Human diacylglycerol acyltransferase 2 (dgat2) family members and uses therefor

Abstract

The present invention relates to compositions and methods for the diagnosis and treatment of obesity and related metabolic disorders. The invention provides isolated nucleic acids molecules, designated DGAT2 family member nucleic acid molecules, which encode diacylglycerol acyltransferase family members. The invention also provides recombinant expression vectors containing DGAT2 family member nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a DGAT2 family member gene has been introduced or disrupted. The invention still further provides isolated DGAT2 family member proteins, fusion proteins, antigenic peptides and anti-DGAT2 family member antibodies. Methods of use of the provided DGAT2 family member compositions for screening, diagnostic and therapeutic methods in connection with obesity disorders are also disclosed.

What is claimed is: 
     
         1 . An isolated nucleic acid molecule selected from the group consisting of:
 a) a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61;   b) a nucleic acid molecule comprising a fragment of at least 300 nucleotides of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61;   c) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:62;   d) a nucleic acid molecule which encodes a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:62, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62;   e) a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising a sequence consisting of SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61 or a complement thereof, under stringent conditions; and   f) a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:61.   
     
     
         2 . The isolated nucleic acid molecule of  claim 1 , which is selected from the group consisting of:
 a) a nucleic acid comprising the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61, and   b) a nucleic acid molecule which encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62.   
     
     
         3 . The nucleic acid molecule of  claim 1  further comprising vector nucleic acid sequences or nucleic acid sequences encoding a heterologous polypeptide. 
     
     
         4 . A host cell which contains the nucleic acid molecule of  claim 1 . 
     
     
         5 . A non-human mammalian host cell containing the nucleic acid molecule of  claim 1 . 
     
     
         6 . An isolated polypeptide selected from the group consisting of:
 a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% Identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61, or a complement thereof;   b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61 or a complement thereof under stringent conditions;   c) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62; and   d) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:62.   
     
     
         7 . The polypeptide of  claim 6  further comprising heterologous amino acid sequences. 
     
     
         8 . An antibody which selectively binds to a polypeptide of  claim 6 . 
     
     
         9 . A method for producing a polypeptide selected from the group consisting of:
 a) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, and SEQ ID NO:62;   b) a polypeptide comprising a fragment of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:20, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:62;   c) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61, or a complement thereof;   d) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:61, or a complement thereof under stringent conditions;   comprising culturing the host cell of  claim 5  under conditions in which the nucleic acid molecule is expressed.   
     
     
         10 . A method for identifying a compound which binds to a polypeptide of  claim 6  comprising the steps of:
 a) contacting a polypeptide, or a cell expressing a polypeptide of  claim 6  with a test compound; and 
 b) determining whether the polypeptide binds to the test compound; 
 
       wherein binding of the test compound to the polypeptide is detected by a method selected from the group consisting of:
 a) detection of binding by direct detecting of test compound/polypeptide binding; 
 b) detection of binding using a competition binding assay; or 
 c) detection of binding using an assay for diacylglycerol acyltransferase activity. 
 
     
     
         11 . A method for modulating the activity of a polypeptide of  claim 6  comprising contacting a polypeptide or a cell expressing a polypeptide of  claim 6  with a compound which binds to the polypeptide in a sufficient concentration to modulate the activity of the polypeptide. 
     
     
         12 . A method for identifying a compound which modulates the activity of a polypeptide of  claim 6 , comprising:
 a) contacting a polypeptide of  claim 6  with a test compound; and   b) determining the effect of the test compound on the diacylglycerol acyltransferase activity of the polypeptide to thereby identify a compound which modulates the activity of the polypeptide.   
     
     
         13 . A method for identifying a compound capable of modulating an adipocyte activity comprising:
 a) contacting an adipocyte with a test compound; and   b) assaying the ability of the test compound to modulate the expression of a DGAT2 family member nucleic acid or the activity of a DGAT2 family member polypeptide; and   c) identifying a compound capable of modulating an adipocyte activity when a test compound modulates the expression of a DGAT2 family member nucleic acid or the activity of a DGAT2 family member polypeptide.   
     
     
         14 . The method of  claim 13 , wherein said adipocyte activity comprises any one of DGAT2 family member nucleic acid expression, diacylglyceroltransferase activity, hyperplastic growth, hypertrophic growth, or lipogenesis. 
     
     
         15 . A method for modulating an adipocyte activity comprising contacting an adipocyte with a DGAT2 family member modulator, thereby modulating said adipocyte activity. 
     
     
         16 . The method of  claim 15 , wherein the DGAT2 family member modulator is selected from the group consisting of:
 a) a DGAT2 family member polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:62, or a fragment thereof;   b) a DGAT2 family member nucleic acid comprising the nucleotide sequence of SEQ ID NO:7, 19, 21, or a fragment thereof;   c) an anti-DGAT2 family member antibody; and   d) an organic small molecule.   
     
     
         17 . A method of determining acyltransferase activity of a polypeptide comprising:
 a) combining a sample comprising an acyltransferase polypeptide with a first fatty acyl coA substrate and a second acylglyceride substrate under conditions suitable for enzyme activity and   b) determining the amount of acylglycerol product formation, thereby determining acyltransferase activity,   wherein one substrate is biotinylated and the other substrate is radiolabeled, and wherein the resulting acylglycerol product is detected using biotin capture and radiometric detection.   
     
     
         18 . The method of  claim 17 , wherein the method detects diacylglycerol acyltransferase activity or monoacylglycerol acyltransferase activity. 
     
     
         19 . The method of  claim 18 , wherein the polypeptide comprises a DGAT2 family member polypeptide selected from the group consisting of:
 a) a polypeptide which is encoded by a nucleic acid molecule comprising a nucleotide sequence which is at least 85% identical to a nucleic acid comprising the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61, or a complement thereof;   b) a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the polypeptide is encoded by a nucleic acid molecule which hybridizes to a nucleic acid molecule comprising SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61 or a complement thereof under stringent conditions;   e) a fragment of a polypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the fragment comprises at least 15 contiguous amino acids of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62; and   f) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:62.   
     
     
         20 . The method of  claim 17 , wherein product formation is detected by a scintillation proximity assay. 
     
     
         21 . The method of  claim 17 , wherein the first substrate is a biotin labeled fatty acyl coA and the second substrate is a radiolabeled diacylglycerol. 
     
     
         22 . The method of  claim 17 , wherein the first substrate is a radiolabeled fatty acyl coA and the second substrate is a biotin labeled diacylglycerol. 
     
     
         23 . A method of identifying a compound which modulates the activity of an acyltransferase, comprising:
 a) combining a sample comprising an acyltransferase polypeptide with a first fatty acyl coA substrate and a second acylglyceride substrate under conditions suitable for enzyme activity;   b) contacting the sample comprising the acyltransferase and substrates with a test compound;   c) comparing the acyltransferase activity in the presence of compound with acyltransferase activity in the absence of compound; and   c) determining the effect of the test compound on the acyltransferase activity,   wherein acyltransferase activity is determined by the method of  claim 17 , and wherein a change in the amount of acyltransferase activity in the presence of test compound thereby identifies a compound which modulates activity of the acyltransferase.   
     
     
         24 . A method for identifying a compound capable of treating a disorder characterized by aberrant DGAT2 family member nucleic acid expression or DGAT2 family member polypeptide activity comprising assaying the ability of the compound to modulate DGAT2 family member nucleic acid expression or DGAT2 family member polypeptide activity, thereby identifying a compound capable of treating a disorder characterized by aberrant DGAT2 family member nucleic acid expression or DGAT2 family member polypeptide activity. 
     
     
         25 . The method of  claim 24 , wherein the disorder is a disorder associated with obesity, aberrant lipogenesis or triglyceride synthesis. 
     
     
         26 . The method of  claim 24 , wherein the ability of the compound to modulate the activity of the DGAT2 family member polypeptide is determined by detecting the diacylglycerol acyltransferase activity. 
     
     
         27 . A method for treating a subject having an obesity disorder characterized by aberrant DGAT2 family member polypeptide activity or aberrant DGAT2 family member nucleic acid expression comprising administering to the subject a DGAT2 family member modulator, thereby treating said subject having an obesity disorder. 
     
     
         28 . The method of  claim 27 , wherein the DGAT2 family member modulator is selected from the group consisting of:
 a) an organic small molecule;   b) an anti-DGAT2 family member antibody;   c) a DGAT2 family member polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 and SEQ ID NO:62, or a fragment thereof; and   d) a DGAT2 family member polypeptide comprising an amino acid sequence which is at least 90 percent identical to the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:62   
     
     
         29 . The method of  claim 27 , wherein the disorder is a disorder associated with obesity, aberrant lipogenesis or triglyceride synthesis.