WO2004050007A2 - Mammalian bt-42 proteins involved in the regulation of energy homeostasis - Google Patents

Abstract

The present invention discloses BT-42 homologous proteins regulating the energy homeostasis and the metabolism of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to the use of these sequences in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic disorders and diseases.

Description

Mammalian BT-42 proteins involved in the regulation of energy homeostasis

This invention relates to the use of nucleic acid sequences encoding mammalian acid phosphatase, in particular to BT-42 and isoforms thereof. Furthermore, the use of (a) BT-42 polypeptide(s) for medical and diagnostic purposes is described and the invention provides for screening methods employing nucleic acid molecules encoding

- BT-42 (or isoforms) and/or screening methods which employ BT-42 polypeptides (or isoforms thereof). In addition, uses of effectors of BT-42 polypeptides and nucleic acid molecules encoding the same (or isoforms of such polypeptides and nucleic acid molecules) in the diagnosis, study, prevention, and treatment of diseases and disorders related to body-weight regulation, for example, but not limited to, metabolic diseases or dysfunctions such as obesity as well as related disorders such as Type 2 diabetes is disclosed.

There are several metabolic diseases of human and animal metabolism, eg., obesity and severe weight loss, that relate to energy imbalance where caloric intake versus energy expenditure is imbalanced. Obesity is one ofthe most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes as a major health problem more and more relevant for western society. Obesity is defined as a body weight more than 20% in excess of the ideal body weight, frequently resulting in a significant impairment of health. Obesity may be measured by body mass index, an indicator of adiposity or fatness. Further parameters for defining obesity are waist circumferences, skinfold thickness and bioimpedance (see, inter alia, Kopelman (1999), loc. cit.). It is associated with an increased risk for cardiovascular disease, hypertension, diabetes mellitus Type π, hyperlipidaemia and an increased mortality rate. Besides severe risks of illness, individuals suffering from obesity are often isolated socially.

Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors and can be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate bound energy and low energy expenditure. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Since obesity is not to be considered as a single disorder but as a heterogeneous group of conditions with (potential) multiple causes, it is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Koltermann, J. Clin. Invest 65, 1980,

1272-1284). A clear involvement of obesity in diabetes mellitus Type π can be confirmed (Kopelman, Nature 404, 2000, 635-643). In diabetes mellitus Type π, liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance). High blood glucose levels (and also high blood lipid levels) lead to an impairment of beta-cell function and to an increase in beta-cell apoptosis. Eventually the application of exogenous insulin becomes necessary in those patients.

Hyperlipidemia and elevation of free fatty acids correlate clearly with the ,Metabolic Syndrome', which is defined as the linkage between several diseases, including obesity an insulin resistance. This often occurs in the same patients and are major risk factors for development of Type 2 diabetes and cardiovascular disease.The concept of 'metabolic syndrome' (syndrome x, insulin-resistance syndrome, deadly quartet) was first described 1966 by Camus and remtroduced 1988 by Reaven (Camus,

1966, Rev Rhum Mai Osteoartic 33: 10-14; Reaven (1988), Diabetes 37: 1595-1607). 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: 286-288). The metabolic syndrome often precedes the development of type II diabetes and cardiovascular disease (Lakka et al, 2002 JAMA 288: 2709-2716). The control of blood lipid levels and blood glucose levels is essential for the treatment of the metabolic syndrome (see, for example, Santomauro et al., 1999 Diabetes, 48: 1836-1841).

The molecular factors regulating food intake and body weight balance are incompletely understood. Even if several candidate genes have been described which are supposed to influence the homeostatic system(s) that regulate body mass/weight, like leptin or the. peroxisome proliferator-activated receptor-gamma co-activator, the distinct molecular mechanisms and/or molecules influencing obesity or body weight/body mass regulations are not known, h addition, several single-gene mutations resulting in obesity have been described in mice, implicating genetic factors in the etiology of obesity. (Friedman and Leibel, 1990, Cell 69: 217-220). In the obese (ob) mouse a single gene mutation (obese) results in profound obesity, which is accompanied by diabetes (Friedman et. al., 1991, Genomics 11: 1054-1062 ).

Therefore, the technical problem underlying the present invention was to provide for means and methods for modulating (pathological) metabolic conditions influencing body-weight regulation andor energy homeostatic circuits. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to genes with novel functions in body- weight regulation, energy honieostasis, metabolism, and obesity. The present invention discloses specific genes involved in the regulation of body-weight, energy homeostasis, metabolism, and obesity, h particular, the present invention describes the human^' BT- 42 genes as being involved in those conditions mentioned above and discloses specific medical and/or diagnostic uses and methods employing nucleic acid molecules encoding BT-42 molecules (or isoforms or fragments thereof) or employing BT-42 polypeptides or isoforms or fragments thereof.

We relate in this invention to BT-42 polypeptides or nucleic acid molecules encoding the same as well as to BT-42 isoforms or fragments. As used herein, the term "BT- 42" relates in particular to nucleic acid molecules or polypeptides deposited under

NM H4659 or AF543190 as well as to sequences shown in appended SEQ ID NOs: 1 to 4 and to sequences which comprise isoforms or (functional) fragments of the same. BT-42 originally represents a nucleotide sequence expressing KIAA0377 protein, a protein not yet further characterized (GenBank Accession number NM_014659). However, the term "BT-42" as used herein also relates to the nucleotide sequence and polypeptide sequence as deposited under AF543190 (NCBI data base) and to isoforms and/or (functional) fragments of said deposited BT-42 form. Such isoforms are, inter alia, disclosed herein and are depicted in appended SEQ ID NOs: 1 to 4. The term "functional fragment" as used herein relates to fragments of BT-42 as defined herein which, comprise at least one biochemical and/or physiological feature as the BT-42 polypeptides or BT-42 polynucleotides disclosed herein. These functions comprise, but are not limited to phosphorylation event, phosphatase activity, in vivo or in vitro interaction with "adipose" and/or direct or indirect regulation of energy honieostasis.

So far, it has not been described that the human BT-42 proteins ofthe invention are involved in the regulation of energy homeostasis and body-weight regulation and related disorders, and thus, no functions in metabolic diseases and dysfunctions and other diseases as listed above have been discussed.

The present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity, fragments of said genes, polypeptides encoded by said genes or fragments thereof, and effectors e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides. Accordingly, the present invention relates to compositions, in particular pharmaceutical and diagnostic compositions which comprise the nucleic acid molecules and polypeptides described herein. Furthermore, compositions are disclosed which comprise effector molecules and/or modifiers ofthe polypeptides and/or nucleic acid molecules disclosed herein.

The term "adipose protein" as employed herein refers to a molecule as defined herein and which is capable of regulating causing or contributing to obesity in an animal or a human, either above or in form of the complex described herein. The human 'adipose' protein is disclosed in WO 01/96371. The adipose protein as described as complex partner herein may be naturally occurring adipose, but also comprises recombinantly produced or biochemically synthesized adipose protein or a fragment or a derivative thereof. Derivatives of the adipose protein or its fragments comprise, but are not limited to naturally occurring and/or genetically engineered variants of adipose, but also to chemically modified adipose, labeled adipose. The adipose protein/fragment thereof comprised in the complex of the invention relates preferably to human adipose. Preferably, but not restricting, the adipose protein/fragment thereof is capable, either alone or in the herein described complex of "regulating, causing or contributing to obesity". The term "regulating, causing or contributing to obesity" relates to the functional properties of a (poly)peptide to modify, either directly or indirectly the physiological status of energy metabolism. Said metabolism may be anabolic or catabolic. In this context, obesity is to be understood as a complex disorder of appetite regulation and/or energy metabolism, influencing body weight/body mass of an individual. Said obesity comprises disorders involving an excess storage of fat. Said obesity may be simple obesity or a certain condition wherein obesity is an associated feature (e.g. genetic syndroms associated with hypogonadism, e.g. Prader-Willi syndrome, hypothroidism, Crashing's syndrome, Stein-Leventhal syndrome, corticosteroid intake, hypothalamic damage, etc.). Further disorders/diseases related to obesity, modified status of energy metabolism and/or body weight/body mass of an individual are disclosed herein below.

The invention is based on the finding that BT-42 polypeptide and their isoforms as well as the polynucleotides encoding the same, are involved in the energy homeostasis. To find genes with novel functions in energy homeostasis, metabolism, and obesity, a yeast two hybrid screen was performed. The present invention is based upon the surprising identification of BT-42 proteins (and isoforms thereof) as an interactor of "adipose" (see WO 01/96371 which is incorporated herein by reference) using interaction assays such as two hybrid screenings (as described in EP-0 963 376, WO 98/25947, WO 00/02911), GST-pull-down columns, co-precipitation assays from cell extracts as described in Kasus-Jacobi, 2000, Oncogene 19, 2052-2059, interaction-trap systems, expression cloning, phage display, in vitro binding assays and the like. BT- 42 proteins were found to form complexes under physiological conditions with adipose (see Examples). These complexes are implicated in modulating the functional activities of adipose and the functional activities of its binding partners.

Such functional activities include, but are not limited to, treating and/or preventing certain diseases and disorders, particularly concerning metabolic conditions, influencing body-weight regulation, thermogenesis, and/or energy homeostatic circuits are also provided.

The present invention discloses that BT-42 isoforms are regulating the energy homeostasis and fat metabolism, and polynucleotides, which identify and encode the proteins disclosed in this invention. The invention also relates to vectors, host cells, antibodies, and recombinant methods for producing the polypeptides and polynucleotides of the invention. The invention also relates to the use of these polynucleotides, polypeptides and effectors thereof in the diagnosis, study, prevention, and treatment of diseases and disorders, for example, but not limited to, metabolic diseases such as obesity and diabetes as well as related disorders.

BT-42 homologous proteins and nucleic acid molecules coding therefore are obtainable from vertebrate species, e.g. mammals. Particularly preferred are nucleic acids encoding the human BT-42 homologs (in particular the human BT-42 isoforms as described in tins invention).

The term "polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number" relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number. The term "GenBank

Accession number" relates to NCBI GenBank database entries (Ref: Benson et al., Nucleic Acids Res. 28 (2000) 15-18). Examples are the entry NM_014659 or AF543190.

The invention particularly relates to compositions as described herem, wherein said compositions preferably comprise a nucleic acid molecule encoding a polypeptide contributing to regulating the energy homeostasis, wherein said nucleic acid molecule comprises (a) a nucleic acid molecule encoding a polypeptide as shown in SEQ ID NOs: 2 or 4_? a polypeptide as deposited under GenBank accession number NM_014659 or under accession number AF543190 or an isoform, fragment or variant of the polypeptide as shown in SEQ ID NOs: 2 or 4 or an isoform, fragment or variant of the polypeptide as deposited under accession numbers NM_014659 or AF543190; (b) a nucleic acid molecule which comprises or is the nucleic acid molecule as shown in SEQ ID NO: 1 or 2, the nucleic acid molecule as deposited under NM_014659 or AF543190; (c) a nucleic acid molecule being degenerate as a result of the genetic code to the nucleic acid sequences as defined in (a) or (b);

(d) a nucleic acid molecule that hybridizes at 65°C in a solution containing 0.2 x SSC and 0.1% SDS to a nucleic acid molecule as defined in claim 2 or as defined in (a) to (c) and/or a nucleic acid molecule which is complementary thereto;

(e) a nucleic acid molecule that encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to a human BT-42 , as defined in claim 2 or to a polypeptide as defined in (a); (f) a nucleic acid molecule that differs from the nucleic acid molecule of (a) to (e) by mutation and wherein said mutation causes an alteration, deletion, duplication or premature stop in the encoded polypeptide.

The terms "hybridizes" and "hybridizing" as employed in context of the present invention preferably relate to stringent conditions as, inter alia, defined herein above, e.g. 0.2 x SSC, 0.1% SDS at 65°. Said conditions comprise hybridization as well as washing conditions. However, it is preferred that washing conditions are more stringent than hybridization conditions. By setting the conditions for hybridization, the person skilled in the art can determine if strictly complementary sequences or sequences with a higher or lower degree of homology are to be detected. The setting of conditions is well within the skill ofthe artisan and to be determined according to protocols described, for example, in Sambrook, Molecular Cloning, A Laboratory

Manual, 2 edition (1989), Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, NY or Hames and Higgins, "Nucleic acid hybridization, a practical approach", IRL Press, Oxford (1985). Non-stringent hybridization conditions for the detection of homologous and not exactly complementary sequences may be set at 6 x

SSC, l% SDS at 65°C. The human BT42 gene that is described in GenBank Accession NM_014659 for nucleotide and amino acid sequences encodes a protein of 1406 amino acids. The . protein belongs to a small, highly conserved KIAA0377 protein family. We could identify two novel isoforms in humans which were not described previously in the prior art, refered to as BT42-I and BT42-H (see FIGURE 1A, B, C, and D for nucleotide and amino acid sequences; SEQ ID NO:l, 2, 3, and 4, respectively). A sequence alignment of the four human BT42 isoforms was performed using the alignment tool ClustalW and is shown in FIGURE IE. This FIGURE shows that both BT42 variants (BT42-I, BT42-II) have a deletion of 40 amino acids in comparison to the BT-42 (KIAA0377). Furthermore, the BT42-H isoform contains one additional exon of 42 amino acids. It is of note that the term "BT-42" as used herein comprises also nucleic acid molecules or polypeptides as deposited under AF543190 and isoforms or f agments thereof.

We found in our analysis that BT42 amino acid sequence contains the central signature of a histidine acid phosphatase ( [L-NM]-x(2)-[L-NMA]-x(2)-[LINM]-x-R- H-[GΝ]-x-R-x-[PAS], pfam00328), with a Histidin amino acid in their active site. This motif is found in BT42 in amino acids 391 to 405 (LRCVIAIIRHGDRTP). Using the InterPro analysis tool, we could identify a histidine acid phosphatase domain in BT-42 (carboxy-terminal amino acids 488 - 947). No conserved domains could be found in the amino-terminal half ofthe protein. The family of histidine acid phosphatases is a heterogeneous group of proteins. They are known to hydrolyse phosphate ester at low pH and are able to use a wide spectrum of substrates.

Our analysis using standard analysis tools (for example, http://www.expf.sy.di/too1s protparam.htm1; Reinhardt & Hubbard, Nucleic Acids Research, 1998, 26:2230-2236) showed that Bt-42-II is a protein of 1408 amino acid with a calculated molecular weight of 156615.6 daltons. The predicted subcellular localization is nuclear and possibly cytoplasmic. The Bt-42-I isoform is a protein of 1366 amino acid with a calculated molecular weight of 152262.7 daltons. The predicted subcellular localization is nuclear and possibly cytoplasmic.

In this invention, we clearly show that mammalian BT42 (or variants thereof) has a function in regulating the metabolism of mature adipocytes. As shown in Figure 2A and described in more detail in the Examples, analysis of the expression of KIAA0377 (BT42)- mRNA in mammalian (mouse) tissues revealed that KIAA0377 (BT42) -protein is expressed in different mammalian tissues, showing highest levels of expression in neural tissues such as cerebellum, hypothalamus, midbrain and cortex, but also high levels of expression in the small intestine, heart, and kidney, and lower levels in muscle, lung and spleen tissues. A clear expression iii brown adipocyte tissue (BAT) and lower expression in white adipocyte tissue (WAT) is seen. Brown adipose tissue is a well characterized tissue which is well developed in newborn mammals, including humans. One important task of BAT is to generate heat and maintain body temperature homeostasis in newborn. Thus, an expression of BT- 42 in adipose tissues is confirming a role in the regulation of energy homeostasis and thermogenesis.

Further, we show that the mammalian BT-42 protein is regulated by fasting and by genetically induced obesity, hi this invention, we used mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, oh (leptin) or db (leptin receptor) mice) to study the expression of the protein of the invention. Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning et al, 1998, Mol. Cell. 2:449-569). We found, for example, that the expression of mouse BT-42 is upregulated in liver of fasted mice (see Figure 2B), and that the expression shows high levels in the muscle of genetically obese mice. In addition, a marked downregulation can be observed in the metabolically active tissue (for example, brown adipose tissue (BAT)) and white adipose tissue (WAT) and several other tissues such as those isolated from brain and small intestine of fasted mice, and also downregulated in BAT and WAT and other tissues including brain of genetically obese mice (see FIGURE 2B). In addition, BT-42 expression in observed in all tissues in the db/db mice model, with marked downregulation in metabolically active tissues (BAT, WAT) (see FIGURE 2C).

The BT42 - protein was also examined in the in vitro differentiation models for the conversion of pre-adipocytes to adipocytes, as described above. We found in different model systems, that BT42 protein is strongly upregulated during adipocyte differentiation in vitro, suggesting a role as modulator of adipocyte lipid accumulation (see FIGURE 2D, 2E, and 2F).

The present invention further describes polypeptides comprising the amino acid sequences of the proteins of the invention and homologous proteins. Based upon homology, the proteins of the invention and each homologous protein or peptide may share at least some activity.

The invention also encompasses polynucleotides that encode the proteins of the invention and homologous proteins. Accordingly, any nucleic acid sequence, which encodes the amino acid sequences of the proteins of the invention and homologous proteins, can be used to generate recombinant molecules that express the proteins ofthe invention and homologous proteins, h a particular embodiment, the invention encompasses a nucleic acid encoding human BT-42 isoforms; referred to herein as the proteins ofthe invention. It will be appreciated by those skilled in the art that as a result ofthe degeneracy ofthe genetic code, a multitude of nucleotide sequences encoding the proteins, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, may be produced. The invention contemplates each and every possible variation of nucleotide sequence that can be made by selecting combinations based on possible codon choices.

The encoded proteins may also contain deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of the protein is retained. Furthermore, the invention relates to peptide f agments 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 described herein, in particular BT-42 proteins, their isoforms and homologous protems. 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 (single nucleotide polymorphism / SNP). Alleles may result in altered mRNAs or polypeptides whose structures or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, are generally ascribed to natural deletions, additions or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others, one or more times in a given sequence.

The nucleic acid sequences encoding the proteins described in the invention and homologous proteins may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.

In order to express a biologically active protem as described in this invention, 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 cont-tining sequences encoding the proteins and the appropriate transcriptional and translational control elements. Regulatory elements include for example a promoter, ah 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 (CMN) promoter/enhancer region, (ϋ) tissue specific promoters such as the insulin promoter (see, Soria et al, 2000, Diabetes 49:157), SOX2 gene promotor (see Li et al, 1998, Curr. Biol. 8:971-4), Msi-1 promotor (see Sakakibara et al., 1997, J. Νeuroscience 17:8300-8312), alpha-cardia myosin heavy chain promotor or human atrial natriuretic factor promotor (Klug et al., 1996, J. clin. Invest 98:216-24; Wu et al., 1989, 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 DΝA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

In a further embodiment ofthe invention, natural, modified or recombinant nucleic acid sequences encoding the proteins in particular the BT-42 proteins described herein and homologous proteins may be ligated to a heterologous sequence to encode a, fusion protein.

A variety of expression vector/host systems may be utilized to contain and express sequences encoding the proteins or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed, with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, adenovirus, adeno-associated virus, lentiverus, retrovirus); plant cell systems transfonned 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 described herein in a sample can be detected by DNA-DNA or DNA-RNA hybridization and or amplification using probes or portions or fragments of said polynucleotides. Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences specific for the gene to detect fransformants containing DNA or RNA encoding the corresponding protein. As used herein 'oligonucleotides' or 'oligomers' refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting polynucleotide sequences include oligo-labeling, nick translation, end-labeling of labeled RNA probes, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).

The presence of proteins described in the invention in a sample can be dete-mined by immunological methods or activity measurement. A variety of protocols for detecting and measuring the expression of protems, 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 immunosofbent assay (ELISA), radiohnmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

Suitable reporter molecules or labels, which may be used, include radionuclides, enzymes, fluorescent, chermlumh escent or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like.

The nucleic acids encoding the proteins described in 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 ofthe invention is altered. Alternatively, a nucleic acid construct is randomly integrated into the genome. Vectors for stable integration include plasmids, retrovirusses and other -inimal 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 protems of the invention to deteπnine 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 ofthe genes ofthe 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 cont-tining 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 -mimals, etc. The transgenic aniinals 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 described herein and related to BT-42 and effector molecules of these nucleic acid molecules and polypeptides are useful, in diagnostic and therapeutic applications implicated, for example but not limited to, in metabolic disorders such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones. Hence, diagnostic and therapeutic uses for the proteins ofthe invention nucleic acids and proteins ofthe invention 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 described herein and effectors thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below. For example, but not limited to, cDNAs encoding the proteins of the invention and particularly their human homologues may be useful in gene therapy, and the proteins ofthe invention and particularly their human homologues may be useful when administered to a subject in need thereof. By way of non-limiting example, the compositions of the present invention will have efficacy for treatment of patients suffering from, for example, but not limited to, in metabolic disorders as described above.

The nucleic acids, isoforms or fragments thereof as described in this invention, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acids or the proteins are to be assessed. Further antibodies that bind immunospecifically to the substances of the invention may be used in therapeutic or diagnostic methods.

For example, in one aspect, antibodies, which are specific for the proteins described herein, in particular the BT-42 and its isoforms and homologous proteins, may he used directly as an effector, e.g. 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, chimeric single chain, Fab fragments, and fragments produced by a Fab expression library.

Neutralising antibodies, (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the protem or any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase hnmunological response. It is preferred that the peptides, fragments or oligopeptides used to induce antibodies to the protein have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.

Monoclonal antibodies to the proteins may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBN-hybridoma technique (Kδhler, G. et al. (1975)

Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol. 62:109-120). In 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. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature 314:452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce single chain antibodies specific for the proteins of the invention and homologous proteins. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for the proteins may also be generated. For example, such fragments include, but are not limited to, the F(ab')₂ fragments which can be produced by Pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 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. (1989) Science 254:1275-1281).

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 utilizing monoclonal antibodies reacive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox, supra). In another embodiment of the invention, the polynucleotides or fragments thereof or nucleic acid effector molecules such as antisense molecules, aptamers, RNAi molecules or ribozym.es may be used for therapeutic purposes. In one aspect, aptamers, i.e. nucleic acid molecules, which are capable of binding to an AOK protein and modulating its activity, may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries.

-h a further aspect, antisense molecules may be used in situations in which it would be desirable to block the transcription (translation) of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding the proteins of the invention and homologous proteins. Thus, antisense molecules may be used to modulate protein activity or to achieve regulation of gene function. Such technology is now well known in the art, and sense or antisense oligomers or larger fragments, can be designed from various locations along the coding or control regions of sequences encoding the proteins. Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding the proteins of the invention and homologous proteins. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra). Genes encoding the proteins of the invention and homologous proteins can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode the proteins of the invention and homologous proteins or fragments thereof. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part ofthe vector system.

As mentioned above, modifications of gene expression can be obtained by designing antisense molecules, e.g. DNA, RNA or nucleic acid analogues such as PNA, to the control regions of the genes encoding the proteins of the invention and homologous proteins, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee, J. E. et al. (1994) In; Huber, B. E. and B. I. Carr, Molecular and hnmunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.). The antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples, which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding the proteins of the invention and homologous proteins. 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 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.

Nucleic acid effector molecules, e.g. antisense molecules and ribozymes described herein may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase -ntracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends ofthe molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g. the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any ofthe 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 aα-ministration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of the nucleic acids and the proteins of the invention and homologous nucleic acids or proteins, antibodies to the proteins of the invention and homologous proteins, mimetics, agonists, antagonists or inhibitors of the proteins of the invention and homologous proteins or nucleic acids. The compositions may be aά-ministered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. The compositions may be administered to a patient alone or in combination with other agents, drugs or hormones. The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means...

hi 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 deteπniiie 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 of the invention or fragments thereof or antibodies, which is sufficient for treating a specific condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration. 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 maybe taken into account, include the severity ofthe disease state, general health ofthe subject, age, weight, and gender of the subject, diet, time and frequency of aά-ministration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be a(-ministered 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.

Accordingly, the invention also provides for use of a nucleic acid molecule as defined in the invention, use of a polypeptide as defined in the invention, use of a vector as defined in the invention, use of a host cell as defined in the invention for the preparation of a pharmaceutical composition for the treatment, alleviation and/or prevention of diseases and disorders, including metabolic diseases such as obesity and other body-weight regulation disorders as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones and other diseases and disorders.

h another embodiment, antibodies which specifically bind to the proteins, in particular the BT-42 and its isoforms as described herein, may be used for the diagnosis of conditions or diseases characterized by or associated with over- or underexpression of the proteins of the invention and homologous proteins or in assays to monitor patients being treated with the proteins of the invention and homologous proteins, or effectors thereof, e.g. agonists, antagonists, or inhibitors. Diagnostic assays include methods which utilize the antibody and a label to detect the protein in human body fluids or extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covaleh ly, with a reporter molecule. A wide variety of reporter molecules which are known in the art may be used several of which are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring proteins are known in the art and provide a basis for diagnosing altered or abnormal levels of gene expression. Normal or standard values for gene expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibodies to the protein under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of protein expressed in control and disease samples e.g. from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.

h another embodiment of the invention, the polynucleotides specific for the proteins described herein, in particular the BT-42 polypeptide and its isoforms and homologous proteins may be used for diagnostic purposes. The polynucleotides, which may be used, include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which gene expression may be correlated with disease. The diagnostic assay may be used to distinguish between absence, presence, and excess gene expression, and to monitor regulation of protein levels during therapeutic intervention.

In one aspect, hybridization with probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding the proteins of the invention and homologous protems or closely related molecules, may be used to identify nucleic acid sequences which encode the respective protein. The hybridization probes ofthe subject invention may be DNA or RNA and are preferably derived from the nucleotide sequence of the polynucleotide encoding the proteins of the invention or from a genomic sequence including promoter, enhancer elements, and introns ofthe naturally occurring gene. Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as ³ P or ³⁵S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avi<--in/biotin coupling systems, and the like.

Polynucleotide sequences specific for the proteins described in the invention and homologous nucleic acids may be used for the diagnosis of conditions or diseases, which are associated with the expression ofthe proteins. Examples of such conditions or diseases include, but are not limited to, pancreatic diseases and disorders, including diabetes. Polynucleotide sequences specific for the proteins of the invention and homologous proteins may also be used to monitor the progress of patients receiving treatment for pancreatic diseases and disorders, including diabetes. The polynucleotide sequences may be used qualitative or quantitative assays, e.g. in Southern or Northern analysis, dot blot or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered gene expression.

hi a particular aspect, the nucleotide sequences specific for the proteins described in the invention and homologous nucleic acids may be useful in assays that detect activation or induction of various metabolic diseases such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones.. The nucleotide sequences may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding the proteins of the invention and homologous proteins 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 -mimal studies, in clinical trials or in monitoring the treatment of an individual patient.

hi order to provide a basis for the diagnosis of a disease associated with expression of the proteins described in the invention and homologous proteins, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence or a fragment thereof, which is specific for the nucleic acids encoding the proteins of the invention and homologous nucleic acids, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease. Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that, which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.

With respect to metabolic diseases such as described above the presence of an unusual amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the metabolic diseases and disorders.

Additional diagnostic uses for oligonucleotides designed from the sequences encoding the proteins of the invention and homologous proteins may involve the use of PCR. Such oligomers may be chemically synthesized, generated enzymatically or produced from a recombinant source. Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5prime.fwdarw.3prime) and another with antisense (3prime.rarw.5prime), employed under optimized conditions for identification of a specific gene or condition. The same two oligomers, nested sets of oligomers or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences. hi another embodiment ofthe invention, the nucleic acid sequences may also be used to generate hybridization probes, which are useful for mapping the naturally occirrring 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 PI constructions or single chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154. FISH (as described in Verma et al. (1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y.). The results may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 198 If). Correlation between the location of the gene encoding the proteins of the invention on a physical chromosomal map and a specific disease or predisposition to a specific disease, may help to delimit the region of DNA associated with that genetic disease.

The nucleotide sequences described herein may be used to detect differences in gene sequences between normal, carrier or affected individuals. An analysis of polymorphisms, e.g. single nucleotide polymorphisms may be carried out. Further, in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to l lq22-23 (Gatti, R. A. et al. (1988) 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 ofthe invention, the proteins described herein, e.g. the BT-42 proteins, isoforms and functional fragments thereof, its 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, ligands or substrates that bind to, modulate or mimic the action of one or more of the proteins ofthe 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 proteins described herein and the agent tested, may be measured. Agents can also, be identified, which, either directly or indirectly, influence the activity ofthe protein ofthe invention. For example the phosphatase activity ofthe protein ofthe invention could be measured in vitro by using recombinantly expressed and purified Bt-42 or fragments thereof by making use of artificial substrates well known in the art, i.e. but not exclusively DiFMUP (Molecular Probes, Eugene, Oregon), which are converted to fluorophores or chromophores upon dephosphorylation. Alternatively, the dephosphorylation of physiological substrates of Bt-42 could be measured by making use of any ofthe well known screening technologies suitable for the detection ofthe phosphorylation status of Bt-42 physiological substates. In addition activity of Bt-42 against its physiological substrate(s) or derivatives thereof could be measured in cell-based assays.

Agents may also interfere with posttranslational modifications ofthe protein, such as phosphorylation and dephosphorylation, famesylation, palmitoylation, acetylation, alkylation, ubiquitinatioή, 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 protems of this invention with other proteins, which are required for protein function, for example, but not exclusively, their downstream signalling. Methods for deteπriining 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, hi 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 ofthe Invention Itself. 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 ofthe functional chemical groups. The candidate agents often comprise carbocyclic or heterocyclic structures and or aromatic or polyaromatic structures substituted with one or more ofthe 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 randomised 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 proteins of the invention large numbers of different small test compounds, e.g. aptamers, peptides, low-molecular weight compounds etc., are provided or synthesized on a solid substrate, such as plastic pins or some other surface. The test compounds are reacted with the proteins or fragments thereof, and washed. Bound proteins are then detected by methods well known in the art. Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding 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.

Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support, hi another embodiment, one may use competitive drug screening assays in which neufrahzing antibodies capable^" of binding the protein specifically compete with a test compound for binding the protein, hi this manner, the antibodies can be used to detect the presence of any peptide, which shares one or more antigenic detenninants with the protein. ^' 5 ^■

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

(a) a BT-42 nucleic acid molecule or a fragment or an isoform thereof;

(b) a BT42 a ino acid sequence or a fragment or an isoform thereof,

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

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

(e) a polypeptide encoded by the nucleic acid of (a), expressed by the vector of (c) or the host cell of (a);

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

(g) an antibody, an aptamer or another receptor against the nucleic acid of 15 . (a) or the polypeptide of (b), (e) or (f), and/or

(h) an anti-sense oligonucleotide ofthe 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.

20

Moreover, the present invention relates to an in vitro metliod for identifying a polymorphism in the BT-42-gene. Said method comprising the steps of:

(a) isolating a polynucleotide or the gene of the invention from a plurality of subgroups of individuals, wherein one subgroup has no prevalence for a

25 body-weight regulation associated disease or disorder and at least one or more further subgroup(s) do have prevalence for a body-weight regulation associated disease or disorder; and

(b) identifying a polymorphism by comparing the nucleic acid sequence of said polynucleotide or said gene of said one subgroup having no prevalence for a

30 body-weight regulation associated disease or disorder with said at least one or more further subgrouρ(s) having a prevalence for a body-weight regulation associated disease or disorder. The term "prevalence" as used herein means that individuals are be susceptible for one or more disease(s) and or disorder(s) which are associated with BT-42 dysfuntion or dysregulation or could already have one or more of said disease(s). Advantageously, polymorphisms according to the present invention which are associated with BT-42 dysfunction or dysregulation or one or more disease(s) based thereon should be enriched in subgroups of individuals which have a prevalence for said diseases versus subgroups which have no prevalence for said diseases. Thus, the above described method allows the rapid and reliable detection of polymorphism which are indicative for one or more body-weight regulation associated disease or disorder or a susceptibility therefor. Advantageously, due to the phenotypic preselection a large number of individuals having no prevalence might be screened for polymorphisms in general. Thereby, a reference sequences comprising polymorphisms which do not correlate to one or more BT-42 associated disease(s) or disorder(s) can be obtained. Based on said reference sequences it is possible to efficiently and reliably detennine the relevant polymorphisms.

As described in detail herein above, the presence of specific alleles of BT-42 carrying at least one SNP in the genome of a cell, tissue, organ or individual may be responsible for a specific expression of the encoded protein. Such alteration may represent an increase as well as a reduction expressed mRNA 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 i a given sequence.

The present invention envisages in an alternative embodiment diagnostic compositions for the detection of SNPs in biological samples. Methods for the detection of SNPs in a biological sample are known in the art; see e.g. Kwok & Chen. Detection of single nucleotide polymorphisms. Curr. Issues Mol. Biol. 5:43-60 (2003); Kwok. Methods for genotyping single nucleotide polymorphisms. Annu. Rev. Genomics Hum. Genet.

2001, 2:235-258; Syvanen,. Accessing genetic variation: Genotyping single nucleotide polymorphisms. Nature Rev. Genet. 2: 930-942 (2001). Hereby, the presence of one or more SNPs in a sample is indicative for a predisposition for or the presence of diseases and disorders related to body-weight regulation.

The above described diagnostic compositions are, inter alia, envisaged to be used for the diagnosis or study of diseases and disorders related to body-weight regulation, for example, but not limited to, metabolic diseases or dysfunctions such as obesity as well as related disorders such as Type 2 diabetes. Thus, methods and uses of diagnostic compositions for the diagnosis or study of diseases and disorders related to body- weight regulation are comprised by the present invention.

Finally, the present invention relates to a diagnostic kit for detection of a single nucleotide polymoφhism comprising the polynucleotide, the gene, the vector, the polypeptide, the antibody, the host cell, the transgenic non-human animal or the solid support of the invention.

The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals. The kit ofthe invention can be used for c-irrying out a method ofthe invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as ^' research tool. The parts of the kit of the invention can be packaged individually in vials or other appropriate means depending on the respective ingredient or in combination in suitable containers or multicontainer units. Manufacture ofthe kit follows preferably standard procedures which are known to the person skilled in the art. The kit may be used for methods for detecting expression of a mutant form of the polypeptides, genes or polynucleotides in accordance with any one of the above- described methods of the invention, employing, for example, immunoassay techniques such as radioimmunoassay or enzymeimmunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples as well as pharmacokinetic studies when using non-human transgenic anhnals ofthe invention.

The Figures show:

FIGURE 1 shows the sequences of human BT-42 nucleic acids and the proteins encoded thereby.

FIG. 1A shows the nucleotide sequence of isoform BT_42-1 (SEQ D NO: 1) FIG. IB shows the amino acid sequence of isoform BT_42-1 (SEQ ID NO: 2) FIG. 1C shows the nucleotide sequence of isoform BT_42-2 (SEQ ID NO: 3) FIG. ID shows the amino acid sequence of isoform BT_42-2 (SEQ ID NO: 4) FIG. IE shows a CLU&TAL W (1.82) multiple sequence alignment ofthe KIAA0377 protein to three other human isoforms.

FIGURE 2 shows the expression of BT-42 in different mammalian models

FIGURE 2A. Expression of KIAA0377 (BT42) i mammalian tissues. Real-time PCR analysis of KIAA0377 (BT42) expression in wildtype mouse tissues. The relative RNA-expression is shown on the left hand side, the tissues tested are given on the horizontal line. WAT = white adipose tissue, BAT = brown adipose tissue

FIGURE 2B. Expression of KIAA0377 (BT42) in mammalian tissues Real-time PCR analysis of KIAA0377 (BT42) expression in different tissues of ob/ob mice and fasted mice in comparison to the expression in the appropriate wildtype tissue. The expression is upregulated in liver of fasted mice. The relative RNA-expression is shown on the left hand side, the tissues tested are given on the horizontal line. WAT = white adipose tissue, BAT = brown adipose tissue

FIGURE 2C. Expression of KIAA0377 (BT42) in mammalian tissues. Real-time

PCR analysis of KIAA0377 (BT42) expression in different tissues of db/db mice in comparison to the expression in the wildtype tissue. The relative RNA-expression is shown on the left hand side, the tissues tested are given on the horizontal line. WAT = white adipose tissue, BAT = brown adipose tissue

FIGURE 2D. Real-time PCR mediated comparison of KIAA0377 (BT42) expression during the differentiation of TA1 cells from preadipocytes to adipocytes. BT42 is upregulated starting on day 4. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (dO = day 0, start ofthe experiment, until dl 0 = day 10)

FIGURE 2E. Real-time PCR mediated comparison of KIAA0377 (BT42) expression during the differentiation of 3T3-L1 cells from preadipocytes to adipocytes. BT42 shows strong upregulation of its expression during this in vitro differentiation. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (dO = day 0, start ofthe experiment, until dlO = day 10)

FIGURE 2F. Real-time PCR mediated comparison of KIAA0377 (BT42) expression during the differentiation of 3T3-F442A cells from preadipocytes to adipocytes. The relative RNA-expression is shown on the left hand side, the days of differention are shown on the horizontal line (dO = day 0, start ofthe experiment, until dlO = day 10)

The examples illustrate the invention :

Example 1. Identification of Molecules Which Interact with Adipose applying the Matchmaker Two Hybrid-System (Clontech)

A standard Yeast-Two-Hybrid Screen was. performed using the pretransformed Human cDNA Library Matchmaker System according to the manufacturer's instructions (Clontech).

All libraries used in the screen are contained in the pACT2 vector as provided by the manufacturer (Clontech). The bait vectors are based on the pGBKT7 vector (Clontech), into which the appropriate part of the human adipose cDNA was cloned in frame with the GAL4 DNA-binding region (GAL4 DNA-BD) into the restriction sites EcoRI (5') and BamHI (3') using standard cloning techniques.

The following two constructs were using in the Yeast-Two-Hybrid screens:

_a. Cloning of pGBKT7-Adp-comp

This construct was generated by amplifying the complete human adipose cDNA with polymerase chain reaction using cloned pfu DNA polymerase (Stratagene) with the forward primer 5'-GATC GAATTC

GGAGGTGGAGCGAAAGTCAACATAACTAG-3' (SEQ ID NO: 7) and the reverse primer 5 'GATC GGATCC CTAGCTGGGCCGGCACTGCACCTG-

3' (SEQ ID NO: 8) on full-length human adipose cDNA, thereby introducing three glycines at the extreme N-terminus in frame with the adipose reading frame in addition to two unique restriction sites (EcoRI/BamHI). The fragment was digested with BamHI and EcoRI and subcloned into the yeast expression vector pGBKT7 in order to generate an expression vector coding for a fusion protein between the GAL4-DNA BD and the complete human adipose protein.

b. Cloning of pGBKT-Adp-TPR

This construct was generated in a similar way using the specific primers 5 '-GATC GAATTC GGAGGTGGACCACCATACCTGGAGCTGG-3' (forward) (SEQ ID NO: 9) and 5'-GATC GGATCC CTAACCAGGTCCCTTCTTCTCC-3' (reverse) (SEQ ID NO: 10) and subcloning the amplified fragment in the EcoRI and BamHI sites of the pGBKT7 yeast expression vector, thereby generating an yeast expression vector coding for a fusion protein between the GAL4-DNA BD and the TPR-domain ofthe human adipose protein .

The cDNA plasmid coding for the insert ofthe isolated yeast clones showing specific interaction with the employed bait constructs were isolated according to the manufacturer's instructions (Clontech, Yeast Protocols Handbook) and analysed by standard sequencing reactions. The BT-42 clones was isolated from a human brain library (pretransformed Human Brain Matchmaker cDNA Library from Clontech), with pGBKT7-Adp-TPR used as bait vector.

The specific association ofthe clones identified in the Yeast-Two-Hybrid screen was tested in a mammalian expression system based on the transient overexpression of full-length human adipose together with the cDNA fragment identified in the Yeast- Two-Hybrid system. For these experiments, the bait and prey constructs had to be transferred to an expression vector driving the expression of the corresponding inserts in mammalian cells.

Example 3: Identification of human BT-42 genes and proteins

BT-42 homologous proteins and nucleic acid molecules coding therefore are obtainable from vertebrate species, e.g. mammals. Particularly preferred are nucleic acids comprising human BT-42 homologs (in particular, the human BT-42, isoform 1, and the human BT-42, isoform 2, the alternative splice variant GenBankAccession Number AF543190, and the human KIAA0377 protein as disclosed in Accession Number NM_014659.1 and NP_ 055474 for the protein).

^' The human BT42 was cloned by polymerase chain reaction on human cDNA (from testis and adipocytes, obtained from clontech) using a proofreading DNA-Polymerase (Herculase, Stratagene), according to the Manufacturer's protocols. The cDNA was cloned in three independent fragments A to C. During the PCR- procedure two new splice variants were identified. The coding region of both isoforms were later joined in the pBluescript KS (Stratagene) and pRK5RS vector using standard cloning techniques, as known to those skilled in the prior art. The complete coding strand of both isoforms were then cloned into a CMV-promoter based eukaryotic expression vector.

Primers used for cloning the human BT42 isoforms (primers were designed using the KIAA0377 sequence, GenBankAccession Number NM_014659.1):

BT42 A.forward primer (SEQ ID NO: 11):

5' GATCTCTAGAGCAGGGATGTGGTCATTGAC 3'

BT42 A reverse primer (SEQ ID NO: 12): 5 ' GATCGAATTCGTCGACGGTAGTAAGTC AATTGTACCTTC 3 '

BT42 B forward primer (SEQ ID NO: 13): : 5' GATCGAGCTCTACTGGAGATGTATGGTCAC 3' BT42 B reverse primer (SEQ ID NO: 14): 5 ' GATCGTCGACCACATGGAACCGTTCCTCTG 3 '

BT42 C forward primer (SEQ ID NO: 15):: 5' CAGAGCTTAACTACATGACC 3' BT42 C reverse primer (SEQ ID NO: 16): 5' GATCAAGCTTCAATTGATTTATCTCCTCAGGGACCTC Example 4: Expression ofthe polypeptides in mammalian tissues

For analyzing the expression of the polypeptides disclosed in this invention in mammalian tissues, several mouse strains (preferably mice strains C57B1/6J, C57B1/6 ob/ob and C57B1/KS db/db which are standard model systems in obesity and diabetes research) were purchased from Harlan Winkelmann (33178 Borchen, Germany) and maintained under constant temperature (preferably 22°C), 40 per cent humidity and a light / dark cycle of preferably 14 / 10 hours. The mice were fed a standard chow (for example, from ssniff Spezialitaten GmbH, order number ssniff M-Z VI 126-000). 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 1993 Jul;92(l):272-80, Mizuno et al. Proc Natl Acad Sci U S A 1996 Apr 16;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 preadipocytes to adipocytes, mammalian fibroblast (3T3-L1) cells (e.g., Green & Kehinde, Cell 1: 113-116, 1974) were obtained from the American Tissue Culture Collection (ATCC, Hanassas, VA, USA; ATCC- CL 173). 3T3-L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g., Qiu. et al., J. Biol. Chem. 276:11988-95, 2001; Slieker et al., BBRC 251: 225-9, 1998). In brief, cells were plated in DMEM/10% FCS (Invitrogen, Karlsruhe, Germany) at 50,000 cells/well in duplicates in 6-well plastic dishes and cultured in a humidified atmosphere of 5% CO₂ at 37°C. At confluence (defined as day 0: dO) cells were transferred to serum-free (SF) medium, containing DMEM/HamF12 (3:1; Invitrogen), Fetuin (300microg/ml; Sigma, Munich, Germany), Transferrin (2microg/ml; Sigma), Pantothenate (17microM; Sigma), Biotin (lmicroM; Sigma), and EGF (0.8nM; Hoffmann-La Roche, Basel, Switzerland). Differentiation was

- induced by adding Dexamethasone (DEX; lmicroM; Sigma), 3-Methyl-Isobutyl-l- Methylxanthine (MIX; 0.5mM; Sigma), and bovine Insulin (5microg/ml; Invitrogen). Four days after confluence (d4), cells were kept in SF medium, containing bovine Insulin (5microg/ml) until differentiation was completed. At various time points of the differentiation procedure, beginning with day 0 (day of confluence) and day 2 (hormone addition; for example, dexamethason and 3 -isobutyl- 1-methylx-mthin), 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: 105-113, 1976) were obtained from the Harvard Medical School, Department of Cell Biology (Boston, MA, USA). Alternatively, mammalian fibroblast TA1 cell line, a murine preadipocyte line derived from T101/2 mouse embryo fibroblasts (Ross et al., 1992), was used. 3T3-F442A cells were maintained as fibroblasts and differentiated into adipocytes as described previously (Djian, P. et al., J. Cell. Physiol., 124:554-556, 1985). At various time points ofthe differentiation procedure, beginning with day 0 (day of confluence and hormone addition, for example, Insulin), up to 10 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.

TaqMan Analysis of the proteins of the invention (Figure 2)

RNA was isolated from mouse tissues or cell culture cells using Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy

Kit (for -example, from Qiagen, Germany) in combination with an DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art. Total RNA was reverse transcribed (preferably using Superscript II RNaseH^"

Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) and subjected to Taqman analysis preferably using the Taqman 2xPCR Master Mix (from Applied

Biosystems, Weiterstadt, Germany; the Mix contains according to the Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, passive reference Rox and optimized buffer components) on a GeneAmp 5700

Sequence Detection System (from Applied Biosystems, Weiterstadt, Germany).

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

For the amplification of mouse KIAA0377 protein (BT42): Mouse KIAA0377 (BT42) forward primer (SEQ ID NO: 17): 5'- CCTGTGGAGAACTGGCCG-3 ';

Mouse KIAA0377 (BT42) reverse primer (SEQ ID NO: 18): 5'- TCGAGAGGAAAGCCTTTGGA-3 ' Taqmanprobe (SEQ ID NO:19): (5/6-FAM)

CCTGCCACTGCCTCATCTCTTTCCA(5/6-TAMRA)

As shown in Figure 2A, real time PCR (Taqman) analysis of the expression of KLAA0377 (BT42)- protein in mammalian (mouse) tissues revealed that KIAA0377 (BT42) - protein is expressed in different mammalian tissues, showing highest levels of expression in neural tissues such as cerebellum, hypothalamus, midbrain and cortex, but also high levels of expression in the small intestine, heart, and kidney, and lower levels in muscle, lung and spleen tissues. A clear expression in brown adipocyte tissue (BAT) and lower expression in white adipocyte tissue (WAT) is seen, confirming a role in the regulation of energy homeostasis and thermogenesis.

Further, we show that the mammalian BT-42 protein is regulated by fasting and by genetically induced obesity. In this invention, we used mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice) to study the expression of the protein of the invention. Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning et al, 1998, Mol. Cell. 2:449-569). We found, for example, that the expression of mouse BT-42 is 3.5 fold upregulated in liver of fasted mice (see Figure 2B), and that the expression shows high levels in the muscle of genetically obese mice. In addition, a marked downregulation can be observed in the metabolically active tissue (for example, brown adipose tissue (BAT)) and white adipose tissue (WAT) and several other tissues such as those isolated from brain and small intestine of fasted mice, and also downregulated in BAT and WAT and other tissues including brain of genetically obese mice (see FIGURE 2B). In addition, BT-

42 expression in observed in all tissues in the db/db mice model, with marked downregulation in metabolically active tissues (BAT, WAT) (see FIGURE 2C). The BT42 - protein was also examined in the in vitro differentiation models for the conversion of pre-adipocytes to adipocytes, as described above. We found in different model systems, that BT42 protein is strongly upregulated during adipocyte differentiation in vitro, suggesting a role as modulator of adipocyte lipid accumulation. For example, as shown in FIGURE 2D, mammalian BT42 - protein shows a 3 to 3.5-fold induction of its expression during differentiation, shows a continuous upregulation starting on day 4 up to 3.5 fold on day 8 in TA1 cells. As shown in FIGURE 2E, BT42 - protein shows a 8- to 11 -fold induction of its expression during differentiation, starting on day 6 of differentiation in 3T3-L1 cells. As shown in FIGURE 2F, BT42 - protein shows a 3- to 6-fold induction of its expression during differentiation, starting on day 4 of differentiation in 3T3-F422A cells.

Thus, we conclude that mammalian BT42 (or variants thereof) has a function in the metabolism of mature adipocytes.

Example 5: In vitro assays for the determination of triglyceride and glycogen levels in BT-42 overexpressing cells

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 infracellular glucose and triglycerides, free or stored as glycogen or lipids as fuel for energy and/or heat production, hi this invention, we therefore show the cellular level of triglycerides and glycogen in cells overexpressing the protein ofthe invention.

Retroviral infection of preadipocytes

Packaging cells were transfected with retroviral plasmids pLPCX carrying mouse Mnk2 transgene 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. 10 min before transfection chloroquine was added directly to the overlying medium (25 microM end concentration). A 250 microl transfection mix consisting of 5 microg plasmid-DNA (candidate:helper- virus in a 1 : 1 ratio) and 250 mM CaCl was prepared in a 15 ml plastic tube. The same volume of 2 x HBS (280 microM NaCl, 50 microM HEPES, 1.5 mM Na₂HPO₄, 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 % CO₂ 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 % CO₂. The supernatant was then filtered through a 0.45 Dm 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 ofthe proteins disclosed in this invention in the in vitro assays for the deteπnination 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 microl HB-buffer (0.5% Polyoxyethylene 10 tridecylethan, 1 mM EDTA, 0.01M NaH₂PO4, 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 FP120, Bio 101 Thermosavant, Holbrock,

USA). Supernatants 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-well 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 microl sample was incubated with 200 microl reagent A for 5 minutes at 37°C. After determination of glycerol (initial absorbance at 540 nm), 50 microl 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-microL samples were incubated with 20-microL amyloglucosidase solution for 15minutes at

60°C to digest glycogen to glucose. The glucose is further metabolised with 100 microL distilled water and 100 microl of enzyme cofactor buffer and 12 microL 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.

Example 6: Generation and analysis of BT-42 transgenic

Generation of the transgenic animals Mouse BT-42 cDNA 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 the transgenic expression vector pTG- O 2004/050007

43 βactin-X-hgh-bgh-polyA. The transgene was microinjected into the male pronucleus of fertilized mouse embryos (preferably strain C57/BL6/CBA Fl (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 Fl mice for analysis. Transgenic mice were continously bred onto the C57/B16 background. The expression ofthe protein ofthe invention can be analyzed by taqman analysis as described above, and further analysis ofthe mice can be done as known to those skilled in the art.

Claims

Claims

A pharmaceutical composition comprising a nucleic acid molecule of the BT- 42 gene family, a nucleic acid molecule encoding a BT42 or an isoform thereof, a polypeptide encoded thereby or encoded by a fragment or a variant of said nucleic acid molecule(s) or of said polypeptide, or an effector of said nucleic acid molecule or said polypeptide, preferably together with pharmaceutically acceptable carriers, diluents and/or adjuvants.

The composition of claim 1, wherein the nucleic acid molecule is a human BT- 42 nucleic acid, particulary encoding the human BT-42 homologs, and/or a nucleic molecule which is complementary thereto or a fragment thereof or a variant thereof.

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

(a) a nucleic acid molecule encoding a polypeptide as shown in SEQ ID NOs: 2 or 4, a polypeptide as deposited under GenBank accession number NM_014659 or under accession number AF543190 or an isoform, fragment or variant of the polypeptide as shown in SEQ ID NOs: 2 or 4 or an isoform, fragment or variant of the polypeptide as deposited under accession numbers NM_014659 or AF543190;

(b) a nucleic acid molecule which comprises or is the nucleic acid molecule as shown in SEQ ID NO: 1 or 2, the nucleic acid molecule as deposited under NM_014659 or AF543190;

(c) a nucleic acid molecule being degenerate as a result ofthe genetic code to the nucleic acid sequences as defined in (a) or (b);

(d) a nucleic acid molecule that hybridizes at 65°C in a solution containing 0.2 x SSC and 0.1% SDS to a nucleic acid molecule as defined in claim

2 or as defined in (a) to (c) and/or a nucleic acid molecule which is complementary thereto;

(e) a nucleic acid molecule that encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to a human BT-42 , as defined in claim 2 or to a polypeptide as defined in (a); (f) a nucleic acid molecule that differs from the nucleic acid molecule of (a) to (e) by mutation and wherein said mutation causes an alteration, deletion, duplication or premature stop in the encoded polypeptide.

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

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

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

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

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

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

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

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

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

13. The composition of any one of claims 1-12 for the manufacture of an agent for detecting and or verifying, for the treatment, alleviation and/or prevention of an disorders, including metabolic diseases such as obesity and other body-weight regulation disorders as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones and others, in cells, cell masses, organs and/or subjects.

14. Use of a nucleic acid molecule of the BT-42 gene family or a polypeptide encoded thereby or a fragment or a variant of said nucleic acid molecule or said polypeptide or an effector of said nucleic or polypeptide for controlling the function of a gene and/or a gene product which is influenced and/or modified by an BT-42 homologous polypeptide.

15. Use of the nucleic acid molecule of the BT-42 gene family or a nucleic acid molecule encoding a BT42 or an isoform thereof, use of a polypeptide encoded by said nucleic acid molecule, use of a fragment or a variant of said nucleic acid molecule or said polypeptide, or use of an effector of said nucleic acid molecule or said polypeptide for identifying substances capable of interacting with an BT-42 homologous polypeptide.

16. A non-human transgenic animal exhibiting a modified expression of an BT-42 homologous polypeptide.

17. The animal of claim 16, wherein the expression of the BT-42 homologous polypeptide is increased and/or reduced.

18. A recombinant host cell exhibiting a modified expression of an BT-42 homologous polypeptide, or a recombinant host cell which comprises a nucleic acid molecule as defined in any one of claims 1 to 6.

19. The cell of claim 18 which is a human cell.

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

(a) contacting a collection of (poly)peρtides with an BT-42 homologous polypeptide or a fragment thereof under conditions that allow binding of said (poly)peptides;

(b) removing (poly)peptides which do not bind and

(c) identifying (poly)peptides that bind to said BT-42 homologous polypeptide.

21. A method of screening for an agent which modulates the interaction of an BT- 42 homologous polypeptide with a binding target/agent, comprising the steps of

(a) incubating a mixture comprising

(aa) an BT-42 homologous polypeptide or a fragment thereof;

(ab) a binding target/agent of said BT-42 homologous polypeptide or fragment thereof; and

(ac) a candidate agent under conditions whereby said BT-42 polypeptide or fragment thereof specifically binds to said binding target/agent at a reference affinity;

(b) defecting the binding affinity of said BT-42 polypeptide or fragment thereof to said binding target to determine a (candidate) agent-biased affinity; and

(c) deteimining a difference between (candidate) agent-biased affinity and reference affinity.

22. A method of screening for an agent which modulates the activity of an BT-42 homologous polypeptide comprising the steps of

(a) incubating a mixture comprising

(aa) an BT-42 homologous polypeptide or a fragment thereof and

(ab) a candidate agent under conditions whereby said BT-42 polypeptide or fragment thereof exhibits a reference activity,

(b) detecting the activity of said BT-42 polypeptide or fragment thereof to determine a (candidate) agent-biased activity and (c) deteπmning a difference between (candidate) agent-biased activity and reference activity.

23. A method of producing a composition comprising the (poly)peptide identified by the method of claim 20 or the agent identified by the method of claim 2 lor 22 with a pharmaceutically acceptable carrier, diluent and/or adjuvant.

24. The method of claim 23 wherein said composition is a pharmaceutical composition for preventing, alleviating or treating of diseases and disorders, includύ g metabolic diseases such as obesity and other body-weight regulation disorders as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones and other diseases and disorders.

25. Use of a (ρoly)peptide as identified by the method of claim 20 or of an agent as identified by the method of claim 21 or 22 for the preparation of a pharmaceutical composition for the treatment, alleviation and/or prevention of diseases and disorders, including metabolic diseases such as obesity and other body-weight regulation disorders as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones and other diseases and disorders. . ^• .

26. Use of a nucleic acid molecule as defined in any one of claims 1 to 6 or 10, use of a polypeptide as defined in any one of claims 1 to 6, 8 or 9, use of a vector as defined in claim 7, use of a host cell as defined in claim 18 or 19 for the preparation of a phaπnaceutical composition for the treatment, alleviation and/or prevention of diseases and disorders, including metabolic diseases such as obesity and other body- weight regulation disorders as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia (dyslipidemia), and gallstones and other diseases and disorders.

27. Use of a nucleic acid molecule of the BT-42 gene family or of a fragment tiiereof for the preparation of a non-human animal which over- or under-expresses the BT-42 gene product.

28. Kit comprising at least one of

(a) a BT-42 nucleic acid molecule or a fragment or an isoform thereof;

(b) a BT42 amino acid sequence 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 (c);

(e) a polypeptide encoded by the nucleic acid of (a), expressed by the vector of (c) or the host cell of (a);

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

(g) an antibody, an aptamer or another receptor against the nucleic acid of (a) or ihe polypeptide of (b), (e) or (f), and/or

(h) an anti-sense oligonucleotide of the nucleic acid of (a).