.1 for the protein, NM_017455 for the cDNA). Drosophila CG31605 also shows homology on protein level to mouse basigin (GenBank Accession
  • the gene product of Drosophila Baldspot (GadFly Accession Number CG3971 , GenBank Accession Numbers NM_140652 for the cDNA, MP_648909 and AAF49430 for the protein) is 63% homologous to human long-chain fatty-acyl elongase (GenBank Accession Number NP_076995.1 for the protein, NM 024090 for the cDNA), 62% homologous to human protein similar to CIG30 (GenBank Accession Number XP 058360.1 for the protein, XM_058360 for the cDNA), and 66% homologous to human Elongation of very long chain fatty acids protein 3 (Cold inducible glycoprotein of 30 kDa; GenBank Accession Number Q9HB03).
  • Drosophila Baldspot also shows homology on protein level to mouse long chain fatty acyl elongase (GenBank Accession Number NM_130450 for the cDNA) and mouse elongation of very long chain fatty acids-like 3 protein (GenBank Accession Number NM 007703 for the cDNA).
  • Drosophila CG13625 The gene product of Drosophila CG13625 (GadFly Accession Number; GenBank Accession Numbers NM 143015 for the cDNA, NP 651272 for the protein) is 50%/39% homologous to human unnamed protein product (GenBank Accession Number BAB71593.1 for the protein, AK057832 for the cDNA) and 42% homologous to human hypothetical protein MGC13125 (GenBank Accession Number NP_1 161 14.1 for the protein, NM_032725 for the cDNA). Drosophila CG13625 also shows homology on protein level to mouse hypothetical protein MGC13125 (GenBank Accession Number XM 134776 for the cDNA).
  • the gene product of Drosophila fusilli (fus; GadFly Accession Number CG8205; GenBank Accession Number NM 079952 for the cDNA, NP_524691 for the protein) is 63%/46% homologous to human protein similar to fusilli; enhancer of cactus (GenBank Accession Number XP_007770.7 for the protein, XM 007770 for the cDNA), and 51 % homologous to human hypothetical protein FLJ21918 (GenBank Accession Number NP 079215.1 for the protein, NM_024939 for the cDNA).
  • Drosophila fusilli also shows homology on protein level to mouse fusilli, enhancer of cactus (GenBank Accession Number XM 131317 for the cDNA) and mouse RIKEN cDNA 9530027K23 gene (GenBank Accession Number XM_134507 for the cDNA).
  • the gene product of Drosophila schnurri (shn; GadFly Accession Number CG7734, GenBank Accession Numbers NM 057375 for the cDNA, NP 476723 for the protein) is 50% homologous to human immunodeficiency virus type 1 enhancer binding protein 1 (GenBank Accession Number NP_002105.1 for the protein, NM_0021 14 for the cDNA), 54% homologous to human immunodeficiency virus type 1 enhancer binding protein 2 (GenBank Accession Number NP_006725.2 for the protein, NM_006734 for the cDNA), 42% homologous to human protein similar to human immunodeficiency virus type 1 enhancer-binding protein 3 (GenBank Accession Number XP_046509.7 for the protein, XM_046509 for the cDNA), 42% homologous to human KIAA1555 protein (GenBank Accession Number BAB13381 .1 for the protein, AB046775 for the cDNA), and
  • cGMP-dependent protein kinase type I is also referred to in patent application WO0255664.
  • Solute carrier family 7, member 1 is also referred to in patent application WO9210506, in patent US5834589, and in patent application WO9325682.
  • Prostate derived STE20-like kinase PSK is also referred to in patent application WO0058473 and KIAA1361 protein is also referred to in patent application WO0222660.
  • Stromal cell derived factor receptor 1 is also referred to in patent application WO0029583.
  • ELOVL3 is also referred to in patent application WO0134643, in patent application
  • ELOVL family member 6 is referred to in patent application WO01 18016, in patent application WO0208401 , in patent application WO0012720, and in patent application WO0065054.
  • Drosophila uncoupling protein dUCPy in a non-vital organ like the eye (Gal4 under control of the eye-specific promoter of the eyeless gene) results in flies with visibly damaged eyes.
  • This easily visible eye phenotype is the basis of a genetic screen for gene products that can modify UCP activity.
  • the activation of the genes therefore occurs in the same cells (eye) that overexpress dUCPy. Since the mutant collection contains several thousand strains with different integration sites of the EP-element it is possible to test a large number of genes whether their expression interacts with dUCPy activity. In case a gene acts as an enhancer of UCP activity the eye defect will be worsened; a suppressor will ameliorate the defect.
  • Adipose is a protein that has been described as regulating, causing or contributing to obesity in an animal or human (see WO 01/96371 ).
  • Transgenic flies containing a wild type copy of the adipose cDNA under the control of the Gal4/UAS system were generated (Brand A.H. and Perrimon N., (1993) Development 1 18: 401 -415; for adipose cDNA, see WO 01 /96371 ).
  • Enhancement of the lethality phenotype was detected by a decreased number of escapers and suppression of the lethality phenotype was detected by increased viability of flies at the restrictive temperature. Mutations changing the lethality phenotype affect genes that modify adipose activity.
  • the inventors have found that the fly line HD-EP(3)31600 is an enhancer of the FB-adp-Gal4 induced lethality phenotype, and that the fly lines HD-EP(3)31 171 , HD-EP(3)30418, and HD-EP(2)22018 are suppressors of the FB-adp-Gal4 induced lethality phenotype.
  • Example 6 Expression of the polypeptides in mammalian (mouse) tissues
  • mice strains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standard model systems in obesity and diabetes research
  • Harlan Winkelmann 33178 Borchen, Germany
  • constant temperature preferrably 22 °C
  • 40 per cent humidity and a light / dark cycle of preferrably 14 / 10 hours.
  • the mice were fed a standard chow (for example, from ssniff Spezialitaten GmbH, order number ssniff M-Z V1 126-000).
  • wild type mice For the fasting experiment (“fasted wild type mice”), wild type mice were starved for 48 h without food, but only water supplied ad libitum (see, for example, Schnetzler B. et al., 1993, J Clin Invest 92: 272-280, Mizuno T.M. et al., 1996, Proc Natl Acad Sci U S A 93: 3434-3438).
  • wild-type (wt) mice were fed a control diet (preferably Altromin C1057 mod control, 4.5% crude fat) or high fat diet (preferably Altromin C1057mod. high fat, 23.5% crude fat). Animals were sacrificed at an age of 6 to 8 weeks. The animal tissues were isolated according to standard procedures known to those skilled in the art, snap frozen in liquid nitrogen and stored at -80°C until needed.
  • mammalian fibroblast (3T3-L1 ) cells e.g., Green H. and Kehinde O., 1974, Cell 1 : 1 13-1 16
  • 3T3-L1 cells were obtained from the American Tissue Culture Collection (ATCC, Hanassas, VA, USA; ATCC- CL 173).
  • 3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g., Qiu Z. et al., 2001 , J. Biol. Chem. 276: 1 1988-1 1995; Slieker L.J.
  • Taqman analysis was performed preferrably using the following primer/probe pairs:
  • mice Prkgl For the amplification of mouse protein kinase, cGMP-dependent, type I (Prkgl ) sequence (GenBank Accession Number NM 01 1 160): Mouse Prkgl forward primer (SEQ ID NO: 1): 5'- GCC TAT CTG CAT TCC AAA GGA A-3'; mouse Prkgl reverse primer (SEQ ID NO: 2): 5'- TGG CAT AGC CTC GAT GAT CTA G-3'; mouse Prkgl Taqman probe (SEQ ID NO: 3): (5/6-FAM)-CAT TTA CAG GGA CCT CAA GCC GGA GAA-(5/6-TAMRA).
  • mice Prkg2 forward primer SEQ ID NO: 4
  • mouse Prkg2 reverse primer SEQ ID NO: 5'- GCT GTG GAG CGA GAC CAA G-3'
  • mouse Prkg2 Taqman probe SEQ ID NO: 6
  • 5/6-FAM TGC CTC TGG ATG TTC ACC GCA AGA C- (5/6-TAMRA).
  • mouse solute carrier family 7 cationic amino acid transporter, y + system
  • member 1 Slc7a1
  • Mouse Slc7a1 forward primer SEQ ID NO: 7
  • mouse Slc7a1 reverse primer SEQ ID NO: 8
  • mouse Slc7a1 Taqman probe SEQ ID NO: 9: (5/6-FAM)-ACG GTA CCA GCC AGA ACA ACC TAA TCT GGT A- (5/6-TAMRA).
  • mouse solute carrier family 7 cationic amino acid transporter, y + system
  • member 2 Slc7a2 sequence
  • Mouse Slc7a2 forward primer SEQ ID NO: 10
  • mouse Slc7a2 reverse primer SEQ ID NO: 1 1
  • mouse Slc7a2 Taqman probe SEQ ID NO: 1 2: (5/6-FAM)- CTC TGC GCT GCC GTC ATT CTG ACC -(5/6-TAMRA) .
  • mouse solute carrier family 7 cationic amino acid transporter, y + system
  • member 3 Slc7a3 sequence
  • Mouse Slc7a3 forward primer SEQ ID NO: 13
  • mouse Slc7a3 reverse primer SEQ ID NO: 14
  • mouse Slc7a3 Taqman probe SEQ ID NO: 1 5): (5/6-FAM)-CAG AAG ATG GCC TCC TGT TCC GTG TC-(5/6-TAMRA).
  • mouse RIKEN cDNA 1 1 10033K02 gene (1 1 10033K02Rik) sequence (GenBank Accession Number XM_133799): Mouse 1 1 10033K02Rik forward primer (SEQ ID NO: 1 6): 5'- GAG CAA CGG GTG GAA GAA GA -3'; mouse 1 1 10033K02Rik reverse primer (SEQ ID NO: 1 7): 5'- AAA CTC CGG ATA CGT TCC GA-3'; mouse 1 1 10033K02Rik Taqman probe (SEQ ID NO: 1 8) : (5/6-FAM)-CTG CTG GCC CTA CAG ACA GGC CG-(5/6-TAMRA).
  • mouse LOC192767 forward primer (SEQ ID NO: 19): 5'- GAA CAT GCA ATG CTA TTG CGA C-3'
  • mouse LOC192767 reverse primer (SEQ ID NO: 20): 5'- CTG ATC AAC TCA CAG CGC ATC-3'
  • mouse LOC192767 Taqman probe (SEQ ID NO: 21): (5/6-FAM)-ACT GGA GTT TCG CCA TCT CAA CAC TAT TCA GA-(5/6-TAMRA).
  • mouse kinesin family member 18A sequence (GenBank Accession Number NM 139303): Mouse Kif18a forward primer (SEQ ID NO: 22): 5'- AAT GGC ACA CAT GAC AGC TCT AG-3'; mouse Kif18a reverse primer (SEQ ID NO: 23): 5'- AAG CAA AGC ATT CAA TAC TGC CT-3'; mouse Kif18a Taqman probe (SEQ ID NO: 24): (5/6-FAM)-TCT CCA GGA ACA GCA GCA CAA GCA AAC T-(5/6-TAMRA).
  • mouse LOC21 1429 For the amplification of mouse similar to phospholipase A2, group IVB (cytosolic) (LOC21 1429) sequence (GenBank Accession Number XM_130446): Mouse LOC21 1429 forward primer (SEQ ID NO: 25): 5'- TGC CAG CTC TGT GGT TCC A-3'; mouse LOC21 1429 reverse primer (SEQ ID NO: 26): 5'- ATG TCA TAC CAG AAA TTC ACA GCA A-3'; mouse LOC211429 Taqman probe (SEQ ID NO: 27): (5/6-FAM)-CAT GTC CAG CAG TCC CAC GGC TG-(5/6-TAMRA).
  • mouse Elovl ⁇ elongation of long chain fatty acids (yeast) sequence (GenBank Accession Number NMJ 30450): Mouse Elovl ⁇ forward primer (SEQ ID NO: 28): 5'- GAA CTA TGG CGT GCA TGC C-3'; mouse Elovl ⁇ reverse primer (SEQ ID NO: 29): 5'- CGG GAG ACT CGG AAA CCC-3'; mouse Elovl ⁇ Taqman probe (SEQ ID NO: 30): (5/6-FAM)-ACT ACG CCT TGC GGG CTG CG-(5/6-TAMRA).
  • RNA-expression is shown on the Y-axis.
  • the tissues tested are given on the X-axis.
  • WAT white adipose tissue
  • BAT brown adipose tissue.
  • the X-axis represents the time axis.
  • “dO” refers to day 0 (start of the experiment)
  • “d2" - "d12” refers to day 2 - day 12 of adipocyte differentiation.
  • the function of the proteins of the invention in metabolism was further validated by analyzing the expression of the transcripts in different tissues and by analyzing the role in adipocyte differentiation.
  • mice carrying gene knockouts in the leptin pathway (for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice) to study the expression of the proteins of the invention.
  • leptin pathway for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • expression of the mRNAs encoding the proteins of the invention was also examined in susceptible wild type mice (for example, C57BI/6) that show symptoms of diabetes, lipid accumulation, and high plasma lipid levels, if fed a high fat diet.
  • Prkgl is expressed in several mammalian tissues, showing highest level of expression in muscle and higher levels in further tissues, e.g. lung, hypothalamus, brain, heart and testis. Furthermore Prkgl is expressed on lower but still robust levels in WAT, BAT and kidney of wild type mice as depicted in Figure 4A. We found, for example, that the expression of Prkgl is up regulated in WAT, BAT and hypothalamus of genetically induced obese mice (ob/ob) compared to wild type mice. Furthermore Prkgl is up regulated in BAT of fasted mice compared to wild type mice (see Figure 4B).
  • Prkgl in metabolic active tissues of wild type mice, as well as the up regulation of Prkgl in different animal models used to study metabolic disorders, suggests that this gene plays a central role in energy homeostasis. This hypothesis is supported by the down regulation during the differentiation from preadipocytes to mature adipocytes,
  • Prkg2 The regulated expression of Prkg2 in different animal models used to study metabolic disorders, together with the down regulation during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.
  • Slc7a1 As regulator of energy metabolism in mammals. Taqman analysis revealed that Slc7a1 is expressed in several mammalian tissues, showing highest level of expression in WAT and higher levels in further tissues, e.g. hypothalamus, brain, heart, lung, small intestine, spleen, colon and testis. Furthermore Slc7a1 is expressed on lower but still robust levels in BAT, muscle, pancreas and kidney of wild type mice as depicted in Figure 9A. We found, for example, that the expression of Slc7a1 is down regulated in WAT and up regulated in BAT and liver of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Slc7a1 is up regulated in liver and down regulated in bone marrow of fasted mice compared to wild type mice (see Figure 9B).
  • the expression of Slc7a1 is up regulated in BAT and muscle and down regulated in the WAT as depicted in Figure 9C.
  • Figure 9D We show in this invention (see Figure 9D) that the Slc7a1 mRNA is expressed and regulated during the differentiation into mature adipocyctes. Therefore, the Slc7a1 protein might play an essential role in adipogenesis.
  • Slc7a1 is strongly regulated in metabolic active tissues (e.g. WAT, BAT, muscle and liver) of different animal models used to study metabolic disorders.
  • WAT metabolic active tissues
  • BAT muscle and liver
  • Slc7a2 As regulator of energy metabolism in mammals. Taqman analysis revealed highest level of Slc7a2 expression in liver of wild type mice. Furthermore Slc7a2 is expressed on lower but still robust levels in WAT, muscle, lung, hypothalamus, brain, spleen and kidney of wild type mice as depicted in Figure 9E. We found, for example, that the expression of Slc7a2 is up regulated in the spleen of genetically induced obese mice (ob/ob) compared to wild type mice. Furthermore Slc7a2 is up regulated in muscle and liver of fasted mice compared to wild type mice (see Figure 9F).
  • 1 1 10033K02Rik is expressed in several mammalian tissues, showing highest level of expression in hypothalamus, brain, and testis and higher levels in further tissues, e.g., lung, WAT, spleen, and kidney. Furthermore 1 1 10033K02Rik is expressed on lower but still robust levels in heart, BAT, muscle, and liver of wild type mice as depicted in Figure 13A. We found, for example, that the expression of 1 1 10033K02Rik is down regulated in WAT of genetically induced obese mice (ob/ob) compared to wild type mice.
  • ob/ob genetically induced obese mice
  • 1 1 10033K02Rik is down regulated in WAT of fasted mice compared to wild type mice (see Figure 13B).
  • the expression of 1 1 10033K02Rik is up regulated in muscle as depicted in Figure 13C.
  • Figure 13D we show in this invention (see Figure 13D) that the 1 1 10033K02Rik mRNA is expressed and down regulated during the differentiation into mature adipocyctes. Therefore, the 1 1 10033K02Rik protein might play an essential role in adipogenesis.
  • LOC192767 is expressed in several mammalian tissues, showing highest level of expression in hypothalamus, brain, and testis and higher levels in further tissues, e.g. BAT, WAT, lung, spleen, muscle, and kidney. Furthermore LOC192767 is expressed on lower but still robust levels in liver, colon, small intestine, pancreas, and bone marrow of wild type mice as depicted in Figure 13E. We found, for example, that the expression of LOC192767 is up regulated in the spleen of genetically induced obese mice (ob/ob) compared to wild type mice.
  • ob/ob genetically induced obese mice
  • LOC192767 is down regulated in bone marrow of fasted mice compared to wild type mice (see Figure 13F).
  • the expression of LOC192767 is up regulated in WAT as depicted in Figure 13G.
  • the LOC192767 mRNA is expressed and down regulated during the differentiation into mature adipocyctes. Therefore, the LOC192767 protein might play an essential role in adipogenesis.
  • Kif 18a is expressed as regulator of energy metabolism in mammals.
  • Taqman analysis revealed highest level of Kif 18a expressed in testis.
  • Kif 18a is expressed on lower but still robust levels in several other tissues, e.g. in spleen, bone marrow, colon, small intestine, WAT, BAT, and muscle of wild type mice as depicted in Figure 16A.
  • the expression of Kif 18a is up regulated in WAT and BAT of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Kif 18a is up regulated in pancreas of fasted mice compared to wild type mice (see Figure 16B).
  • Kif 18a In wild type mice fed a high fat diet, the expression of Kif 18a is up regulated in BAT and muscle as depicted in Figure 16C. We show in this invention (see Figure 16D) that the Kif 18a mRNA is expressed and regulated during the differentiation into mature adipocyctes. Therefore, the Kif 18a protein might play an essential role in adipogenesis.
  • Kif 18a The regulated expression of Kif 18a in metabolic active tissues of different animal models used to study metabolic disorders, together with the regulated expression of Kif 18a during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.
  • LOC21 1429 is expressed in several mammalian tissues, showing highest level of expression in hypothalamus and testis, and higher levels in further tissues, e.g. BAT, heart, lung, spleen, brain, and kidney.
  • LOC21 1429 is expressed on lower but still robust levels in WAT, muscle, liver, colon, small intestine, and bone marrow of wild type mice as depicted in Figure 19A.
  • LOC21 1429 is down regulated in the bone marrow of genetically induced obese mice (ob/ob) compared to wild type mice.
  • the LOC21 1429 mRNA is expressed and down regulated during the differentiation into mature adipocyctes. Therefore, the LOC21 1429 protein might play an essential role in adipogenesis.
  • LOC21 1429 in metabolic active tissues of wild type mice, together with the down regulation of LOC21 1429 expression during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.
  • Elovl6 is expressed in several mammalian tissues, showing highest level of expression in BAT and higher levels in further tissues, e.g. small intestine, hypothalamus, brain WAT, liver, and kidney. Furthermore Elovl6 is expressed on lower but still robust levels in muscle of wild type mice as depicted in Figure 29A.
  • the expression of Elovl ⁇ is strongly up regulated in the liver and down regulated in BAT of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Elovl ⁇ is down regulated in BAT, muscle, and liver of fasted mice compared to wild type mice (see Figure 29B).
  • Elovl ⁇ expression in the liver of genetically obese mice suggests that this gene plays a central role in energy homeostasis.
  • This hypothesis is supported by the regulated expression of Elovl ⁇ in further metabolic active tissues (WAT, BAT and muscle) of different animal models used to study metabolic disorders and by the regulated expression of Elovl ⁇ during the differentiation from preadipocytes to mature adipocytes.
  • Elovl3 is expressed in specific mammalian tissues, showing highest level of expression in the liver. Furthermore Elovl3 is expressed on lower but still robust levels in BAT and muscle of wild type mice as depicted in Figure 29E.
  • the expression of Elovl3 is down regulated in WAT, BAT, and liver of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Elovl3 is down regulated in liver of fasted mice compared to wild type mice (see Figure 29F).
  • Elovl3 in metabolic active tissues (WAT, BAT and liver) of different animal models used to study metabolic disorders, suggests that this gene plays a central role in energy homeostasis. This hypothesis is supported by the strong up regulation of Elovl3 expression during the differentiation from preadipocytes to mature adipocytes.
  • Example 7 Analysis of the differential expression of transcripts of the proteins of the invention in human tissues
  • RNA preparation from human primary adipose tissues was done as described in Example 6.
  • the hybridization and scanning was performed as described in the manufactures manual (see Affymetrix Technical Manual, 2002, obtained from Affmetrix, Santa Clara, USA).
  • the X-axis represents the time axis, shown are day 0 and day 12 of adipocyte differentiation.
  • the Y-axis represents the flourescent intensity.
  • the expression analysis (using Affymetrix GeneChips) of the cGMP-dependent protein kinase, type II (PRKG2), stromal cell derived factor receptor 1 (SDFR1 ), basigin (BSG), ELOVL family member 6, elongation of long chain fatty acids (ELOVL6), and immunodeficiency virus type I enhancer binding protein 1 (HIVEP1 ) genes using primary human abdominal adipocycte differentiation clearly shows differential expression of human PRKG2, SDFR1 , BSG, ELOVL6, and HIVEP1 genes in adipocytes. Several independent experiments were done.
  • the experiments further show that the PRKG2, SDFR1 , and HIVEP1 transcripts (see Figures 5, 26A, and 37) are most abundant at day 0 compared to day 12 during differentiation, and that the BSG and ELOVL6 transcripts (see Figures 26B and 30) are most abundant at day 12 compared to day 0 during differentiation.
  • the PRKG2, SDFR1 , and HIVEP1 proteins have to be significantly decreased and the BSG and ELOVL6 proteins have to be significantly increased in order for the preadipocyctes to differentiate into mature adipocycte. Therefore, the PRKG2, SDFR1 , and HIVEP1 proteins in preadipocyctes have the potential to inhibit, and the BSG and ELOVL6 proteins in preadipocyctes have the potential to enhance adipose differentiation. Therefore, PRKG2, SDFR1 , BSG, ELOVL6, and HIVEP1 proteins might play an essential role in the regulation of human metabolism, in particular in the regulation of adipogenesis and thus it might play an essential role in obesity, diabetes, and/or metabolic syndrome.

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Abstract

La présente invention concerne de nouvelles utilisations de protéines régulatrices de l'homéostase énergétique, et des polynucléotides codant ces protéines dans le diagnostic, l'étude, la prévention et le traitement de maladies ou troubles du métabolisme.
PCT/EP2003/008826 2002-08-08 2003-08-08 Proteines intervenant dans la regulation de l'homeostase energetique WO2004016641A2 (fr)

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