Site-specific and redox-controlled S-nitrosation of thioredoxin
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Tannenbaum-2011-Aug-Site-Specific and Redox.pdf
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Author(s)
Barglow, Katherine T.
Knutson, Charles G. F.
Wishnok, John S.
Tannenbaum, Steven Robert
Marletta, Michael A.
Date Issued
August 2011
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publisher
National Academy of Sciences (U.S.)
Citation
Barglow, K. T. et al. “Site-specific and Redox-controlled S-nitrosation of Thioredoxin.” Proceedings of the National Academy of Sciences 108.35 (2011): E600–E606. Web.
Version
Final published version
Abstract
Protein S-nitrosation on cysteine residues has emerged as an important posttranslational modification in mammalian cells. Previous studies have suggested a primary role for thioredoxin (Trx) in controlling protein S-nitrosation reactions. Human Trx contains five conserved Cys, including two redox-active catalytic Cys (Cys32 and Cys35) and three non-active-site Cys (Cys62, Cys69, and Cys73), all of which have been reported as targets of S-nitrosation. Prior reports have studied thermodynamic end points of nitrosation reactions; however, the kinetics of Trx nitrosation has not previously been investigated. Using the transnitrosation agent, S-nitrosoglutathione, a kinetic analysis of the selectivity and redox dependence of Trx nitrosation at physiologically relevant concentrations and times was performed, utilizing a mass spectrometry-based method for the direct analysis of the nitrosated Trx. Reduced Trx (rTrx) was nitrosated 2.7-times faster than oxidized Trx (oTrx), and rTrx was nitrosated selectively on Cys62, whereas oTrx was nitrosated only on Cys73. These sites of nitrosation were confirmed at the peptide level using a novel modification of the biotin-switch technique called the reductive switch. These results suggest separate signaling pathways for Trx-SNO under different cellular redox states.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1073/pnas.1110736108
