Kar2 modification as a response to reactive oxygen species generated from protein folding machinery inside the endoplasmic reticulum

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Reactive oxygen species are generated as a byproduct of protein folding inside the endoplasmic reticulum. Hydrogen peroxide is a particular form of reactive oxygen that can damage and modify DNA as well as cellular proteins. Previous studies in our lab identified Kar2, a chaperone protein in the family of Heat Shock Protein 70kDa (HSP70), as a target for peroxide modification which also conferred the cell’s resistance against oxidative stress. Kar2 contains a conserved cysteine thiol that is susceptible to redox modification by several candidate modifiers including hydrogen peroxide and glutathione, but it still remains elusive which modification of Kar2 provides protection against oxidative stress. In this thesis, I utilized a recently described enzyme-linked immunosorbent assay to detect peroxide and glutathione modifications of Kar2 in vitro. Results of my experiments show that Kar2 can be modified by glutathione (glutathionylation) through a sulfenic acid intermediate (sulfenylation) that requires the presence of Kar2’s conserved cysteine residue. Similar experiments also show that glutaredoxin 6 and 7, thiol oxidoreductases present in the early secretory pathway, have the ability to de-glutathionylate Kar2 in vitro, and thus may be involved in this signaling pathway. In a related but distinct project, I also show in this thesis that human CYB5R genes, sequential homologs of yeast PGA3 whose product serves as a potential electron acceptor alternative to oxygen, cannot complement for the loss of PGA3 in yeast.
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Biological sciences honors program; oxidative stress; reactive oxygen species; glutathione; Kar2; Glutaredoxin; Pga3
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Biological Sciences
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B.A. of Biological Sciences
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Bachelor of Arts
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Government Document
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dissertation or thesis
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