WO2003061681A2

WO2003061681A2 - Proteins involved in the regulation of energy homeostasis and organelle metabolism

Info

Publication number: WO2003061681A2
Application number: PCT/EP2003/000738
Authority: WO
Inventors: Arnd Steuernagel; Andreas Molitor; Karsten Eulenberg; Günter BRÖNNER
Original assignee: DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
Priority date: 2002-01-25
Filing date: 2003-01-24
Publication date: 2003-07-31
Also published as: WO2003061681A3; AU2003214048A1

Abstract

This invention relates to the use of nucleic acid and amino acid sequences of Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1, escargot homolog, human KIAA1585 protein, CG11940 homolog, dappled homolog, CG11753 homolog, human KIAA0095 protein, formin-binding protein 21, and/or homologous proteins in pharmaceutical compositions, and to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation and thermogenesis.

Description

Proteins involved in the regulation of energy homeostasis and organelle metabolism

Description

This invention relates to the use of nucleic acid and amino acid sequences of Optic atrophy 1 protein (OPA1 ), comichon-Iike, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CGl 1940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 , or a homologous protein in pharmaceutical compositions, and to the use of these sequences and to the use of effectors thereof in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis and gallstones, and disorders related to ROS defence, such as diabetes mellitus and neurodegenerative disorders.

Mitochondria are the energy suppliers of animal cells. Most of the energy available from metabolising foodstuffs like carbohydrates, fats etc. is used to create a proton gradient across the inner mitochondrial membrane. This proton gradient drives the enzyme ATP synthetase that produces ATP, the cells major fuel substance (Mitchell P, Science 206, 1 979, 1 148-1 1 59) . In the mitochondria of brown adipose tissue exists a protein (Uncoupling Protein 1 ) that tunnels protons through the inner mitochondrial membrane (review in Klingenberg et al., 1 999, Biochim. Biophys. Acta, 141 5(2):271 -96). The energy stored in the proton gradient is thereby released as heat and not used for ATP synthesis. When the energy intake of an animal exceeds expenditure surplus energy can be stored as fat in adipose tissue. The generation of a proton leak across the inner mitochondrial membrane by the activation of uncoupling proteins would reduce caloric efficiency and thus avoid the accumulation of excess body fat (obesity) that is detrimental to the animals health. In human, however, brown adipose tissue is almost absent in adults. Therefore, UCP1 was not considered to be a major factor in the formation or prevention of human obesity. Recently, the discovery of further proteins of similar sequence (UCP2-UCP5) that are widely expressed in human tissues (e.g. white adipose tissue, muscle) made this members of the UCP family to important targets for pharmaceutical research (reviewed in Adams 2000, Nutr., 130(4) :71 1 -4) . Interestingly, and as reviewed in Ricquier, 2000, Biochem J. 345, 1 61 -1 79, further homologues have been identified, like, inter alia, the plant UCPs StUCP (from Solanum tuberculosum) and AtUCP (Arabidopsis thaliana). Although the in vivo function of these proteins is still unknown, the possibility to influence UCP activity would be a conceivable therapy for the treatment or prevention of obesity and related diseases.

There are several metabolic diseases of human and animal metabolism, e.g., 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 a still poorly understood human disease that becomes more and more relevant for western society. Obesity is defined as an excess of body fat, frequently resulting in a significant impairment of health. Besides severe risks of illness such as diabetes, hypertension and heart disease, individuals suffering from obesity are often isolated socially. Human obesity is strongly influenced by environmental and genetic factors, whereby the environmental influence is often a hurdle for the identification of (human) obesity genes. Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Obese individuals are particularly prone to ailments including: diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis and gallstones.

Hyperlipidemia and elevation of free fatty acids correlate clearly with the 'Metabolic Syndrome'. The concept of metabolic syndrome (syndrome x, insulin-resistance syndrome, deadly quartet) was first described 1 966 by Camus and reintroduced 1 988 by Reaven (Camus JP, 1 966, Rev Rhum Mai Osteoartic 33(1 ): 10-14; Reaven et al. 1 988, Diabetes, 37(1 2) : 1 595-1 607) . Today "metabolic syndrome" is commonly defined as clustering of cardiovascular risk factors like hypertension, abdominal obesity, high blood levels of triglycerides and fasting glucose as well as low blood levels of HDL cholesterol. Insulin resistance greatly increases the risk of developing the metabolic syndrome (Reaven, 2002, Circulation 106(3): 286-8 reviewed) . The metabolic syndrome often precedes the development of type II diabetes and cardiovascular disease (McCook, 2002, JAMA 288:2709-271 6) .

Obesity is not to be considered as a single disorder but a heterogeneous group of contitions with (potential) multiple causes. Obesity is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Koltermann, J. Clin. Invest 65, 1 980, 1 272-1 284) and a clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman, Nature 404, 2000, 635-643) .

Even if several candidate genes have been described which are supposed to influence the homeostatic system(s) that regulate body mass/weight, like leptin, VCPI, VCPL, 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. Mitochondria have a very specialized function in energy conversion and said function is reflected in their morphological structure, namely the distinct internal membrane. This internal membrane does not only provide the framework for electron-transport processes but also creates a large internal compartment in each organelle in which highly specialized enzymes are confined. Therefore, there is a strong relationship between mitochondrial energy metabolism and the biochemical/biophysical properties of these organelles.

The technical problem underlying the invention was to provide for means and methods for modulating the biological/biochemical activities of mitochondria and, thereby, modulating metabolic conditions in eukaryotic cells which influence energy expenditure, body temperature, thermogenesis, cellular metabolism to an excessive or deficient supply of substrate(s) in order to regulate the ATP level, the NADVNADH ratio, and/or superoxide production. The solution to this technical problem is achieved by providing the embodiments characterized in the claims.

As shown in the appended examples, this invention discloses genes that can suppress the eye defect induced by the activity of dUCPy. These genes are coding for cornichon (GadFly Accession Number CG5855), neuralized (GadFly Accession Number CG1 1988), dco (GadFly Accession

Number CG2048), kraken (GadFly Accession Number CG3943), escargot

(GadFly Accession Number CG3758), GadFly Accession Number CG1 1940, dappled (GadFly Accession Number CG1624), GadFly

Accession Number CG1 1753, GadFly Accession Number CG7262, GadFly

Accession Number CG4291 . In addition, as shown in the appended examples, this invention discloses genes that can enhance the eye defect induced by the activity of dUCPy. These genes are coding for GadFly Accession Number CG8479, Imp (GadFly Accession Number CG1691 ),

GadFly Accession Number CG831 1 , Gdh (GadFly Accession Number

CG5320), Sir2 (GadFly Accession Number CG5216), msI-2 (GadFly Accession Number CG3241 ). It is envisaged that mutations in one or several of these genes affect the activity of uncoupling proteins (UCPs) thereby leading to an altered mitochondrial activity. The present invention provides for specific genes involved in the regulation of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesteroiemia, dyslipidemia, osteoarthritis and gallstones and disorders related to ROS defence, such as diabetes mellitus and neurodegenerative disorders.

The term 'GenBank Accession number' relates to NCBI GenBank database entries (Benson et al, Nucleic Acids Res. 28, 2000, 1 5-1 8).

The Drosophila gene with GadFly Accession Number CG8479 encodes for a protein which is most homologous to human OPA1 , optic atrophy 1 (KIAA0567) protein (SEQ ID NO: 4; predicted coding nucleotide sequence; SEQ ID NO: 5; protein; GenBank Accession Number XP_039926.2) and to mouse large GTP binding protein (Accession Number BAB59000.1 ) . Dominant optic atrophy is the commonest form of inherited optic neuropathy.

The Drosophila gene with GadFly Accession Number CG5855 encodes for protein which is most homologous to human cornichon-like protein (SEQ ID NO: 6; predicted coding nucleotide sequence; SEQ ID NO:7; protein; GenBank Accession Number NP_005767) and to mouse gene Accession Number sp035372. Comichon, a transmembrane protein, has a crucial but so far undefined role in epidermal growth factor (EGF) signaling during Drosophila embryogenesis. Human comichon which is expressed in a variety of human tissues functions in similar signaling establishing vectorial re-localization and concentration of signaling events in T-cell activation (Utku, 1 999, Biochim Biophys Acta; 1 449(3) :203-1 0) . The Drosophila gene with GadFly Accession Number CG 1 691 encodes for a protein which most homologous to human IGF-II mRNA-binding protein 3 (SEQ ID NO: 8; predicted coding nucleotide sequence; SEQ ID NO: 9; protein; GenBank Accession Number NP_006538.1 ) and to mouse gene with GenBank Accession Number NP_034081 .1 . Human IGF (insulin growth factor)-ll mRNA binding proteins are major fetal growth factors implicated in rRNA localization and translational control vertebrate development.

The Drosophila gene neuralized (neur) with GadFly Accession Number CG 1 1 988 encodes for a protein which is homologous to human neuralized-like protein (GenBank Accession Number NP_004201 .1 for the protein (SEQ ID NO: 1 1 ), NM_004210 for the cDNA (SEQ ID NO: 10)) . The Drosophila neurogenic gene neuralized is expressed in precursors of larval and adult neurons, embryonic mesoderm and specific follicle cells in the ovary (Boulianne G.L. et al., 1 991 , EMBO J 1 0(10) :2975-2983). The protein neuralized is necessary for Notch activation. In Drosophila, neuralized encodes a peripheral membrane protein involved in delta signaling and endocytosis (Pavlopoulos E. et al., 2001 , Dev Cell 1 (6):807-81 6). Xenopus neuralized (Xneur) is a ubiquitin ligase that interacts with Xdelta 1 and regulates Notch signaling (Deblandre G.A. et ai, 2001 , Dev Cell 1 (6) :795-806) . XNeur plays a conserved role in Notch activation by regulating the cell surface levels of the Delta ligands via ubiquitination. h-neu (human neuralized) encodes a protein with strong homology to the Drosophila neuralized (D-neu) protein. The h-neu gene plays a role in determination of cell fate in the human central nervous system and may act as a tumor suppressor whose inactivation could be associated with malignant progression of astrocytic tumors (Nakamura H. et ai., 1 998, Oncogene 1 6(8): 1009-101 9) .

The Drosophila gene with GadFly Accession Number CG831 1 encodes for a protein, which is most homologous to human KIAA1094 protein (SEQ ID NO: 1 3; GenBank Accession Number NP_055723.1 for the protein (SEQ ID NO: 1 2, NM_01 4908 for the cDNA), which is a transmembrane protein located in the plasma membrane (Psortll prediction, 74%). No functional data have been published for this protein.

The casein kinase I (CKI) family of protein kinases is a group of highly related, ubiquitously expressed serine/threonine kinases found in all eukaryotic organisms from protozoa to man. (Vielhaber and Virshup, 2001 , IUBMB Life 51 (2):73-78) Recent advances in diverse fields, including developmental biology and chronobiology, have elucidated roles for CKI in regulating critical processes such as Wnt signaling, circadian rhythm, nuclear import, and Alzheimer's disease progression. Casein kinase I is a serine/threonine-specific protein kinase that constitutes most of the kinase activity in eukaryotic cells, where it is mainly localized in the nucleus, cytoplasm, and several membranes. The monomeric enzyme phosphorylates hierarchically a variety of substrates without the involvement of the second messenger in signal transduction.

Drosophila double-time (dbt) gene, which encodes a protein similar to vertebrate epsilon and delta isoforms of casein kinase I, is essential for circadian rhythmicity because it regulates the phosphorylation and stability of period (per) protein (Bao et ai. 2001 , J Neurosci 21 (1 8):71 1 7-26) . Lee et al have provided in vivo evidence that, in addition to casein kinase I epsilon, casein kinase I delta is a second clock relevant kinase (2001 , Cell 107(7) :855-67) . The human casein kinase I delta nucleotide sequence is shown in SEQ ID NO: 14; the amino acid sequence is shown in SEQ ID NO: 1 5. The human casein kinase I epsilon nucleotide sequence is shown in SEQ ID NO: 1 6; the amino acid sequence is shown in SEQ ID NO: 1 7.

The canonical Wnt-signaling pathway is critical for many aspects of development, and mutations in components of the Wnt pathway are carcinogenic. Sufficiency tests identified casein kinase I epsilon (CKIepsilon) as a positive component of the canonical Wnt/beta-catenin pathway, and necessity tests showed that CKIepsilon is required in vertebrates to transduce Wnt signals (McKay et al., 2001 , Dev Biol 235(2) :388-396). In addition to CKIepsilon, the CKI family includes several other isoforms (alpha, beta, gamma, and delta) and their role in Wnt sufficiency tests had not yet been clarified. All wild-type CKI isoforms activate Wnt signaling.

Casein kinase I delta (CKIdelta) and casein kinase I epsilon (CKIepsilon) have been implicated in the response to DNA damage, but the understanding of how these kinases are regulated remains incomplete. In vitro, these kinases rapidly autophosphorylate, predominantly on their carboxyl-terminal extensions, and this autophosphorylation markedly inhibits kinase activity (Cegielska et al., 1 998, J. Biol. Chem. 273: 1 357-1 364) .

Glutamate dehydrogenase (GDH) is an enzyme catalyzing the oxidative deamination of glutamate to alpha-ketoglutarate using NAD or NADP as cofactors. In mammalian brain, GDH is located predominantly in astrocytes, where it is involved in the metabolism of neurotransmitter glutamate (see, for example, Plaitakis and Zaganas, 2001 , J Neurosci Res 1 ;66(5) :899-908) . In human, GDH exists in two isoforms, encoded by the GLUD1 (referred to as housekeeping) and GLUD2 (referred to as nerve tissue-specific) genes which differ in their catalytic and allosteric properties. The housekeeping GDH is regulated primarily by GTP, the nerve tissue GDH activity depends largely on available ADP or L-leucine levels. Interestingly, the uncoupling protein - 1 (referred to as UCP-1 ) is also regulated by these nucleotides but adversly to the nerve tissue-specific GDH; ADP inactivates and GTP activates UCP-1 . The human glutamate dehydrogenase I nucleotide sequence is shown in SEQ ID NO: 1 8; the amino acid sequence is shown in SEQ ID NO: 1 9. The human glutamate dehydrogenase II nucleotide sequence is shown in SEQ ID NO: 20; the amino acid sequence is shown in SEQ ID NO: 21 .

Glutamate is the precursor of the inhibitory neurotransmittor GABA. Disruptions of glutamate metabolism have been implicated in clinical disorders, such as, for example congenital hyperinsulinism and pyridoxine-dependent seizures. The hyperinsulinism/hyperammonemia syndrome is a form of congenital hyperinsulinism in which children have hypoglycemia together with elevations of plasma ammonium levels. The disorder is caused by dominant mutations of the mitochondrial GDH, that impair sensitivity to the allosteric inhibitor GTP (see, for example, MacMuIlen et al., 2001 , J Clin Endocrinol Metab 86(4) : 1 782-7). Congenital hyperinsulinism is thus implicating a role of glutamate oxidation by GDH in beta-cell insulin secretion and in hepatic and CNS ammonia detoxification (see, for example, Kelly and Stanley, 2001 , Ment Retard Dev Disabil Res Rev 2001 ;7(4) :287-95).

Dietary-induced obesity in rats showed a stable, higher body weight than controls, and key enzymes of alpha-amino nitrogen metabolism, including glutamine synthetase and GDH, showed reduced activities in brown adipose tissue of obese rats (see, for example, Serra et al., 1 994, Biochem Mol Biol Int 32(6):1 1 73-1 188) . These adaptations in amino acid metabolism were dependent on the obese status of the rats.

The Drosophila gene kraken with GadFly Accession Number CG3943 encodes for a protein which is most homologous to protein encoded by a novel human gene mapping to chromosome 22 (SEQ ID NO:23; GenBank Accession Number CAC1 6804.1 for the protein, SEQ ID NO: 22; AL450314 for the cDNA) . No functional data are available for this protein.

The Drosophila gene with GadFly Accession Number CG521 6 encodes for Sir2 (also referred to as sirtuin) protein. Sir2 protein is most homologous to human Sirtuin 1 protein (SEQ ID NO: 24; predicted coding nucleotide sequence; SEQ ID NO:25; protein; GenBank Accession Number NP_036370) and to mouse Sirtuin 1 protein (GenBank Accession Number NPJD62786.1 ) . Sirtuins (silent mating type information regulation) are a large family of NAD-dependent deacetylase enzymes. These proteins are conserved from prokaryotes to eukaryotes, but most remain uncharacterized, including all seven human sirtuins (Grotzinger et al., 2001 , J Biol Chem 276(42) :38837-43).

The Drosophila esg gene with GadFly Accession Number CG3758 encodes for escargot (also referred to as Esgarot) protein, a specific RNA polymerase II transcription factor which is a component of the nucleus. Drosophila esg is a key regulator of cell adhesion and motility in tracheal morphogenesis. Esg is most homologous to human hypothetical protein, similar to gonadotropin protein (SEQ ID NO: 26; predicted coding nucleotide sequence; SEQ ID NO:27; protein; GenBank Accession Number XP_030528) and to mouse gene with the Accession Number NP_035545. No functional data are available for the mammalian proteins.

The Drosophila gene with GadFly Accession Number CG3241 encodes for msl-2 (male specific lethal 2) protein. Msl-2 protein is most homologous to human hypothetical KIAA1585 protein (SEQ ID NO: 28; predicted coding nucleotide sequence; SEQ ID NO:29; protein; GenBank Accession Number AB046805) and to mouse protein with GenBank Accession Number BF471 233. The Drosophila male-specific lethal (MSL) genes regulate transcription from the male X chromosome in a dosage compensation pathway that equalizes X-linked gene expression in males and females. Drosophila Msl-2 is part of a protein complex that regulates gene activities by altering the chromatin structure (Kageyama et al., 2001 , EMBO J 20(9) :2236-45). Zhou et al. described that the Drosophila male-specific lethal 2 (msl-2) gene is involved in dosage compensation (1 995, EMBO J 14(1 2) :2884-95) . The encoded protein (MSL-2) has a RING finger (C3HC4 zinc finger) and a metallothionein-like domain and undergoes sex-specific regulation. The protein Sex-lethal (SXL) controls dosage compensation in Drosophila by inhibiting splicing and subsequently translation of male-specific-lethal-2 (msl-2) transcripts (Forch et al., 2001 , RNA 7(9): 1 1 85-91 ) .

The Drosophila gene with GadFly Accession Number CG1 1 940 encodes for alsin protein. Alsin protein is most homologous to human Alsin aslrcr9 protein (SEQ ID NO: 30; predicted coding nucleotide sequence; SEQ ID NO:31 ; protein; GenBank Accession Number XP_028059.1 ) and to mouse Alsin protein (GenBank Accession Number AAH03991 ). Alsin, a protein with three guanine-nucleotide (GTP) exchange factor domains, has been identified to be responsible for amytrophic lateral sclerosis which is a neurodegenerative condition that affects large motor neurons of the central nervous system.

The Drosophila gene dappled (dpld) with GadFly Accession Number CG1624 encodes for a protein which is most homologous to human protein (SEQ ID NO:33; GenBank Accession Number XP_067369.1 for the protein, SEQ ID NO: 32; XM_067369 for the cDNA), similar to C1 2C8.3b.p. No functional data are available for the human protein. C1 2C8.3b.p is a Caenorhabditis elegans protein with GenBank Accession Number NP_492488.

The Drosophila gene with GadFly Accession Number CG1 1 753 encodes for a protein which is most homologous to human protein (SEQ ID NO:35; GenBank Accession Number XP_029849.1 for the protein, SEQ ID NO: 34; XM_029849 for the cDNA), encoded by a gene similar to mouse RIKEN cDNA 261 0042014 gene (GenBank Accession Number NM_025575) . No functional data are available for these proteins. The Drosophila gene with GadFly Accession Number CG7262 encodes for a protein which is most homologous to human KIAA0095 protein (SEQ ID NO:37; GenBank Accession Number NP_055484.1 for the protein; SEQ ID NO: 36; NM_014669 for the cDNA (Nagase et al., 1 995, DNA Res. 2 ( 1 ) :37-43); GenBank Accession Number AX306779, Sequence 1 2 from Patent WO001 8961 ) . No functional data are available for this protein. The KIAA0095 gene is related to S. cerevisia NIC96 gene (GenBank Accession Number P34077) which is part of the nucleoporin complex and is required for protein transport in the nucleus. The KIAA0095 protein also shows homologies to Xenopus An4a protein (GenBank Accession Number AAB49669) and Zebrafish hi4 "dead eye" protein (GenBank Accession Number AAB61 1 37).

The Drosophila gene with GadFly Accession Number CG4291 encodes for a protein which is most homologous to human WW domain binding protein

4 (formin binding protein 21 (FBP21 ); SEQ ID NO: 38; predicted coding nucleotide sequence; SEQ ID NO:39; protein; GenBank Accession Number

XP_049375) and to mouse WW domain binding protein 4 (formin binding protein 21 ) gene with the Accession Number NP_061 235. The WW domain is a protein module with two highly conserved tryptophans that binds proline-rich peptide motifs in vitro. The Drosophila gene CG4291 encodes a small nuclear ribonucleoprotein involved in mRNA splicing which is a component of the snRNP U2e. Human FBP21 is present in highly purified spliceosomal complex A, is associated with U2 snRNPs, and colocalizes with splicing factors in nuclear speckle domains. FBP21 may play a role in cross-intron bridging of snRNPs in the mammalian A complex.

So far, it has not been described that Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-Iike, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, formin-binding protein 21 , or a homologous protein is involved in the regulation of body-weight and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, and gallstones, and disorders related to ROS defence, such as diabetes mellitus and neuro- degenerative disorders, and thus, no functions in metabolic diseases and other diseases as listed aboved have been discussed in the prior art.

In this invention we demonstrate that the correct gene dose of the Drosophila melanogaster homologues of SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 is essential for maintenance of energy homeostasis and for the activity of mitochondral uncoupling protein. A genetic screen was designed to identify factors that modulate activity of uncoupling protein. We discovered that mutation of these genes caused a reduction of the activity of uncoupling protein, thereby leading to an altered mitochondrial activity. Thus, the invention is also based on the finding that homologues of the above Drosophila genes, particularly the human homologues as described in SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38 are contributing to membrane stability and/or function of organelles, preferably mitochondria and thus represent targets for diagnostic and/or therapeutic applications in medicine, particularly in human medicine.

The function of the proteins of the invention in metabolic disorders is further validated by data obtained from additional screens. For example, the content of triglycerides and glycogen of a pool of flies with the same genotype was analyzed using a triglyceride and a glycogen assay. Additionally expression profiling studies (see Examples for more detail) confirm the particular relevance of the proteins of the invention as regulators of energy metabolism in mammals. 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.

Polynucleotides encoding proteins as shown in SEQ ID NO:5, 7, 9, 1 1 , 13, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39 are suitable to investigate, to treat, to prevent or to diagnose diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders as described above. Molecules related to SEQ ID NO:5, 7, 9, 1 1 , 13, 15, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 provide new compositions useful in diagnosis, treatment, and prognosis of diseases and disorders related to body-weight regulation and thermogenesis as described above.

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, which 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, which 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 the proteins as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39 and homologous proteins are directly or indirectly involved in membrane stability and/or function of organelles, in particular mitochondria, and polynucleotides, which identify and encode the proteins are 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 and effectors thereof, e.g. antibodies, aptamers or other receptors recognizing the nucleic acid molecules or polypeptides encoded thereby in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis and gallstones and disorders related to ROS defence, such as diabetes mellitus and neurodegenerative disorders.

The invention relates to a pharmaceutical composition comprising a nucleic acid molecule of the Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, or formin-binding protein 21 gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or a polypeptide encoded thereby together with pharmaceutically acceptable carriers, diluents and/or adjuvants. Proteins as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39 and homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds. Particularly preferred are human nucleic acid molecules as shown in SEQ ID NO:4, 6, 8, 1 0, 1 2, 1 4, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 (Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG 1 1753 homolog, KIAA0095 protein, formin-binding protein 21 , and homologous proteins), i.e. nucleic acids encoding a the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39.

The invention particularly relates to a nucleic acid molecule encoding a polypeptide contributing to regulating the energy homeostasis, and/or contributing to membrane stability and/or function of organelles, wherein said nucleic acid molecule comprises

(a) a nucleotide sequence as shown in SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 and/or a nucleotide sequence complementary thereto,

(b) a nucleotide sequence which hybridizes at 50°C in a solution containing 1 x SSC to a nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 and/or a nucleic acid molecule complementary thereto,

(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 as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39;

(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 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 at least 1 5 bases, preferably at least 20 bases, more preferably at least 25 bases and most preferably at least 50 bases.

The present invention discloses that the proteins as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39 and homologous proteins are directly or indirectly involved in membrane stability and/or function of organelles, in particular mitochondria, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention describes the use of these compositions for the diagnosis, study, prevention, or treatment of diseases and disorders related to body-weight regulation and thermogenesis as described above.

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 and the modified expression of a fly gene can identify the association of the human ortholog with the particular human disease. Accordingly, the present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity. To find genes with novel functions in energy homeostasis, metabolism, and obesity, a functional genetic screen was performed with the model organism Drosophila melanogaster (Meigen). One resource for screening was a proprietary Drosophila melanogaster stock collection of EP-lines. The P-vector of this collection has Gal4-UAS-binding sites fused to a basal promoter that can transcribe adjacent genomic Drosophila sequences upon binding of GaI4 to UAS-sites. This enables the EP-line collection for overexpression of endogenous flanking gene sequences. In addition, without activation of the UAS-sites, integration of the EP-element into the gene is likely to cause a reduction of gene activity, and allows determining its function by evaluating the loss-of-function phenotype.

It is preferred that the nucleic acid molecule encodes a polypeptide contributing to membrane stability and/or function of organelles and represents a protein of Drosophila which has been found to be able to modify UCPs, see also appended examples. As demonstrated in the appended examples, the here described polypeptide (and encoding nucleic acid molecule) was able to modify, e.g. suppress or enhance a specific eye phenotype in Drosophila which was due to the overexpression of the Drosophila melanogaster gene dUCPy. The overexpression of dUCPy (with homology to human UCPs) in the compound eye of Drosophila led to a clearly visible eye defect which can be used as a 'read-out' for a genetical 'modifier Screen'.

In said "modifier screen" thousands of different genes are mutagenized to modify their expression in the eye. Should one of the mutagenized genes interact with dUCPy and modify its activity an enhancement or suppression of the eye defect will occur. Since such flies are easily to discern they can be selected to isolate the interacting gene. As shown in the appended examples, genes were deduced that can enhance or suppress the eye defect induced by the activity of dUCPy. The identified genes have high homologies to the human proteins shown in SEQ ID NO:5, 7, 9, 1 1 , 13, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39, as described above. It is envisaged that mutations in the herein described proteins (and corresponding genes) lead to phenotypic and/or physiological chances which may comprise a modified and altered mitochondrial activity. This, in turn, may lead to, inter alia, an altered energy metabolism, altered thermogenesis and/or altered energy homeostasis.

As shown in the appended examples, new genes were found that can enhance or suppress the eye defect induced by the activity of dUCPy. The genes suppressing the eye defect are comichon (GadFly Accession Number CG5855), neuralized (GadFly Accession Number CG 1 1 988), dco (GadFly Accession Number CG2048), kraken (GadFly Accession Number CG3943), escargot (GadFly Accession Number CG3758), GadFly Accession Number CG 1 1 940, dappled (GadFly Accession Number CG 1 624), GadFly Accession Number CG1 1 753, GadFly Accession Number CG7262, and GadFly Accession Number CG4291 ; and the genes enhancing the eye defect induced by UCP activity are GadFly Accession Number CG8479, Imp (GadFly Accession Number CG 1 691 ), GadFly Accession Number CG831 1 , Gdh (GadFly Accession Number CG5320), Sir2 (GadFly Accession Number CG521 6), and msl-2 (GadFly Accession Number CG3241 ) . The invention also encompasses polynucleotides that encode the proteins as shown in SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, and 39 or homologous proteins. Accordingly, any nucleic acid sequence, which encodes the amino acid sequences of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 can be used to generate recombinant molecules that express the corresponding mRNA and protein.

In an additional screen using Drosophila mutants, the content of triglycerides and glycogen was analyzed after feeding for six days using a triglyceride and a glycogen assay. Male flies homozygous for the integration of vectors for Drosophila lines HD-EP20292, HD-35207, HD-EP20506, HD-EP2081 7, HD-EP26792, HD-EP25097, and HD-EP10934 were analyzed in assays measuring the triglyceride and glycogen contents of these flies, illustrated in more detail in the EXAMPLES section. The results of the triglyceride and glycogen content analysis are shown in FIGURES 6, 1 6, 20, 22, and 23D.

Expression profiling studies (see Examples for more detail) confirm the particular relevance of the proteins of the invention as regulators of energy metabolism in mammals. OPA1 is expressed in different mammalian tissues, showing 2 to 3 fold higher levels of expression in BAT, hypothalamus, brain, muscle and heart when compared to other tissues (see FIGURE 4A) . BAT, brain, muscle and heart represent tissues with the major catabolic activity in the body. The high experession levels of OPA-1 in these tissues indicate, that OPA-1 is involved in the metabolism of tissues relevant for the metabolic syndrome. Neuralized-like is highly expressed in muscle, hypothalamus, brain and testis (see FIGURE 9). The high expression levels in muscle when compared to other tissues is indicative for a role in the metabolism in one of the major catabolic tissues of the body. The CG831 1 homologous protein shows highest expression levels in brown adipose tissue compared to several other mouse tissues and organs (see FIGURE 1 1 ) .

In a particular embodiment, the invention encompasses a polynucleotide comprising the nucleic acid sequence of SEQ ID N0:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38. 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 of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39, 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. These combinations are made in accordance with the standard triplet genetic code as applied to the nucleotide sequences as shown in SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38, and all such variations are to be considered as being specifically disclosed. Although nucleotide sequences which encode the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 and variants thereof are preferably capable of hybridising to the nucleotide sequences of the naturally occurring nucleic acids of SEQ ID N0:4, 6, 8, 1 0, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, formin-binding protein 21 , or homologous proteins or their derivatives possessing a substantially different codon usage. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilised by the host. Other reasons for substantially altering the nucleotide sequence without altering the encoded amino acid sequences include the production of RNA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequences. The invention also encompasses production of DNA sequences, or portions thereof, which encode the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 and derivatives, entirely by synthetic chemistry. After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art at the time of the filing of this application. Moreover, synthetic chemistry may be used to introduce mutations into the sequence in any portion thereof.

Also encompassed by the invention are polynucleotide sequences that are capable of hybridising to the claimed nucleotide sequences, under various conditions of stringency. Hybridisation 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 of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 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 a functionally equivalent Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1 094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, formin-binding protein 21 , or homologous protein. 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 is at least partially retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine. Furthermore, the invention relates to peptide fragments of the proteins or derivatives thereof such as cyclic peptides, retro-inverso peptides or peptide mimetics having a length of at least 4, preferably at least 6 and up to 50 amino acids.

Also included within the scope of the present invention are alleles of the genes encoding the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39. 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 SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 may be extended utilising 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. 2:318-322). Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia, T. et al. (1 988) Nucleic Acids Res. 1 6:81 86) . Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (PCR Methods Applic. 1 : 1 1 1 -1 1 9) . Another method which may be used to retrieve unknown sequences is that of Parker, J. D. et al. (1 991 ; 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 another embodiment of the invention, polynucleotide sequences or fragments thereof which encode the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules for the expression of the proteins in appropriate host cells.

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 the appropriate transcriptional and translational control elements. Regulatory elements include for example a promoter, an initiation codon, a stop codon, a mRNA stability regulatory element, and a polyadenylation signal. Expression of a polynucleotide can be assured by (i) constitutive promoters such as the Cytomegalovirus (CMV) promoter/enhancer region, (ii) tissue specific promoters such as the insulin promoter (see, Soria et al., 2000, Diabetes 49: 1 57), SOX2 gene promotor (see Li et al., (1998) Curr. Biol. 8:971 -4), Msi-1 promotor (see Sakakibara et ai., (1 997) J. Neuroscience 17:8300-8312), alpha-cardia myosin heavy chain promotor or human atrial natriuretic factor promotor (Klug et al., ( 1 996) J. clin. Invest 98:21 6-24; Wu et al., (1 989) J. Biol. Chem. 264:6472-79) or (iii) inducible promoters such as the tetracycline inducible system. Expression vectors can also contain a selection agent or marker gene that confers antibiotic resistance such as the neomycin, hygromycin or puromycin resistance genes. 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.

In another 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.

In order to express biologically active proteins, the nucleotide sequences coding therefor or for 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 utilised to contain and express a sequence encoding the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g. baculovirus, adenovirus, adeno-associated virus, lentivirus, retrovirus); 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 presence of polynucleotide sequences encoding the protein can be detected by DNA-DNA or DNA-RNA hybridisation and/or amplification using probes or portions or fragments of polynucleotides encoding the protein. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences encoding the protein to detect transformants containing DNA or RNA encoding the 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.

The presence of proteins of the invention in a sample can be determined by immunological methods or activity measurement. A variety of protocols for detecting and measuring the expression of the proteins using either polyclonal or monoclonal antibodies specific for the protein or reagents for determining protein activity 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 utilising 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. (1 983; 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 labelled hybridisation or PCR probes for detecting sequences related to polynucleotides encoding the protein include oligo-labelling, nick translation, end-labelling or PCR amplification using a labelled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

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 synthesise RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labelled 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.

The nucleic acids encoding the proteins of the invention can be used to generate transgenic animal or site specific gene modifications in cell lines. Transgenic animals may be made through homologous recombination, where the normal locus of the genes encoding the proteins of the invention is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retrovirusses and other animal virusses, YACs, and the like. The modified cells or animal are useful in the study of the function and regulation of the proteins of the invention. For example, a series of small deletions and/or substitutions may be made in the genes that encode the proteins of the invention to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.

Specific constructs of interest include anti-sense molecules, which will block the expression of the proteins of the invention, or expression of dominant negative mutations. A detectable marker, such as for example lac-Z, may be introduced in the locus of the genes of the invention, where upregulation of expression of the genes of the invention will result in an easily detected change in phenotype.

One may also provide for expression of the genes of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development. In addition, by providing expression of the proteins of the invention in cells in which they are not normally produced, one can induce changes in cell behavior.

DNA constructs for homologous recombination will comprise at least portions of the genes of the invention with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration need not include regions of homology to mediate recombination. Conveniently, markers for positive and/or negative selection are included. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For 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 or grown in presence of leukemia inhibiting factor (LIF). When ES or 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 detected 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 manipulation 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 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.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids and proteins of the invention and effector molecules thereof are useful in diagnostic and therapeutic applications implicated, for example but not limited to, in metabolic disorders like obesity, diabetes, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions, arteriosclerosis, coronary artery disease (CAD), and other diseases and disorders. Hence, diagnostic and therapeutic uses for the proteins of the invention, e.g. the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 are, for example but not limited to, the following: (i) protein therapeutic, (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 are useful in diagnostic and therapeutic applications implicated in various diseases and disorders described below and/or other pathologies and disorders. 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 like obesity, diabetes, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions, arteriosclerosis, coronary artery disease (CAD), and other diseases and disorders.

The nucleic acid encoding the proteins of the invention, or 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 the protein may be used directly as an antagonist, or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein. The antibodies may be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimerical, single chain, Fab fragments, and fragments produced by a Fab expression library. Neutralizing 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 immunised 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. Such adjuvants include, but are not limited to, Freund's, mineral gels such as aluminium hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.

Among adjuvants used in human, BCG (Bacille Calmette-Guerin) and

Corynebacterium parvum are especially preferable. It is preferred that the peptides, fragments, or oligopeptides used to induce antibodies to proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 15, 17, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35,

37, or 39 have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.

Monoclonal antibodies to the protein may be prepared using any technique which 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 (Kδhler, 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 ai. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62: 109-120). 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 31 2:604-608; 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 protein-specific single chain antibodies. 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 1 20-3) . 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 ai. (1 991 ) Nature 349:293-299).

Antibody fragments, which contain specific binding sites for the protein, 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: 1275-1 281 ).

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 epitopes is preferred, but a competitive binding assay may also be employed (Maddox, supra) .

In another embodiment of the invention, the polynucleotides encoding the proteins of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39, or effector nucleic acids such as aptamers, antisense molecules, ribozymes or RNAi molecules may be used for therapeutic purposes. In one aspect, aptamers, i.e. nucleic acid molecules capable of binding to a target protein and modulating its activity may be obtained by known methods, e.g. by affinity selection of combinatorial nucleic acid libraries.

In a further aspect, antisense to the polynucleotide encoding the protein 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 the protein. Thus, antisense molecules may be used to modulate the 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 protein. 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 gene encoding the protein. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra) . Genes encoding the protein and can be turned off by transforming a cell or tissue with expression vectors which express high levels of polynucleotide or fragment thereof which encodes the protein. 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, DNA, RNA, or nucleic acid analogues such as PNA, to the control regions of the gene encoding the 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 ai. (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 catalyse the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridisation 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 catalyse endonucleolytic cleavage of sequences encoding the 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 hybridisation with complementary oligonucleotides using ribonuclease protection assays.

Nucleic acid effector molecules such as 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 synthesising 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 the 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 synthesise 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 recognised 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 comprise the protein, antibodies to the protein, mimetics, agonists, antagonists, or inhibitors of the protein. The compositions may be administered alone or in combination with at least one other agent, such as stabilising 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 utilised 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.).

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 does 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 the nucleic acids or the proteins or fragments thereof or antibodies against the protein which are effective against 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 being employed, the 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 100,000 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 the protein may be used for the diagnosis of conditions or diseases characterised by or associated with over- or underexpression of the protein, or in assays to monitor patients being treated with the 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 for the protein include methods, which utilise 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 labelled 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 the protein are known in the art and provide a basis for diagnosing altered or abnormal levels of protein expression. Normal or standard values for protein expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody 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 the 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 encoding the 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 expression of the protein 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 gene expression levels during therapeutic intervention.

In one aspect, hybridisation with probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding the protein and closely related molecules, may be used to identify nucleic acid sequences which encode the protein. The specificity of the probe, whether it is made from a highly specific region, e.g. unique nucleotides in the 5' regulatory region, or a less specific region, e.g. especially in the 3' coding region, and the stringency of the hybridisation or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding the protein, alleles, or related sequences. Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the protein-encoding sequences. The hybridisation probes of the subject invention may be DNA or RNA and are preferably derived from the nucleotide sequence of SEQ ID N0:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38, or from the genomic sequence including promoter, enhancer elements, and introns of the naturally occurring gene. Means for producing specific hybridisation probes for DNAs encoding the protein include the cloning of nucleic acid sequences encoding protein derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, commercially available, and may be used to synthesise RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labelled nucleotides. Hybridisation probes may be labelled 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 encoding the protein may be used for the diagnosis of conditions or diseases, which are associated with expression of the protein. Examples of such conditions or diseases include, but are not limited to, pancreatic diseases and disorders, including diabetes.

Polynucleotide sequences encoding the protein may also be used to monitor the progress of patients receiving treatment for pancreatic diseases and disorders, including diabetes. The polynucleotide sequences encoding the protein 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 utilising 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 encoding the protein may be useful in assays that detect activation or induction of various metabolic diseases and disorders, including obesity, diabetes, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions, arteriosclerosis, coronary artery disease (CAD), disorders related to ROS production, and neurodegenerative diseases. The nucleotide sequences encoding the protein may be labelled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridisation 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 target nucleotide sequences 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 disease associated with expression of the sequence of SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38, 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, which encodes the protein, or a fragment thereof, under conditions suitable for hybridisation or amplification. Standard hybridisation 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, hybridisation 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 and disorders, including obesity, diabetes, eating disorders, wasting syndromes (cachexia), pancreatic dysfunctions, arteriosclerosis, coronary artery disease (CAD), disorders related to ROS production, and neurodegenerative diseases 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 pancreatic diseases and disorders.

Additional diagnostic uses for oligonucleotides designed from the sequences of SEQ ID NO:4, 6, 8, 10, 1 2, 1 4, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, or 38 may involve the use of PCR. Such oligomers may be chemically synthesised, generated enzymatically, or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'.fwdarw.3') and another with antisense (3'.rarw.5'), employed under optimised 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 gene expression include radiolabelling 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 ai. (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 encode the protein, may also be used to generate hybridisation 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. (1 991 ) Trends Genet. 7: 149-1 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 1 994 Genome Issue of Science (265: 1 981 f). Correlation between the location of the gene encoding SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39 on a physical chromosomal map and a specific disease, or predisposition to a specific disease, may help to delimit the region of DNA associated with that genetic disease.

The nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals. In situ hybridisation of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localised 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, 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 in any of a variety of drug screening techniques. One can identify effectors, e.g. receptors, enzymes, proteins, ligands, or substrates that bind to, modulate or mimic the action of one or more of the proteins of the invention. The protein or fragment thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between the protein and the agent tested, may be measured. Agents could also, either directly or indirectly, influence the activity of the proteins of the invention.

Candidate agents may also be found in kinase assays where a kinase substrate such as a protein or a peptide, 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, 1998, Antibodies, CSH Lab Press, NY. If the analyte molecule is not an antibody, it may be detected by virtue of its chemical, physical or immunological properties, being endogenously associated with it or engineered to it. 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. (1 999) Prog. Biomed. Optics (SPIE) 3603:261 ; Parker, G. J. et al. (2000) J. Biomol. Screen. 5:77-88; Wu, P. et al. (1 997) 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 know to those skilled in the art as indirect fluorescence polarization.

In vivo, the enzymatic kinase activity of the unmodified polypeptides of casein kinase delta and epsilon and dolichol kinase (CG831 1 homologous protein) towards a substrate can be enhanced by appropriate stimuli, triggering the phosphorylation of casein kinase delta and epsilon and dolichol kinase. This 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 activated casein kinase delta and epsilon and dolichol kinase, may it be an organism, a tissue, a culture of cells or cell-free environment, by exogenously applying this stimulus or by mimicking this stimulus by a variety of the techniques, some of them described further below. A system containing activated casein kinase delta and epsilon and dolichol kinase may be produced (i) for the purpose of diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, and gallstones.

In addition activity of Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, or formin-binding protein 21 against its physiological substrate(s) or derivatives thereof could be measured in cell-based assays. Agents may also interfere with posttranslational modifications of the protein, 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, 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 the interacting protein to the protein of the invention, 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 Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, or formin-binding protein 21 .

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.

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 the protein of the invention large numbers of different small test compounds are synthesised on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the protein, 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 immobilise it on a solid support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding the protein 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. In additional embodiments, the nucleotide sequences which encode the protein may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.

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, 1 998, 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 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 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). ES or embryonic cells may be transfected and can then be used to produce transgenic animals. After transfection, the ES cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be selected by employing a selection medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination. Colonies that are positive may then be used for embryo manipulation and morula aggregation. Briefly, morulae are obtained from 4 to 6 week old superovulated females, the Zona Pellucida is removed and the morulae are put into small depressions of a tissue culture dish. The ES cells are trypsinized, and the modified cells are placed into the depression closely to the morulae. On the following day the aggregates are transfered into the uterine horns of pseudopregnant females. Females are then allowed to go to term. Chimeric offsprings can be readily detected by a change in coat color and are subsequently screened for the transmission of the mutation into the next generation (F1 -generation). Offspring of the F1 -generation are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogenic or congenic grafts or transplants, or in vitro culture. The transgenic animals may be any non-human mammal, such as laboratory animal, domestic animals, etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and others. The transgenic animals may be used in functional studies, drug screening, and other applications and are useful in the study of the function and regulation of the proteins of the invention in vivo.

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

(a) an Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, or formin-binding protein 21 nucleic acid molecule or a fragment thereof;

(b) an Optic atrophy 1 protein (OPA1 ), cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1 753 homolog, K1AA0095 protein, or formin-binding protein 21 amino acid molecule or a 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 (b); (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 against 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.

Figures

Figure 1 . Drosophila UCPy Figure 1 A. Full length cDNA sequence of Drosophila UCPy (SEQ ID NO: 1 ) Figure 1 B. Open reading frame of the deduced protein of Drosophila UCPy (SEQ ID NO:2) . Figure 1 C. Amino acid sequence of Drosophila UCPy (SEQ ID NO:3) .

Figure 2. The human homolog of CG8479 Figure 2A. Blastn search result for CG8479

Figure 2B. Predicted coding nucleotide sequence for the human homolog of CG8479 (SEQ ID NO:4) Figure 2C. Predicted amino acid sequence for the human homolog of CG8479 (SEQ ID NO:5) .

Figure 3. Multiple Sequence alignment (ClustllW 1 .83) of Drosophila protein with Gadfly Accession Number CG8479 (referred to as CG8479 Dm), mouse (XP 4801 6 Mm), and human (OPA1 -5 Hs) homologs. The sequences are shown in the one letter code. Figure 4. Expression of OPA1 in mammalian tissues

Figure 4A. Real time PCR analysis of OPA1 expression in wildtype mouse tissues.

Figure 5. The human homolog of CG5855 (comichon) Figure 5A. Blastn search result for CG5855

Figure 5B. Predicted coding nucleotide sequence for the human homolog (SEQ ID NO:6)

Figure 5C. Predicted amino acid sequence for the human homolog of CG5855 (SEQ ID NO:7) .

Figure 6. Energy storage metabolites (ESM; triglyceride (TG) and glycogen) content of a comichon (Gadfly Accession Number CG5855) mutant. Shown is the change of triglyceride content of HD-EP20292 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1 ) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2). Also shown is the change of glycogen content of HD-EP20292 flies caused by integration of the P-vector into the annotated transcription unit (column 5) in comparison to controls (referred to as 'control (glycogen)' column 4) .

Figure 7. The human homolog of CG1691 (Imp) Figure 7A. Blastn search result for CG1 691

Figure 7B. Predicted coding nucleotide sequence for the human homolog (SEQ ID NO:8)

Figure 7C. Predicted amino acid sequence for the human homolog of CG 1 691 (SEQ ID NO:9) .

Figure 8. Human homolog of CG1 1 988

Figure 8A. BlastP search result for CG1 1 988 (neuralized) Figure 8B. Predicted coding nucleotide sequence for the human homolog of CG1 1 988; length of the sequence in base pairs (SEQ ID NO: 10) Figure 8C. Predicted amino acid sequence for the human homolog of CG 1 1 988; length of the sequence in amino acids (SEQ ID NO: 1 1 ) .

Figure 9. Expression of neuralized-like in mammalian tissues - Real time PCR analysis of neuralized-like expression in wildtype mouse tissues (DCT Pancreas = 23,34) .

Figure 10. The human homolog of CG831 1 Figure 1 0A. BlastP search result for CG831 1

Figure 10B. Predicted coding sequence for the human homolog; length of the sequence in base pairs, referred to as bp. (SEQ ID NO:1 2) Figure 1 0C. Predicted amino acid sequence for the human homolog of CG831 1 ; length of the sequence in amino acids, referred to as aa. (SEQ ID NO: 1 3) Figure 10D. Transmembrane prediction for the human homolog protein.

Figure 1 1 . Expression of CG831 1 homolog in mammalian tissues - Real-time PCR analysis of the murine CG831 1 homolog protein shows strongest expression in brown adipose tissue.

Figure 1 2. A human homolog of CG2048 (dco)

Figure 1 2A. BlastP search result for CG2048 Figure 12B. Predicted coding nucleotide sequence for the human homolog,

Casein Kinase 1 , delta; length of the sequence in base pairs, referred to as bp. (SEQ ID NO: 1 4)

Figure 1 2C. Predicted amino acid sequence for the human homolog of

CG2048; length of the sequence in amino acids, referred to as aa. (SEQ ID NO: 1 5) .

Figure 13. A human homolog of CG2048 (dco) Figure 1 3A. BlastP search result for CG2048

Figure 13B. Predicted coding nucleotide sequence for the human homolog, Casein Kinase 1 , epsilon; length of the sequence in base pairs, referred to as bp. (SEQ ID NO: 1 6) Figure 1 3C. Predicted amino acid sequence for the human homolog of CG2048; length of the sequence in amino acids, referred to as aa. (SEQ ID NO: 17)

Figure 1 3D. ClustaW alignment of Drosophila GadFly Accession Number CG2048 (referred to as ^'dCK I ^'), human casein kinase 1 , delta (GenBank Accession Number NM_001 893.1 ; referred to as ^'hCK I delta ^'), human casein kinase 1 , epsilon (GenBank Accession Number XM_009983.4; referred to as 'hCK I epsilon '), mouse casein kinase 1 , delta (Accession Number AB028241 .1 ; referred to as 'mCK I delta ^'), mouse casein kinase 1 , epsilon (Accession Number NM_01 3767.2; referred to as ^'mCK I epsilon ') .

Figure 14. A human homolog of CG5320 (Gdh) Figure 14A. BlastP search result for CG5320

Figure 14B. Predicted coding nucleotide sequence for the human homolog with Accession Number NM_005271 .1 (Glutamate dehydrogenase I); length of the sequence in base pairs, referred to as bp. (SEQ ID NO: 1 8) Figure 14C. Predicted amino acid sequence for the human homolog with Accession Number NM_005271 .1 (Glutamate dehydrogenase I); length of the sequence in amino acids, referred to as aa. (SEQ ID NO: 1 9) .

Figure 1 5. A human homolog of CG5320 (Gdh) Figure 1 5A. BlastP search result for CG5320

Figure 1 5B. Predicted coding nucleotide sequence for the human homolog with Accession Number NT_01 1 746.5 (Glutamate dehydrogenase II); length of the sequence in base pairs, referred to as bp. (SEQ ID NO:20) Figure 1 5C. Predicted amino acid sequence for the human homolog with Accession Number NT_01 1 746.5 (Glutamate dehydrogenase ll); length of the sequence in amino acids, referred to as aa. (SEQ ID NO:21 ).

Figure 1 6. Energy storage metabolites (ESM; triglyceride (TG) and glycogen) content of a Drosophila Gdh (Gadfly Accession Number CG5320) mutant. Shown is the change of triglyceride content of HD-EP35207 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1 ) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2). Also shown is the change of glycogen content of HD-EP35207 flies caused by integration of the P-vector into the annotated transcription unit (column 5) in comparison to controls (referred to as 'control (glycogen)' column 4).

Figure 1 7. Human homolog of CG3943 (kraken)

Figure 1 7A. tBlastN search result for CG3943

Figure 1 7B. Predicted coding nucleotide sequence for the human homolog of CG3943; length of the sequence in base pairs (SEQ ID NO:22)

Figure 1 7C. Predicted amino acid sequence for the human homolog of CG3943; length of the sequence in amino acids (SEQ ID NO:23) Figure 1 7D. ClustalW alignment of Drosophila protein with GadFly Accession Number CG3943 (referred to as "drosophila") and the mouse (referred to as "mS0273353.1 ") and human (referred to as "HSC1401 79.1 ") homologs. The sequences are shown in the one-letter-code; shaded residues match exactly.

Figure 1 8. The human homolog of CG521 6 (Sir2) Figure 1 8A. Blastn search result for CG521 6

Figure 1 8B. Predicted coding nucleotide sequence for the human homolog (SEQ ID NO:24) Figure 1 8C. Predicted amino acid sequence for the human homolog of CG521 6 (SEQ ID NO:25).

Figure 1 9. The human homolog of CG3758 (escargot)

Figure 1 9A. Blastn search result for CG3758

Figure 1 9B. Predicted coding nucleotide sequence for the human homolog

(SEQ ID NO:26)

Figure 1 9C. Predicted amino acid sequence for the human homolog of

CG3758 (SEQ ID NO:27).

Figure 20. Triglyceride content of Drosophila escargot (Gadfly Accession Number CG3758) mutants. Shown is the change of triglyceride content of HD-EP20506 (column 2), HD-EP2081 7 (column 3), and HD-EP26792 (column 4) flies caused by integration of the P-vector into the annotated transcription unit in comparison to controls containing all fly lines of the proprietary EP collection (ΕP-controI)', column 1 ).

Figure 21 . The human homolog of CG3241 (msl-2)

Figure 21 A. BlastP search result for CG3241 Figure 21 B. Predicted coding nucleotide sequence for the human homolog with Accession Number AB046805.1 encoding hypothetical protein

KIAA1 585; length of the sequence in base pairs, referred to as bp. (SEQ ID

NO:28)

Figure 21 C. Predicted amino acid sequence for the human homolog of CG3241 ; length of the sequence in amino acids, referred to as aa. (SEQ ID

NO:29)

Figure 21 D. ClustaW alignment of Drosophila msl-2 (GadFly Accession

Number CG3241 ; referred to as 'd Msl-2'), human msl-2 (GenBank

Accession Number AB046805.1 ; referred to as 'hH!A1 585'), and mouse msl-2 (GenBank Accession Number BF471 233; referred to as

'mBF471 233') . The sequences are shown in the one-letter-code; shaded residues match exactly. Figure 22. Triglyceride content of a Drosophila msl-2 (Gadfly Accession Number CG3241 ) mutant. Shown is the change of triglyceride content of HD-EP25097 flies caused by integration of the P-vector into the annotated transcription unit (column 2) in comparison to controls containing all fly lines of the proprietary EP collection (ΕP-control)', column 1 ) .

Figure 23. The human homolog of CG 1 1 940 and triglyceride content of a

Drosophila CG1 1940 mutant

Figure 23A. Blastn search result for CG1 1 940 Figure 23B. Predicted coding nucleotide sequence for the human homolog

(SEQ ID NO:30)

Figure 23C. Predicted amino acid sequence for the human homolog of

CG 1 1 940 (SEQ ID NO:31 )

Figure 23D. Triglyceride content of a Drosophila CG 1 1 940 (Gadfly Accession Number) mutant. Shown is the change of triglyceride content of

HD-EP10934 flies caused by integration of the P-vector into the annotated transcription unit (column 2) in comparison to controls containing all fly lines of the proprietary EP collection (ΕP-control)', column 1 ) .

Figure 24. Human homolog of CG 1 624 (dappled) Figure 24A. tBlastN search result for CG 1 624

Figure 24B. Predicted coding nucleotide sequence for the human homolog of CG 1 624; length of the sequence in base pairs (SEQ ID NO:32) Figure 24C. Predicted amino acid sequence for the human homolog of CG 1 624; length of the sequence in amino acids (SEQ ID NO:33).

Figure 25. Human homolog of CG 1 1 753 Figure 25A. tBlastN search result for CG 1 1 753

Figure 25B. Predicted coding nucleotide sequence for the human homolog of CG 1 1 753; length of the sequence in base pairs (SEQ ID NO:34)

Figure 25C. Predicted amino acid sequence for the human homolog of CG 1 1 753; length of the sequence in amino acids (SEQ ID NO:35) Figure 25D. ClustalW alignment of Drosophila protein with GadFly Accession Number CG 1 1 753 (referred to as "dCG 1 1 753") and the human (referred to as "hCG1 1753") and mouse (referred to as "mCG1 1753") homologs. The sequences are shown in the one-letter-code; shaded residues match exactly.

Figure 26. Human homolog of CG7262 Figure 26A. tBlastN search result for CG7262

Figure 26B. Predicted coding nucleotide sequence for the human homolog of CG7262; length of the sequence in base pairs (SEQ ID NO: 36)

Figure 26C. Predicted amino acid sequence for the human homolog of CG7262; length of the sequence in amino acids (SEQ ID NO: 37) .

Figure 27. The human homolog of CG4291 Figure 27A. Blastn search result for CG4291

Figure 27B. Predicted coding nucleotide sequence for the human homolog (SEQ ID NO:38)

Figure 27C. Predicted amino acid sequence for the human homolog of CG4291 (SEQ ID NO:39) .

The Examples illustrate the invention:

Example 1 : Cloning of a Drosophila melanogaster gene with homology to human Uncoupling Proteins (UCPs)

A BLAST homology search was performed in a public database (NCBI/NIH) looking for Drosophila genes with sequence homology to the human UCP2 and UCP3 genes. The search yielded sequence fragments of a family of Drosophila genes with UCP homology. They are clearly different to the next related mitochondrial proteins (oxoglutarate carrier) . Using the sequence fragment of one of this genes (here called dUCPy), a PCR primer pair was generated (Upper

5'-CTAAACAAACAATTCCAAACATAG (SEQ ID NO: 40), Lower 5'-AAAAGACATAGAAAATACGATAGT (SEQ ID NO: 41 )) and a PCR reaction performed on Drosophila cDNA using standard PCR conditions. The amplification product was radioactively labeled and used to screen a cDNA library made from adult Drosophila flies (Stratagene) . A full-length cDNA clone was isolated, sequenced and used for further experiments. The nucleotide sequence of UCPy is shown in SEQ ID NO: 1 (see FIGURE 1 A), the coding sequence in SEQ ID NO:2 (see FIGURE 1 B), and the deduced open reading frame is shown as SEQ ID NO:3 (see FIGURE 1 C) .

Example 2: Cloning of the dUCPy cDNA into an Drosophila expression vector

In order to test the effects of dUCPy expression in Drosophila cells the dUCPy cDNA was cloned into the expression vector pUAST (Ref.: Brand A & Perrimon N, Development 1 993, 1 1 8:401 -41 5) using the restriction sites Notl and Kpnl. The resulting expression construct was injected into the germline of Drosophila embryos and Drosophila strains with a stable integration of the construct were generated. Since the expression vector pUAST is activated by the yeast transcription factor Gal4 which is normally absent from Drosophila cells dUCPy is not yet expressed in these transgenic animals. If pUAST-dUCPy flies are crossed with a second Drosophila strain that expresses Gal4 in a tissue specific manner the offspring flies of this mating will express dUCPy in the GaI4 expressing tissue.

The cross of pUAST-dUCPy flies with a strain that expresses Gal4 in all cells of the body (under control of the actin promoter) showed no viable offspring. This means that dUCPy overexpression in all body cells is lethal. This finding is consistent with the assumption that dUCPy overexpression could lead to a collapse of the cellular energy production.

Expression of dUCPy in a non-vital organ like the eye (Gal4 under control of the eye-specific promoter of the "eyeless" gene) results in flies with visibly damaged eyes. This easily visible eye phenotype is the basis of a genetic screen for gene products that can modify UCP activity.

Example 3: dUCPy modifier screen

Parts of the genomes of the strain with GaI4 expression in the eye and the strain carrying the pUAST-dUCPy construct were combined on one chromosome using genomic recombination. The resulting fly strain has eyes that are permanently damaged by dUCPy expression. Flies of this strain were crossed with flies of a large collection of mutagenized fly strains. In this mutant collection a special expression system (EP-element, Ref. : Rorth P, Proc Natl Acad Sci U S A 1 996, 93(22) : 1 241 8-1 2422) is integrated randomly in different genomic loci. The yeast transcription factor Gal4 can bind to the EP-element and activate the transcription of endogenous genes close the integration site of the EP-element. The activation of the genes therefore occurs in the same cells (eye) that overexpress dUCPy. Since the mutant collection contains several thousand strains with different integration sites of the EP-element it is possible to test a large number of genes whether their expression interacts with dUCPy activity. In case a gene acts as an enhancer of UCP activity the eye defect will be worsened; a suppressor will ameliorate the defect.

Using this screen genes with suppressing activity were discovered that were found to be the comichon (GadFly Accession Number CG5855), neuralized (GadFly Accession Number CG1 1 988), dco (GadFly Accession Number CG2048), kraken (GadFly Accession Number CG3943), escargot (GadFly Accession Number CG3758), GadFly Accession Number CG1 1 940, dappled (GadFly Accession Number CG1 624), GadFly Accession Number CG1 1753, GadFly Accession Number CG7262, and GadFly Accession Number CG4291 genes in Drosophila. Using this screen genes with enhancing activity were discovered that was found to be the GadFly Accession Number CG8479, Imp (GadFly Accession Number CG1 691 ), GadFly Accession Number CG831 1 , Gdh (GadFly Accession Number CG5320), Sir2 (GadFly Accession Number CG521 6), and msl-2 (GadFly Accession Number CG3241 ) genes in Drosophila.

Example 4: Identification of human homologous genes and proteins

Genomic DNA neighbouring to the respective eye-defect rescuing EP-element was cloned by inverse PCR and sequenced. These sequences were used for BLAST searches in a public Drosophila gene database.

The database search indicated that the EP-element EP20761 which is enhancing the eye-phenotype is integrated in a predicted transcript annotated as CG8479 (Drosophila Genome Project), located on chromosome 2R, encoding for a protein with 65% homologies to human optic atrophy 1 protein (see FIGURE 2; SEQ ID NO: 4 and 5; GenBank Accession Number XP_039926.2) .

The database search indicated that the EP-element EP20292 which is suppressing the eye-phenotype is integrated in a predicted transcript annotated as FlyBase Symbol CG5855 (Drosophila Genome Project; gene cni), located on chromosome 2L, encoding for a protein with 76% homologies to human Cornichon-like protein (see FIGURE 5; SEQ ID NO:6 and 7; GenBank Accession Number NP 005767.1 ). The database search indicated that the EP-elements EP10858 and EP1 0570 which are enhancing the eye-phenotype are integrated in a predicted transcript annotated as CG 1 691 (Drosophila Genome Project), located on chromosome X, encoding for a protein with 63% homologies to human IGF-II mRNA binding protein 3 (see FIGURE 7; SEQ ID NO:8 and 9; GenBank Accession Number XP_004780.2) .

The database search indicated that the EP-element EP31 874 which is suppressing the eye-phenotype is integrated in a predicted transcript annotated as CG 1 1 988 (Drosophila Genome Project; gene neur), located on chromosome 3R, encoding for a protein with 46% homology / 50% homology to human neuralized-like protein (see FIGURE 8; SEQ ID NO: 10 and 1 1 ; GenBank Accession Number NM_004210).

The database search indicated that the EP-element EP20700 which is enhancing the eye-phenotype is integrated in a predicted transcript annotated as CG831 1 (Drosophila Genome Project), located on chromosome 2R, encoding for a protein with homologies to human KIAA1094 protein (GenBank Accession Number NM_014908.1 ; SEQ ID NO: 1 2 and 1 3; see FIGURE 1 0); corresponding to patent WO01 53486 (Sequence 69) . Human KIAA1094 is 46% homologous and 29% identical to Drosophila CG831 1 over 405 amino acids (see FIGURE 10A), and Human KIAA1 094 ia 50% homologous and 31 % identical to Saccharomyces cerevisiae Sec59p (Accession Number NP_01 3726.1 ) over 267 amino acids. The transmembrane prediction of the CG831 1 homolog is shown in FIGURE 10D. The protein shows according to the THMM prediction program (Krogh et al., 2001 , Journal of Molecular Biology 305(3):567-580; for example see http://www.cbs.dtu.dk/services/ TMHMM-2.0/) 1 4 transmembrane domains, shown as black peaks in FIGURE 10D. The human protein is most likely (74%) located in the plasma membrane, according to the publicly available prediction program Psortll (Horton and Nakai, 1 996, Proc Int Conf Intell Syst Mol Biol. 4:109-1 5; for example see http://psort.nibb.ac.jp) . Drosophila CG831 1 shows also homologies to mouse gene with the Accession Number AW553567.

The database search indicated that the EP-element EP31 834 which is suppressing the eye-phenotype leads to the overexpression of a predicted transcript annotated as FlyBase Symbol CG2048 (Drosophila Genome Project), located on chromosome 3R, encoding for a protein with 93% homologies over 281 amino acids to human casein kinase delta (see FIGURE 1 2; SEQ ID NO: 14 and 1 5; GenBank Accession Number NM_001 893.1 ; corresponding to patent US5846764 (Sequence 43), US5728806 (Sequence 43), and US568641 2 (Sequence 34). CG2048 also shows high homologies to human casein kinase epsilon (see FIGURE 1 3; SEQ ID NO: 1 6 and 1 7; GenBank Accession Number XM_009983.4) . Drosophila CG2048 shows also homologies to mouse genes with the Accession Numbers BAA88082 (murine casein kinase 1 delta), and NM_01 3767 (murine casein kinase 1 , epsilon) . A Clusta-W alignment of Drosophila CG2048, both human homolog casein kinases, and the two homolog murine casein kinases was conducted and is shown in FIGURE 1 3D.

The database search indicated that the EP-element EP31710 which is enhancing the eye-phenotype is integrated in the promoter opposite to the driving direction of the predicted transcript annotated as CG5320 (Drosophila Genome Project), located on chromosome 3R, encoding for a protein with 78% homologies to 553 amino acids of human glutamate dehydrogenase GdH protein (GLUD1 ; see FIGURE 14; Seq ID NO: 1 8 and 1 9; GenBank Accession Number NM_005271 .1 ) ); corresponding to patent WO0073801 A2 (Sequence 453) . CG5320 also shows high homologies (85% over 404 amino acids) to a second human glutamate dehydrogenase GdH protein (GLUD2; see FIGURE 1 5; Seq ID NO:20 and 21 ; GenBank Accession Number XP_010438). Drosophila CG5320 shows also homologies to a mouse gene with the Accession Number NM 0081 33.1 . The database search indicated that the EP-element EP20903 which is suppressing the eye-phenotype leads to the overexpression of a predicted transcript annotated as FlyBase Symbol CG3943 (Drosophila Genome Project; gene kraken), located on chromosome 2L, encoding for a protein with 54% homologies over 289 amino acids to a human hypothetical protein (see FIGURE 1 7; Seq ID NO:22 and 23; GenBank Accession Number CAC1 6804.1 . A ClustalW alignment of Drosophila kraken and the mouse and human homologs was conducted and is shown in FIGURE 1 7D) .

The database search indicated that the EP-element EP20105 which is enhancing the eye-phenotype is integrated in a predicted transcript annotated as CG521 6 (Drosophila Genome Project; gene Sir2), located on chromosome 2L, encoding for a protein with 71 % homologies to human Sirtuin protein (sirtuin 1 ; see FIGURE 1 8; SEQ ID NO:24 and 25; GenBank Accession Number XP_008902.2).

The database search indicated that the EP-element EP20506 which is suppressing the eye-phenotype is integrated in a predicted transcript annotated as FlyBase Symbol CG3758 (Drosophila Genome Project; gene escargot), located on chromosome 2L, encoding for a protein with 85% homologies to human hypothetical protein, similiar to Gonadotropin (see FIGURE 1 9; SEQ ID NO:26 and 27; GenBank Accession Number XP_030528.1 ) .

The database search indicated that the EP-element EP25097 which is enhancing the eye-phenotype is integrated in a predicted transcript annotated as FlyBase Symbol CG3241 (Drosophila Genome Project; gene msl-2), located on chromosome 2L, encoding for a protein with 58% homologies over 66 amino acids to human hypothetical protein KIAA1 585 (see FIGURE 21 ; SEQ ID NO:28 and 29; GenBank Accession Number AB046805.1 . A Clusta-W alignment of Drosophila msl-2 and the human homolog was conducted and is shown in FIGURE 21 D). Drosophila CG3241 shows also homologies to a mouse gene with the Accession Number BF471 233.

The database search indicated that the EP-element EP1 1 934 which is suppressing the eye-phenotype is integrated within the first (13kb) intron of a predicted transcript annotated as CG1 1 940 (Drosophila Genome Project), located on chromosome X, encoding for a protein with 61 % homologies to 226 amino acids of human alsin aslcr2 protein (see FIGURE 23; Seq ID NO:30 and 31 ; GenBank Accession Number XP_028059.1 ) .

The database search indicated that the EP-element EP35393 which is suppressing the eye-phenotype is integrated in 3'-5' direction in a predicted transcript annotated as CG1624 (Drosophila Genome Project; gene dappled)), located on chromosome 3R, encoding for a protein with 68% homology to 1 71 amino acids, with 55% homology to 1 71 amino acids, and with 66% homology to 83 amino acids of a human protein (see FIGURE 24; Seq ID NO: 32 and 33; GenBank Accession Number XM_067369) .

The database search indicated that the EP-element EP32534 which is suppressing the eye-phenotype leads to the overexpression of a predicted transcript annotated as FlyBase Symbol CG1 1 753 (Drosophila Genome Project), located on chromosome 3R, encoding for a protein with 61 % homologies over 144 amino acids to a human protein (see FIGURE 25; SEQ ID NO:34 and 35; GenBank Accession Number XP_029849.1 ) . A ClustalW alignment of Drosophila CG1 1 753 and the human and the mouse homolog was conducted and is shown in FIGURE 25D.

The database search indicated that the EP-element EP35056 which is suppressing the eye-phenotype is integrated in 3'-5' direction in the first intron of a predicted transcript annotated as CG7262 (Drosophila Genome Project), located on chromosome 3R, encoding for a protein with homologies to human KIAA0095 protein (GenBank Accession Number NM_014669; SEQ ID NO: 36 and 37; see FIGURE 26); corresponding to patent WO001 8961 (Sequence 1 2) . Human KIAA0095 is 55% homologous and 36% identical to Drosophila CG7262 over 823 amino acids (see FIGURE 26A). The protein shows according to the THMM prediction program (Krogh et al., 2001 , Journal of Molecular Biology 305(3) :567-580; for example see http://www.cbs.dtu.dk/services/ TMHMM-2.0/) no transmembrane domains. The human protein is most likely (52%) located in the plasma membrane, according to the publicly available prediction program Psortll (Horton and Nakai, 1 996, Proc Int Conf Intell Syst Mol Biol. 4: 109-1 5; for example see http://psort.nibb.ac.jp) .

The database search indicated that the EP-element EP20903 which is suppressing the eye-phenotype is integrated in a predicted transcript annotated as FlyBase Symbol CG4291 (Drosophila Genome Project), located on chromosome 2L, encoding for a protein with 45% homologies to human formin binding protein 21 (FBP21 ; see FIGURE 27; SEQ ID NO:38 and 39; GenBank Accession Number XP 049375.1 ) .

Example 5: Measurement of energy storage metabolites (ESM) content

Mutant flies are obtained from a fly mutation 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 for the EP-lines HD-EP20292, HD-35207, HD-EP20506, HD-EP2081 7, HD-EP26792, HD-EP25097, and HD-EP10934. The average change of triglyceride and glycogen (herein referret to as energy storage metabolites, ESM) content of Drosophila containing the EP-vector as homozygous or hemizygous viable integration was investigated in comparison to control flies, respectively (see FIGURES 6, 1 6, 20, 22, and 23D) . For determination of ESM content, 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 336-1 0 or -20) assay by measuring changes in the optical density according to the manufacturer's protocol, and the glycogen content of the flies extract was determined using Roche (Starch UV-method Cat. No. 0207748) assay by measuring changes in the optical density according to the manufacturer's protocol. As a reference the protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol. These experiments and assays were repeated several times.

The average triglyceride level ((μg triglyceride/μg protein) of all flies of the EP collection (referred to as 'EP-control') is shown as 100% in the first column in FIGURES 20, 22, and 23D. The average triglyceride level ((μg triglyceride/μg protein) of 2108 fly lines of the proprietary EP-collection (referred to as 'HD-control (TG)') is shown as 100% in the first column in FIGURES 6 and 1 6. The average triglyceride level ((μg triglyceride/μg protein) of Drosophila wildtype strain Oregon R flies determined in 84 independent assays (referred to as 'WT-control (TG)') is shown as 1 02% in the second column in FIGURES 6 and 1 6. The average glycogen level ((μg glycogen/μg protein) of an internal assay control consisting of two different wildtype strains and an inconspicuous EP-line of the HD stock collection (referred to as 'control (glycogen)') is shown as 1 00% in the fourth column in FIGURES 6 and 1 6. Standard deviations of the measurements are shown as thin bars.

HD-EP20292 homozygous flies show constantly a lower triglyceride content (μg triglyceride/μg protein) than the controls (column 3 in FIGURE 6, 'HD-EP20292 (TG)') . HD-EP20292 homozygous flies also show a lower glycogen content (μg glycogen/μg protein) than the controls (column 5 in FIGURE 6, 'HD-EP20292 (glycogen)') . Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage metabolites.

HD-35207 homozygous flies show constantly a lower triglyceride content (μg triglyceride/μg protein) than the controls (column 3 in FIGURE 1 6, 'HD-35207 (TG)'). HD-35207 homozygous flies also show a lower glycogen content (μg glycogen/μg protein) than the controls (column 5 in FIGURE 1 6, 'HD-35207 (glycogen)'). Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage metabolites.

HD-EP20506, HD-EP2081 7, and HD-EP26792 homozygous flies show constantly a higher triglyceride content (μg triglyceride/μg protein) than the controls (column 2 in FIGURE 20, 'HD-EP20506'; column 3 in FIGURE 20 'HD-EP2081 7', and column 4 in FIGURE 20, 'HD-EP26792'). Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage triglycerides.

HD-EP25097 homozygous flies show constantly a higher triglyceride content (μg triglyceride/μg protein) than the controls (column 2 in FIGURE 22, 'HD-EP25097') . Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage triglycerides.

HD-EP1 0934 hemizygous flies show constantly a higher triglyceride content (μg triglyceride/μg protein) than the controls (column 3 in FIGURE 23D, 'HD-EP10934'). Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage triglycerides.

Example 6: Expression profiling experiments

To analyze the expression of the polypeptides disclosed in this invention in mammalian tissues, several mouse strains (preferably 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 (331 78 Borchen, Germany) and maintained under constant temperature (perferrably 22°C), 40 percent humidity and a light / dark cycle of preferably 14 / 10 hours. The mice were fed a standard diet (for example, from ssniff Spezialitaten GmbH, order number sniff 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 Nati 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.

For analyzing the role of the proteins disclosed in this invention in the in vitro differentiation of different mammalian cell culture cells for the conversion of pre-adipocytes to adipocytes, mammalian fibroblast (3T3-L1 ) cells (e.g., Green & Kehinde, Cell 1 : 1 1 3-1 1 6, 1 974) were obtained from the American Tissue Culture Collection (ATCC, Hanassas, VA, USA; ATCC- CL 1 73) . 3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g., Qiu. et al., J. Biol. Chem. 276: 1 1 988-95, 2001 ; Slieker et al., BBRC 251 : 225-9, 1 998) . At various time points of the differentiation procedure, beginning with day 0 (day of confluence) and day 2 (hormone addition; for example, dexamethasone and 3-isobutyl-1 -methyIxanthine), up to 10 days of differentiation, suitable aliquots of cells were taken every two days. Alternatively, mammalian fibroblast 3T3-F442A cells (e.g., Green & Kehinde, Cell 7: 105-1 13, 1 976) were obtained from the Harvard Medical School, Department of Cell Biology (Boston, MA, USA) . 3T3-F442A cells were maintained as fibroblasts and differentiated into adipocytes as described previously (Djian, P. et al., J. Cell. Physiol., 124:554-556, 1 985) . At various time points of the differentiation procedure, beginning with day 0 (day of confluence and hormone addition, for example, Insulin), up to 1 0 days of differentiation, suitable aliquots of cells were taken every two days. 3T3-F442A cells are differentiating in vitro already in the confluent stage after hormone (insulin) addition.

RNA was isolated from mouse tissues or cell culture cells using Trizol Reagent (e.g. from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy Kit (for example, from Qiagen, Germany) in combination with a DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art. Total RNA was reverse transcribed (Superscript II RNaseH- Reverse Transcriptase, e.g. from Invitrogen, Germany) and subjected to Taqman analysis using the Taqman 2xPCR Master Mix (e.g. 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 (e.g. from Applied Biosystems, Weiterstadt, Germany) .

The Taqman analysis of the CG8479 homologous protein (OPA1 ) was performed using the following primer/probe pair: mouse OPA1 forward primer (SEQ ID NO: 42) : 5'- GCC TGG GAG ACT CTA CAA GAG G -3'; mouse OPA1 reverse primer (SEQ ID NO: 43): 5'- AAT ATG TCG TCG TGT TCC TTT CC -3'; Taqman probe (SEQ ID NO: 44) : (5/6-FAM) (5/6-FAM) TTT CCC GCT TCA TGA CAG AAC CCA A (5/6-TAMRA).

The Taqman analysis of the neuralized homologous protein was performed using the following primer/probe pair: mouse neuralized forward primer (SEQ ID NO: 45) : 5'- TCA AGG ACA TCA TCA AGA CCT ACC-3'; mouse neuralized reverse primer (SEQ ID NO: 46) : δprime- GGG AGA CGT TGT GCA GGT G -3'; Taqman probe (FAM/TAMRA) (SEQ ID NO: 47): 5'- CAG CTC CTA GCC CAC TGC AGA GCC -3'. The Taqman analysis of the CG831 1 homologous protein was performed using the following primer/probe pair: mouse forward primer (SEQ ID NO: 48) : 5'-GGAGGCCACAGTATCACCCA-3'; mouse reverse primer (SEQ ID NO : 49) : 5'-AAGGAGCAAGAGCCCTGGTC-3' ; Taqman probe 5 (FAM/TAMRA) (SEQ ID NO: 50): 5'-ACCCACAGCCAAGACCCCAGCA-3' .

As shown in Figure 4, real time PCR (Taqman) analysis of the expression of the OPA-1 RNA in mammalian (mouse) tissues revealed revealed that o OPA-1 is expressed in different mammalian tissues, showing 2 to 3 fold higher levels of expression in BAT, hypothalamus, brain, muscle and heart when compared to other tissues. BAT, brain, muscle and heart represent tissues with the major catabolic activity in the body. The high experession levels of OPA-1 in these tissues indicate, that OPA-1 is involved in the 5 metabolism of tissues relevant for the metabolic syndrome.

As shown in Figure 9, real time PCR (Taqman) analysis of the expression of the neuralized RNA in mammalian (mouse) tissues revealed revealed that neuralized is highly expressed in muscle, hypothalamus, brain and testis. 0 The high expression levels in muscle when compared to other tissues is indicative for a role in the metabolism in one of the major catabolic tissues of the body.

The Taqman analysis revealed that transcript levels of the CG831 1 5 homologous protein show a prominent peak in brown adipose tissue compared to several other mouse tissues and organs. In Figure 1 1 , Rel. RNA refers to relative RNA expression in the corresponding tissue, expressed as levels in percent [%]. The pancreas tissue was set as reference level to zero. The mouse tissue tested are shown on the vertical o line; BAT refers to brown adipose tissue; WAT refers to white adipose tissue. Example 7: In vitro assays for the determination of triglyceride and glycogen storage

Obesity is known to be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate bound energy and low energy expenditure. For example, an increase in energy expenditure (and thus, lowering the body weight) would include the elevated utilization of both circulating and intracellular glucose and triglycerides, free or stored as glycogen or lipids as fuel for energy and/or heat production. In this invention, we therefore show the cellular level of triglycerides and glycogen in cells overexpressing the protein of the invention.

Retroviral infection of preadipocytes Packaging cells were transfected with retroviral plasmids pLPCX carrying the mouse transgene encoding a protein of the invention and a selection marker using calcium phosphate procedure. Control cells were infected with pLPCX carrying no transgene. Briefly, exponentially growing packaging cells were seeded at a density of 350,000 cells per 6-well in 2 ml DMEM + 10 % FCS one day before transfection. 1 0 min before transfection chloroquine was added directly to the overlying medium (25 μM end concentration) . A 250 μl transfection mix consisting of 5 μg plasmid-DNA (candidate:helper-virus in a 1 : 1 ratio) and 250 mM CaCI₂ was prepared in a 1 5 ml plastic tube. The same volume of 2 x HBS (280 μM NaCl, 50 μM HEPES, 1 .5 mM Na₂HP0₄, pH 7.06) was added and air bubbles were injected into the mixture for 15 sec. The transfection mix was added drop wise to the packaging cells, distributed and the cells were incubated at 37°C, 5 % C0₂ for 6 hours. The cells were washed with PBS and the medium was exchanged with 2 ml DMEM + 10 % CS per 6-well. One day after transfection the cells were washed again and incubated for 2 days of virus collection in 1 ml DMEM + 10 % CS per 6-well at 32°C, 5 % C0₂. The supernatant was then filtered through a 0.45 μm cellulose acetate filter and polybrene (end concentration 8 μg/ml) was added. Mammalian fibroblast (3T3-L1 ) cells in a sub-confluent state were overlaid with the prepared virus containing medium. The infected cells were selected for 1 week with 2 μg/ml puromycin. Following selection the cells were checked for transgene expression by western blot and immunofluorescence. Over expressing cells were seeded for differentiation.

3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art and supra. For analysing the role of the proteins disclosed in this invention in the in vitro assays for the determination of triglyceride storage, synthesis and transport were performed.

Preparation of cell lysates for analysis of metabolites Starting at confluence (dO), cell media was changed every 48 hours. Cells and media were harvested 8 hours prior to media change as follows. Media was collected, and cells were washed twice in PBS prior to lyses in 600 μl HB-buffer (0.5% polyoxyethylene 10 tridecylethane, 1 mM EDTA, 0.01 M NaH₂P0₄, pH 7.4) . After inactivation at 70°C for 5 minutes, cell lysates were prepared on Bio 101 systems lysing matrix B (0.1 mm silica beads; Q-Biogene, Carlsbad, USA) by agitation for 2 x 45 seconds at a speed of 4.5 (Fastprep FP1 20, Bio 101 Thermosavant, Holbrock, USA) . Supematants of lysed cells were collected after centrifugation at 3000 rpm for 2 minutes, and stored in aliquots for later analysis at -80°C.

Changes in cellular triglyceride levels during adipogenesis Cell lysates and media were simultaneously analysed in 96-weII plates for total protein and triglyceride content using the Bio-Rad DC Protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturer's instructions and a modified enzymatic triglyceride kit (GPO-Trinder; Sigma) briefly final volumes of reagents were adjusted to the 96-well format as follows: 10 μl samples were incubated with 200 μl reagent A for 5 minutes at 37 °C. After determination of glycerol (initial absorbance at 540 nm), 50 μl reagent B was added followed by another incubation for 5 minutes at 37 °C (final absorbance at 540 nm) . Glycerol and triglyceride concentrations were calculated using a glycerol standard set (Sigma) for the standard curve included in each assay.

Changes in cellular glycogen levels during adipogenesis

Cell lysates and media were simultaneously analysed in triplicates in 96-well plates for total protein and glycogen content using the Bio-Rad DC Protein assay reagent (Bio-Rad, Munich, Germany) according to the manufacturer's instructions and an enzymatic starch kit from Hoffmann-La Roche (Basel, Switzerland). 10-μl samples were incubated with 20-μl amyloglucosidase solution for 1 5 minutes at 60°C to digest glycogen to glucose. The glucose is further metabolised with 1 00 μl distilled water and 1 00 μl of enzyme cofactor buffer and 1 2 μl of enzyme buffer (hexokinase and glucose phosphate dehydrogenase) . Background glucose levels are determined by subtracting values from a duplicate plate without the amyloglucosidase. Final absorbance is determined at 340 nm. HB-buffer as blank, and a standard curve of glycogen (Hoffmann-La Roche) were included in each assay. Glycogen content in samples were calculated using a standard curve.

Synthesis of lipids during adipogenesis During the terminal stage of adipogenesis (day 1 2) cells were analysed for their ability to metabolise lipids. A modified protocol to the method of Jensen et al (2000) for lipid synthesis was established. Cells were washed 3 t i m e s w i t h P B S p r i o r t o s e r u m s t a r v a t i o n i n Krebs-Ringer-Bicarbonate-Hepes buffer (KRBH; 1 34 nM NaCl, 3.5 mM KCI, 1 .2 mM KH₂P0₄, 0.5 mM MgS0₄, 1 .5 mM CaCI₂, 5 mM NaHC0₃, 10 mM Hepes, pH 7.4), supplemented with 0.1 % FCS for 2.5h at 37°C. For insulin-stimulated lipid synthesis, cells were incubated with 1 μM bovine insulin (Sigma; carrier: 0.005N HCl) for 45 min at 37 °C. Basal lipid synthesis was determined with carrier only. ¹⁴C(U)-D-Glucose (NEN Life Sciences) in a final activity of 1μCi/WeIl/ml in the presence of 5 mM glucose was added for 30 min at 37°C. For the calculation of background radioactivity, 25 μM Cytochalasin B (Sigma) was used. All assays were performed in duplicate wells. To terminate the reaction, cells were washed 3 times with ice cold PBS, and lysed in 1 ml 0.1 N NaOH. Protein concentration of each well was assessed using the standard Biuret method (Protein assay reagent; Bio-Rad). Total lipids were separated from aqueous phase after overnight extraction in Insta-Fluor scintillation cocktail (Packard Bioscience) followed by scintillation counting.

Transport and metabolism of free fatty acids during adipogenesis During the terminal stage of adipogenesis (d1 2) cells were analysed for their ability to transport long chain fatty acid across the plasma membrane. A modified protocol to the method of Abumrad et al (1991 ) (Proc. Natl. Acad. Sci. USA, 1 991 : 88; 6008-1 2) for cellular transportation of fatty acid was established. In summary, cells were washed 3 times with PBS prior to serum starvation. This was followed by incubation in KRBH buffer supplemented with 0.1 % FCS for 2.5h at 37°C. Uptake of exogenous free fatty acids was initiated by the addition of isotopic media containing non radioactive oleate and (³H)oleate (NEN Life Sciences) complexed to serum albumin in a final activity of 1μCi/Well/ml in the presence of 5 mM glucose for 30min at room temperature (RT) . For the calculation of passive diffusion (PD) in the absence of active transport (AT) across the plasma membrane 20mM of phloretin in glucose free media (Sigma) was added for 30 min at RT. All assays were performed in duplicate wells. To terminate the active transport 20mM of phloretin in glucose free media was added to the cells. Cells were lysed in 1 ml 0.1 N NaOH and the protein concentration of each well were assessed using the standard Biuret method (Protein assay reagent; Bio-Rad). Esterified fatty acids were separated from free fatty acids using overnight extraction in Insta-Fluor scintillation cocktail (Packard Bioscience) followed by scintillation counting.

Example 8: Glucose uptake assay

For the determination of glucose uptake, cells were washed 3 times with PBS prior to serum starvation in KRBH buffer supplemented with 0.1 % FCS and 0.5mM glucose for 2.5h at 37°C. For insulin-stimulated glucose uptake, cells were incubated with 1 μM bovine insulin (Sigma; carrier: 0.005N HCl) for 45 min at 37°C. Basal glucose uptake was determined with carrier only. Non-metabolizable 2-deoxy-³H-D-glucose (NEN Life Science, Boston, USA) in a final activity of 0,4 μCi/Well/ml was added for 30 min at 37°C. For the calculation of background radioactivity, 25 μM cytochalasin B (Sigma) was used. All assays were performed in duplicate wells. To terminate the reaction, cells were washed 3 times with ice cold PBS, and lysed in 1 ml 0.1 N NaOH. Protein concentration of each well was assessed using the standard Biuret method (Protein assay reagent; Bio-Rad), and scintillation counting of cell lysates in 1 0 volumes Ultima-gold cocktail (Packard Bioscience, Groningen, Netherlands) was performed.

Example 9: Generation and analysis of transgenic mice

Generation of the transgenic animals Mouse cDNA encoding OPA1 , cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta and epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, K1AA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, or formin-binding protein 21 , was isolated from mouse brown adipose tissue (BAT) using standard protocols as known to those skilled in the art. The cDNA was amplified by RT-PCR and point mutations were introduced into the cDNA. The resulting mutated cDNA was cloned into a suitable transgenic expression vector. The transgene was microinjected into the male pronucleus of fertilized mouse embryos (preferably strain C57/BL6/CBA F1 (Harlan Winkelmann). Injected embryos were transferred into pseudo-pregnant foster mice. Transgenic founders were detected by PCR analysis. Two independent transgenic mouse lines containing the construct were established and kept on a C57/BL6 background. Briefly, founder animals were backcrossed with C57/BL6 mice to generate F1 mice for analysis. Transgenic mice were continously bred onto the C57/BI6 background. The expression of the proteins of the invention can be analyzed by taqman analysis as described above, and further analysis of the mice can be done as known to those skilled in the art.

All publications and patents mentioned in the above specification are herein incorporated by reference.

Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1 . A pharmaceutical composition comprising a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or a said polypeptide encoded thereby together with pharmaceutically acceptable carriers, and/or diluents and/or adjuvants.

2. The composition of claim 1 , wherein the nucleic acid molecule is a vertebrate or insect nucleic acid, particularly a human nucleic acid as shown in SEQ ID NO:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, and/or 38 or a nucleic acid having a nucleotide sequence complementary thereto or a fragment or a variant thereof.

3. The composition of claim 1 or 2, wherein said nucleic acid molecule

(a) hybridizes at 50°C in a solution containing 1 x SSC and 0.1 % SDS to a nucleic acid molecule encoding an amino acid sequence of SEQ ID NO:5, 7, 9, 1 1 , 1 3, 1 5, 1 7, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39; and/or a nucleic acid molecule complementary thereto;

(b) is degenerate with respect to the nucleic acid molecule of (a); (c) 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 protein as shown in SEQ ID NO:5, 7, 9, 1 1 , 13, 1 5, 17, 1 9, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, or 39;

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

(e) comprises a partial sequence of any of the nucleotide sequences of (a) to (d) having a length of at least 15 bases.

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 to membrane stability and/ or function in organelles such as mitochondria and/or peroxisomes.

6. The composition of claim 5, wherein said polypeptide participates in the maintenance of said membrane.

7. The composition of any one of claims 1 to 6, wherein said polypeptide is a transporter molecule and/or a regulator of a transporter molecule.

8. The composition of any one of claims 1 to 7, wherein said polypeptide is a modifying polypeptide.

9. The composition of claim 8, wherein said modifying polypeptide is a modifier of mitochondrial proteins.

10. The composition of claim 9, wherein said mitochondrial protein is a member of the UCP family.

1 . The composition of claim 10, wherein said member of the UCP family is UCP1 , UCP2, UCP3, UCP4, UCP5, StUCP or AtUCP.

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

3. The composition of any one of claims 1 -1 2, wherein the nucleic acid molecule is a vector, particularly an expression vector.

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

5. The composition of claim 14, wherein said recombinant polypeptide is a fusion polypeptide.

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

7. The composition of any one of claims 1 -1 6 which is a diagnostic composition.

8. The composition of any one of claims 1 -17 which is a pharmaceutical composition.

9. The composition of any one of claims 1 -1 8 for the manufacture of an agent for detecting and/or verifying, for the treatment, alleviation and/or prevention of diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis and gallstones and disorders related to ROS defence, such as diabetes mellitus, neurodegenerative disorders, mitochondrial disorders and others, in cells, cell masses, organs and/or subjects.

20. A vector comprising a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog,

CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family operatively linked to an expression control sequence.

21 A host transformed with the vector of claim 20.

22. A method for producing a polypeptide comprising culturing the host of claim 21 under suitable conditions and isolating the polypeptide produced.

23. An antibody, fragment or derivative thereof or an aptamer or another receptor specifically recognizing a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1 094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a polypeptide encoded thereby.

24. An anti-sense oligonucleotide, primer or hybridization probe for a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, K1AA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family.

25. A non-human transgenic animal expressing a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family, which is transfected with the vector of claim 20.

26. A non-human transgenic animal, wherein expression of a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a homolog, paralog or ortholog thereof is silenced and/or mutated.

27. The non-human animal of claim 25 or 26 which is selected from the group consisting of mouse, rat, sheep, hamster, pig, dog, monkey, rabbit, calf, horse, nematodes, fly and fish.

28. Use of a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or a polypeptide encoded thereby for controlling the function of a gene and/or a gene product which is influenced and/or modified by said polypeptide.

29. The use of claim 28, wherein said gene and/or gene product is a gene and/or gene product expressed in organelles.

30. The use of claim 29, wherein said organelle is a mitochondrium or a peroxisome.

31 . The use of any one of claims 28 to 30, wherein said gene and/or gene product is a member of the UCP family.

32. Use of a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, K1AA1 094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an antibody, an aptamer or another receptor recognizing said nucleic acid molecule or a polypeptide encoded thereby for identifying substances capable of interacting with said polypeptide.

33. The use of claim 32, wherein said substance(s) capable of interacting with said polypeptide is/are (an) antagonist(s) or (an) agonist(s).

34. A method of identifying a polypeptide or (a) substance(s) involved in cellular metabolism in an animal or capable of modifying homeostasis comprising the steps of: (a) testing a collection of polypeptides or substances for interaction with a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CG1 1940 homolog, dappled homolog,

CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or (a) fragment(s) thereof using a readout system; and

(b) identifying polypeptides or substances which test positive for interaction in step (a) .

35. A method of identifying a polypeptide or (a) substance(s) involved in cellular metabolism in an animal or capable of modifying homeostasis comprising the steps of (a) testing a collection of polypeptides or substances for interaction with the polypeptide identified by the method of claim 34; and (b) identifying polypeptides that test positive for interaction in step (a); and optionally (c) repeating steps (a) and (b) with the polypeptides identified one or more times wherein the newly identified polypeptide replaces the previously identified polypeptide as a bait for the identification of a further interacting polypeptide.

36. The method of claim 34 or 35 further comprising the step of identifying the nucleic acid molecule(s) encoding the one or more interacting (poly)peptides.

37. A method of identifying a (poly)peptide involved in the regulation of body weight in a mammal comprising the steps of

(a) contacting a collection of (poly)peptides with a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1 094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, K1AA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or (a) fragment(s) thereof under conditions that allow binding of said (poly)peptides;

(b) removing (poly)peptides from said collection of (poly)peptides that did not bind in step (a); and (c) identifying (poly)peptides that bind.

38. The method of claim 37 wherein said polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1 094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CG1 1940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family is fixed to a solid support.

39. The method of claim 38 wherein said solid support is a gel filtration or an affinity chromatography material.

40. The method of any one of claims 37 and 39 wherein, prior to said identification in step (c), said binding (poly)peptides are released.

41 . The method of claim 40 wherein said release is effected by elution.

42. The method of any one of claims 37 to 41 further comprising the step of identifying the nucleic acid molecule(s) encoding the one or more binding (poly)peptides.

43. A method of identifying a compound influencing the expression of a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family comprising the steps of

(a) contacting a host cell or a non-human host carrying an expression vector of claim 20 or the nucleic acid molecule identified by the method of claim 36 or 42 operatively linked to a readout system with a compound or a collection of compounds;

(b) assaying whether said contacting results in a change of signal intensity provided by said readout system; and, optionally,

(c) identifying a compound within said collection of compounds that induces a change of signal in step (b); wherein said change in signal intensity correlates with a change of expression of said nucleic acid molecule.

44. A method of identifying a compound influencing the activity of a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family comprising the steps of

(a) contacting a non-human host or a host cell carrying an expression vector of claim 20 operatively linked to a readout system and/or carrying a (poly)peptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family linked to a readout system with a compound or a collection of compounds;

(b) assaying whether said contacting results in a change of signal intensity provided by said readout system; and, optionally

(c) identifying a compound within said collection of compounds that induces a change of signal in step (b); wherein said change in signal correlates with a change in activity of said (poly)peptide.

45. The method of claim 43 or 44 wherein said host cell is a eukaryotic host cell, particularly a mammalian host cell.

46. The method of claim 43 or 44 wherein said host cell is a unicellular organism, particularly a bacterium or a yeast.

47. The method of any one of claims 43 to 46 wherein said change in signal intensity is an increase or decrease in signal intensity.

48. A method of assessing the impact of the expression of one or more polypeptides of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family in an animal comprising the steps of (a) overexpressing a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CG1 1940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a nucleic acid molecule of claim 36 or 42 in said animal; and

(b) determining whether the weight of said animal has increased, decreased, whether metabolic changes are induced and/or whether the eating behaviour is modified.

49. A method of assessing the impact of the expression of one or more polypeptides of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CG1 1940 homolog, dappled homolog, CG 1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family in an animal comprising the steps of

(a) underexpressing the nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1585 protein, CG1 1940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family or a nucleic acid molecule of claim 36 or 42 in said animal; and

0. A method of screening for an agent which modulates the interaction of a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family with a binding target/agent, comprising the steps of

(a) incubating a mixture comprising (aa) said polypeptide or a fragment thereof;

(ab) a binding target/agent of said (poly)peptide or fragment thereof; and

(ac) a candidate agent under conditions whereby said (poly)peptide, or fragment thereof specifically binds to said binding target/agent at a reference affinity;

(b) detecting the binding affinity of said (poly)peptide, or fragment thereof to said binding target to determine an (candidate) agent-biased affinity; and (c) determining a difference between (candidate) agent-biased affinity and the reference affinity.

1 . A method of screening for an agent which modulates the activity of a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog, CG1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family comprising the steps of

(a) incubating a mixture comprising

(aa) said polypeptide or a fragment thereof; and (ab) a candidate agent under conditions whereby said (poly)peptide, or fragment thereof has a reference activity,

(b) detecting the activity of said (poly)peptide, or fragment thereof to determine an (candidate) agent-biased activity; and

(c) determining a difference between (candidate) agent-biased activity and the reference activity.

52. A method of refining the compound identified by the method of any one of claims 43 to 47 or the agent identified by the method of claim 50 or 51 comprising

(a) modeling said compound by peptidomimetics; and

(b) chemically synthesizing the modeled compound.

53. A method of producing a composition comprising formulating the compound identified by the method of any one of claims 43-47 or the agent identified by the method of claim 50 or 51 or the compound refined by the method of claim 52 with a pharmaceutically acceptable carrier and/or diluent.

54. A method of producing a composition comprising the compound identified by the method of any one of claims 43 to 47 or the agent identified by the method of claim 50 or 51 comprising the steps of

(a) modifying a compound identified by the method of any one of claims 43 to 47 or the agent of claim 50 or 51 as a head compound to achieve

(i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or

(iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route, and (b) formulating the product of said modification with a pharmaceutically acceptable carrier.

55. The method of claim 53 or 54 wherein said composition is a pharmaceutical composition.

56. The method of claim 55, wherein said composition is a pharmaceutical composition for preventing, alleviating or treating diseases and disorders related to body-weight regulation and thermogenesis, for example, but not limited to, metabolic diseases such as obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, and gallstones, and disorders related to ROS defence, such as diabetes mellitus, neurodegenerative disorders, mitochondrial disorders and others.

57. A composition comprising (a) an inhibitor or stimulator of the (poly)peptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG 1 1 940 homolog, dappled homolog, CG1 1753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family or identified by the method of any one of claims 34, 35 or 37 to 41 , 50 or 51 or refined by the method of claim 52;

(b) an inhibitor of the expression of the gene identified by the method of claim 36 or 42; and/or

(c) a compound identified by the method of claim 43 or 44.

58. The composition of claim 57 which is a pharmaceutical composition.

59. Use of

(a) an inhibitor or stimulator of the (poly)peptide identified by the method of any one of claims 34, 35, 37 to 41 or 43 to 47,

50 or 51 or refined by the method of claim 52;

(b) an inhibitor or stimulator of the expression of the gene identified by the method of claim 36 or 42; and/or

(c) a compound identified by the method of claim 47; for the preparation of a pharmaceutical composition for the treatment of obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis and gallstones and disorders related to ROS defence, such as diabetes mellitus, neurodegenerative disorders, mitochondrial disorders and others.

60. Use of an agent as identified by the method of claim 50 or 51 for thepreparation of a pharmaceutical composition for the treatment, alleviation and/or prevention of obesity, as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis and gallstones and disorders related to ROS defence, such as diabetes mellitus, neurodegenerative disorders, mitochondrial disorders and others.

61 . Use of a nucleic acid molecule as depicted in SEQ ID N0:4, 6, 8, 10, 1 2, 14, 1 6, 1 8, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 38 or of (a) fragment(s) thereof for the preparation of a non-human animal which over- or underexpresses the gene product as encoded by said nucleic acid.

62. Kit comprising at least one of

(a) a nucleic acid molecule of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog,

KIAA1585 protein, CG1 1 940 homolog, dappled homolog, CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 gene family;

(b) a vector of claim 20; (c) a host of claim 21 ;

(d) a polypeptide of the Optic atrophy 1 protein, cornichon-like, IGF-II mRNA-binding protein 3, neuralized-like, KIAA1094 protein, casein kinase (delta or epsilon), glutamate dehydrogenase, kraken homolog, sirtuin 1 , escargot homolog, KIAA1 585 protein, CG1 1 940 homolog, dappled homolog,

CG 1 1 753 homolog, KIAA0095 protein, and/or formin-binding protein 21 family;

(e) a fusion protein of the polypeptide (d);

(f) an antibody or a fragment or derivative thereof or an antiserum, an aptamer or another receptor of claim 23; and

(g) a hybridization probe, primer or anti-sense oligonucleotide of claim 24.