WO2003103704A2 - Proteines impliquees dans la regulation de l'homeostasie energetique - Google Patents

Proteines impliquees dans la regulation de l'homeostasie energetique Download PDF

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WO2003103704A2
WO2003103704A2 PCT/EP2003/006080 EP0306080W WO03103704A2 WO 2003103704 A2 WO2003103704 A2 WO 2003103704A2 EP 0306080 W EP0306080 W EP 0306080W WO 03103704 A2 WO03103704 A2 WO 03103704A2
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nucleic acid
polypeptide
acid molecule
protein
homologous
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Karsten Eulenberg
Martin Meise
Günter BRÖNNER
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DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/53—Ligases (6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
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    • A—HUMAN NECESSITIES
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • This invention relates to the use of l(2)44DEa, wunen-2, grapes, CG2221 , CG 1 172, rutabaga, or CG1 1940 homologous proteins, to the use of polynucleotides encoding these, and to the use of effectors/modulators 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, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes as a major health problem 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. Besides severe risks of illness, individuals suffering from obesity are often isolated socially.
  • 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 (Koltermann J., (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 (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 Drosophila gene l(2)44DEa (GadFly Accession Number CG8732) encodes an acetate-CoA ligase. As shown in this invention, l(2)44DEa is most homologous to the human and mouse long-chain fatty-acid-CoA ligase 3 (LACS3) and 4 (LACS4) isoforms.
  • LACS3 long-chain fatty-acid-CoA ligase 3
  • LACS4 LACS4
  • the family of iong-chain-fatty-acid-CoA ligases consists of at least 6 members that seem to have specificity for different fatty acids.
  • Long-chain-fatty-acid-CoA ligases catalyze the activation of short-, medium and long chain fatty acids by catalyzing the esterification of fatty acids (FA) and coenzyme A to acyl-CoA esters using ATP.
  • Acyl-CoA formation is the prerequisite for the use of fatty acids (FA) within the cell.
  • Acyl-CoAs are primary key substrates for different anabolic and catabolic pathways such as beta-oxidation, triacylglycerol synthesis, phospholipid synthesis, cholesterol ester synthesis and fatty acid elongation.
  • Acyl-CoA synthetase enhance the uptake of fatty acids (FA) into liver by catalyzing their activation for subsequent use in metabolic pathways .
  • LACS Iong-chain-fatty-acid-CoA ligases
  • ACS Acyl-CoA synthetase
  • LACS long chain Acyl-CoA synthetase
  • ACS4 Acyl-CoA synthetase 4
  • an arachidonate preferring enzyme - modulates female fertility and uterine prostaglandin production.
  • female mice heterozygote for ACS4 show reduced fertility aasociated with morphological changes including enlarged uteri filled proliferative cysts. (Cho Y.-Y. et al., 2001 , Biochem Biophys Res Comm 284: 993-997).
  • acyl-CoA synthetase ACS
  • mRNA expression levels are increased in adipose tissue compared to control mice while ACS expression in liver is not altered.
  • the distribution of ACS activity in liver is altered in ob/ob mice.
  • more ACS activity is associated with microsomes whereas less ACS activity is associated with mitochondria.
  • the change in distribution in ob/ob mouse liver makes more fatty acid available for esterification, rather than oxidation.
  • Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), an animal model of NIDDM characterized by obesity with visceral fat accumulation, show increased hepatic ACS mRNA expression levels and ACS activity suggesting that enhanced expression of ACS might be associated with visceral fat accumulation and the pathogenesis of hyperlipidemia in obese animal models with non-insulin-dependent diabetes mellitus (Kuriyama H. et al., 1 998, Hepatology 27(2): 557-562) .
  • Lipopolysaccharide decreases ACS activity in adipose tissue, heart, and muscle.
  • LPS and cytokines decrease ACS1 mRNA expression and ACS activity in tissues where FA uptake and/or oxidation are decreased during sepsis.
  • LPS and cytokines decrease ACS1 mRNA and mitochondrial ACS activity, which may inhibit FA oxidation, but increase microsomal ACS activity, which may support the re-esterification of peripherally derived FA for triglyceride synthesis (Memon R. A. et al., 1 998, Am J Physiol 275(1 Pt 1 ): E64-72).
  • the Drosophila gene wun2 (wunen-2; GadFly Accession Number CG8805, GenBank Accession Number NM_80252) encodes a phosphatidate phosphatase (EC:3.1 .3.4) involved in dephosphorylation which is most likely located in the plasma membrane. As shown in this invention, wun2 is most homologous to the human and mouse phosphatidic acid (phosphatidate) phosphatase (PAP) family. wun2 acts as a repellent for migrating primordial germ cells, wun and wun2 cooperate to guide the germ cells by acting as repellants (Starz-Gaiano et al., 2000, A. Dros. Res. Conf. 41 : 85). wun and wun2 appear to act redundantly (Starz-Gaiano et al., 2001 , Development 1 28(6) : 983-991 ).
  • Phosphatidic acid phosphatases are involved in triacylglycerol metabolism. They catalyze the reaction of phosphatidic acid (diacylglycerol phosphate) to diacylglycerol by removing a phosphate residue.
  • Diacylglycerol (DAG) is a metabolic intermediate for triacylglycerol (or phospholipid synthesis.
  • a synonym for PAP is phosphatidate phosphohydrolase.
  • Type2 PAPs are Mg2 + independent and insensitive to inhibition by N-ethylmaleimide.
  • LPP lipid phosphate phosphatases
  • LPPs hydrolyse e.g. lysophosphatidate (LPA), ceramide-1 -phosphate and sphingosine-1 -phoshate (S1 P) and thereby affect signalling by these bioactive lipids and generate further molecules with biological activity.
  • LPA is able to activate preadipocytes by interacting with a specific G-protein coupled receptor (Pages et al., 2001 JBC 276 (1 1 599-1 1 605) .
  • LPPs serve as regulators of strength and duration of LPA signal and thereby attenuate its effects on cell signalling (Pilquil et al., Prostaglandins 2001 64 83-92) .
  • LPP1 -3 expression is downregulated after differentiation of 3T3F442A preadipocytes into adipocytes (Simon et al., 2002 JBC 277 (23131 -23136). Based on this, LPPs may participate in the control of adipose tissue development.
  • the Drosophila gene grapes (GadFly Accession Number CG17161 , GenBank Accession Numbers NM 057663 and AF057042) encodes a protein serine/threonine kinase involved in DNA damage checkpoint. As shown in this invention, grapes is most homologous to the human and mouse serine/threonine-protein kinases Chk1 (Checkpoint kinase 1 ).
  • Cyclin-dependent kinases are main regulators of cell cycle progression.
  • modulators of cdks may have a role in the treatment of human malignancies.
  • the cdk modulator UCN-01 blocks cell cycle progression and promotes apoptosis.
  • UCN-01 may abrogate checkpoints induced by genotoxic stress due to inhibition of chkl kinase (Senderowicz A. M., 2000, Oncogene 19(56): 6600-6606).
  • the Drosophila gene CG2221 (GadFly Accession Number) encodes a protein with unknown molecular function, which contains a plexin/semaphorin/integrin (PSI) domain.
  • Plexin is involved in the development of neural and epithelial tissues, semaphorins induce the collapse and paralysis of neuronal growth cones, and integrins may mediate adhesive or migratory functions of epithelial cells.
  • CG2221 is most homologous to the human and mouse tumor endothelian marker 7-related precursor.
  • TEMs tumor endothelial markers
  • TEMs are associated with the cell surface membrane, and TEM 1 , TEM5, TEM7, and TEM8 contain putative transmembrane domains.
  • TEM5 appears to be a seven-pass transmembrane receptor, whereas TEM1 , TEM7, and TEM8 span the membrane once (Carson-Walter E. B. et al., 2001 , Cancer Res 61 (1 8): 6649-6655) .
  • the Drosophila gene CG 1 1 72 (GadFly Accession Number) encodes a protein with unknown molecular function, which contains a Ubiquitin-like domain.
  • Ubiquitin is a protein of 76 amino acid residues, found in all eukaryotic cells and whose sequence is extremely well conserved from protozoan to vertebrates. It plays a key role in a variety of cellular processes, such as ATP-dependent selective degradation of cellular proteins, maintenance of chromatin structure, regulation of gene expression, stress response and ribosome biogenesis.
  • CG1 172 is most homologous to human and mouse protein similar to hypothetical protein FLJ1 1 807 and hypothetical protein FLJ 1 1 807 No functional data are available for the human and murine FLJ1 1807 and protein similar to FLJ1 1807.
  • the Drosophila gene rut (rutabaga; GadFly Accession Number CG9533; GenBank Accession Number NM_078601 ) encodes an adenylate cyclase (integral plasma membrane protein) involved in cAMP biosynthesis in Drosophila.
  • adenylate cyclases mediate signaling of G protein-coupled receptors.
  • GTP exchanges with GDP bound to a subunit of the G-protein. This complex binds adenylate cyclase, thereby activating the enzyme.
  • rut is most homologous to the human and mouse adenylate cyclase type I (AC type I).
  • Type I adenylyl cyclase (AC1 ) is a neurospecific enzyme that is stimulated by Ca 2+ and calmodulin (CaM) (Wayman G. A., 1 996, Mol Cell Biol 1 6(1 1 ):6075-6082). This enzyme couples the Ca 2+ and cyclic AMP (cAMP) regulatory systems in neurons. Ca 2+ and neurotransmitter stimulation of type I adenylyl cyclase may play a role in synaptic plasticity by generating optimal cAMP signals for regulation of transcription (see, for example, Impey S. et al., 1 994, Mol Cell Biol 14(1 2) : 8272-8281 ) .
  • AC1 might act as the center of a gating mechanism between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm (Tzavara E. T. et al., 1996, Proc Natl Acad Sci U S A 93(20): 1 1208-1 1212). Mutant mice lacking type I adenylyl cyclase show deficiencies in spatial memory and altered long-term potentiation (see, for example, Wu Z. L., 1 995, Proc Natl Acad Sci U S A 92(1 ): 220-224) .
  • the Drosophila gene CG 1 1 940 (GadFly Accession Number) encodes for a protein that contains a Ras-associated domain (RA domain) and a
  • Pleckstrin-homology domain (PH domain). Proteins with a RA domain are mostly RasGTP effectors and include guanine-nucleotide releasing factor in mammals. This factor stimulates the dissociation of GDP from the
  • Ras-related RALA and RALB GTPases which allows GTP binding and activation of the GTPases. It interacts and acts as as effector molecule for
  • the domain is also present in a number of other proteins among them the sexual differentiation protein in yeast that is essential for mating and meiosis and yeast adenylate cyclase. These proteins contain repeated leucine-rich (LRR) segments.
  • LRR leucine-rich
  • the 'pleckstrin homology' (PH) domain is a domain of about 100 residues that occurs in a wide range of proteins involved in intracellular signaling or as constituents of the cytoskeleton. The function of this domain is not clear, several putative functions have been suggested, like for example binding to the beta/gamma subunit of heterotrimeric G proteins, to lipids, or to phosphorylated Serine/Threonine residues.
  • CG1 1940 is most homologous to human KIAA1681 isoforms and mouse protein similar to Mig-10 protein.
  • the human KIAA1681 protein might be a signalling molecule acting as GTPase regulator.
  • Human KIAA1681 protein partial deletion leads to juvenile familial amyotrophic lateral sclerosis (ALS) 2, a hereditary form of progressive motor neuron disease (Hadano S. et al., 2001 , Nat. Genet. 29(2):166-173).
  • ALS familial amyotrophic lateral sclerosis
  • ALS hereditary form of progressive motor neuron disease
  • the affected motor neurons undergo shrinkage, often with accumulation of lipofuscin.
  • a protein of the invention or a homologous protein is involved in the regulation of energy homeostasis and body-weight regulation and related disorders, and thus, no functions in metabolic diseases and other diseases as listed above have been discussed.
  • this invention we demonstrate that the correct gene dose of a protein of the invention is essential for maintenance of energy homeostasis.
  • a genetic screen was used to identify that mutations of a gene encoding a protein of the invention or a homologous gene causes changes in the metabolism, in particular related to obesity, which is reflected by a significant change in the triglyceride content, the major energy storage substance.
  • the present invention discloses that l(2)44DEa (GadFly Accession Number CG8732), wunen-2 (GadFly Accession Number CG8805), grapes (GadFly Accession Number CG17161 ), GadFly Accession Number CG2221 , GadFly Accession Number CG1 172, rutabaga (GadFly Accession Number CG9533), or GadFly Accession Number CG1 1940 homologous proteins (herein referred to as "proteins of the invention” or "a protein of the invention") or polypeptides encoded by a member of the FACL gene family are regulating the energy homeostasis and fat metabolism especially the metabolism and storage of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention.
  • the invention also relates to vectors, host cells, antibodies, and recombinant methods for producing the polypeptides and polynucleotides of the invention.
  • the invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of metabolic diseases and dysfunctions, including metabolic syndrome, obesity, or diabetes as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones.
  • 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
  • (f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 1 5-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.
  • the present invention is based on the finding that l(2)44DEa, wunen-2, grapes, CG2221 , CG1 172, rutabaga, or CG1 1 940 and/or homologous proteins and proteins of the FACL family 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, 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 fragments thereof, 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 polynucleotides or polypeptides.
  • model organisms such as the fly Drosophila melanogaster
  • the ability to manipulate and screen the genomes of model organisms provides a powerful tool to analyze biological and biochemical processes that have direct relevance to more complex vertebrate organisms due to significant evolutionary conservation of genes, cellular processes, and pathways (see, for example, Adams M. D. et al., (2000) Science 287: 2185-2195).
  • 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
  • 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, Johnston Nat Rev Genet 3: 176-188 (2002); 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 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 or hemizygous for the integration of vectors for Drosophila EP-lines were analyzed in assays 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, 1 1 1 , 16, 21 , 25, and 29, respectively.
  • Genomic DNA sequences were isolated that are localized adjacent to the EP or PX 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, 12, 17, 22, 26, and 30, respectively. An additional screen using Drosophila mutants with modifications of the eye phenotype identified an interaction of GadFly Accession Number CG1 172 with adipose, a protein regulating, causing or contributing to obesity.
  • 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 et al, 1998, Mol. Cell. 2:449-569).
  • 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 6).
  • 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. et al. (1 995) U.S. Patent No.
  • 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.
  • 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.
  • therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • FACL1 , FACL2, FACL3, FACL4, PPAP2A, PPAP2B, PPAP2C, CHEK1 , TEM7, FLJ1 1807 and APBB1 IP show differential expression in human primary adipocytes.
  • FACL1 , FACL2, FACL3, FACL4, PPAP2A, PPAP2B, PPAP2C, CHEK1 , TEM7, FLJ1 1807 and APBB1 IP 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 nucleic acids encoding Drosophila l(2)44DEa, wunen-2, grapes, CG2221 , CG1 172, rutabaga, or CG1 1940, or human l(2)44DEa, wunen-2, grapes, CG2221 , CG1 172, rutabaga, or CG1 1940 homologs or nucleic acids of the FACL gene family; 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 could 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 l(2)44DEa, wunen-2, grapes, CG2221 , CG1 172, rutabaga, or CG1 1940, 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. and S. L. Berger (1987: Methods Enzymol. 152:399-407) and Kimmel, A. R.
  • 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 of such fragments such as cyclic peptides, retro-inverso peptides or peptide mimetics, wherein the peptides or derivatives usually have a length of at least four, preferably at least six 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. (1 993) 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. (1 988) Nucleic Acids Res. 1 6:81 86) .
  • 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. (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.
  • the nucleotide sequence encoding the proteins 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 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.
  • nucleic acid sequences encoding the sequences of the invention 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. 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, e.g. mammalian 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,
  • polynucleotide sequences encoding a protein of the invention or a homologous protein can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or portions or fragments of polynucleotides encoding a protein of the invention or a homologous protein.
  • 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.
  • a variety of protocols for detecting and measuring the expression of proteins, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:121 1 -1216).
  • labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and protein, e.g. immunological assays.
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding a protein of the invention or a homologous protein include oligo-labeling, nick translation, end-labeling of RNA probes or PCR amplification using a labeled nucleotide. 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 the protein may be cultured under conditions suitable for the expression and recovery of the 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 The inclusion of 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.
  • cleavable linker sequences such as those specific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.)
  • nucleic acids and proteins of the invention and effectors/modulators thereof are useful in diagnostic and therapeutic applications implicated, for example but not limited to, in metabolic diseases or dysfunctions, including metabolic syndrome, obesity, or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones.
  • 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 are useful in diagnostic and therapeutic applications implicated in various applications as described below.
  • cDNAs encoding the proteins 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 acid sequence encoding a protein of the invention or 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 for use in therapeutic or diagnostic methods.
  • antibodies which are specific for a protein of the invention or a homologous protein, may be used directly as an antagonist 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, chimerical, 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 ( ⁇ hler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1 985) J. Immunol. Methods 81 :31 -42; Cote, R. J. et al. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1 984) Mol. Cell Biol. 62: 109-1 20) .
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1991 ) Proc. Natl. Acad. Sci. 88:1 1 120-3). 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. et al. (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 254: 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 reactive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox, supra).
  • the polynucleotides or fragments thereof, or nucleic acid effector molecules such as antisense molecules, aptamers, RNAi molecules or ribozymes may be used for therapeutic purposes.
  • nucleic acid effector molecules such as antisense molecules, aptamers, 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.
  • antisense molecules e.g. DNA, RNA or nucleic acid analogues such as PN A
  • PN A nucleic acid analogues
  • 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.
  • 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 1 5 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.
  • 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 the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • 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.
  • compositions may consist of a 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 protein of the invention or a homologous protein or nucleic acid sequence.
  • 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.
  • compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • the pharmaceutical 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 functional fragment thereof, antibodies, 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, agonists, antagonists or inhibitors.
  • the antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutics. 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 photometry, means. Quantities of protein expressed in control and disease, samples 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 PCR 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 are preferably derived from the nucleotide sequence of the polynucleotide encoding a l(2)44DEa, wunen-2, grapes, CG2221 , CG1 172, rutabaga, or CG1 1940 homologous protein, preferably a human homologous protein as described in Table 1 or from a genomic sequence including promoter, enhancer elements, and introns of the naturally occurring gene or from the nucleotide sequence of a number of the FACL gene family.
  • 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 proteins.
  • Examples of such conditions or diseases include, but are not limited to, metabolic diseases and disorders, including obesity and 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 and diabetes.
  • the polynucleotide sequences may be used 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, or diabetes.
  • 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.
  • the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding a protein of the invention or a homologous protein in the sample indicates the presence of the associated disease.
  • Such 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, C. M. (1993) Blood Rev. 7: 127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
  • FISH FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y.) 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 (265:1981f). Correlation between the location of the gene encoding a protein of the invention or a homologous protein 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.
  • In situ hybridisation 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. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • 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 the protein and the agent tested, may be measured. Agents could also, either directly or indirectly, influence the activity of the proteins of the invention.
  • the phosphatase activity of the protein of the invention could be measured in vitro by using recombinantly expressed and purified phosphatidic acid phosphatase type 2A, 2B, or 2C, or fragments thereof by making use of artificial phosphatase substrates well known in the art, i.e. but not exclusively DiFMUP or FDP (Molecular Probes, Eugene, Oregon), which are converted to fluorophores or chromophores upon dephosphorylation.
  • DiFMUP DiFMUP
  • FDP Molecular Probes, Eugene, Oregon
  • the dephosphorylation of physiological substrates of phosphatidic acid phosphatase type 2A, 2B, or 2C could be measured by making use of any of the well known screening technologies suitable for the detection of the phosphorylation status of phosphatidic acid phosphatase type 2A, 2B, or 2C substrates, i.e. in a procedure similar as described for the inositol phosphatase SHIP2 (T. Habib et al. (1998), JBC 273, 18605-18609).
  • phosphatidic acid phosphatase type 2A, 2B, or 2C against its physiological substrate(s) or derivatives thereof could be measured in cell-based assays, thereby determining activity of the phosphatase at the level of their downstream signalling.
  • the enzymatic kinase activity of the unmodified polypeptides of CHEK1 kinase, or homologues thereof 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 CHEK1 kinase, or homologues thereof, 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 CHEK1 kinase, or homologues thereof 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.
  • agents could influence the dimerization or oligomerization of the proteins of the invention or, in a heterologous manner, of the proteins 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 interaction of the proteins of the invention with cellular proteins could be the basis for a cell-based screening assay, in which both proteins are fluorescently labeled and interaction of both proteins is detected by analysing cotranslocation of both proteins with a cellular imaging reader, as has been developed for example, but not exclusively, by Cellomics or EvotecOAI.
  • 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 l(2)44DEa, wunen-2, grapes, CG2221 , CG 1 172, rutabaga, or CG1 1 940 or FACL and/or 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.
  • Candidate agents may also be found in kinase assays where a 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.
  • a therapeutic candidate agent may be identified by its ability to increase or decrease the enzymatic activity of the proteins 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 and Lane, 1998, Antibodies, 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. (1 999) 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.
  • 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.
  • non-neutralizing antibodies can be used to capture the peptide and immobilise 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 a protein of the invention.
  • the nucleic acids encoding the proteins 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 the proteins of the invention 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 effectors/modulators of the proteins of the invention.
  • Misexpression for example, overexpression or lack of expression
  • 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 et al, 1998, Mol. Cell. 2:449-569).
  • 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 effector/modulator 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.
  • 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. For example, 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
  • 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 l(2)44DEa (Gadfly Accession Number CG8732) mutant. Shown is the change of triglyceride content of HD-EP(2)26805 flies caused by integration of the P-vector into an intron of the l(2)44DEa gene (referred to as 'HD-EP26805', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 2 shows the molecular organization of the mutated l(2)44DEa (Gadfly Accession Number CG8732) gene locus.
  • Figure 3 shows the BLASTP search result for the CG8732 gene product (Query) with the four best human homologous matches (Sbjct) .
  • Figure 4 shows the expression of members of the Facl family in mammalian tissues.
  • Figure 4A shows the real-time PCR analysis of Facl3 expression in wild-type mouse tissues.
  • Figure 4B shows the real-time PCR analysis of Facl3 expression in different mouse models.
  • Figure 4C shows the real-time PCR analysis of Facl3 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 Facl4 expression in wild-type mouse tissues.
  • Figure 4E shows the real-time PCR analysis of Facl4 expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 4F shows the real-time PCR analysis of fatty acid Coenzyme A ligase, long chain 2 (Facl2) expression in wild type mouse tissues.
  • Figure 4G shows the real-time PCR analysis of fatty acid Coenzyme A ligase, long chain 2 (Facl2) expression in different mouse models.
  • Figure 4H shows the real-time PCR analysis of fatty acid Coenzyme A ligase, long chain 2 (Facl2) expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 41 shows the real-time PCR analysis of fatty acid Coenzyme A ligase, long chain 5 (Facl ⁇ ) expression in wild type mouse tissues.
  • Figure 4J shows the real-time PCR analysis of fatty acid Coenzyme A ligase, long chain 5 (Facl ⁇ ) expression in different mouse models.
  • Figure 5 shows the expression of human members of the FACL family in mammalian (human) tissue.
  • Figure 5A shows the quantitative analysis of fatty-acid-Coenzyme A ligase, long-chain 1 (FACL1 ) expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 5B shows the quantitative analysis of fatty-acid-Coenzyme A ligase, long-chain 2 (FACL2) expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 5C shows the quantitative analysis of fatty-acid-Coenzyme A ligase, long-chain 3 (FACL3) expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 5D shows the quantitative analysis of human fatty-acid-Coenzyme A ligase, long-chain 4 (FACL4) expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 6 shows the triglyceride content of a Drosophila wun2 (Gadfly Accession Number CG8805) mutant. Shown is the change of triglyceride content of HD-EP(2)25261 flies caused by integration of the P-vector into the cDNA of the wun2 (referred to as 'HD-EP25261 ', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 7 shows the molecular organization of the mutated wun2 (Gadfly Accession Number CG8805) gene locus.
  • Figure 8 shows the BLASTP search result for the CG8805 gene product (Query) with the three best human homologous matches (Sbjct).
  • Figure 9 shows the expression of the wunen-2 homologs in mammalian tissues.
  • Figure 9A shows the real-time PCR analysis of phosphatidic acid phosphatase 2a (Ppap2a) expression in wild-type mouse tissues.
  • Figure 9B shows the real-time PCR analysis of Ppap2a expression in different mouse models.
  • Figure 9C shows the real-time PCR analysis of Ppap2a 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 phosphatidic acid phosphatase 2b (Ppap2b) expression in wild-type mouse tissues.
  • Figure 9E shows the real-time PCR analysis of Ppap2b expression in different mouse models.
  • Figure 9F shows the real-time PCR analysis of Ppap2b expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 9G shows the real-time PCR analysis of phosphatidic acid phosphatase 2c (Ppap2c) expression in wild-type mouse tissues.
  • Figure 9H shows the real-time PCR analysis of Ppap2c expression in different mouse models.
  • Figure 10 shows the expression of the wunen-2 homologs in mammalian (human) tissue.
  • Figure 10A shows the quantitative analysis of PPAP2A expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 10B shows the quantitative analysis of PPAP2B expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 10C shows the quantitative analysis of PPAP2C expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 1 1 shows the triglyceride content of a Drosophila grapes (Gadfly Accession Number CG17161) mutant. Shown is the change of triglyceride content of HD-EP(2) 25268 flies caused by integration of the P-vector into the cDNA of the grapes gene (referred to as 'HD-EP25268', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 1 2 shows the molecular organization of the mutated grapes (Gadfly Accession Number CG17161 , GenBank Accession Numbers NM_057663, or AF057041 ) gene locus.
  • Figure 1 3A shows the BLASTP search result for the grapes gene product (Query) with the best human homologous match (Sbjct).
  • Figure 1 3B shows the amino acid sequence of the Drosophila grapes protein (SEQ ID NO: 1 ).
  • Figure 14 shows the expression of the grapes homolog in mammalian tissues.
  • Figure 14A shows the real-time PCR analysis of checkpoint kinase 1 homolog (S. pombe) (Checkl ) expression in wild-type mouse tissues.
  • Figure 14B shows the real-time PCR analysis of Checkl expression in different mouse models.
  • Figure 14C shows the real-time PCR analysis of Checkl expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 1 5 shows the expression of the grapes homolog in mammalian (human) tissue. Shown is the quantitative analysis of CHECK1 expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 16 shows the triglyceride content of a Drosophila Gadfly Accession Number CG2221 mutant. Shown is the change of triglyceride content of PX960.1 flies caused by integration of the P-vector into the cDNA of the CG2221 gene (referred to as 'PX 960.1 ', column 2) in comparison to controls (referred to as 'PX control 2', column 1 ).
  • Figure 1 7 shows the molecular organization of the mutated CG2221 (Gadfly Accession Number) gene locus.
  • Figure 18 shows the BLASTP search result for the CG2221 gene product (Query) with the best human homologous match (Sbjct).
  • Figure 19 shows the expression of a CG2221 homolog in mammalian tissues.
  • FIG 19A shows the real-time PCR analysis of tumor endothelial marker 7-related precursor (Tem7R) expression in wild-type mouse tissues.
  • Figure 19B shows the real-time PCR analysis of Tem7R expression in different mouse models.
  • Figure 19C shows the real-time PCR analysis of Tem7R expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 20 shows the expression of a CG2221 homolog in mammalian (human) tissue. Shown is the quantitative analysis of tumor endothelial marker 7 precursor (TEM7) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • TEM7 tumor endothelial marker 7 precursor
  • Figure 21 shows the triglyceride content of a Drosophila Gadfly Accession Number CG1 172 mutant. Shown is the change of triglyceride content of HD-EP(3)35037 flies caused by integration of the P-vector into the promoter/enhancer of the CG1 172 gene (referred to as 'HD-EP35037', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 22 shows the molecular organization of the mutated CG1 172 (Gadfly Accession Number) gene locus.
  • Figure 23 shows the BLASTP search result for the CG1172 gene product (Query) with the two best human homologous matches (Sbjct).
  • Figure 24 shows the expression of a CG1 172 homolog in mammalian (human) tissue. Shown is the quantitative analysis of FLJ1 1807 expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 25 shows the triglyceride content of a Drosophila rut (Gadfly Accession Number CG9533) mutant. Shown is the change of triglyceride content of PX9610.1 flies caused by integration of the P-vector into the cDNA of the rut gene (referred to as 'PX 9610.1 ', column 2) in comparison to controls (referred to as 'PX-control 1 ', column 1 ).
  • Figure 26 shows the molecular organization of the mutated rut (Gadfly Accession Number CG9533) gene locus.
  • Figure 27A shows the BLASTP search result for the CG9533 gene product (Query) with the four best human homologous matches (Sbjct).
  • Figure 27B shows the amino acid sequence of the Drosophila rutabaga gene product (GadFly Accession Number CG9533) (SEQ ID NO:2).
  • Figure 28 shows the expression of rutabaga homologs in mammalian
  • Figure 28A shows the real-time PCR analysis of adenylate cyclase 6
  • Figure 28B shows the quantitative analysis of human ADCY6 expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 28C shows the real-time PCR analysis of adenylate cyclase 1
  • ADCY1 (ADCY1 ) expression in human tissues.
  • Figure 28D shows the quantitative analysis of human ADCY1 expression in human abdominal adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 29 shows the triglyceride content of a Drosophila Gadfly Accession Number CG1 1940 mutant. Shown is the change of triglyceride content of HD-EP(X) 10934 flies caused by integration of the P-vector into the first intron of the CG1 1940 gene ('HD-EP10934', column 2) in comparison to controls (referred to as 'EP-control', column 1 ).
  • Figure 30 shows the molecular organization of the mutated CG1 1940 (Gadfly Accession Number) gene locus.
  • Figure 31 shows the BLASTP search result for the CG1 1940 gene product (Query) with the best human homologous match (Sbjct).
  • Figure 32 shows the expression of a CG 1 1940 homolog in mammalian tissues.
  • Figure 32A shows the real-time PCR analysis of amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein
  • Figure 32B shows the real-time PCR analysis of Apbbl ip-pending expression in different mouse models.
  • Figure 32C shows the real-time PCR analysis of Apbbl ip-pending expression in mice fed with a high fat diet compared to mice fed with a standard diet.
  • Figure 33 shows the expression of CG1 1940 homologs in mammalian (human) tissue. Shown is the quantitative analysis of amyloid beta (A4) precursor protein-binding, family B, member 1 interacting protein (APBB1 IP) expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • A4 amyloid beta
  • APBB1 IP member 1 interacting protein
  • Example 1 Measurement of triglyceride content
  • Mutant flies are obtained from proprietary and 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 and PX-vectors in homozygous or hemizygous viable integration was investigated in comparison to control flies (see Figures 1 , 6, 1 1 , 16, 21 , 25, and 29).
  • flies in case of the PX-lines PX9610.1 and PX960.1 , ten flies in several independent assays, respectively
  • flies were incubated for 5 min at 90°C (in case of the PX-lines at 70°C) in an aqueous buffer using a waterbath, followed by hot extraction.
  • 90°C in case of the PX-lines at 70°C
  • 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.
  • HD-EP(2) 26805 HD-EP(2)25261 , HD-EP(2)25268, HD-EP(X) 10934, or HD-EP(3)35037
  • BIO-RAD DC Protein Assay according to the manufacturer's protocol for the EP-lines. The assays were repeated several times.
  • the average triglyceride level of all flies of the EP collections (referred to as 'EP-control') is shown as 100% in the first columns in Figures 1 , 6, 1 1 , 21 , and 29, respectively.
  • the average triglyceride levels of 179 lines of the PX collection (referred to as 'PX-control 2') or of 22 lines of the PX collection (referred to as 'PX-control 1 ') are shown as 100% (relative amount of triglyceride per fly) in the first column in Figures 16 and 25. Standard deviations of the measurements are shown as thin bars.
  • HD-EP(2)26805 homozygous flies (column 2 in Figure 1 ), PX9610.1 hemizygous flies (column 2 in Figure 25), and HD-EP(X) 10934 hemizygous flies (column 2 in Figure 29) show constantly a higher triglyceride content than the controls
  • HD-EP(2)25261 homozygous flies (column 2 in Figure 6), HD-EP(2)25268 homozygous flies (column 2 in Figure 1 1 ), PX960.1 hemizygous flies (column 2 in Figure 16), and HD-EP(3)35037 homozygous flies (column 2 in Figure 21 ) show constantly a lower triglyceride content than the controls.
  • 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)26805, HD-EP(2)25261 , HD-EP(2)25268, PX960.1 , HD-EP(3)35037, PX9610.1 , or HD-EP(X) 10934) integration.
  • public databases like Berkeley Drosophila Genome Project (GadFly) were screened, thereby identifying the integration sites of the vectors, and the corresponding genes. The molecular organization of these gene loci is shown in Figures 2, 7, 12, 17, 22, 26, and 30.
  • 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(2) 26805, HD-EP(2)25261 , HD-EP(2)25268, PX960.1 , HD-EP(3)35037, or HD-EP(X) 10934. 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 + " or "P-elements -" lines.
  • ESTs Transcribed DNA sequences (ESTs) are shown as grey bars in the "EST + “ and/or the "EST -” 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 introns are shown as light grey bars.
  • the HD-EP(2)26805 vector is homozygous viable integrated into an intron of a Drosophila gene in sense orientation, identified as l(2)44DEa (GadFly Accession Number CG8732).
  • the chromosomal localization site of the integration of the vector of HD-EP(2)26805 is at gene locus 2R, 44E2.
  • the coordinates of the genomic DNA are starting at position 3697795 on chromosome 2R, ending at position 3722795.
  • the insertion site of the P-element in Drosophila HD-EP(2)26805 is shown as triangle in the "P Elements + " line and is labeled.
  • the predicted cDNA of the l(2)44DEa gene shown in the "cDNA + " line is labeled, the corresponding ESTs are shown in the "EST + " line.
  • the HD-EP(2)25261 vector is homozygous viable integrated into the cDNA of a Drosophila gene in sense orientation, identified as wun2 (wunen-2, GadFly Accession Number CG8805, GenBank Accession Number NM_080252).
  • the chromosomal localization site of the integration of the vector of HD-EP(2)25261 is at gene locus 2R, 45D8. In Figure 7, the coordinates of the genomic DNA are starting at position 4449000 on chromosome 2R, ending at position 4455250.
  • the insertion site of the P-element in Drosophila HD-EP(2)25261 line is shown as box in the "P Elements + " line and is labeled.
  • the predicted cDNA of the wun2 gene is shown in the "cDNA + " line, the corresponding ESTs are shown in the "EST + " line and are labeled. According to the ESTs, the gene might extend further in 5prime direction than the annotated wun2 gene.
  • the HD-EP(2)25268 vector is homozygous viable integrated into the cDNA of a Drosophila gene in antisense orientation, identified as grapes (grp; GadFly Accession Number CG17161 , GenBank Accession Numbers NM 057663 and AF057041).
  • the chromosomal localization site of the integration of the vector of HD-EP(2)25268 is at gene locus 2L, 36A10. In the upper half of Figure 12, the coordinates of the genomic DNA are starting at position 16508590 on chromosome 2L, ending at position 16533590.
  • the insertion site of the P-element in Drosophila HD-EP(2)25268 is shown as triangle in the "P Elements -" line and is labeled .
  • the predicted cDNA of the grapes gene is shown in the "cDN A + " line, the is corresponding ESTs are shown in the "EST + " line.
  • the molecular organization of the grapes (grp) gene locus according to Flybase is shown. According to this map exons 1 and 2 of the annotated CG17161 might not belong to the grapes gene.
  • the chromosomal localization site of the integration of the vector of PX960.1 is at gene locus X, 9B14. In Figure 17, the coordinates of the genomic DNA are starting at position 10149046 on chromosome X, ending at position 10174046.
  • the insertion site of the P-element in Drosophila PX960.1 line is shown as triangle in the "P Elements + " line and is labeled.
  • the predicted cDNA of the CG2221 gene shown in the the "cDNA -" line is labeled, the corresponding ESTs are shown in the "EST -” line.
  • the HD-EP(3) 35037 vector is homozygous viable integrated into the promoter/enhancer of a Drosophila gene in sense orientation, identified as CG1 172 (GadFly Accession Number).
  • the chromosomal localization site of the integration of the vector of HD-EP(3)35037 is at gene locus 3R, 83A1 .
  • the coordinates of the genomic DNA are starting at position 1218394 on chromosome 3R, ending at position 1224644.
  • the insertion site of the P-element in Drosophila HD-EP ⁇ 3)35037 line is shown as triangle in the "P Elements + " line and is labeled.
  • the predicted cDNA of the CG1 172 gene is shown in the "cDNA + " line, and the corresponding ESTs are shown in the "EST + " line and are labeled.
  • genomic DNA sequence is represented by the assembly as a black scaled arrow in the middle that includes the integration sites of the vector for line PX9610.1.
  • Ticks represent the length of the genomic DNA (100000 base pairs per tick).
  • the upper half of the figure represents the sense strand, the lower half represents the antisense strand.
  • the grey arrows in the upper part of the figure represent BAG clones, the black arrow in the topmost part of the figure represents the section of the chromosome.
  • the insertion site of the P-element in the Drosophila line is shown as grey triangle and as black vertical line, and is labeled.
  • Grey bars, linked by black lines represent cDNA sequences.
  • Predicted genes are shown as black bars (exons), linked by black lines (introns), and are labeled (see also key at the bottom of the figure).
  • the PX9610.1 vector is hemizygous viable integrated 270 base pairs upstream of the 5prime-end of a Drosophila gene in antisense orientation, identified as the published mRNA-sequence of rut (rutabaga, GadFly Accession Number CG9533, GenBank Accession Number NM_078601 , see also Levin L. R. et al, 1992, Cell 68 (3): 479-489).
  • the chromosomal localization site of the integration of the vector of PX9610.1 is at gene locus X, 12F5-1.
  • the cDNA of the rut gene is labeled.
  • the PX9610.1 vector is integrated 270 base pairs ⁇ prime of rut (GadFly Accession Number CG9533) in antisense orientation.
  • the HD-EP(X) 10934 vector hemizygous viable integrated into the first intron of a Drosophila gene in sense orientation, identified as CG1 1940 (GadFly Accession Number).
  • the chromosomal localization site of the integration of the vector of HD-EP(X) 10934 is at gene locus X, 18F2-3.
  • the coordinates of the genomic DNA are starting at position 19568000 on chromosome X, ending at position 19893000.
  • the insertion site of the P-element in Drosophila HD-EP(X) 10934 line is shown as triangle in the "P Elements -" line and is labeled.
  • the predicted cDNA of the CG1 1940 gene is shown in the "cDNA -" line, and the corresponding ESTs are shown in the "EST -” line. According to the ESTs, the gene might extend further in ⁇ prime direction than the annotated CG1 1940 gene.
  • 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, 13, 18, 23, 27, and 31 ).
  • FACL1 FACL2, FACL3, FACL4, FACL5, FACL6
  • 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 l(2)44DEa (GadFly Accession Number CG8732) is 60% homologous to human protein similar to Long-chain-fatty-acid-CoA ligase 3 (GenBank Accession Number XP 048262.1 for the protein, XM_048262 for the cDNA), 60% homologous to long-chain fatty-acid-Coenzyme A ligase 3 (GenBank Accession Number NP_004448.1 for the protein, NM_00457 for the cDNA), 60% homologous to long-chain fatty-acid-Coenzym A ligase 4, isoform 2 (GenBank Accession Number NP_075266.1 for the protein, NM_022977 for the cDNA), and 62% homologous to long-chain fatty-acid-Coenzyme A ligase 4, isoform 1 (GenBank Accession Number NP 004449.1 for the protein, NM_004458 for the cDNA).
  • CG8732 also shows 59% homology on protein level to mouse Long-chain-fatty-acid-CoA ligase 3 (GenBank Accession Number Q9CZW4), and 62% homology to mouse fatty acid-Coenzyme A ligase, long chain 4 (GenBank Accession Number NP_062350.1 ).
  • gene product of wun2 (GenBank Accession Number NM_0802 ⁇ 2) is 56% homologous to human protein similar to type-2 phosphatidic acid phosphatase alpha-2 (GenBank Accession Number XP_042105.1 for the protein, XM_042105 for the cDNA), 56% homologous to human phosphatidic acid phosphatase type 2B (GenBank Accession Number AA09196.1 for the protein, AAH09196 for the cDNA), and 53% homologous to human phosphatidic acid phosphatase type 2C (GenBank Accession Number NP_003703.1 for the protein, NM_003712 for the cDNA).
  • CG8805 also shows ⁇ 7% homology on protein level to mouse phosphatidic acid phosphatase type 2B (GenBank Accession Number ).
  • gene product of grapes (GadFly Accession Number CG17161 , GenBank Accession Numbers NM_0 ⁇ 7663 and AF067041 ) is 63% homologous to human CHK1 checkpoint homolog (S. pombe) (GenBank Accession Number NP 001266.1 for the protein, NM_001274 for the cDNA; see also sequence 1 from patent US 6,218,109). grapes also shows 62% homology on protein level to mouse checkpoint kinase 1 homolog (S. pombe) (GenBank Accession Number NP_031717.1 ).
  • gene product of GadFly Accession Number CG2221 is 51 % homologous to human tumor endothelian marker 7-related precursor (GenBank Accession Number NP_1 16201.6 for the protein, NM 032812 for the cDNA). CG2221 also shows 52% homology on protein level to mouse tumor endothelian marker 7-related precursor (GenBank Accession Number NP 080438.1 ).
  • gene product of GadFly Accession Number CG1 172 is 65% homologous to human hypothetical protein FLJ1 1807 (GenBank Accession Number NP 079230.1 for the protein, NM_024954 for the cDNA) and 70% homologous to human protein similar to hypothetical protein FLJ 1 1807 (GenBank Accession Number XP_043394.3 for the protein, XM 043394 for the cDNA).
  • CG1 172 also shows 46% homology on protein level to mouse protein similar to hypothetical protein FLJ 1 1807 (GenBank Accession Number XP 129290.1 ) .
  • gene product of rut is 66% homologous to human adenylate cyclase 6, isoform b (KIAA0422; GenBank Accession Number NP 066193.1 for the protein, NM 020983 for the cDNA), 66% homologous to human adenylate cyclase 6, isoform a (KIAA0422; GenBank Accession Number NP_05608 ⁇ .1 for the protein, NM_015270 for the cDNA), 64% homologous to human adenylate cyclase 8 (GenBank Accession Number NP 001 106.1 for the protein, NM_001 1 15 for the cDNA), and 58% homologous to human adenylate cyclase, type I (GenBank Accession Number Q08828).
  • Drosophila rut also shows 74% homology on protein level to mouse adenylyl cyclase type I (GenBank Accession Number Q08828).
  • Drosophila rut also shows 74% homo
  • gene product of CG1 1940 (GadFly Accession Number) is 40% homologous to human KIAA1681 protein (GenBank Accession Number BAB21772.1 for the protein, AB051468 for the cDN A. CG1 1940 also shows 46% homology on protein level to mouse protein similar to Mig-10 protein (GenBank Accession Number XP_130045.1 ).
  • LACS3 is also referred to as prostate cancer-associated protein 84 in patent application WO 02/30268-A2.
  • PPAP2A is also referred to as
  • PPAP2B is also referred to as GenBank Accession Number BC009196 and as human phosphatidic acid phosphatase beta in patent application WO 98/46730-A1 .
  • CHEK1 is also referred to as human checkpoint protein chkl in patent US 6,307,016-B1 ,
  • TEM7 is also referred to as human tumour endothelial marker polypeptide
  • TEM7R is also referred to as human tumour endothelial marker polypeptide SEQ ID NO 230 in patent application WO 02/10217-A2, TEM7R is also referred to as human tumour endothelial marker polypeptide SEQ ID NO 230 in patent application WO 02/10217-A2, TEM7R is also referred to as human tumour endothelial marker polypeptide SEQ ID NO 230 in patent application WO 02/10217-A2, TEM7R is also referred to as human tumour endothelial marker polypeptide SEQ ID NO
  • FLJ1 1807 is also referred to as human protein sequence SEQ ID NO: 13666 in Patent
  • ADCY1 is also referred to as GenBank Accession Number Q08828, and ADCY6 is also referred to as Human cardiac adenylcyclase VI (ACVI) isoform 2 in patent application WO 01 /48164-A2.
  • 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 and Perrimon, 1993, Development 1 18:401 -415; for adipose cDNA, see WO 01 /96371 ) .
  • Chromosomal recombination of these transgenic flies with an eyeless-Gal4 driver line has been used to generate a stable recombinant fly line over-expressing adipose in the developing Drosophila eye.
  • 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.
  • Example 6 Expression of the polypeptides in mammalian (mouse and human) tissues
  • mice 6 ob/ob and C57BI/KS db/db which are standard model systems in obesity and diabetes research) were purchased from Harlan Winkelmann (33178 Borchen, Germany) and maintained under 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 et al., (1993) J Clin Invest 92(1 ):272-280, Mizuno et al., (1996) Proc Natl Acad Sci U S A 93(8):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 C10 ⁇ 7mod. high fat, 23. ⁇ % 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.
  • Trizol Reagent for example, from Invitrogen, Düsseldorf, Germany
  • RNeasy Kit for example, from Qiagen, Germany
  • Taqman analysis was performed preferrably using the following primer/probe pairs:
  • long chain 4 (Facl4) (SEQ ID NO: 6): 5'- TGA CAG GCC AGT GTG AAC GT -3'; mouse Facl4 reverse primer (SEQ ID NO: 7): 5'- CAG CAC ATG AGC CAA AGG TAA G -3'; Taqman probe (SEQ ID NO: 8): ( ⁇ /6-FAM) TCC CTG GAC TAG GAC CGA AGG ACA CAT ATA TT (5/6-TAMRA).
  • mouse phosphatidic acid phosphatase 2a (SEQ ID NO: 9): 5'- CCA TGT TCG ACA AGA CGC G -3'; mouse Ppap2a reverse primer (SEQ ID NO: 10) : 5'- AGC CAG CAA CAC GCA AAT C -3'; Taqman probe (SEQ ID NO: 1 1 ): ( ⁇ /6-FAM) CTG CCG TAC GTG GCC CTC GAT ( ⁇ /6-TAMRA).
  • mouse phosphatidic acid phosphatase 2b (SEQ ID NO: 1 2): ⁇ '- GTG GCA GCT CTC TAT AAG CAA GTG -3'; mouse Ppap2b reverse primer (SEQ ID NO: 13) : ⁇ '- GAT GTC GGT GAA CTG GC -3'; Taqman probe (SEQ ID NO: 14): ( ⁇ /6-FAM) ATG CTT CCT TTT CGG CTG TGC CAT T (5/6-TAMRA).
  • mouse phosphatidic acid phosphatase 2c (SEQ ID NO: 1 ⁇ ): ⁇ '- TGT GAG CCA GTC TCT CAC CG -3'; mouse Ppap2c reverse primer (SEQ ID NO: 16): 5'- GGG TCA CAG ACA GCC AAG AAA -3'; Taqman probe (SEQ ID NO: 17): (5/6-FAM) AAG TAC ATG ATT GGC CGT CTT CGA CCC ( ⁇ /6-TAMRA).
  • mouse checkpoint kinase 1 homolog S. pombe
  • ChekD SEQ ID NO: 18
  • mouse Chekl reverse primer SEQ ID NO: 19
  • Taqman probe SEQ ID NO: 20
  • mice tumor endothelial marker 7-related precursor (SEQ ID NO: 21 ): 5'- GCC AGC ATC TTC TTC ATT GAG A -3'; mouse Tem7R reverse primer (SEQ ID NO: 22): ⁇ '- CCT GAG CCT CTT CGA AAC TTC A -3'; Taqman probe (SEQ ID NO: 23): (5/6-FAM) ACG CCC AAG CAG ATG GCC AGC (5/6-TAMRA).
  • mouse amyloid beta (A4) precursor protein-binding, family B member 1 interacting protein (Apbbl ip-pending) (SEQ ID NO: 24): 5'- GGG ACT GTT GGT ACA CCG ATG -3'; mouse Apbbl ip-pending reverse primer (SEQ ID NO: 25): 5'-. GCT GGC TGG TGC CTG TTT -3'; Taqman probe (SEQ ID NO: 26): (5/6-FAM) CAG CTC AGC CTT CTA CAG TTT CCT CAG GAC TT (5/6-TAMRA).
  • Facl2 For the amplification of mouse fatty acid Coenzyme A ligase, long chain 2 (Facl2) (SEQ ID NO: 27): ⁇ '- ACG GAG AAA GCT TGC AGG C -3'; mouse Facl2 reverse primer (SEQ ID NO: 28): ⁇ '- TGC CCA GGA CGG TAG GC - 3'; Taqman probe (SEQ ID NO: 29): ( ⁇ /6-FAM) TCA TAG CAG TTG TGG TAC CCG ACG TTG ( ⁇ /6-TAMRA).
  • long chain 5 (SEQ ID NO: 30): ⁇ '- TTC CAA CTC CGG CCC TG -3'; mouse Facl ⁇ reverse primer (SEQ ID NO: 31 ): 5'- ACT GGC TGA GGT CTG TTG ATC A -3'; Taqman probe (SEQ ID NO: 32): (5/6-FAM) CCT CCT GAC GTT TGG AAC GGC CAT (5/6-TAMRA)
  • ADCY6 human adenylate cyclase 6
  • SEQ ID NO: 33 5'- GGA ACA CAG TGA ATG TCT CTA GTC GT -3'
  • human ADCY6 reverse primer SEQ ID NO: 34
  • Taqman probe SEQ ID NO: 36
  • ADCY1 human adenylate cyclase 1
  • SEQ ID NO: 36 human adenylate cyclase 1
  • human ADCY1 reverse primer SEQ ID NO: 37
  • Taqman probe SEQ ID NO: 38
  • 5/6-FAM CGC CCA GCA CTT CCT CAT GTC CA
  • RNA-expression is shown on the Y-axis.
  • the tissues tested are given on the X-axis.
  • WAT refers to white adipose tissue
  • BAT refers to brown adipose tissue.
  • the X-axis represents the time axis. 'dO' refers to day 0 (start of the experiment), 'd2' - 'd14' refers to day 2 - day 14 of adipocyte differentiation).
  • Facl3 is highly expressed in hypothalamus and brain, and on lower levels in further tissues, e.g. BAT, kidney and small intestine.
  • Facl3 is three fold down regulated in BAT of ob /ob mice and in the kidney of fasted animals.
  • Facl3 is nearly three fold up regulated in BAT and more than four fold up regulated in the small intestine of palmitatdiet mice as shown in Figure 4C.
  • Facl4 is highy expressed in hypothalamus, brain and lung, and on lower but robust levels in further tissue, e.g. WAT, BAT, kidney, liver and muscle of wild type animals.
  • Facl4 is more than two fold up regulated in BAT and small intestine of palmitatdiet mice.
  • Facl3 and Facl4 play central roles in the metabolism.
  • Facl2 is expressed in different mammalian tissues, showing highest level of expression in BAT, testis, heart and WAT, and higher levels in further tissues, e.g. muscle, liver, kidney and lung.
  • Facl2 is more than two fold up regulated in kidney of fasted animals and in the muscle of ob /ob mice.
  • Facl2 is three fold up regulated in muscle of high fat diet mice as shown in Figure 4H.
  • Facl ⁇ is highly expressed in small intestine, BAT, liver and lung, and on lower but robust levels in further tissue, e.g. WAT, kidney, colon and brain of wild type animals.
  • Facl ⁇ is more than six fold down regulated in BAT of fasted animals and ob/ob mice.
  • Facl2 The high expression level of Facl2 in metabolic active tissues and the regulated expression of Facl ⁇ in BAT of fasted animals and in BAT of the genetic model for obesity, suggests that Facl2 and Facl ⁇ play central roles in the metabolism.
  • Ppap2a is highly expressed in testis, lung and small intestine, and in lower level in further tissues, e.g. WAT, BAT, muscle and liver.
  • Ppap2a is more than two fold up regulated in the pancreas of fasted animals and more than three fold up regulated in WAT of fasted animals.
  • Ppap2a is also more than two fold up regulated in WAT of ob/ob mice.
  • Ppap2a is more than two fold up regulated in muscle of high fat diet mice and three fold up regulated in the lung, WAT and BAT of high fat diet mice. In the liver of high fat diet mice Ppap2a expression is nearly three fold down regulated.
  • Ppap2b is highy expressed in brain and hypothalamus of wild type animals, and on lower but robust levels in further tissues e.g. liver, WAT, BAT, kidney, small intestine and muscle.
  • Ppap2b is more than two fold up regulated in BAT and colon of fasted animals as well as in muscle of fasted animals and ob/ob mice.
  • the robust expression of Ppap2b in the liver is more than two fold decreased in fasted animals and ob/ob mice.
  • Ppap2b is more than two fold up regulated in BAT and small intestine of palmitatdiet mice as shown in Figure 9F.
  • Ppap2c is highy expressed in small intestine and colon of wild type animals, and on lower levels in further tissues e.g. kidney, liver, WAT and BAT.
  • the low, but detectable, expression levels of Ppap2c in heart and muscle of wild type animals are clearly up regulated in the heart of fasted animals and in the muscle ofob/ob mice as shown in Figure 9H.
  • Ppap2a, Ppap2b, and Ppap2c are expressed and slightly regulated during the differentiation of 3T3L1 cells (data not shown).
  • Ppap2a, Ppap2b, and Ppap2c are expressed in several tissues, the main expression of each is found in distinct tissues. This tissue specific expression peak together with the tissue specific regulation of each gene in the different mouse models used, suggests distinct roles for Ppap2a, Ppap2b, and Ppap2c in the different metabolic requirements of various tissues.
  • Tem7R is highly expressed in kidney, lung, hypothalamus and brain, and on lower levels in further tissues, e.g. WAT, BAT, heart and muscle.
  • Tem7R is nearly five fold up regulated in the colon and more than 2 fold up regulated in BAT of ob /ob mice.
  • Tem7R is more than two fold up regulated in the pancreas and three fold up regulated in liver of fasted animals.
  • Tem7R is more than three fold up regulated in BAT and the small intestine of palmitatdiet mice as shown in Figure 19C.
  • Tem7R plays a central role in the metabolism.
  • ADCY6 is expressed in different mammalian (human) tissues, showing highest level of expression in Adipose tissue and higher levels in brain, lung and liver.
  • the expression of ADCY6 is up regulated during the adipogensis, showing the highest level in the mature adipocyte as shown in Figure 28B.
  • FIG. 28C real time PCR (Taqman) analysis of the expression of the ADCY1 RNA in human tissues revealed that ADCY1 is highly expressed in brain, liver, testis and muscle, and on lower levels in further tissues, e.g. Adipose tissue and pancreas.
  • Figure 28D shows that expression of ADCY 1 is up regulated during early adipogenesis.
  • ADCY6 The high expression of ADCY6 in Adipose tissue and the upregulation of ADCY1 and ADCY 6 during the differentiation from human preadipocytes to mature adipocyte suggest that ADCY1 and ADCY 6 play a central role in the metabolism.
  • Apbbl ip is up regulated in several metabolic active tissues of both, mice fed a high fat diet and genetically obese mice. This together with the converse regulation in BAT of fasted animals suggests that Apbbl ip plays a central role in the metabolism.
  • 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 Affymetrix, 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 FACL1 , FACI2, FACL3, FACL4, PPAP2A, PPAP2B, PPAP2C, CHEK1 , TEM7, FLJ 1 1807 and APBB1 IP genes using primary human abdominal adipocycte differentiation clearly shows differential expression of human FACL1 , FACL2, FACL3, FACL4, PPAP2A, PPAP2B, PPAP2C, CHEK1 , TEM7, FLJ1 1807 and APBB1 IP genes in adipocytes.
  • the FACL1 , FACI2, FACL3, FACL4, PPAP2A, PPAP2B, FLJ1 1807, and APBB1 IP proteins have to be significantly increased in order for the preadipocyctes to differentiate into mature adipocycte.
  • the FACL1 , FACL2, FACL3, FACL4, PPAP2A, PPAP2B, FLJ 1 1807 and APBB1 IP proteins in preadipocyctes have the potential to enhance adipose differentiation at a very early stage.
  • the PPAP2C, CHEK1 and TEM7 proteins have to be significantly decreased in order for the preadipocyctes to differentiate into mature adipocycte. Therefore, the PPAP2C, CHEK1 and TEM7 proteins in preadipocyctes have the potential to inhibit adipose differentiation at a very early stage.
  • FACL1 , FACL2, FACL3, FACL4, PPAP2A, PPAP2B, PPAP2C, CHEK1 , TEM7, FLJ1 1807 and APBB1 IP 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

L'invention concerne des nouvelles utilisations de protéines régulant l'homéostasie énergétique et de polynucléotides codant ces protéines pour le diagnostic, l'étude, la prévention et le traitement de maladies et de troubles du métabolisme.
PCT/EP2003/006080 2002-06-10 2003-06-10 Proteines impliquees dans la regulation de l'homeostasie energetique WO2003103704A2 (fr)

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DATABASE WPI Section Ch, Week 200163 Derwent Publications Ltd., London, GB; Class B04, AN 2001-565582 XP002261391 & WO 01/066706 A (HUMAN GENOME SCI INC) 13 September 2001 (2001-09-13) *
DATABASE WPI Section Ch, Week 200214 Derwent Publications Ltd., London, GB; Class B04, AN 2002-106464 XP002261389 & WO 01/096371 A (DEVELOGEN AG) 20 December 2001 (2001-12-20) *
DATABASE WPI Section Ch, Week 200217 Derwent Publications Ltd., London, GB; Class B04, AN 2002-130151 XP002261388 & WO 01/018210 A (GENENTECH INC) 15 March 2001 (2001-03-15) *
DATABASE WPI Section Ch, Week 200224 Derwent Publications Ltd., London, GB; Class B04, AN 2002-188506 XP002261392 & WO 02/05810 A (JOSLIN DIABETES CENT) 24 January 2002 (2002-01-24) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016526888A (ja) * 2013-06-28 2016-09-08 アキュメン リサーチ ラボラトリーズ プライヴェット リミテッドＡｃｕｍｅｎ Ｒｅｓｅａｒｃｈ Ｌａｂｏｒａｔｏｒｉｅｓ Ｐｔｅ． Ｌｔｄ． 敗血症バイオマーカー及びそれらの使用
CN112618703A (zh) * 2020-12-24 2021-04-09 上海交通大学医学院附属瑞金医院 Acsl4作为制备2型糖尿病药物的用途

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