Regulation of Hsp70 chaperone cycle via reversible methionine oxidation of the nucleotide exchange factor Fes1

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Elevated levels of reactive oxygen species (ROS) are associated with the pathologies of many degenerative disorders (like aging, Alzheimer’s disease, atherosclerosis, and diabetes). Cells employ a network of regulatory pathways to manage intracellular ROS levels. Identification and characterization of these fundamental pathways is necessary to determine what triggers the decline in anti-oxidant defenses associated with disease progression. Key to this understanding are the emerging roles of ROS as signaling molecules. ROS-based signaling is initiated by activation of sulfur-based redox switches, in which protein cysteine or methionine residues are post-translationally modified (oxidized) to transiently alter protein function in a manner appropriate for the physiological environment. Hsp70s are a conserved family of molecular chaperones that serve critical roles in maintaining cellular proteostasis under oxidative stress. In this dissertation, I have uncovered that a co-chaperone (Fes1) of the cytoplasmic Hsp70 system in yeast undergoes reversible methionine oxidation during excessively oxidizing cellular conditions. I have determined that three clustered methionine residues in Fes1 are oxidized to methionine sulfoxide, which inhibits Fes1 activity (and correspondingly Hsp70 chaperone activity) during stress. Importantly, I establish that Fes1 oxidation is a reversible signaling event that is regulated enzymatically by methionine sulfoxide reductase (Msr). These studies have revealed that the two yeast Msr proteins (Mxr1 and Mxr2) are both expressed in the cytosol, in contrast to prior reports of Mxr2 suggesting sole mitochondrial localization. I demonstrate that a cytoplasmic isoform of Mxr2 co-operates with Mxr1 to regulate the redox state of Fes1. Together, these studies provide a new perspective on how activities of the well-studied Hsp70 family can be attuned by ROS, and further define how methionine redox-switches, which are largely under-studied in comparison to their cysteine counterparts, are used as a means for cells to sense and respond to oxidative stress. I speculate that Fes1 oxidation may be conserved in higher eukaryotes and could serve to beneficially support cell viability during periods of oxidative stress. Elucidation of a methionine-based signaling pathway involving Fes1 furthers a general understanding of the types of events mediated by this emerging post-translational modification, for which there are few examples characterized in mechanistic detail.
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70 kilodalton heat shock protein (Hsp70); Fes1; methionine oxidation; methionine sulfoxide reductase; nucleotide exchange factor (NEF); reactive oxygen species (ROS); Cellular biology; Biochemistry
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Sevier, Carolyn S.
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Brown, William J.
Fromme, Joseph Chris
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Biochemistry, Molecular and Cell Biology
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Ph.D., Biochemistry, Molecular and Cell Biology
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Doctor of Philosophy
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Government Document
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dissertation or thesis
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