title: Distinction of thioredoxin transnitrosylation and denitrosylation target proteins by the ICAT quantitative approach.
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.
authors.affiliation: Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School Cancer Center, Newark, NJ 07103, USA.
authors.forename: Changgong
authors.initials: C
authors.lastname: Wu
authors.forename: Andrew Myles
authors.initials: AM
authors.lastname: Parrott
authors.forename: Tong
authors.initials: T
authors.lastname: Liu
authors.forename: Mohit Raja
authors.initials: MR
authors.lastname: Jain
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Yang
authors.forename: Junichi
authors.initials: J
authors.lastname: Sadoshima
authors.forename: Hong
authors.initials: H
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002478:Cells, Cultured; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D008954:Models, Biological; D015538:Nitrosation / Q000502:physiology; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013879:Thioredoxins / Q000378:metabolism* / Q000502:physiology*
chemical_list: D011506:Proteins; D013879:Thioredoxins