Doctor of Philosophy, Case Western Reserve University, 2019, Biochemistry

The majority of nitric oxide's (NO) biological effect across cell types and tissues occurs through protein S-nitrosylation, the oxidative, posttranslational modification of cysteine thiols in proteins by NO to form S-nitrosothiols (SNOs). Through S-nitrosylation, NO controls protein activity, stability, interactions with other proteins, and subcellular localization to alter cellular function. NO also modifies small molecule thiols, primarily glutathione and coenzyme A, to serve as SNO-based signaling molecules. S-nitroso-glutathione (GSNO) and S-nitroso-coenzyme A (SNO-CoA) signal through S-nitrosylation of target proteins and are regulated by enzymatic denitrosylases, proteins that metabolize GSNO and SNO-CoA. SNO-CoA was recently identified as a conserved metabolic signal transducer regulated by the activity of S-nitroso-coenzyme A reductases (SCoRs). The discovery of SNO-CoA generates numerous, fundamental questions regarding how SNOs derive specificity in signaling, how enzymes recognize different SNOs, and endogenous roles of SNO-CoA in mammals. This thesis addresses these questions by providing a mechanistic understanding of how aldo-keto reductase 1A1 (AKR1A1), the mammalian SCoR, specifically recognizes SNO-CoA among myriad S-nitrosothiols, identifying targets of SCoR-dependent denitrosylation in mammalian cells and tissues, and exploring metabolic processes regulated by the SCoR/SNO-CoA system of S-nitrosylation and denitrosylation. Here we use a combination of in silico molecular modeling, in vitro biochemical assays, cell culture models, and animal models to address these questions. We find that SCoR binds SNO-CoA through both the CoA moiety and NO group; the latter finding provides the first evidence of direct SNO recognition by enzymes and explains how SCoR can identify SNO-CoA among the multitude of SNOs and CoA derivatives. SCoR also controls the S-nitrosylation of metabolic proteins and alters mitochondrial metabolism. Finally, SCoR regulates mammali (open full item for complete abstract)

Committee: Jonathan Stamler (Advisor); Focco van den Akker (Committee Chair); Mukesh Jain (Committee Member); George Dubyak (Committee Member); Hung-Ying Kao (Committee Member) Subjects: Biochemistry