Thesis advisor: Eranthie Weerapana / A chemical-proteomic platform to monitor cysteine sensitivity to transnitrosation Yani Zhou Dissertation advisor: Dr. Eranthie Weerapana Abstract S-nitrosation has emerged as a ubiquitous endogenous protein posttranslational modification that significantly impacts cellular protein function through a variety of mechanisms. Despite the advent of chemical and proteomic methods to study S-nitrosation, the subset of cellular cysteine residues that show uniquely high reactivity to endogenous transnitrosation donors is poorly characterized. To further these existing global studies, a cysteine-reactivity profiling strategy was applied herein to rank ~600 cysteine residues by sensitivity to S-nitrosoglutathione. These proteomic studies revealed several previously uncharacterized sites of S-nitrosation, including Cys58 in HADH2. Further characterization revealed that HADH2 catalytic activity is allosterically regulated by S-nitrosation, and this modification occurs in cells at (patho)physiological levels of nitrosative stress. Functional role of Cys58 and its regulation by S-nitrosation facilitated the identification of RB-21-CA as a potential covalent Cys58 inhibitor. Global analysis of GSNO, S-nitroso-Coenzyme A and Thioredoxin-C73-SNO transnitrosation identified 756 cysteines with different sensitivity to each of three SNO donors. Systematic evaluation on transnitrosation selectivity revealed that specific interaction of transnitrosation donor with its protein target is a key component governing the selective transnitrosation of a specific cysteine residue. Together, these studies illustrated the potential of cysteine-reactivity profiling strategy for evaluating the substrate specificity of transnitrosation donors and enable the identification of previously uncharacterized, functionally relevant sites of S-nitrosation. Another cysteine oxoform, S-glutathionylation, is the disulfide formation of a protein cysteine residue with glutathione. Although glucose starvation is known to induce redox-disturbance, global and individual protein S-glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. By using a clickable glutathione approach, which forms clickable glutathione by the use of a mutant of glutathione synthetase, we found that protein S-glutathionylation is readily induced in response to glucose starvation when mitochondrial reactive oxygen species are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Application of a proteomic mass spectrometry platform identified over 1,300 S-glutathionylated. Confirmation of S-glutathionylation for selected proteins by in gel analysis further validated the mass spectrometry results, and highlights the dynamic change of S-glutathionylation on an individual protein level. In order to expand on the understanding of the functional role of the cysteine residues in biological systems, we evaluated a panel of 1,3,5-triazine- and 4-aminopiperidine-based cysteine-reactive small-molecules on two proteins, apoptosis signal-regulating kinase 1 (ASK1) and peroxiredoxin 1 (Prdx1), between which a intermolecular disulfide forms and results in the activation of mitogen-activated protein kinase pathway. In-gel fluorescence revealed that RB-11-CA and SMC-1, both of which contain an n-octyl group as a diversity element, showed greatest selectivity and potency to ASK1 and Prdx1, respectively. Further mass spectrometry analysis identified cysteine 225 of ASK1 and cysteine 173 of Prdx1 are the sites of covalent probe modification. / Thesis (PhD) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_106814 |
| Date | January 2016 |
| Creators | Zhou, Yani |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author. This work is licensed under a Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ |
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