Date of Award
Chemistry and Biochemistry
CC BY-NC-ND 4.0
S-nitrosation is the covalent attachment of nitric oxide (NO) moiety to cysteine thiol side chain. This reversible modification represents an important mechanism of post-translational regulation for a large number of proteins. In the cellular environment, S-nitrosoglutathione (GSNO) can transfer its NO group to reactive cysteine residues within proteins via transnitrosation reactions. Similarly, S-nitrosated protein can transfer its NO moiety to reduced glutathione (GSH). Due to the existence of this equilibrium, the GSNO metabolizing enzyme GSNO reductase (GSNOR) indirectly drives protein de-nitrosation. To date, aberrant GSNOR activity has been implicated in a large spectrum of human diseases. In this dissertation, we report the synthesis and characterization of O-aminobenzoyl-S-nitrosoglutathione (OAbz-GSNO), a novel fluorogenic substrate for GSNOR. OAbz-GSNO reduction mediated by GSNOR results in significant increases in fluorescence; and this increase in fluorescence is attenuated by GSNOR inhibitor treatment. In addition, OAbz-GSNO is cell membrane permeable and can be used to monitor endogenous GSNOR activity in cultured cells. Overall, our work demonstrates that OAbz-GSNO is a useful tool for assessing GSNOR activity, both in vitro and in cells. Site-directed mutagenesis and kinetic studies conducted using recombinant GSNOR suggest acetylation of Lys101 negatively affects enzyme activity; while computational simulations uncovered a putative allosteric GSNO binding site. We have experimental evidence supporting our model that GSNO binding to this allosteric site enhances GSNOR activity. Both lysine acetylation and allosteric substrate binding represent potential mechanisms involved in the post-translation regulation of GSNOR activity. Neutral sphingomyelinase II (NSMase II) is a mediator of cellular stress response. It catalyzes the hydrolysis of plasma membrane sphingomyelin to generate bioactive ceramide and phosphocholine. This project looks into whether chronic cortisol exposure (as a stressor) affects NSMase II expression/activity. Experimental results demonstrate exposure to cortisol leads to increased cell size, but NSMase II expression and activity are unaffected. However, NSMase II over-expressing cells appear to have less cholesterol in the plasma membrane. Since cholesterol is important for the formation of lipid rafts, these finding suggest that in addition to ceramide generation, modulation of plasma membrane cholesterol content may represent an alternative mechanism by which NSMase II exerts its biological effects.
Sun, Bei, "Biochemical and Functional Studies of S-nitrosoglutathione Reductase and Neutral Sphingomyelinase II" (2017). Electronic Theses and Dissertations. 5953.