Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach

Changgong Wu, Andrew Myles Parrott, Tong Liu, Mohit Raja Jain, Yanfei Yang, Junichi Sadoshima, Hong Li

Research output: Contribution to journalArticlepeer-review

58 Scopus citations

Abstract

S-Nitrosylation is a reversible PTM for regulating protein function. Thioredoxin-1 (Trx1) catalyzes either transnitrosylation or denitrosylation of specific proteins, depending on the redox status of the cysteines within its conserved oxidoreductase CXXC motif. With a disulfide bond formed between the two catalytic cysteines, Trx1 is not only inactive as a denitrosylase, but it may also be nitrosylated at Cys73 and serve as a transnitrosylating agent. Identification of Trx1-mediated transnitrosylation or denitrosylation targets will contribute to a better understanding of Trx1's function. Previous experimental approaches based on the attenuation of CXXC oxidoreductase activity cannot readily distinguish Trx1 transnitrosylation targets from denitrosylation targets. In this study, we used the ICAT method in conjunction with the biotin switch technique to differentiate Trx1 transnitrosylation targets from denitrosylation target proteins from neuroblastoma cells. We demonstrate that the ICAT approach is effective for quantitative identification of putative Trx1 transnitrosylation and denitrosylation target peptides. From these analyses, we confirmed reports that peroxiredoxin 1 is a Trx1 transnitrosylation, but not a denitrosylation target, and we found several other proteins, including cyclophilin A to be modulated in this manner. Unexpectedly, we found that many nitrosylation sites are reversibly regulated by Trx1, suggesting a more prominent role for Trx1 in regulating S-nitrosylation.

Original languageEnglish (US)
Pages (from-to)2498-2509
Number of pages12
JournalJournal of Proteomics
Volume74
Issue number11
DOIs
StatePublished - Oct 19 2011

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry

Keywords

  • Denitrosylation
  • ICAT
  • SH-SY5Y
  • Thioredoxin
  • Transnitrosylation

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