The yeast Saccharomyces cerevisiae transcription factor Yap1 mediates an adaptive response to oxidative stress by regulating protective genes. H(2)O(2) activates Yap1 through the Gpx3-mediated formation of a Yap1 Cys303-Cys598 intramolecular disulfide bond. Thiol-reactive electrophiles can activate Yap1 directly by adduction to cysteine residues in the C-terminal domain containing Cys598, Cys620, and Cys629. H(2)O(2) and N-ethylmaleimide (NEM) showed no cross-protection against each other, whereas another thiol-reactive chemical, acrolein, elicited Yap1-dependent cross-protection against NEM, but not H(2)O(2). Either Cys620 or Cys629 was sufficient for activation of Yap1 by NEM or acrolein; Cys598 was dispensable for this activation mechanism. To determine whether Yap1 activated by H(2)O(2) or thiol-reactive chemicals elicits distinct adaptive gene responses, microarray analysis was performed on the wild-type strain or its isogenic single-deletion strain Δyap1 treated with control buffer, H(2)O(2), NEM, or acrolein. Sixty-five unique H(2)O(2) and 327 NEM and acrolein Yap1-dependent responsive genes were identified. Functional analysis using single-gene-deletion yeast strains demonstrated that protection was conferred by CTA1 and CTT1 in the H(2)O(2)-responsive subset and YDR042C in the NEM- and acrolein-responsive subset. These findings demonstrate that the distinct mechanisms of Yap1 activation by H(2)O(2) or thiol-reactive chemicals result in selective expression of protective genes.
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http://dx.doi.org/10.1016/j.freeradbiomed.2010.10.697 | DOI Listing |
Nat Protoc
September 2020
State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences • Beijing, Beijing Institute of Lifeomics, Beijing, China.
Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved.
View Article and Find Full Text PDFMethods Mol Biol
March 2020
School of Science and the Environment, University of Worcester, Worcester, UK.
Oncol Res
September 2019
Department of Thoracic Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, P.R. China.
Cysteine oxidation occurs at the active site of deubiquitinases (DUBs) during many biologic signaling cascades. Here we report that hepatocellular carcinoma cells (HCCs) generated higher levels of endogenous reactive oxygen species (ROS). This elevated ROS production was inhibited by NADPH oxidase inhibitor diphenylene iodonium (DPI) and mitochondria electron chain inhibitor rotenone in HCC cells.
View Article and Find Full Text PDFFree Radic Biol Med
July 2017
Department of Plant Physiology, RWTH Aachen University, 52056 Aachen, Germany. Electronic address:
Unlabelled: Allicin is a thiol-reactive sulfur-containing natural product from garlic with a broad range of antimicrobial effects against prokaryotes and eukaryotes. Previous work showed that the S. cerevisiae OSI1 gene is highly induced by allicin and other thiol-reactive compounds, and in silico analysis revealed multiple Yap1p binding motifs in the OSI1 promoter sequence.
View Article and Find Full Text PDFFree Radic Biol Med
July 2017
Department of Medical Microbiology, Maastricht University Medical Center, PO Box 5800, 6202 AZ Maastricht, The Netherlands. Electronic address:
Introduction: Airway epithelial cells have been described to release extracellular vesicles (EVs) with pathological properties when exposed to cigarette smoke extract (CSE). As CSE causes oxidative stress, we investigated whether its oxidative components are responsible for inducing EV release and whether this could be prevented using the thiol antioxidants N-acetyl-l-cysteine (NAC) or glutathione (GSH).
Methods: BEAS-2B cells were exposed for 24h to CSE, HO, acrolein, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), bacitracin, rutin or the anti-protein disulfide isomerase (PDI) antibody clone RL90; with or without NAC or GSH.
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