Enzyme regulating ether lipid signaling pathways
A multidimensional profiling strategy that combines activity-based proteomics and metabolomics was used to determine that an active protein, which is a previously uncharacterized enzyme highly elevated in aggressive cancer cells, serves as a central node in an ether lipid signaling network that bridges platelet-activating factor and the lysophospholipids. Biochemical studies confirmed that the active protein regulates this pathway by hydrolyzing the metabolic intermediate 2-acetyl monoalkylglycerol. Inactivation of the active protein disrupted ether lipid metabolism in cancer cells and impaired cell migration and tumor growth in vivo.
1. A method of identifying an inhibitor of neutral cholesterol ester hydrolase 1 (abbreviated as “KIAA1363”) comprising:
a) providing cells that express an amino acid sequence set forth in SEQ ID NO: 2;
b) contacting the cells of step (a) with a first agent;
c) determining the rate of 2-acetyl monoalkylglycerol (MAGE) hydrolysis, or a derivative thereof, in the presence of the agent;
wherein when the rate of 2-acetyl MAGE hydrolysis decreases in the presence of the first agent, the decrease is indicative of an inhibitory effect of the first agent.
2. The method of claim 1 , further comprising contacting the cells with a second agent, wherein the second agent modulates the expression of KIAA1363.
3. A method for identifying an inhibitor of KIAA1363 comprising
a) isolating the membrane proteome from cells expressing KIAA1363;
b) contacting the membrane proteome with a thiol-reactive fluorescent reagent and 2-thioacetyl monoalkylglycerol (2-thioacetyl MAGE), in the presence and absence of a test agent under conditions which allow hydrolysis of the 2-thioacetyl MAGE by KIAA1363; and
c) determining the absorbance, wherein a decrease in the absorption in the presence of test compound as compared to the absorbance in the absence of test agent is indicative of an inhibitory effect of the test agent.
4. The method of claim 1 , wherein the cells expressing KIAA1363 are host cells transfected with nucleic acid encoding KIAA1363.
5. The method of claim 4 , wherein the nucleic acid encodes the amino acid sequence set forth in SEQ ID NO:2.
6. The method of claim 3 , wherein the thiol-reactive fluorescent reagent is 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB).