Structure and regulation of lipid metabolism and transport

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PROJECT SUMMARY: We are interested in understanding how lipid-metabolizing enzymes and lipid transport proteins function and are regulated at the molecular and structural level. Previously, we focused on two enzymes in phosphatidic acid (PA) metabolism: lipin and phospholipase D (PLD). Lipins are PA phosphatases that dephosphorylate PA to generate diacylglycerol, which is the penultimate step in triglyceride biosynthesis. Lipins regulate phospholipid and lipoprotein synthesis, fat storage as triglycerides, fatty acid synthesis, and insulin sensitivity with implications to obesity, diabetes, and cardiovascular disease. PLDs hydrolyze phosphatidylcholine to produce the lipid second messenger PA in response to extracellular stimuli. Receptor- mediated activation of PLD regulates vesicular trafficking, cell proliferation, and cell migration, which has established them as therapeutic targets for cancer. Despite extensive studies on these highly regulated, multi- domain enzymes, significant gaps remain in our knowledge of their molecular mechanisms of action. During the past funding period, we determined the first structures of lipin and PLD, identified a new type of membrane- binding domain in lipins, and provided insight into PLD activation by lipids and protein effectors. These discoveries provide an excellent foundation for this application, where our main goals include 1) continuing to understand how PLD is activated by protein effectors and lipids, and 2) examining the molecular mechanisms regulating lipin PA phosphatase activity and PA substrate specificity. We will also 3) biochemically and structurally characterize the nuclear envelope protein phosphatase complex CTDNEP1-NEP1R1, which dephosphorylates lipin to control lipin PA phosphatase activity; and 4) study the structure and function of Nir lipid transfer proteins, which transfer PA and phosphatidylinositol between membrane contact sites to replenish pools of plasma membrane phosphoinositides after hydrolysis by PLC. Structural studies will be complemented by an array of lipid biochemistry and lipid-protein interaction assays that we are well versed in, hydrogen-deuterium exchange mass spectrometry, and cellular studies in mammalian cells. A network of collaborators who are leaders in their respective fields supports these studies. We aim to answer several major questions: (1) How does the structure of lipid-modifying enzymes allow them to recognize their lipid substrates and interact with the membrane? (2) What role do structurally and/or functionally uncharacterized domains play in the action of these proteins? (3) How are these enzymes/proteins regulated? Specifically, are they autoinhibited? how do protein effectors, lipids, ions, and post-translational modifications regulate activity? and what conformational changes occur during activation? Overall, this work will improve our understanding of biological mechanisms and provide information on lipid-protein interactions of physiological and pharmacological significance.