Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

Wu, C., Liu, T., Chen, W., Oka, S., Fu, C. X., Jain, M. R., Parrott, A. M., Baykal, A. T., Sadoshima, J., Li, H. (October 2010) Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin. Molecular & Cellular Proteomics, 9 (10). pp. 2262-2275. ISSN 1535-9476

URL: https://www.ncbi.nlm.nih.gov/pubmed/20660346
DOI: 10.1074/mcp.M110.000034

Abstract

Transnitrosylation and denitrosylation are emerging as key post-translational modification events in regulating both normal physiology and a wide spectrum of human diseases. Thioredoxin 1 (Trx1) is a conserved antioxidant that functions as a classic disulfide reductase. It also catalyzes the transnitrosylation or denitrosylation of caspase 3 (Casp3), underscoring its central role in determining Casp3 nitrosylation specificity. However, the mechanisms that regulate Trx1 transnitrosylation and denitrosylation of specific targets are unresolved. Here we used an optimized mass spectrometric method to demonstrate that Trx1 is itself nitrosylated by S-nitrosoglutathione at Cys(73) only after the formation of a Cys(32)-Cys(35) disulfide bond upon which the disulfide reductase and denitrosylase activities of Trx1 are attenuated. Following nitrosylation, Trx1 subsequently transnitrosylates Casp3. Overexpression of Trx1(C32S/C35S) (a mutant Trx1 with both Cys(32) and Cys(35) replaced by serine to mimic the disulfide reductase-inactive Trx1) in HeLa cells promoted the nitrosylation of specific target proteins. Using a global proteomics approach, we identified 47 novel Trx1 transnitrosylation target protein candidates. From further bioinformatics analysis of this set of nitrosylated peptides, we identified consensus motifs that are likely to be the determinants of Trx1-mediated transnitrosylation specificity. Among these proteins, we confirmed that Trx1 directly transnitrosylates peroxiredoxin 1 at Cys(173) and Cys(83) and protects it from H2O2-induced overoxidation. Functionally, we found that Cys(73)-mediated Trx1 transnitrosylation of target proteins is important for protecting HeLa cells from apoptosis. These data demonstrate that the ability of Trx1 to transnitrosylate target proteins is regulated by a crucial stepwise oxidative and nitrosative modification of specific cysteines, suggesting that Trx1, as a master regulator of redox signaling, can modulate target proteins via alternating modalities of reduction and nitrosylation. Molecular & Cellular Proteomics 9:2262-2275, 2010.

Item Type: Paper
Uncontrolled Keywords: PROTEIN S-NITROSYLATION CYSTEINE-SULFINIC ACID ACTIVE-SITE CYSTEINE NITRIC-OXIDE ENDOTHELIAL-CELLS OXIDATIVE STRESS GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE BIOCHEMICAL-CHARACTERIZATION 2-CYS PEROXIREDOXINS PROTEOMIC ANALYSIS
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > apoptosis
Investigative techniques and equipment > spectroscopy > mass spectrometry
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression > post-translational modification
CSHL Authors:
Communities: CSHL labs > Pappin lab
Depositing User: CSHL Librarian
Date: October 2010
Date Deposited: 20 Oct 2011 13:40
Last Modified: 13 Mar 2018 15:50
PMCID: PMC2953919
Related URLs:
URI: http://repository.cshl.edu/id/eprint/15586

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