Oxidative protein folding: investigating new enzymes, new assays, and new locations
Date
2019
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Publisher
University of Delaware
Abstract
Oxidative protein folding is the process of inserting disulfide bonds into unfolded reduced proteins as the native structure of the protein is acquired. In addition to being strongly stabilizing structures thermodynamically, a number of disulfides serve catalytic or signaling functions. Two types of oxidoreductases implicated in oxidative folding are featured in this Dissertation. The first is Quiescin sulfhydryl oxidase (QSOX), a facile disulfide-generating enzyme that was discovered in this laboratory. Two isoforms of QSOX have been identified in vertebrates: QSOX1 and QSOX2. QSOX1 has been extensively studied with an initial emphasis on the chicken enzyme, and later the recombinant human QSOX1. Chapter 2 of this Dissertation describes our efforts to express recombinant human QSOX2 in multiple systems in attempts to illuminate QSOX2 catalytic activity and physiological functions. The second enzyme utilized in this work is protein disulfide isomerase; this thiol/disulfide oxidoreductase is capable of shuffling mispaired disulfide bonds in proteins undergoing oxidative protein folding. We envisaged a new class of assays for this family of enzymes using Gaussia princeps luciferase (GLuc). GLuc generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. In Chapter 3 of this Dissertation we show that this 5-disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative folding. Reduced GLuc is completely inactive as a luciferase, but greater than 60% bioluminescence activity can be recovered using a range of oxidizing regimens. When reduced GLuc is reoxidized under denaturing conditions, the resulting scrambled protein can be employed in a sensitive bioluminescence assay for protein disulfide isomerase activity. ☐ In the final Chapter of this work, the thiol reductive output of several cell types has been investigated. We found that small molecule thiols represent the great majority of the reductive capacity of conditioned media in HeLa cells. We have developed two methods for continuously following release of thiols, using either fluorescence or absorbance formats, in a 96-well plate reader. We discuss thiol/disulfide exchange reactions between reduced glutathione and oxidized cystine with the cycles of reactions that connect them between intracellular and extracellular environments. We propose two benefits of the secretion of reduced glutathione. Firstly, it represents a potential way to dispose of intracellular reducing equivalents to the more aerobic stromal regions surrounding the tumor. Secondly, it would deliver protons out of the cell and consequently might lower the acidosis associated with a tumor metabolism that relies heavily on glycolysis