We are interested in understanding how lipid-metabolizing enzymes function and are regulated at the molecular and structural level. Our current focus is on two enzymes in phosphatidic acid (PA) metabolism: Lipin and phospholipase D (PLD). Lipins are lipid phosphatases that dephosphorylate PA to generate diacylglycerol, which is the penultimate step in triglyceride biosynthesis. Lipins regulate triglyceride biosynthesis, triglyceride catabolism, 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. Both Lipin and PLD are highly regulated, multi-domain proteins that are structurally uncharacterized. Determining the three-dimensional structures of these enzymes alone, and in complex with their lipid substrates, lipid activators, and protein activators is essential to understanding their function and regulation. In preliminary data, we have used innovative bioinformatics and protein engineering to identify endogenous homologs and constructs of mouse/human Lipin and PLD that are more amenable for structural studies, demonstrated these constructs are fully functional in cells and in vitro, and obtained diffraction quality crystals. 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 and yeast. A network of collaborators who are leaders in their respective fields supports these studies.
We aim to answer several major questions: (1) How do lipid-modifying enzymes recognize their hydrophobic substrates and interact with the membrane during interfacial catalysis? (2) What role do novel structurally and functionally uncharacterized domains play in the action of these enzymes? (3) How do lipids activate these enzymes? (4) How are these enzymes regulated? Specifically, are they autoinhibited? How do protein effectors activate them? 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. In addition, this work will aid our long-term interest to develop and improve small molecule modulators of these enzymes, which would have potential therapeutic applications for the treatment of cancer, cardiovascular disease, and diabetes.

Public Health Relevance

Alterations in lipid metabolism play important roles in the onset and progression of various diseases including cancer, heart disease, and diabetes. For example, increased levels of triglycerides have long been associated with increased risk of cardiovascular disease and diabetes, while activation of phospholipase D has been implicated in cancer. This proposal seeks to understand the molecular basis for the action of two enzymes: Lipins, which play key roles in triglyceride synthesis and breakdown, and the Phospholipase D isozymes, which are novel cancer therapeutic targets.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
3R35GM128666-02S1
Application #
10085314
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Barski, Oleg
Project Start
2018-08-01
Project End
2023-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794