Nonenzymatic Cysteine Modification Regulates Pro-Apoptotic BAX
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Cohen, Daniel Thomas
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CitationCohen, Daniel Thomas. 2020. Nonenzymatic Cysteine Modification Regulates Pro-Apoptotic BAX. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractThe BCL-2 family are central regulators of mitochondrial apoptosis, maintaining the delicate balance between cellular life and death in all multicellular organisms. These key regulatory proteins communicate with many different cellular signaling pathways to sense and transduce cellular stress and are modulated by protein-protein and protein-lipid interactions, as well as a variety of post-translational modifications. In this dissertation, I investigate the role of the sphingolipid metabolite trans-2-hexadecenal (t-2-hex) in potentiating apoptosis through its activation of the pro-apoptotic executioner protein BAX. I investigate the biochemical mechanism to discover that t-2-hex activates BAX through an uncatalyzed Michael addition with C126, a cysteine within a key regulatory region. I then use dynamic structural biology techniques to show that t-2-hex induces BAX to take an atypical conformational route toward activation. Using a combination of artificial and natural membranes, chemical probes, and cell biology techniques, I validate the functional interaction between t-2-hex and BAX in vitro and in mitochondrial and cellular settings. Finally, I use mass spectrometry-based high-throughput screening to identify a covalent inhibitor of BAX activation that also targets C126. Together, these biochemical, structural, and cellular experiments present a model of C126 as a regulatory site that is accessible through nonenzymatic chemical modification. This work may inform future drug-development and treatment efforts for cancer and diseases arising from inborn errors of lipid metabolism.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37365911
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