No description available.

No description available.

Cysteine dioxygenase (CDO) is a non-heme iron enzyme that can be found in mammalian tissue. It is mainly localized in the liver but is also present in the brain, kidney, and adipose tissue. CDO converts cysteine to cysteine sulfinic acid, which is the first step in cysteine metabolism in the human body. CDO contains a novel cofactor located near the metal binding site that is present in another enzyme, galactose oxidase, where it is essential for redox function. This suggests that the linkage may play an important role in CDO as well. The cofactor consists of Y157 and C93. Mutation of the C93S causes a drop in activity to 57.1% and a mutation of the Y157F causes a drop to 8.1%. The metal center was studied using XAS revealing that the addition of cysteamine, an activator of CDO, changes the conformation of the binding site significantly. CDO differs from the rest of the cupin super family in that it does not contain a 2-his-1-carboxylate binding motif but rather the carboxylate is replaced with another histidine. A mutation of one of the binding residues, H140D, caused the enzyme to be non-active. Also the mechanism of the CDO was studied by conducting activity assays with various inhibitors and activators that yielded contradicting results with previously published work.

Thesis advisor: Eranthie Weerapana / Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS. / Thesis (PhD) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Oxidative stress is commonly associated with diet-induced metabolic syndrome (MetS) and left ventricular cardiac remodeling, but much remains unknown about the role of redox signaling, sensors, and switches in mediating the effects of high fat and sugar intake. In this work, I describe and apply an optimized method for quantifying changes in reversible protein-cysteine oxidation in the heart. This method uses isobaric tagging of cysteine thiols and mass spectrometry in a modified biotin switch on whole tissue lysate. Analyzing the resulting data with systems biology approaches helped delineate redox pathways playing a role in disease development, while cysteine-specificity provided exact targets for mutation-based mechanistic studies. Initial findings in a mouse model for MetS, wherein C57Bl6J mice were fed a high fat/high sucrose diet, identified energy pathways as the primary target of changing reversible cysteine oxidation. In follow-up studies, our collaborators helped validate the pathophysiological role of two particular cysteines in complex II; their early reversible oxidation and later irreversible oxidation contributed to decreased ATP output from cardiac mitochondria. A subsequent, more robust study revealed a weakness in our original method. While investigating the role of hydrogen peroxide-induced oxidative post-translational modifications (OPTMs) in the development of MetS sequelae, analysis of four mouse groups, each with an n=5, revealed that measurements of reversibly oxidized cysteine thiols were highly variable compared to those of all available thiols. Thus, I developed a strategy to address the source of variability and, in the process, improved many additional steps in the switch protocol. Finally, in an effort to clarify the role of the most stable reversible OPTM, glutathionylation (RSSG), we characterized the HFHS diet response in mice engineered to have more or less RSSG via genetic manipulation of glutaredoxin-1 expression. Those with more RSSG suffered worsened cardiac function, making them an ideal model for future studies with the methods optimized in this work. Studying the progression from poor diet to cardiac involvement in these and other mouse models using the methods described herein will aid in the design of diagnostics and targeted therapies against the growing burden of metabolic CVD.

No description available.

No description available.

Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases. We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all. A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I. Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 124-142).

Thesis (Ph.D.)--University of Alberta, 2009. / A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Chemistry. Title from pdf file main screen (viewed on November 8, 2009). Includes bibliographical references.