Characterization of Diacylglycerol Metabolizing Enzyme Functional Specificity in Native Biological Systems

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Ware, Timothy, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hsu, Ku-Lung, University of Virginia

Diacylglycerols (DAGs) are a poorly understood signaling lipid system where specificity is expected to serve a pivotal role in its functionality, yet few examples have been reported in literature. Many DAG metabolizing enzymes regulate distinct biological processes throughout human biology however, what remains unclear are the underlying mechanisms which impart unique functionality while exhibiting minimal structural diversity. Lipid specificity offers an explanation why certain lipids of the same class are used more over others in biological processes. The work presented in this dissertation leverages the implementation of advanced bioanalytical approaches to elucidate lipid-protein interplay in living cell systems to provide critical insight into how DAG metabolizing enzymes regulate fundamental biological processes. The goal of this dissertation is to apply recently advanced analytical methodology towards discovering novel specificity and functionality of DAG metabolizing enzymes and their associated signaling networks. In Chapter 2, we utilize high-resolution liquid chromatography mass spectrometry (LC-MS/MS) and engineering of recombinant proteins to elucidate the structure-function relationship of diacylglycerol lipid kinases (DGK) regulatory domains which impart substrate specificity to in a cellular context. Chapter 3 discusses the approaches used to characterize the novel substrate specificity of diacylglycerol lipase beta (DAGLĪ²), expanding our understanding of how these enzymes may regulate immune responses and energy catabolism. Chapter 4 examines a proposed convergence of inflammatory signaling networks attenuated by DAGLĪ² expression and activity involving both inflammatory lipid production and protein kinase signaling cascades. Lastly, Chapter 5 examines the body of work presented in this dissertation and discusses limitations of implemented approaches along with future directions for deeper exploration of DAG metabolizing enzyme signaling pathways.

PHD (Doctor of Philosophy)
diacylglycerol, lipid metabolism, metabolomics, chemoproteomics, substrate specificity
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