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Access Type

WSU Access

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Young-Hoon Ahn

Abstract

The reactive oxygen species (ROS) are involved in extensive cellular damage causing different pathological conditions, but also regulate important physiological functions through cell signaling pathways. The balance between oxidants and antioxidants determines the biological outcomes of ROS in cells. ROS production can lead to oxidative modifications of proteins, whereas redox enzymes such as thioredoxin 1 (Trx1) and glutaredoxin1 (Grx1) are the major antioxidant enzymes that are important for restoring redox-homeostasis, and regulating stress-relevant signaling pathways. In this thesis work, I sought to develop chemical tools and methods for investigating redox-mediated protein oxidative modifications and Trx1-signaling pathways.

Protein S-glutathionylation is a well-known protein oxidative modification, which forms a disulfide bond between protein cysteine and glutathione (GSH). A novel chemical tool was developed for detection of a protein glutathionylation, while overcoming some of the limitations of existing methods. We developed a mutant of the glutathione synthetase enzyme that catalyzes L-allyl-Gly as a glycine surrogate during the biosynthesis of glutathione (GSH), which resulted in metabolic synthesis of allyl-GSH. An optimized iEDDAC reaction between L-allyl-Gly and tetrazine probes was employed to detect glutathionylated proteins.

Trx1 is one of the major enzymes that maintains redox balance in the cell and regulates important cellular signaling pathways by interacting with other proteins. To understand the redox regulation of cell signaling pathways, I developed a non-thiol-reactive peptide, namely 2GTP1 (ETRWPNT), which selectively disrupts the Trx1-Ask1 interaction. We further demonstrated that a cell permeable derivative of 2GTP1 could activate the MAP kinase pathway and apoptosis signaling in cancer cells without affecting other stress signaling pathways.

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