WO2003084566A2

WO2003084566A2 - Proteins involved in the regulation of energy homeostasis

Info

Publication number: WO2003084566A2
Application number: PCT/EP2003/003747
Authority: WO
Inventors: Karsten Eulenberg; Thomas HÄDER; Arnd Steuernagel; Günter BRÖNNER
Original assignee: DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
Priority date: 2002-04-10
Filing date: 2003-04-10
Publication date: 2003-10-16
Also published as: WO2003084566A3; AU2003224062A1

Abstract

The present invention discloses novel uses for energy homeostasis regulating proteins and polynucleotides encoding these in the diagnosis, study, prevention, and treatment of metabolic diseases and disorders.

Description

Proteins involved in the regulation of energy homeostasis

Description

This invention relates to the use of slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous proteins, to the use of polynucleotides encoding these, and to the use of effectors/modulators in the diagnosis, study, prevention, and treatment of obesity, and/or diabetes mellitus, and/or metabolic syndrome.

There are several metabolic diseases of human and animal metabolism, eg., obesity and severe weight loss, that relate to energy imbalance where caloric intake versus energy expenditure is imbalanced. 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 (see, inter alia, Kopelman (1999), loc. cit.). 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., ( 1 980) Clin. Invest 65, 1 272-1 284) . A clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman P.G., (2000) Nature 404, 635-643) .

Hyperlipidemia and elevation of free fatty acids correlate clearly with the metabolic syndrome, which is defined as the linkage between several diseases, including obesity and insulin resistance. This often occurs in the same patients and are major risk factors for development of Type 2 diabetes and cardiovascular disease. It was suggested that the control of lipid levels and glucose levels is required to treat Type 2 Diabetes, heart disease, and other occurances of Metabolic Syndrome (see, for example, Santomauro A. T. et al., (1 999) Diabetes, 48(9) : 1 836-1 841 and McCook, 2002, JAMA 288:2709-2716).

The molecular factors regulating food intake and body weight balance are incompletely understood. Even if several candidate genes have been described which are supposed to influence the homeostatic system(s) that regulate body mass/weight, like leptin or the peroxisome proliferator-activated receptor-gamma co-activator, the distinct molecular mechanisms and/or molecules influencing obesity or body weight/body mass regulations are not known. In addition, several single-gene mutations resulting in obesity have been described in mice, implicating genetic factors in the etiology of obesity (Friedman and Leibel, 1 990, Cell 69: 21 7-220) . In the ob mouse a single gene mutation (obese) results in profound obesity, which is accompanied by diabetes (Friedman et. al., 1991 , Genomics 1 1 : 1 054-1 062 ) .

Therefore, 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. Accordingly, 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 useful in diagnosis, treatment, and prognosis of metabolic diseases and disorders as described.

Membrane peptidases are a multifunctional group of ectoenzymes that have been implicated in the control of growth and differentiation of many cellular systems (Riemann D. et al, 1 999, Immunol Today 20(2):83-8) . The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits a high degree of substrate specificity (Heuer H. et al., ( 1 998) Thyroid 8( 10) :91 5-920) . TRH-DE is a member of the M 1 family of Zn-dependent aminopeptidases. The stringent regulation of the TRH-degrading ectoenzyme suggests that this enzyme represents an important regulatory element, controlling the stimulation of TRH target cells and, thus, adenohypophyseal hormone secretion (Schomburg L. and Bauer K., ( 1 995) Endocrinology 1 36(8):3480-3485) .

Alanyl aminopeptidase (APN) is highly expressed in human monocytes. APN contributes to the regulation and realisation of lymphocyte growth and function by modulating the mRNA expression of IL-2, IL-1 receptor antagonist, and TGF-beta1 and increasing the activity of MAP kinase p42/Erk2 (Lendeckel U. et at., 1 999, Int J Mol Med 4(1 ) : 1 7-27) . Protease-induced leukocyte chemotaxis and activation: roles in host defense and inflammation. The migration of leukocytes such as neutrophils, monocytes and lymphocytes into inflamed lesions is one of the critical events of inflammation. Aminopeptidase N and endothelin were shown to induce chemotactic migration of leukocytes. Thus, protease-induced leukocyte chemotaxis and activation may play an important role in immunologic events of inflammatory and allergic diseases (Tani K. et al., 2001 , J Med Invest 48(3-4): 1 33-41 ).

DNA polymerase delta is a 3'-5' exodeoxyribonuclease involved in leading strand elongation. Chang L.S. et al. identified the structure of the gene for the catalytic subunit of human DNA polymerase delta, POLD1 ( 1 995, Genomics 28(3) :41 1 -9). DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination, and DNA repair. The cell has developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. In addition DNA polymerases show a large degree of cross-functionality of in the different pathways. DNA polymerase delta functions as a dimer and is responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase zeta, for mutagenesis. DNA polymerase delta suffices for the repair of UV-induced damage (Burgers P.M., 1 998, Chromosoma 107(4):21 8-27).

Protein kinase C (PKC) is a family of multifunctional isoenzymes, which play a central role in signal transduction and intracellular crosstalk by phosphorylating at serine/threonine residues an array of substrates, including cell-surface receptors, enzymes, contractile proteins, transcription factors and other kinases. In liver, muscle and adipose tissue, PKC isozymes have been implicated both as mediators and inhibitors of insulin action (Idris I. et al., 2001 , Diabetologia 44(6):659-73) . The protein kinase C inhibitor, like protein kinase C itself, is a zinc-binding protein, although the sequence does not reveal a "zinc finger" structure (Pearson J.D. et al., 1 990, J Biol Chem 265(8):4583-91 ). The zinc-binding region of an endogenous protein inhibitor of protein kinase C contains three closely positioned histidine residues, a characteristic histidine triad (HIT) (Mozier N.M. et al., 1 991 , FEBS Lett 279( 1 ) : 14-8) . The HIT protein family is present in prokaryotes, yeast and mammals (Seraphin B., 1 992, DNA Seq 1 992;3(3) : 1 77-9) . Hint (histidine triad nucleotide-binding protein)-related proteins, found in all forms of life, and fragile histidine triad (Fhit)-related proteins, found in animals and fungi, represent the two main branches of the HIT superfamily. Hint homologs are intracellular receptors for purine mononucleotides (Brenner C. et al., 1 999, J Cell Physiol 1 81 (2): 1 79-87). Levels of nucleoside diphosphate kinase B, Rab GDP-dissociation inhibitor beta and histidine triad nucleotide-binding protein are significantly reduced in fetal Down syndrome brain (Weitzdoerfer R. et al., 2001 , J Neural Transm Suppl (61 ):347-59).

Drosophila melanogaster gene Indy (for I'm not dead yet), is most closely related to a mammalian sodium dicarboxylate cotransporter, a membrane protein that transports Krebs cycle intermediates. Indy is most abundantly expressed in the fat body, midgut, and oenocytes (Rogina B. et al., 2000, Science 290(5499):2137-2140). Rogina et al (supra) found that independent insertional mutations in the Indy gene in Drosophila resulted in a near doubling of the average adult life-span without a decline in fertility or physical activity. The secondary structure model of the Na( ⁺ )/dicarboxylate cotransporter, NaDC-1 , contains 1 1 transmembrane domains. The carboxy terminus of the protein is located extracellularly and contains an N-glycosylation site. The N-terminus and hydrophilic loop 4 of NaDC-1 are located intracellularly (Zhang F.F. and Pajor A.M., 2001 , Biochim Biophys Acta 1 51 1 ( 1 ) :80-9) . The substrate recognition domain in the Na⁺/dicarboxylate cotransporter is located in the carboxy-terminal portion of the protein (Pajor A.M. et al., 1 998, Biochim Biophys Acta 1 370(1 ):98-106).

The cDNA coding for a rabbit renal Na⁺/dicarboxylate cotransporter (NaDC-1 ) is abundant in kidney and small intestine. The transport of succinate by NaDC-1 is sodium-dependent, sensitive to inhibition by lithium, and inhibited by a range of di- and tricarboxylic acids. This transporter also carries citrate (Pajor 1 995, J Biol Chem 270(1 1 ) :5779-5785). The rabbit and human Na(⁺)-dicarboxylate cotransporters, NaDC-1 and hNaDC-1 , have similar affinities for succinate and glutarate, and differ in their handling of citrate. The human transporter is more sensitive to pH than the rabbit (Pajor & Sun, 1 996, Am J Physiol 271 (5 Pt 2) :F1093-1099) .

Myeloid differentiation primary response (MyD) genes play a role in negative growth control, including growth suppression and apoptosis in many cell types (Liebermann & Hoffman, 1 998, Oncogene 1 7(25):3319-29) . The analysis of knockout mice revealed a role for Myeloid differentiation primary response gene 88 (MyD88) in the signaling of the Toll-like receptors (TLR) / interleukin-1 receptor (IL-1 R) family (Takeuchi O. and Akira S., 2001 , Int Immunopharmacol 1 (4):625-35; Akira S. et al., 2001 , Nat Immunol 2(8):675-80)) . The original function of this TLR/IL-1 R family is to mediate responses in the immune system (Hultmark D., 1 994, Biochem Biophys Res Commun 199(1 ): 144-6).

The Drosophila gene Beached l (BeachD with GadFly Accession Number CG 14001 encodes for a protein (SEQ ID NO: 1 ) which is most homologous to human novel protein (SEQ ID NO:3, Ensembl Accession Number ENSP00000295892 for the protein; encoded by SEQ ID NO:2, ENSG0000001 6628 for the cDNA) . No functional data are available for the human protein.

Nuclear fallout (nuf) is a maternal effect mutation that specifically disrupts the cortical syncytial divisions during Drosophila embryogenesis. The nuf gene encodes a highly phosphorylated protein. During prophase of the late syncytial divisions, Nuf concentrates at the centrosomes and is generally cytoplasmic throughout the rest of the nuclear cycle (Rothwell et al., 1 998, Development 1 25(7): 1 295-1 303) . Nuf is required for recruiting a membrane associated protein, to furrows in the early embryo (Rothwell, 1 999, J Cell Sci 1 1 2 ( Pt 1 7) :2885-2893). No functional data are available for the homolog human proteins described in this invention.

A group of genes sharing similar motifs, the basic Helix-Loop-Helix (bHLH) proteins are involved at different steps of the development of the peripheral nervous system (PNS) of Drosophila (Dambly-Chaudiere C. and Vervoort M., 1 998, Int J Dev Biol 42(3 Spec No) :269-73) . Hairy-related proteins are a distinct subfamily of basic helix-loop-helix (bHLH) proteins that generally function as DNA-binding transcriptional repressors. In the development of the Drosophila peripheral nervous system, Hairy-related genes function at multiple steps during neurogenesis and in the establishment and restriction of other types of equivalence groups. This general function in cell fate specification has been conserved from Drosophila to vertebrates and has implications for human disease pathogenesis (Fisher A. and Caudy M., 1 998, Bioessays 20(4) :298-306) . In human neural stem cells the HLH transcription factor HES1 regulates proliferation. The suppression of HES1 expression initiates differentiation of neural stem cells into neurons, the majority of which develop the GABAergic phenotype. Therefor the HLH network, and HES1 in particular, play an important role in guiding the phenotypic development of neural stem cells (Kabos P. et al., 2002, J Biol Chem 277( 1 1 ):8763-6) .

The expression of the insulin gene is highly specific to pancreatic beta cells and is downregulated in pancreatic HIT-T1 5 cells by dexamethasone (DEX), blocking the glucose-dependent insulin promoter activity. After the addition of DEX to HIT-T1 5 cells, a decrease of insulin mRNA and insulin protein was observed. HES-1 , a potent negative regulator of bHLH-type transcription factors, is expressed in HIT-T1 5 cells, and its expression was increased after the addition of DEX. Overexpression of HES-1 suppressed the insulin promoter activity in a dose-dependent manner. These results suggest that enhancement of HES-1 expression is involved in impairment of insulin synthesis (Shinozuka Y. et al., 2001 , Biochem Biophys Res Commun 287(1 ):229-35) .

So far, it has not been described that 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. In 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 mutation 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 of triglyceride content, the major energy storage substance.

The function of Indy in metabolic disorders is further validated by data obtained from additional screens. For example, an additional screen using Drosophila mutants with modifications of the eye phenotype identified an interaction of Indy with adipose, a protein regulating, causing or contributing to obesity. These findings suggest the presence of similar activities of these described homologous proteins in humans that provides insight into diagnosis, treatment, and prognosis of metabolic disorders.

Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies that are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure.

The present invention discloses that slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous proteins (herein referred to as "proteins of the invention" or "a protein of the invention") are regulating the energy homeostasis and fat metabolism especially the metabolism and storage of triglycerides, and 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 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.

Slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan 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

(a) the nucleotide sequence of slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, deadpan, or homologous nucleic acids, particularly nucleic acids encoding a human protein as described in TABLE 1 , and/or a sequence complementary thereto,

(b) a nucleotide sequence which hybridizes at 50°C in a solution containing 1 x SSC and 0.1 % SDS to a sequence of (a),

(c) a sequence corresponding to the sequences of (a) or (b) within the degeneration of the genetic code,

(d) a sequence which encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the amino acid sequences of a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan homologous protein, preferably of a human homologous protein as described in TABLE 1 , (e) a sequence which differs from the nucleic acid molecule of (a) to (d) by mutation and wherein said mutation causes an alteration, deletion, duplication and/or premature stop in the encoded polypeptide or

(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 invention is based on the finding that slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan and/or homologous proteins and the polynucleotides encoding these, are involved in the regulation of triglyceride storage and therefore energy homeostasis. The invention describes the use of these compositions for the diagnosis, study, prevention, or treatment of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones.

Accordingly, 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, 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.

The ability to manipulate and screen the genomes of model organisms such as the fly Drosophila melanogaster 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: 2185-21 95). Identification of novel gene functions in model organisms can directly contribute to the elucidation of correlative pathways in mammals (humans) and of methods of modulating them. A correlation between a pathology model (such as changes in triglyceride levels as indication for metabolic syndrome including obesity) and the modified expression of a fly gene can identify the association of the human ortholog with the particular human disease.

In one embodiment, 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: 1 76-1 88 (2002); Rorth P., (1 996) Proc Natl Acad Sci U S A 93: 1 2418-1 2422) . In this invention, we have used a genetic screen to identify mutations of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan gene, or homologous genes that cause changes in the body weight, which are reflected by a significant change of triglyceride levels.

Obese people mainly show a significant increase in the content of triglycerides. Triglycerides are the most efficient storage for energy in cells. In order to isolate genes with a function in energy homeostasis, several thousand proprietary EP-lines were tested for their triglyceride content after a prolonged feeding period (see Examples 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.

In this invention, 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 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 , 5, 9, 13, 1 6, 20, 23, and 26.

Genomic DNA sequences were isolated that are localized to the EP vector integration. Using those isolated genomic sequences public databases like Berkeley Drosophila Genome Project (GadFly; see also FlyBase (1 999) Nucleic Acids Research 27:85-88) were screened thereby identifying the integration site of the vectors, and the corresponding gene, described in more detail in the Examples section. The molecular organization of the gene is shown in Figures 2, 6, 10, 14, 17, 21 , 24, and 27. An additional screen using Drosophila mutants with modifications of the eye phenotype identified an interaction of Indy with adipose, a protein regulating, causing or contributing to obesity.

The 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.

The function of the mammalian homologs in energy homeostasis was further validated in this invention by analyzing the expression of the transcripts in different tissues and by analyzing the role in adipocyte differentiation. Expression profiling studies (see Examples for more detail) confirm the particular relevance of the protein(s) of the invention as regulators of energy metabolism in mammals. Further, we show that the proteins of the invention are regulated by fasting and/or by genetically induced obesity. In this invention, we used 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) to study the expression of the protein of the invention. Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning et al, 1 998, 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 4). 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. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, 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. (1995) U.S. Patent No. 5,474,796- Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93: 10614-1061 9; Baldeschweiler et al. (1 995) PCT application WO95/251 1 1 6; Shalon, D. et al. (1995) PCT application WO95/35505; Heller, R.A. et al. (1 997) Proc. Natl. Acad. Sci. USA 94:21 5021 55; Heller, M.J. et al. ( 1 997) U.S. Patent No. 5,605,662) . Various types of microarrays are well known and thoroughly described in Schena, M., ed. ( 1 999; DNA Microarrays: A Practical Approach, Oxford University Press, London) .

In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray. The microarray can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below. The microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents, which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.

As determined by Microarray analysis, thyrotropin-releasing hormone degrading ectoenzyme, alanyl (membrane) aminopeptidase, and Myd88 show differential expression in human primary adipocytes. Thus, thyrotropin-releasing hormone degrading ectoenzyme, alanyl (membrane) aminopeptidase, and Myd88 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 present invention further describes polypeptides comprising the amino acid sequences of the proteins of the invention and homologous proteins. Based upon homology, the proteins of the invention and each homologous protein or peptide may share at least some activity. No functional data described the regulation of body weight control and related metabolic diseases are available in the prior art for the genes of the invention.

The invention also encompasses polynucleotides that encode the proteins of the invention and homologous proteins. Accordingly, any nucleic acid sequence, which encodes the amino acid sequences of the proteins of the invention and homologous proteins, can be used to generate recombinant molecules that express the proteins of the invention and homologous proteins. In a particular embodiment, the invention encompasses a nucleic acid encoding Drosophila slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan, or human slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologs; referred to herein as the proteins of the invention. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding the proteins, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of nucleotide sequence that could be made by selecting combinations based on possible codon choices.

Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those of the polynucleotides encoding slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, deadpan 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 ( 1 987: Methods Enzymol. 1 52:399-407) and Kimmel, A. R. ( 1 987; Methods Enzymol. 1 52:507-51 1 ), and may be used at a defined stringency. Preferably, 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. Furthermore, the invention relates to peptide fragments of the proteins or derivates of such peptide fragments such as cyclic peptides, retro-inverso peptides or peptide mimetics wherein the peptide fragments or peptide derivatives preferably have a length of at least 4, more preferably of at least 6 and up to 50 amino acids.

Also included within the scope of the present invention are alleles of the genes encoding a protein of the invention or a homologous protein. As used herein, 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.

The nucleic acid sequences encoding the proteins of the invention and homologous proteins 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. For example, 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. 2:31 8-322) . 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 6:8186). Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (PCR Methods Applic. 1 : 1 1 1 -1 1 9) . Another method which may be used to retrieve unknown sequences is that of Parker, J. D. et al. (1991 ; Nucleic Acids Res. 1 9:3055-3060). 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.

In order to express a biologically active protein, the nucleotide sequences encoding the proteins or functional equivalents, may be inserted into appropriate expression vectors, i.e., a vector, which contains the necessary elements for the transcription and translation of the inserted coding sequence. 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. (1 989) Molecular Cloning,

A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. et al. ( 1 989) Current Protocols in Molecular Biology, John

Wiley & Sons, New York, N.Y.

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 cell systems. The "control elements" or "regulatory sequences" are those non-translated regions of the vector-enhancers, promoters, 5' and 3' untranslated regions which interact with host cellular proteins to carry out transcription and translation. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, may be used. The baculovirus polyhedrin promoter may be used in insect cells. Promoters and enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO; and storage protein genes) or from plant viruses (e.g., viral promoters and leader sequences) may be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of the sequences encoding the protein, vectors based on SV40 or EBV may be used with an appropriate selectable marker.

In a further embodiment of the invention, natural, modified or recombinant nucleic acid sequences encoding the proteins of the invention and homologous proteins may be ligated to a heterologous sequence to encode a fusion protein.

The presence of polynucleotide sequences encoding a protein of the invention or a homologous protein can be detected by DNA-DNA or DNA-RNA hybridization and/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. As used herein "oligonucleotides" or "oligomers" refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 1 5 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). 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. (1 990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 1 58: 1 21 1 -1 21 6) .

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid 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 or PCR amplification using a labeled nucleotide.

Alternatively, the sequences encoding the protein, or any portions thereof may be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. 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, 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. As will be understood by those of skill in the art, 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.) 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. In addition to recombinant production, functional fragments of the proteins may be produced by direct peptide synthesis using solid-phase techniques (Merrifield J. (1 963) J. Am. Chem. Soc. 85:2149-21 54) . Protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using Applied Biosystems 431 A peptide synthesizer (Perkin Elmer). Various fragments of the proteins may be chemically synthesized separately and combined using chemical methods to produce the full length molecule. Diagnostics and Therapeutics

The data disclosed in this invention show that the 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. Hence, diagnostic and therapeutic uses for the nucleic acids and proteins of the invention are, for example but not limited to, the following: (i) protein therapy, (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi) research tools, and (vii) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues).

The nucleic acids and proteins of the invention and effectors/modulators thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below. For example, but not limited to, 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. By way of non-limiting example, 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.

The nucleic acids or functional fragments 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.

For example, in one aspect, antibodies which are specific for a protein of the invention or a homologous protein may be used directly as an effector, e.g. 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.

For the production of antibodies, 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. Depending on the host species, 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 (Kόhler, G. et al. ( 1 975) 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). ln addition, techniques developed for the production of "chimeric antibodies", the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison, S. L. et al. (1 984) Proc. Natl. Acad. Sci. 81 :6851 -6855; Neuberger, M. S. et al ( 1 984) Nature 312:604-608; Takeda, S. et al. (1 985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce single chain antibodies specific for a protein of the invention or a homologous protein. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1 991 ) 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. (1 989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1 991 ) Nature 349:293-299).

Antibody fragments which contain specific binding sites for the proteins may also be generated. For example, such 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. Alternatively, 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. (1 989) Science 254: 1 275-1 281 ) .

Various 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 utilising monoclonal antibodies reactive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox, supra) .

In another embodiment of the invention, 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. In one aspect, 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.

In a further aspect, antisense molecules, may be used for therapeutic purposes. In one aspect, antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding a protein of the invention or a homologous protein. Thus, antisense molecules may be used to modulate / effect protein activity, or to achieve regulation of gene function. Such technology is now well know in the art, and 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 polynucleotide which encodes 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.

As mentioned above, modifications of gene expression can be obtained by designing antisense molecules, e.g. DNA, RNA, or nucleic acid analogues such as PNA, to the control regions of the genes encoding a protein of the invention or a homologous protein, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and + 10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1 994) In; Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). 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. Once identified, short 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. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding a protein of the invention or a homologous protein. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues. RNA molecules may be modified to increase intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases. Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of a protein of the invention or homologous nucleic acids or proteins, 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 acids. The compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone, or in combination with other agents, drugs or hormones. 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.

In addition to the active ingredients, 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.).

The pharmaceutical 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, including but not limited to, hydrochloric, sulphuric, acetic, lactic, tartaric, malic, succinic, etc. 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.

Pharmaceutical 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. For any compounds, 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, a nucleic acid or an antibody, which is sufficient for treating a specific condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population) . The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. 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 1 00,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.

In another embodiment, antibodies which specifically bind to a protein of the invention or a homologous protein 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 photometric means. Quantities of protein expressed in control and disease, samples e.g. from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

In another embodiment of the invention, 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. ln one aspect, 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 slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan 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. Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as ³²P or ³⁵S, 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.

In a particular aspect, the 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 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.

In order to provide a basis for the diagnosis of a disease associated with expression of a protein of the invention or a homologous protein, 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 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. With respect to metabolic diseases or dysfunctions, including metabolic syndrome, obesity, or diabetes., the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the metabolic diseases and disorders. Additional diagnostic uses for 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 n ucleotid e seq uences, o ne with sense orientatio n (5prime.fwdarw.3prime) and another with antisense (3prime.rarw.5prime), 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. (1 993) J. Immunol. Methods, 1 59:235-244; Duplaa, C. et al. (1 993) Anal. Biochem. 21 2:229-236). 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. In another embodiment of the invention, the nucleic acid sequences which are sprecific 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. (1 993) Blood Rev. 7: 1 27-1 34, and Trask, B. J. (1991 ) Trends Genet. 7: 1 49-1 54. FISH (as described in Verma et al. (1 988) 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: 1 981 f). 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. An analysis of polymorphisms, e.g. single nucleotide polymorphisms may be carried out. Further, in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 1 1 q22-23 (Gatti, R. A. et al. (1 988) Nature 336:577-580), 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.

In another embodiment of the invention, the proteins of the invention or homologous proteins, their catalytic or immunogenic fragments or oligopeptides thereof, an in vitro model, a genetically altered cell or animal, 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. One can identify modulators/effectors, e.g. receptors, enzymes, proteins, ligands, or substrates that bind to and/or modulate or mimic the action of one or more of the proteins of the invention. The protein or fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between a protein of the invention or a homologous protein and the agent tested, may be measured . Agents could also, either directly or indirectly, influence the activity of the proteins of the invention.

Protein kinase C (PKC) is a group of calcium and phospholipid-dependent enzymes, which plays a pivotal role in cell signaling systems. PKC transduces the cellular signals that promote lipid hydrolysis. This 80kDa enzyme is recruited to the plasma membrane by diacylglycerol and, in many cases, by calcium. PKC phosphorylates a variety of target proteins which control growth and cellular differentiation. The structure of PKC is not known, but the isozymes of PKC are homologous with cAMP-dependent protein kinase (protein kinase A). Interactions were identified between PKCI-1 (HINT1 ) and the regulatory domain of protein kinase C-beta (Lima CD. et al., ( 1 996) Proc Natl Acad Sci U S A 93(1 1 ):5357-5362). In vivo, the enzymatic kinase activity of the unmodified polypeptides of PKC, 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 PKC and PKCI-1 , 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 PKC and PKCI-1 , 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.

In one embodiment of the invention, one may produce activated PKC independent of the natural stimuli for the above said purposes by, for example, but not limited to, (i) an agent that mimics the natural stimulus; (ii) an agent, that acts downstream of the natural stimulus, such as activators of the PKC, phorbol ester, anisomycin, constitutive active alleles of the PKC itself as they are described or may be developed; (iii) by introduction of single or multiple amino acid substitutions, deletions or insertions within the sequence of PKC to yield constitutive active forms; (iv) by the use of isolated fragments of PKC. In addition, one may generate enzymatically active PKC in an ectopic system, prokaryotic or eukaryotic, in vivo or in vitro, by co-transfering to this system the activating components.

In addition activity of the proteins of the invention against their physiological substrate(s) or derivatives thereof could be measured in cell-based assays. Agents may also interfere with posttranslational modifications of the proteins of the invention, such as phosphorylation and dephosphorylation, farnesylation, palmitoylation, acetylation, alkylation, ubiquitination, proteolytic processing, subcellular localization and degradation. Moreover, 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.

Methods for determining protein-protein interaction are well known in the art. For example binding of a fluorescently labeled peptide derived from a protein of the invention to the interacting protein (or vice versa) could be detected by a change in polarisation. In case that both 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. In addition, a variety of commercially available assay principles suitable for detection of protein-protein interaction are well known In the art, for example but not exclusively AlphaScreen (PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham. Alternatively, 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. In all cases 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 slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous proteins.

Assays for determining enzymatic and carrier activity of the proteins of the invention are well known in the art. Well known in the art are also a variety of assay formats to measure receptor-ligand binding.

Of particular interest are screening assays for agents that have a low toxicity for mammalian cells. The term "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. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, 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. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Where 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.

Yet in another example, the change of mass of the substrate due to its phosphorylation may be detected by mass spectrometry techniques. One could also detect the phosphorylation status of a substrate with an analyte discriminating between the phosphorylated and unphosphorylated status of the substrate. 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.

Such an 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, 1 998, 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.

Yet in another example 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.

In one example, the kinase substrate may be labelled with a fluorochrome. The binding of the analyte to the labelled substrate in solution may be followed by the technique of fluorescence polarization as it is described in the literature (see, for example, Deshpande, S. et al. (1999) Prog. Biomed. Optics (SPIE) 3603:261 ; Parker, G. J. et al. (2000) J. Biomol. Screen. 5:77-88; Wu, P. et al. (1997) Anal. Biochem. 249:29-36). In a variation of this example, 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, which may be used, provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, as applied to a protein of the invention or a homologous protein large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the proteins, or fragments thereof, and washed. Bound proteins are then detected by methods well known in the art. Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding a protein of the invention specifically compete with a test compound for binding the protein. In this manner, the antibodies can be used to detect the presence of any peptide, which shares one or more antigenic determinants with the protein of the invention.

The nucleic acids encoding the proteins of the invention can be used to generate transgenic cell lines and animals. 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 modulators of the protein of the invention. Misexpression (for example, overexpression or lack of expression) of the protein of the invention, particular feeding conditions, and/or administration of biologically active compounts can create models of metablic disorders.

In one embodiment of the invention, 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). 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. In addition to testing the expression of the proteins of the invention in such mouse strains (see EXAMPLES section), these mice could be used to test whether administration of a candidate 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 the protein of the invention is mutated. Alternatively, a nucleic acid construct encoding the protein is injected into oocytes and is randomly integrated into the genome. One may also express the genes of the invention or variants thereof in tissues where they are not normally expressed or at abnormal times of development. Furthermore, variants of the genes of the invention like specific constructs expressing anti-sense molecules or expression of dominant negative mutations, which will block or alter the expression of the proteins of the invention may be randomly integrated into the genome. A detectable marker, such as lac Z or luciferase may be introduced into the locus of the genes of the invention, where upregulation of expression of the genes of the invention will result in an easily detectable change in phenotype. Vectors for stable integration include plasmids, retroviruses and other animal viruses, yeast artificial chromosomes (YACs), and the like.

DNA constructs for homologous recombination will contain at least portions of the genes of the invention with the desired genetic modification, and will include regions of homology to the target locus. Conveniently, markers for positive and negative selection are included. DNA constructs for random integration do not need to contain regions of homology to mediate recombination. 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 field. 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).

When non-human ES or non-human embryonic cells or somatic pluripotent stem cells have been transformed, they may be used to produce transgenic animals. After transformation, 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 blastocyst injection.

Blastocysts are obtained from 4 to 6 week old superovulated females. The

ES cells are trypsinized, and the modified cells are injected into the blastocoel of the blastocyst. After injection, the blastocysts are returned to each uterine horn of pseudopregnant females. Females are then allowed to go to term and the resulting offspring is screened for the construct. By providing for a different phenotype of the blastocyst and the genetically modified cells, chimeric progeny can be readily detected. The chimeric animals 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. The transgenic animals may be used in functional studies, drug screening, etc.

Finally, the invention also relates to a kit comprising at least one of

(a) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous nucleic acid molecule or a functional fragment thereof;

(b) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous amino acid molecule or a functional fragment or an isoform thereof;

(c) a vector comprising the nucleic acid of (a);

(d) a host cell comprising the nucleic acid of (a) or the vector of (c);

(e) a polypeptide encoded by the nucleic acid of (a); (f) a fusion polypeptide encoded by the nucleic acid of (a);

(g) an antibody, an aptamer or another effector/modulator against/of the nucleic acid of (a) or the polypeptide of (b), (e) or (f) and (h) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or for screening applications as described above. The kit may further contain user instructions. The Figures show:

Figure 1 shows the triglyceride content of a Drosophila slamdance (GadFly Accession Number CG551 8) mutant. Shown is the change of triglyceride 5 content of EP(3)3289 flies caused by integration of the P-vector into the promoter of the slamdance gene (referred to as 'EP(3)3289', column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ).

ιo Figure 2 shows the molecular organization of the mutated slamdance (Gadfly Accession Number CG551 8) gene locus.

Figure 3 shows the homology of Drosophila slamdance (GadFly Accession Number CG 55 1 8) to human alanyl aminopeptidase and i s thyrotropin-releasing hormone degrading ectoenzyme.

Figure 3A shows the BLASTP search result for the slamdance gene product (Query) with the best human homologous match (Sbjct). Figure 3B shows the comparison (ClustalW (1 .83) protein sequence alignment analysis) of human and Drosophila proteins. Gaps in the 0 alignment are represented as -. In the figure 'Slamdance Dm' refers to Drosophila protein encoded by slamdance, 'TRHDE Hs' refers to human thyrotropin-releasing hormonedegrading ectoenzyme, and 'Aminopeptidase Hs' refers to human alanyl (membrane) aminopeptidase.

25 Figure 4 shows the expression of slamdance homologs in mammalian (human) tissue.

Figure 4A shows the quantitative analysis of THRDE expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.

30 Figure 4B shows the quantitative analysis of alanyl (membrane) aminopeptidase expression in human abdominal adipocyte cells, during the differentiation from preadipocytes to mature adipocytes. Figure 5 shows the triglyceride content of a Drosophila DNA-polymerase-delta (GadFly Accession Number CG5949) mutant. Shown is the change of triglyceride content of EP(3)3292 flies caused by homozygous viable integration of the P-vector into the cDNA of the DNA-polymerase-delta gene (referred to as 'EP(3)3292', column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ).

Figure 6 shows the molecular organization of the mutated DNA-polymerase-delta (Gadfly Accession Number CG5949) gene locus.

Figure 7 shows the BLASTP search result for the DNA-polymerase-delta gene product (Gadfly Accession Number CG5949) (Query) with the best human homologous match (Sbjct).

Figure 8 shows the expression of the DNA-polymerase-delta homologs in mammalian tissues.

Figure 8A shows the real-time PCR analysis of DNA polymerase delta 1 expression in wild-type mouse tissues. Figure 8B shows the real-time PCR analysis of DNA polymerase delta 1 expression in different mouse models.

Figure 9 shows the triglyceride content of a Drosophila protein kinase C inhibitor (GadFly Accession Number CG2862) . Shown is the change of triglyceride content of HD-EP(2)21 1 47 flies caused by homozygous viable integration of the P-vector into the cDNA of the GadFly Accession Number CG2862 gene (referred to as 'HD-EP21 147', column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ) .

Figure 10 shows the molecular organization of the mutated protein kinase C inhibitor (Gadfly Accession Number CG2862) gene locus. Figure 1 1 shows homology of Drosophila protein kinase C inhibitor (GadFly Accession Number CG2862) to human histidin triad proteins. Figure 1 1 A shows the BLASTP search result for the GadFly Accession Number CG2862 gene product (Query) with the best human homologous match (Sbjct).

Figure 1 1 B shows the comparison (ClustalW (1 .83) protein sequence alignment analysis) of human and Drosophila proteins. Gaps in the alignment are represented as -. In the figure 'CG2862 Dm' refers to Drosophila protein kinase C inhibitor protein encoded by CG2862, 'HINT1 Hs' refers to human histidine triad nucleotide binding protein 1 , 'HIT Hs' refers to human histidine triad protein, and 'HINT2 Hs' refers to human histidine triad nucleotide binding protein 2.

Figure 1 2 shows the expression of protein kinase C inhibitor (PKCI) in mammalian tissues.

Figure 1 2A shows the real-time PCR analysis of PKCI expression in wild-type mouse tissues.

Figure 1 2B shows the real-time PCR analysis of PKCI expression in mice fed with a high fat diet compared to mice fed with a standard diet.

Figure 1 3 shows the triglyceride content of a Drosophila Indy (Gadfly Accession Number CG3979) mutant. Shown is the change of triglyceride content of HD-EP(3)37224 flies caused by integration of the P-vector into the annotated transcription unit (referred to as 'HD-EP37224', column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ).

Figure 14 shows the molecular organization of the mutated Indy (Gadfly Accession Number CG3979) gene locus.

Figure 1 5 shows the homology of Drosophila Indy (GadFly Accession Number 3979) to human solute carrier family 13, members 1 , 2, 3, and 4 Figure 1 5A shows the BLASTP search result for the Indy gene product (Query) with the best human homologous match (Sbjct) . Figure 1 5B shows the comparison (ClustalW (1 .83) protein sequence alignment analysis) of human and Drosophila proteins. Gaps in the alignment are represented as -. In the figure 'Indy Dm' refers to Drosophila protein encoded by Indy, 'SC-1 3-1 Hs' refers to human solute carrier family 1 3, member 1 , 'SC-1 3-4 Hs' refers to human solute carrier family 1 3, member 4, 'SC-1 3-2 Hs' refers to human solute carrier family 13, member 2, and 'SC-1 3-3 Hs' refers to human solute carrier family 1 3, member 3.

Figure 1 6 shows the triglyceride content of Drosophila Myd88 (GadFly Accession Number CG2078) mutants. Shown is the change of triglyceride content of HD-EP(2)251 57 flies caused by integration of the P-vector into the annotated transcription unit (referred to as 'HD-EP25275', column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ), and by ectopic expression of the Myd88 gene mainly in the fatbody of these flies (referred to as 'HD-EP251 57/FB', column 4) in comparison to controls with integration of this vector type (referred to as 'random EP/FB', column 3) . No change of triglyceride content is shown by ectopic expression of the Myd88 gene mainly in the neurons of these flies (referred to as 'HD-EP251 57/elav', column 6) in comparison to controls with integration of this vector type (referred to as 'random EP/elav', column 5) .

Figure 1 7 shows the molecular organization of the mutated Myd88 (Gadfly Accession Number CG2078) gene locus.

Figure 1 8 shows the BLASTP search result for the Drosophila Myd88 gene product (Gadfly Accession Number CG2078) (Query) with the best human homologous match (Sbjct) . Figure 1 9 shows the expression of the mammalian Myd88 homologs in mammalian tissues.

Figure 1 9A shows the real-time PCR analysis of myd88 expression in wild-type mouse tissues. Figure 1 9B shows the quantitative analysis of MYD88 expression in human abdominal adipocyte.cells, during the differentiation from preadipocytes to mature adipocytes.

Figure 20 shows the triglyceride content of a Drosophila Beachl (GadFly Accession Number CG 14001 ) mutant. Shown is the change of triglyceride content of HD-EP(2)251 94 flies caused by integration of the P-vector into the first intron of Beachl (referred to as 'HD-EP251 94', column 2) flies in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ) .

Figure 21 shows the molecular organization of the mutated Beachl (Gadfly Accession Number CG14001 ) gene locus.

Figure 22 shows the homology of Drosophila Beach l (GadFly Accession Number CG 14001 ) to the human ALFY protein.

Figure 22A shows the BLASTP search result for the Drosophila Beachl gene product (GadFly Accession Number CG 14001 ) (Query) with the best human homologous match (Sbjct).

Figure 22B shows the amino acid sequence of the Drosophila Beach l protein (SEQ ID NO: 1 ).

Figure 22C shows the nucleotide sequence of the human ALFY gene (SEQ

ID NO:2).

Figure 22D shows the amino acid sequence of the human ALFY gene (SEQ

ID NO:3).

Figure 23 shows the triglyceride content of a Drosophila nuf (GadFly Accession Number CG7867) mutant. Shown is the change of triglyceride content of EP(3)3324 flies caused by integration of the P-vector into an intron of the nuf gene (referred to as 'EP(3)224' column 2) in comparison to controls containing all flies of the EP-collection (referred to as 'EP control', column 1 ).

Figure 24 shows the molecular organization of the mutated nuf (Gadfly Accession Number CG7867) gene locus.

Figure 25 shows the homlogy of Drosophila nuf (GadFly Accession Number CG7867) to human rabl 1 -family interacting proteins 3 (Rabl 1 -FIP3) and 4 (Rab1 1 -FIP4).

Figure 25A shows the BLASTP search results for the nuf gene product (Query) with the two best human homologous matches (Sbjct) . Figure 25B shows the comparison (ClustalW (1 .83) protein sequence alignment analysis) of human and Drosophila proteins. Gaps in the alignment are represented as -. In the figure 'CG7867 Dm' refers to Drosophila protein encoded by nuf, 'Rab1 1 -FIP3 Hs' refers to human rab1 1 -family interacting protein 3, and 'Rab1 1 -FIP4 Hs' refers to human rabl 1 -family interacting protein 4.

Figure 26 shows the triglyceride content of a Drosophila deadpan mutant. Shown is the change of triglyceride content of HD-EP(2)20750 flies caused by extopic expression of deadpan (referred to as 'HD-EP(2)2075O/elav', column 2) in comparison to controls with integration of this vector type (referred to as 'random EP/elav', column 1 ) mainly in the neurons of these flies.

Figure 27 shows the molecular organization of the mutated deadpan (Gadfly Accession Number CG8704) gene locus.

Figure 28 shows the homology of Drosophila deadpan (GadFly Accession Number CG8704) to human HES-1 , HES-2, and HES-4. Figure 28A shows the BLASTP search result for the Drosophila deadpan gene product (Query) with the best human homologous match (Sbjct) . Figure 28B shows the comparison (ClustalW (1 .83) protein sequence alignment analysis) of human and Drosophila proteins. Gaps in the alignment are represented as -. In the figure 'CG8704 Dm' refers to Drosophila protein encoded by deadpan, 'HES-1 Hs' refers to human hairy and enhancer of split (Drosophila), 'HES-4 Hs' refers to human bHLH factor Hes4, and 'HES-2 Hs' refers to human transcription factor HES-2.

The examples illustrate the invention:

Example 1 : Measurement of triglyceride content

Mutant flies are obtained from a proprietary fly mutation stock collection and a publicly available stock collection. The flies are grown under standard conditions known to those skilled in the art. In the course of the experiment, additional feedings with bakers yeast (Saccharomyces cerevisiae) are provided. The average change of triglyceride content of Drosophila containing the EP-vectors in homozygous viable integration was investigated in comparison to control flies (see Figures 1 , 5, 9, 13, 1 6, 20, 23, and 26). For determination of triglyceride, flies were incubated for 5 min at 90°C in an aqueous buffer using a waterbath, followed by hot extraction. After another 5 min incubation at 90°C and mild centrifugation, 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. As a reference protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol. The assay was 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 , 5, 9, 1 3, 1 6, 20, and 23, respectively. The average triglyceride level of all flies containing the FB- Gal4 vector (referred to as 'random EP/FB') is shown as 100% in the third column in Figure 16. The average triglyceride level of all flies containing the elav-Gal4 vector (referred to as 'random EP/elav') is shown as 100% in the fifth column in Figure 1 6 and the first column in Figure 26. Standard deviations of the measurements are shown as thin bars.

HD-EP(3)37224 homozygous flies (column 2 in Figure 13) and HD-EP(2)251 94 homozygous flies (column 2 in Figure 20) show constantly a lower triglyceride content than the controls. EP(3)3289 homozygous flies (column 2 in Figure 1 ), EP(3)3292 homozygous flies (column 2 in Figure 5), HD-EP(2)21 1 47 homozygous flies (column 2 in Figure 9), and EP(3)3324 homozygous flies (column 2 in Figure 23) show constantly a higher triglyceride content than the controls. Therefore, the loss of gene activity in the loci where the EP-vectors are viably integrated, is responsible for changes in the metabolism of the energy storage triglycerides.

HD-EP(2)251 57 homozygous flies show constantly a slightly lower triglyceride content than the controls (column 2 in Figure 1 6). The offspring of HD-EP(2)251 57 males that are crossed to FB-Gal4 and elav-Gal4 virgins, carrying a copy of the HD-EP(2)251 57 vector and a copy of the FB-Gal4 ('HD-EP(2)251 57/FB') or elav-Gal4 ('HD-EP(2)251 57/elav') vector, leading to ectopic expression of adjacent genomic DNA sequences 3' of the HD-EP(2)25157 integration locus, mainly in the fatbody or neurons of these flies. 'HD-EP(2)251 57/FB' flies show constantly a higher triglyceride content (column 4 in Figure 16) than the control EP-collection that is crossed to FB-Gal4 (referred to as 'random EP/FB', column 3 in Figure 1 6). 'HD-EP(2)251 57/elav' flies show no significant change in triglyceride content (column 6 in Figure 1 6) in comparison to the control EP-collection that is crossed to elav-Gal4 (referred to as 'random EP/elav', column 5 in Figure 1 6). Therefore, the gain of gene activity in the locus, where the EP-vector of HD-EP(2)25157 flies is homozygous viable integrated δprime of the Myd88 gene, is responsible for changes in the metabolism of the energy storage triglycerides.

The offspring of HD-EP(2)20750 males that are crossed to elav-Gal4 virgins, carrying a copy of the HD-EP(2)20750 vector and a copy of the elav-Gal4 vector ('HD-EP(2)20750/elav'), leading to ectopic expression of adjacent genomic DNA sequences 3' of the HD-EP(2)20750 integration locus, mainly in the neurons of these flies. 'HD-EP(2)20750/elav' flies show constantly a higher triglyceride content (column 2 in Figure 26) than the control EP-collection that is crossed to elav-Gal4 (referred to as 'random EP/elav', column 1 in Figure 26) . Therefore, the the dominant-negative expression in the locus, where the EP-vector of HD-EP(2)20750 flies is heterozygous lethal integrated in the enhancer region of the deadpan gene, is responsible for changes in the metabolism of the energy storage triglycerides.

Example 2: Identification of the Drosophila genes involved in the regulation of energy homeostasis

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 EP(3)3289, EP(3)3292, HD-EP(2)21 147, HD-EP(3)37224, HD-EP(2)251 57, HD-EP(2)25194, EP(3)3324, or HD-EP(2)20750) integration. Using those isolated genomic sequences 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, 6, 10, 14, 1 7, 21 , 24, and 27. The EP(3)3289 vector is homozygous viably integrated into the promoter of a Drosophila gene in sense orientation, identified as slamdance (BcDNA:GH07466; GadFly Accession Number CG551 8). Figure 2 shows the molecular organization of this gene locus. The chromosomal localization site of the integration of the vector of EP(3)3289 is at gene locus 3R, 97D8-9. In Figure 2, genomic DNA sequence is represented by the assembly as a thin black scaled line in the middle that includes the integration sites of the vector for line EP(3)3289. Numbers and ticks represent the length of the genomic DNA (10000 base pairs per tick). The upper part of the figure represents the sense strand, the lower part represent the antisense strand. The dark grey boxes in the topmost part of the figure represent BAC clones, the light grey box in the middle of the figure represents the section of the chromosome. The insertion sites of the P-elements in the Drosophila lines are shown as grey triangles; the P-insertion in Drosophila EP(3)3289 line is labeled. Grey bars, linked by black lines represent cDNA sequences. Predicted genes are shown as black bars (exons), linked by light grey lines (introns), and labeled with CG-numbers represent the predicted genes (see also key at the bottom of the figure) . The predicted slamdance gene is referred to as BcDNA:GH07466 and is labeled.

In Figures 6, 10, 14, 17, 21 , 24, and 27, genomic DNA sequence is represented by the assembly as a dotted black line in the middle that includes the integration sites of the vectors for lines HD-EP(3)37224, EP(3)3292, HD-EP(2)21 147, HD-EP(2)251 57, HD-EP(2)251 94, EP(3)3324, and HD-EP(2)20750. Numbers represent the coordinates of the genomic DNA. The upper parts of the figures represent the sense strand " + ", the lower parts represent the antisense strand "-". The insertion sites of the P-elements in the Drosophila lines are shown as triangles or boxes in the "P-elements + " and/or "P-elements -" lines. Transcribed DNA sequences (ESTs) are shown as grey bars in the "EST + " 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 EP(3)3292 vector is homozygous viably integrated into the cDNA of a Drosophila gene in sense orientation, identified as DNA-polymersase-delta (GadFly Accession Number CG5949). The chromosomal localization site of the integration of the vector of EP(3)3292 is at gene locus 3L, 72C1 . In Figure 6, coordinates of the genomic DNA are starting at position 1 5881 756 on chromosome 3L, ending at position 1 5888006. The insertion site of the P-element in Drosophila EP(3)3292 line is shown in the "P-elements -" line and is labeled. The predicted cDNA of the DNA-polymersase-delta gene is shown in the "cDNA -" line and is labeled.

The HD-EP(2)21 147 vector is homozygous viably integrated into the cDNA of a Drosophila gene in sense orientation, identified as protein kinase C inhibitor (GadFly Accession Number CG2862). The chromosomal localization site of the integration of the vector of HD-EP(2)21 147 is at gene locus 2L, 23B1 . In Figure 10, coordinates of the genomic DNA are starting at position 268841 3 on chromosome 2L, ending at position 2689976. The insertion site of the P-element in Drosophila HD-EP(2)21 1 47 line is shown in the "P-elements + " line and is labeled. The predicted cDNA of the CG2862 gene is shown in the "cDNA + " line.

The HD-EP(3)37224 vector is homozygous viably integrated into an intron of a Drosophila gene in antisense orientation, identified as Indy (GadFly

Accession Number CG3979) . The chromosomal localization site of the integration of the vector of HD-EP(3)37224 is at gene locus 3L, 75D8-E1 .

In Figure 14, coordinates of the genomic DNA are starting at position

1871 2000 on chromosome 3L, ending at position 18732000. The insertion site of the P-element in Drosophila HD-EP(3)37224 line is shown in the

"P-elements + " line and is labeled. The predicted cDNA of the Indy gene is shown in the "cDNA -" line and is labeled. The HD-EP(2)251 57 vector is homozygous viably integrated into the promoter of a Drosophila gene in sense orientation, identified as Myd88 (GadFly Accession Number CG2078). The chromosomal localization site of the integration of the vector of HD-EP(2)251 57 is at gene locus 2R, 45C4. In Figure 1 7, coordinates of the genomic DNA are starting at position 4339801 on chromosome 2R, ending at position 4346051 . The insertion site of the P-element in Drosophila HD-EP(2)251 57 line is shown in the "P-elements -" line and is labeled. The predicted cDNA of the Myd88 cDNA is shown in the "cDNA -" line and is labeled.

The HD-EP(2)251 94 vector is homozygous viably integrated into the first intron of a Drosophila gene in antisense orientation, identified as Beach l (GadFly Accession Number CG 14001 ). The chromosomal localization site of the integration of the vector of HD-EP(2)251 94 is at gene locus 2L, 26A6. In Figure 21 , coordinates of the genomic DNA are starting at position 5824500 on chromosome 2L, ending at position 5827625. The insertion site of the P-element in Drosophila HD-EP(2)251 94 line is shown in the "P-elements -" line and is labeled. The predicted cDNA of the Beach l gene is shown in the "cDNA -" line and is labeled.

The EP(3)3324 vector is homozygous viably integrated into an intron of a Drosophila gene in sense orientation, identified as nuf (GadFly Accession Number CG7867). The chromosomal localization site of the integration of the vector of EP(3)3324 is at gene locus 3L, 70D2-3. In Figure 24, coordinates of the genomic DNA are starting at position 14082076 on chromosome 3L, ending at position 141 32076. The insertion site of the P-element in Drosophila EP(3)3324 line is shown in the "P-elements + " line and is labeled. The predicted cDNA of the nuf gene is shown in the "cDNA + " line and is labeled.

The HD-EP(2)20750 vector is heterozygous lethal integrated into the first intron of a Drosophila gene in sense orientation, identified as deadpan (GadFly Accession Number CG8704). The chromosomal localization site of the integration of the vector of HD-EP(2)20750 is at gene locus 2R, 97D8-9. In Figure 27, the coordinates of the genomic DNA are starting at position 3265505 on chromosome 2R, ending at position 3269005. The insertion site of the P-element in Drosophila HD-EP(2)20750 line is shown in the "P-elements -" line and is labeled. The predicted cDNA of the deadpan gene is shown as bars in the "cDNA -" line and is labeled.

Expression of the genes described above could be affected by integration of the vectors into the transcription units, leading to a change in the amount of the energy storage triglycerides.

Example 3: Identification of human homologous genes and proteins

The 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, 6, 9, 1 3, 1 7, 21 , 24, and 27). The term "polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number" relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number. The term "GenBank Accession number" relates to NCBI GenBank database entries (Ref.: Benson et al., Nucleic Acids Res. 28 (2000) 1 5-1 8). Table 1:

Slamdance, DNA-polymerase-delta, protein kinase C inhibitor, Indy, Myd88, Beachl , nuf and deadpan homologous proteins and 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 .

The human slamdance homologous protein alanyl (membrane) aminopeptidase is also referred to as sequence 201 from patent US 6, 1 80,084 (GenBank Accession Number AAE58390.1 ) . The human CG2862 homologous protein HINT1 is also referred to as sequence 3 from patent US 6,218, 1 13. The human Beachl homologous protein ALFY is also referred to as Ensembl Accession Number ENSG000001 63628 for the cDNA (SEQ ID NO: 2) and ENSP00000295892 for the protein (SEQ ID NO: 3) . The human nuf homologous protein rabl 1 -family interacting protein 4 is also referred to as KIAA1 821 protein with GenBank Accession Number XM_050101 for the cDNA and XP_050101 .1 for the protein.

The gene product of Drosophila DNA-polymerase-delta is homologous to mouse DNA-polymerase delta 1 , catalytic domain (GenBank Accession Number AAH091 28.1 ), the gene product of Drosophila CG2862 shows homology to mouse protein kinase C-inhibitor 1 (GenBank Accession Number P70349), the gene product of Drosophila Indy is homologous to mouse slc1 3a2 (GenBank Accession Number NP 071 856.1 ), the gene product of Drosophila Myd88 shows homology on protein level to mouse myeloid differentiation primary response gene 88 (GenBank Accession Number NP_034981 .1 ), and the gene product of Drosophila deadpan is homologous to mouse transcription factor HES-1 (GenBank Accession Number NP 032261 .1 ). Example 4: Genetic adipose pathway screen

Adipose (adp) 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, 1 993, Development 1 18:401 -41 5; 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. Animals receiving transgenic adipose activity under these conditions developed into adult flies with a visible change of eye phenotype. Virgins of the recombinant driver line were crossed with males of the mutant EP-line collection in single crosses and kept for preferably 1 2 to 1 5 days at 29°C. The offspring was checked for modifications of the eye phenotype (enhancement or suppression). Mutations changing the eye phenotype affect genes that modify adipose activity. The inventors have found that the fly line HD-EP(3)37224 is an enhancer of the eye-adp-Gal4 induced eye phenotype. This result is strongly suggesting an interaction of the Indy gene with adipose since the integration of HD-EP(3)37224 was found to be located at the Indy locus. This is supporting the function of Indy and homologous proteins in the regulation of the energy homeostasis.

Example 5: Expression of the polypeptides in mammalian (mouse) tissues

For analyzing the expression of the polypeptides disclosed in this invention in mammalian tissues, several mouse strains (preferrably mice strains C57BI/6J, C57BI/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 1 26-000) . 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. J Clin Invest 1 993 Jul;92(1 ):272-80, Mizuno et al. Proc Natl Acad Sci U S A 1 996 Apr 1 6;93(8):3434-8). 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.

RNA was isolated from mouse tissues using Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy Kit (for example, from Qiagen, Germany) in combination with an DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art. Total RNA was reverse transcribed (preferrably using Superscript II RNaseH- Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) and subjected to Taqman analysis preferrably using the Taqman 2xPCR Master Mix (from Applied Biosystems, Weiterstadt, Germany; the Mix contains according to the Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, passive reference Rox and optimized buffer components) on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems, Weiterstadt, Germany) .

Taqman analysis was performed preferrably using the following primer/probe pairs:

For the amplification of mouse DNA polymerase delta 1 (DNApol-deltaD (SEQ ID NO: 4): 5'- CAT GAC AAA ATG GAC TGC AAG G -3'; mouse

DNApol-deltal reverse primer (SEQ ID NO: 5) : 5'- AAC GAG GTT GGC CAC CAG -3'; Taqman probe (SEQ ID NO: 6): (5/6-FAM) CCT GGA GGC TGT GCG CAG GG (5/6-TAMRA)

For the amplification of mouse protein kinase C-inhibitor (mPKCl) (SEQ ID NO: 7) : 5'- GGC AAG AAA TGT GCT GCA GA -3'; mouse mPKCl reverse primer (SEQ ID NO: 8) : 5'- TCA TTC ACC ACC ATC CGG T -3'; Taqman probe (SEQ ID NO: 9) : (5/6-FAM) CTG GGC CTG AAG CGC GGG (5/6-TAMRA)

For the amplification of mouse myd88 (SEQ ID NO: 10): 5'- GTG TGT CCG ACC GTG ACG T -3'; mouse myd88 reverse primer (SEQ ID NO: 1 1 ): 5'- AAT TAG CTC GCT GGC AAT GG -3'; Taqman probe (SEQ ID NO: 12): (5/6-FAM) CTG CCG GGC ACC TGT GTC TGG (5/6-TAMRA)

In the figures, the relative RNA-expression is shown on the Y-axis. In Figures 8A and B, 1 2A and B, and 1 9A, the tissues tested are given on the X-axis. "WAT" refers to white adipose tissue, "BAT" refers to brown adipose tissue.

As shown in Figure 8A, real time PCR (Taqman) analysis of the expression of the DNApol deltal RNA in mammalian (mouse) tissues revealed that DNApol deltal is widely expressed. As shown in Figure 8B, the expression is up-regulated more than 2-fold in WAT and more than 2-fold down regulated in muscle of ob/ob mice. Ob/ob mice represent an excellent model for metabolic syndrome. The opposite direction of gene regulation seen in this genetic model of obesity is indicative for a role of DNApol deltal in tissues most relevant for the development of a metabolic syndrome.

As shown in Figure 1 2A, real time PCR (Taqman) analysis of the expression of the mPKCl RNA in mammalian (mouse) tissues revealed that mPKCl is ubiquitously expressed. As shown in Figure 1 2B the expression is up regulated nearly 3-fold in muscle and up to two fold in other tissues under high fat diet.

As shown in Figure 1 9A, real time PCR (Taqman) analysis of the expression of the myd88 RNA in mammalian (mouse) tissues revealed that myd88 is expressed with high levels in mainly the inner organs, eg. WAT, the gut, lung, spleen, kidney and BAT with the interesting exception of a comparable low expression level in liver. Also in all muscle and brain tissues analysed the expression is comparably lower. Myd88 seems to play a role in mainly the metabolism of the inner organs.

Example 6. 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 5. The hybridization and scanning was performed as described in the manufactures manual (see Affymetrix Technical Manual, 2002, obtained from Affmetrix, Santa Clara, USA) .

In Figure 4A and B, and 1 9B, the X-axis represents the time axis, shown are day 0 and day 1 2 of adipocyte differentiation. The Y-axis represents the flourescent intensity. The expression analysis (using Affymetrix GeneChips) of the thyrotropin-releasing hormone degrading ectoenzyme, alanyl (membrane) aminopeptidase, and Myd88 genes using primary human abdominal adipocycte differentiation clearly shows differential expression of human thyrotropin-releasing hormone degrading ectoenzyme, alanyl (membrane) aminopeptidase, and Myd88 genes in adipocytes. Several independent experiments were done. The experiments show that the thyrotropin-releasing hormone degrading ectoenzyme transcripts are most abundant at day 1 2 compared to day 0 during differentiation (see Figure 4A) . The experiments further show that the alanyl (membrane) aminopeptidase (see Figure 4B) and Myd88 (see Figure 1 9B) transcripts are most abundant at day 0 compared to day 1 2 during differentiation.

Thus, the thyrotropin-releasing hormone degrading ectoenzyme has to be significantly increased in order for the preadipocyctes to differentiate into mature adipocycte. The thyrotropin-releasing hormone degrading ectoenzyme in preadipocyctes has the potential to enhance adipose differentiation at a very early stage. The alanyl (membrane) aminopeptidase or Myd88 proteins have to be significantly decreased in order for the preadipocyctes to differentiate into mature adipocycte. Therefore, the alanyl (membrane) aminopeptidase and Myd88 proteins in preadipocyctes have the potential to inhibit adipose differentiation at a very early stage.

Therefore, the thyrotropin-releasing hormone degrading ectoenzyme, alanyl (membrane) aminopeptidase, or Myd88 proteins play an essential role in the regulation of human metabolism, in particular in the regulation of adipogenesis and thus, having importing functions in obesity, diabetes, and/or metabolic syndrome.

For the purpose of the present invention, it will understood by the person having average skill in the art that any combination of any feature mentioned throughout the specification is explicitly disclosed herewith.

Claims

1 . A pharmaceutical composition comprising a nucleic acid molecule of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor,

Indy, Myd88, Beachl , nuf, or deadpan gene family or a polypeptide encoded thereby and/or a functional fragment or an effector/ modulator of said nucleic acid molecule and/or a polypeptide encoded thereby, preferably together with pharmaceutically acceptable carriers, diluents and/or additives.

2. The composition of claim 1 , wherein the nucleic acid molecule is a vertebrate or insect slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan nucleic acid, particulary encoding a human protein as described in Table 1 , and/or a nucleic acid molecule which is complementary thereto, or a functional fragment thereof or a variant thereof.

3. The composition of claim 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of

(a) a nucleic acid molecule encoding a polypeptide as shown in Table 1 and/or a nucleic acid molecule which is complementary thereto;

(b) a nucleic acid molecule which comprises or is the nucleic acid molecule as shown in Table 1 and/or a nucleic acid molecule which is complementary thereto;

(c) a nucleic acid molecule degenerate with as a result of the genetic code to the nucleic acid sequences as defined in (a) or (b), and/or a nucleic acid molecule which is complementary thereto;

(d) a nucleic acid molecule that hybridizes at 50°C in a solution containing 1 x SSC and 0.1 % SDS to a nucleic acid molecule as defined in claim 2 and/or a nucleic acid molecule which is complementary thereto;

(e) a nucleic acid molecule that encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to a human protein as described in Table 1 or as defined in claim 2;

(f) a nucleic acid molecule that differs from the nucleic acid molecule of (a) to (e) by mutation and wherein said mutation causes an alteration, deletion, duplication or premature stop in the encoded polypeptide.

4. The composition of any one of claims 1 -3, wherein the nucleic acid molecule is a DNA molecule, particularly a cDNA or a genomic DNA.

5. The composition of any one of claims 1 -4, wherein said nucleic acid encodes a polypeptide contributing to regulating the energy homeostasis and/or the metabolism of triglycerides.

6. The composition of any one of claims 1 -5, wherein said nucleic acid molecule is a recombinant nucleic acid molecule.

7. The composition of any one of claims 1 -6, wherein the nucleic acid molecule is a vector, particularly an expression vector.

8. The composition of any one of claims 1 -5, wherein the polypeptide is a recombinant polypeptide.

9. The composition of claim 8, wherein said recombinant polypeptide is a fusion polypeptide.

10. The composition of any one of claims 1 -7, wherein said nucleic acid molecule is selected from hybridization probes, primers and anti-sense oligonucleotides.

1 1 . The composition of any one of claims 1 -10 which is a diagnostic composition.

1 2. The composition of any one of claims 1 -1 0 which is a therapeutic composition.

1 3. The composition of any one of claims 1 -1 2 for the manufacture of an agent for detecting and/or verifying, for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones, in cells, cell masses, organs and/or subjects.

14. Use of a nucleic acid molecule of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88,

Beach l , nuf, or deadpan gene family or a polypeptide encoded thereby or a functional fragment or a variant of said nucleic acid molecule or said polypeptide and/or an effector/modulator of said nucleic acid or polypeptide for controlling the function of a gene and/or a gene product which is influenced and/or modified by a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous polypeptide.

1 5. Use of the nucleic acid molecule of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88,

Beachl , nuf, or deadpan gene family or a polypeptide encoded thereby or a functional fragment or a variant of said nucleic acid molecule or said polypeptide or use of an effector/modulator of said nucleic acid molecule or said polypeptide for identifying substances in vitro capable of interacting with with a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan homologous polypeptide.

1 6. A non-human transgenic animal exhibiting a modified expression of a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan homologous polypeptide.

1 7. The animal of claim 1 6, wherein the expression of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous polypeptide is increased and/or reduced.

1 8. A recombinant host cell exhibiting a modified expression of a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan homologous polypeptide.

1 9. The cell of claim 1 8 which is a human cell.

20. A method of identifying a (poly)peptide involved in the regulation of energy homeostasis and/or metabolism of triglycerides in a mammal comprising the steps of (a) contacting a collection of (poly)peptides with a slamdance,

DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan homologous polypeptide or a functional fragment thereof under conditions that allow binding of said (poly)peptides; (b) removing (poly)peptides which do not bind and (c) identifying (poly)peptides that bind to said slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan homologous polypeptide.

21. A method of screening for an agent which modulates/effects the interaction of a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan homologous polypeptide with a binding target, comprising the steps of

(a) incubating a mixture comprising (aa) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan homologous polypeptide, or a functional fragment thereof;

(ab) a binding target/agent of said slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy,

Myd88, Beachl, nuf, or deadpan homologous polypeptide or functional fragment thereof; and

(ac) a candidate agent under conditions whereby said slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy,

Myd88, Beachl, nuf, or deadpan polypeptide or functional fragment thereof specifically binds to said binding target at a reference affinity;

(b) detecting the binding affinity of said slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy,

Myd88, Beachl, nuf, or deadpan polypeptide or functional fragment thereof to said binding target to determine an affinity in the presence of the candidate agent; and

(c) determining a difference between affinity in the presence of the candidate agent and the reference affinity.

22. A method of screening for an agent which modulates/effects the activity of a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan homologous polypeptide comprising the steps of (a) incubating a mixture comprising

(aa) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan homologous polypeptide, or a functional fragment thereof, and (ab) a candidate agent under conditions whereby said slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan polypeptide or functional fragment thereof has a reference activity; (b) detecting the activity of said slamdance,

DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl, nuf, or deadpan polypeptide or functional fragment thereof to determine an activity in the presence of the candidate agent, and (c) determining a difference between activity in the presence of the candidate agent and reference activity.

23. A method of producing a composition comprising the (poly)peptide identified by the method of claim 20 or the agent identified by the method of claim 21 or 22 with a pharmaceutically acceptable carrier, diluent and/or additive.

24. The method of claim 23 wherein said composition is a pharmaceutical composition for preventing, alleviating or treating of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones.

25. Use of a (poly)peptide as identified by the method of claim 20 or of 5 an agent as identified by the method of claim 21 or 22 for the preparation of a pharmaceutical composition for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity, and/or diabetes, as well as related disorders such as eating disorder, cachexia, hypertension, o coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, or gallstones.

26. Use of a nucleic acid molecule as defined in any one of claims 1 to 6 or 10 for the preparation of a medicament for the treatment, 5 alleviation and/or prevention of metabolic diseases or dysfunctions, including obesity, diabetes, and/or metabolic syndrome.

27. Use of a polypeptide as defined in any one of claims 1 to 6, 8 or 9 for the preparation of a medicament for the treatment, alleviation o and/or prevention of metabolic diseases or dysfunctions, including obesity, diabetes, and/or metabolic syndrome.

28. Use of a vector as defined in claim 7 or the preparation of a medicament for the treatment, alleviation and/or prevention of 5 metabolic diseases or dysfunctions, including obesity, diabetes, and/or metabolic syndrome.

29. Use of a host cell as defined in claim 1 8 or 1 9 for the preparation of a medicament for the treatment, alleviation and/or prevention of 0 metabolic diseases or dysfunctions, including obesity, diabetes, and/or metabolic syndrome.

0. Use of a nucleic acid molecule of the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan family or of a functional fragment thereof for the production of a non-human transgenic animal which over- or under-expresses the slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan gene product.

1 . Kit comprising at least one of (a) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beachl , nuf, or deadpan nucleic acid molecule or a functional fragment thereof;

(b) a slamdance, DNA-polymerase-delta, Protein kinase C inhibitor, Indy, Myd88, Beach l , nuf, or deadpan amino acid molecule or a functional fragment thereof;

(c) a vector comprising the nucleic acid of (a);

(d) a host cell comprising the nucleic acid of (a) or the vector of (c);