Dgat1/2-independent enzyme synthesizing storage lipids (diesl)
AssigneeStichting Het Nederlands Kanker Instituut - Antoni van Leeuwenhoek Ziekenhuis
InventorsGian-Luca MCLELLAND, Thijn Reinout BRUMMELKAMP
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Abstract
The present invention is in the field of triacylglyccrol synthesis. In particular the invention relates to the identification of DIESL as a new triacylglycerol synthase. The invention also relates to the identification of TMX1 as a negative regulator of DIESL mediated triacylglycerol synthesis. Screening assays for identifying agents that modulate the activity of DIESL and/or of TMX1, or that modulate the interaction between DIESL and TMX1 are also provided.
Claims40
An isolated DGAT1/2-Independent Enzyme Synthesizing storage Lipids (DIESL) polypeptide, wherein the DIESL polypeptide comprises an amino acid sequence that has at least 75% amino acid sequence identity with the amino acid sequence as defined in SEQ ID NO:1, wherein the DIESL polypeptide exhibits triacylglycerol synthase activity.
Use of a DIESL polypeptide in the production of triacylglycerol.
A screening method for identifying an agent that modulates the activity of a DIESL polypeptide, wherein the method comprises
- (a) contacting the DIESL polypeptide with a candidate agent;
- and
- (b) detecting a change in activity of the DIESL polypeptide compared to a control to determine the candidate agent's modulatory activity.
A screening method for identifying an agent that modulates interaction of a DIESL polypeptide with a Thioredoxin-related transmembrane protein 1 (TMX1) polypeptide, wherein the method comprises:
- (a) providing a cell that expresses the DIESL polypeptide and the TMX1 polypeptide;
- (b) providing a candidate agent;
- (c) contacting the candidate agent with the cell that expresses the DIESL polypeptide and the TMX1 polypeptide under conditions that allow interaction of the candidate agent with the DIESL polypeptide and/or the TMX-1 polypeptide;
- (d) determining the amount of DIESL monomer, TMX-1 monomer and/or the amount of DIESL/TMX-1 dimer;
- (e) comparing the amounts obtained in step (d) to a control to determine the candidate agent's modulatory activity.
A screening method for identifying mutants of a DIESL polypeptide, wherein said mutant has altered activity and/or altered substrate preference, wherein the method comprises
- (a) providing a mutant of the DIESL polypeptide;
- and
- (b) detecting activity of the mutant of the DIESL polypeptide compared to a control and/or detecting substrate preference of the mutant of the DIESL polypeptide compared to a control to determine the mutant of the DIESL polypeptide's altered activity and/or substrate preference.
A method for producing triacylglycerol, wherein the method comprises contacting a DIESL polypeptide with a diglyceride and a source of fatty acids under conditions sufficient for the triacylglycerol to be produced, preferably wherein the DIESL polypeptide is expressed in a cell.
A method for determining triacylglycerol production and/or DIESL activity in a cell wherein the method comprises determining DIESL polypeptide and/or TMX1 polypeptide expression, DIESL polypeptide and/or TMX1 polypeptide level, amount of DIESL/TMX1 dimer, and/or DIESL polypeptide and/or TMX1 polypeptide activity in the cell.
An isolated Thioredoxin-related transmembrane protein 1 (TMX1) polypeptide, wherein the TMX1 polypeptide comprises an amino acid sequence that has at least 75% amino acid sequence identity with the amino acid sequence as defined in SEQ ID NO:2, wherein the TMX1 polypeptide exhibits inhibition of DIESL mediated triacylglycerol synthase activity, TAG production and/or TAG accumulation.
Use of a TMX1 polypeptide in inhibiting the production or accumulation of triacylglycerol, preferably in inhibiting DIESL polypeptide mediated triacylglycerol accumulation.
A screening method for identifying mutants of a TMX1 polypeptide, wherein said mutant has altered modulation of DIESL mediated triacylglycerol synthase activity, wherein the method comprises
- (a) providing a mutant of theTMX1 polypeptide and providing a DIESL polypeptide;
- (b) allowing the mutant TMX1 polypeptide to contact the DIESL polypeptide, preferably under conditions that allow the DIESL polypeptide to produce triacylglycerol;
- and
- (c) detecting activity of the mutant TMX1 polypeptide compared to a control and/or detecting activity of the DIESL polypeptide compared to control to determine the mutant of the TMX1 polypeptide's altered regulation of DIESL mediated triacylglycerol synthase activity.
Use of an oligonucleotide to knock down or knock out DIESL and/or TMX1 for modulating triglyceride production in a cell or organism, preferably wherein the use comprises CRISPR/Cas technology, RNAi or antisense technology.
A cell that:
- is a non-animal cell that expresses a DIESL polypeptide and/or a TMX1 polypeptide;
- is a non-human cell that expresses human DIESL polypeptide and/or TMX1 polypeptide;
- is a cell that has been modified to not express a DIESL polypeptide and/or a TMX1 polypeptide, and that has been modified to not express a DGAT1 polypeptide;
- is a cell that has been modified to not express a DIESL polypeptide and/or a TMX1 polypeptide, and that has been modified to not express a DGAT2 polypeptide;
- is a cell that has been modified to not express a DIESL polypeptide and/or a TMX1 polypeptide, and that has been modified to not express a DGAT1 polypeptide and a DGAT2 polypeptide.
A non-human transgenic knockout animal, in which the gene encoding for a DIESL polypeptide is knocked out, preferably wherein the polypeptide has at least 75% amino acid sequence identity with the amino acid sequence as defined in SEQ ID NO:1 and exhibits triacylglycerol synthase activity.
Dependencies are inferred from the claim text.