Thiol-disulfide oxidoreductases: assays, inhibitors, and metabolic roles
Date
2019
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Publisher
University of Delaware
Abstract
Disulfide bonds are common post-translational modifications which serve to stabilize or impart function onto proteins. Although this product of oxidation between two cysteine thiols is relatively stable, disulfide bonds are able to undergo isomerization, to interact with thiol substrates, and to allow for protein conformational changes. The dynamic nature of thiol-disulfide exchange with respect to cellular processes such as oxidative protein folding and modulation of the extracellular environment is the focus of this work. ☐ Oxidative protein folding occurs primarily in the endoplasmic reticulum where the oxidizing nature of the compartment supports disulfide bond formation of cysteine-containing nascent peptide chains. Quiescin sulfhydryl oxidase (QSOX) is an enzyme, discovered in our laboratory, that can facilely install disulfide bonds into unfolded reduced substrates. QSOX can also function as an extracellular oxidase to support matrix assembly. Chapter 2 of this dissertation describes our efforts, in collaboration with University of Chicago, to isolate an inhibitory antibody fragment against QSOX through phage display screening. Protein disulfide isomerase (PDI) is an oxidoreductase that is also proficient in oxidizing reduced clients; reduced PDI can also function as an isomerase to rearrange mis-paired cysteines. A novel PDI reductase assay is presented in Chapter 4, which utilizes a commercially-available fluorogenic disulfide to dynamically monitor enzyme activity in the presence of cellularly-relevant concentrations of glutathione. This work highlights the challenges of assessing the potency of PDI inhibitors which are themselves reactive towards thiols. ☐ Conserved catalytic vicinal thiol motifs of enzymes involved in oxidative protein folding have historically been the target of arsenic (III) species. Following previous work from this laboratory in the development of multivalent arsenic inhibitors against in vitro oxidative protein folding models, Chapter 5 discusses efforts to construct a multivalent arsenical intracellularly. Novel small molecule arsenicals and bivalent linkers using the trans-cyclooctene tetrazine biorthogonal ligation chemistry were synthesized and their efficacy in inducing cell death were observed. ☐ The redox state of the cell surface has been described to modulate thioldisulfide interactions between exofacial enzymes and extracellular proteins, and to contribute to the cell’s overall reductive capability. The work presented in Chapter 6 describes a study of the rate of cellular thiol secretion and an identification of the small molecular weight thiols secreted. Secretion was found to vary significantly between the cell lines tested and to depend on the presence of extracellular cystine. These findings provide a new perspective into methods of effectively assaying the cell surface for redox state and insight into a source of reducing equivalents for thiol/disulfide oxidoreductases at the cell exoface.
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Keywords
Assays, Disulfides, Extracellular thiols, Intracellular thiols, Oxidative protein folding, Thiol