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US 2003/0157513 A1Published application
Novel triacylgl ycerol biosynthesis in the cytosol of eukaryotes
AssigneeIndian Institute of Science
InventorRam Rajasekharan
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AssigneeIndian Institute of Science
InventorRam Rajasekharan
Estimating the term…
This invention describes novel catalytically active cytosolic enzymes for triacylglycerol biosynthesis from eukaryotic systems. The complex from oleaginous yeast was enzymatically characterized, and was found to contain lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl-acyl carrier protein synthetase, superoxide dismutase and acyl carrier protein. The triacylglycerol biosynthetic machinery rapidly incorporates free fatty acids as well as fatty acyl-coenzyme A into triacylglycerol and its biosynthetic intermediates. Lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase and diacylglycerol acyltransferase from the complex were microsequenced. Acyl carrier protein, superoxide dismutase and diacylglycerol acyltransferase genes were cloned and expressed in bacterial system. The soluble triacylglycerol biosynthetic enzymes (lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase) in yeast, rat adipocytes and human hepatocyte cell-line (HepG2) exist in the cytosol either as free enzymes or as a multienzyme complex.
Novel soluble triacylglycerol (TAG) biosynthetic enzymes in eukaryotic cells comprising of lysophosphatidic acid acyltransferase with peptide SEQ ID 1 XALELQADDFNK and peptide SEQ ID 3 XXVNNVXPGXIEQ, phosphatidic acid phosphatase with peptide SEQ ID 4 NALTGLHMGGGK and peptide SEQ ID 5 sequence YVEGARPXK, diacylglycerol acyltransferase with peptide SEQ ID 7 XLWAVVGAQPFGGARGS, acyl-acyl carrier protein synthetase with peptide SEQ ID 8 sequence VHLAVALYGLAAVRVSRIVR, superoxide dismutase encoded by the gene sequence as in SEQ ID 9, and acyl carrier protein encoded by the gene sequence as in SEQ ID 10 and diacylglycerol acyltransferase encoded by the gene sequence as in SEQ ID 11.
The uses of novel soluble TAG biosynthetic enzymes to identify compounds that are capable of altering for TAG synthesis and accumulation.
Polypeptide of superoxide dismutase (SOD) as encoded by SEQ ID 9.
Polypeptide of acyl carrier protein (ACP), as encoded by SEQ ID 10.
Polypeptide of diacylglycerol acyltransferase, as encoded by SEQ ID 11.
The newly identified novel TAG biosynthetic enzyme sequence can be used as a target site for controlling fat.
The identified individual enzymes can also be used as a target for controlling fat.
An oligopeptide of at least 5 to 20 amino acids (peptide SEQ ID 1 to 8) capable of specifically identifying with a unique sequence of proteins present within a triacylglycerol biosynthetic complex (TBC).
Antibodies to at least 5 to 20 amino acids (peptide SEQ ID 1 to 8) from the isolated polypeptides from TBC, which are capable of specifically identifying the proteins present within a triacylglycerol biosynthetic complex.
An oligonucleotide of at least 15 nucleotides selected from SEQ ID 9 capable of specifically hybridizing with a unique sequence of nucleotides present within a nucleic acid, which encodes a superoxide dismutase.
An oligonucleotide of at least 15 nucleotides selected from SEQ ID 10 capable of specifically hybridizing with a unique sequence of nucleotides present within a nucleic acid, which encodes an acyl carrier protein.
A nucleic acid having a sequence complementary to the sequences (SEQ ID 9 to 11) of the isolated nucleic acid, which encode superoxide dismutase and acyl carrier protein and diacylglycerol acyltransferase.
An in vitro method of detecting the soluble triacylglycerol biosynthetic enzymes, which exist either as free or as complex in the cytosol.
A method for determining whether a subject known to have an imbalance in triacylglycerol has the imbalance due to a defect in the synthesis of triacylglycerol.
A method for treating a subject who has an imbalance in triglyceride (triacylglycerol) levels due to a defect in the synthesis of soluble triglyceride, which comprises introducing the isolated nucleic acid which encodes lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl carrier protein (ACP), and acyl-ACP synthetase into the subject under conditions such that the nucleic acid expresses the soluble triacylglycerol biosynthetic enzymes individually or in combination, so as to thereby treat the subject.
A method for inhibiting the soluble triacylglycerol biosynthetic enzymes in a subject which comprises transforming appropriate cells from the subject with a vector which expresses the nucleic acid which encodes the components in TBC, and introducing the transformed cells into the subject so as to thereby inhibit the soluble enzyme and/or complex.
A method for inhibiting the soluble triacylglycerol biosynthetic enzymes in a subject which comprises introducing the any of the above-described oligonucleotides of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of nucleotides present within a nucleic acid which encode lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, acyl carrier protein (ACP), acyl-ACP synthetase and superoxide dismutase into the subject so as to thereby inhibit the triglyceride formation and accumulation.
A method of obtaining soluble triacylglycerol (TAG) biosynthetic enzymes using polyacrylmide gel electgrophoresis, chromatographic procedures or density gradient centrifugation, said method comprising subjecting blue sepharose, DEAE matrix octail sepharose, antibodies raised from the peptide SEQ ID 1 to 8 and identifying and isolating the complex as a whole or individual components thereof.
Novel Lysophosphatidic Acid Acyltransferase
Novel Phosphatidic Acid Phosphatase
Novel Diacylglycerol Acyltransferase
Novel Acyl-Acyl Carrier Protein Synthetase
Dependencies are inferred from the claim text.