Investigating Diacylglycerol Kinase Function and Regulation Using Chemical Proteomics and MassSpectrometry Lipidomics

Author: ORCID icon
Mendez, Roberto, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Hsu, Ku-Lung (Ken), Chemistry, University of Virginia
Cafiso, David, Chemistry, University of Virginia
Hilinski, Michael, Chemistry, University of Virginia
Lazo, John, Chemistry, University of Virginia
Harris, Thurl, Pharmacology, University of Virginia

Diacylglycerol kinases (DGKs) regulate cell metabolism and signaling through poorly defined mechanisms with regards to lipid specificity. My thesis work aims to bridge gaps in our knowledge by investigating isoform-specific functions of DGKs using chemoproteomic and lipidomic methods. We aim to apply novel technologies to explore DGKs to unravel how individual members of this enzyme family can regulate distinct cell biology. We have made progress in determining DGKθ-mediated alterations in the lipidome. These studies have provided key insights into molecular lipid composition at the level of fatty acyl chain length and unsaturation, using tandem LC-MS/MS techniques. We discovered a putative role for this DGK isoform in the Lands cycle.

We applied modern technologies for perturbing protein function, such as CRISPR/Cas9, PROTAC, and autoSTOMP, along with mass spectrometry techniques, to probe DGK metabolic function. We have demonstrated that targeted protein degradation (using dTAG method) can be used to probe lipid substrate specificity of DGK isoforms. This allowed us to then focus our chemoproteomic efforts to identify important/critical sites on all ten DGK enzymes with importance for metabolic function. Collectively, the ligandable sites identified represent the first step towards developing potent and selective inhibitors for individual DGK isoforms. Based on my collective work, we provide evidence for distinct ligand binding pockets for individual DGK isoforms, which is reflected in specific lipid substrates regulated in cells.

PHD (Doctor of Philosophy)
DGK, diacylglycerol kinase, lipidomic, chemoproteomic, proteomic, activity based protein profiling (ABPP), proteolysis targeting chimeras (PROTAC), degradation tag (dTAG), mass spectrometry (LC-MS/MS)

It has been a journey. It was a pleasure being part of the Wahoos experience at UVa!

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