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.

Identification of Novel Nuclear Targets of Human Thioredoxin 1

“…Yano et al used monobromobimane to label free thiols in proteins reduced by Trx that were resolved via two-dimensional gel electrophoresis and identified five Trx targets in peanut seeds (26). The Trx affinity technique was subsequently developed to identify Trx targets in diverse cell types, including spinach (27)(28)(29)(30)(31), Arabidopsis (32)(33)(34)(35), Escherichia coli (36,37), wheat seeds (38,39), Synechocystis (40 -42), and HeLa cells (16,43). The most successful of these studies identified up to 80 putative Trx targets (37).…”

“…In 2008, Hagglund et al used a quantitative proteomics approach based on the isotope-coded affinity tag method to identify 40 putative Trx targets in the seeds of malting barley (44). More recently, we used the isotope-coded affinity tag approach to identify over 70 targets of Trx1 transnitrosylation and 50 targets of Trx1-mediated denitrosylation (43). Although a Trx mutant has been used to identify Trx targets in non-mammalian cells (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)44), its application in the identification of mammalian targets has been rare, presumably because of the presence of highly abundant cytosolic targets in the total cell lysates, such as Prx1, that compete for a limited number of binding sites on the Trx1 C35S mutant.…”

Specificity in S-Nitrosylation: A Short-Range Mechanism for NO Signaling?

“…S-Nitrosylation of nonactive Cys has also been described, particularly Cys62, Cys69, and Cys73, in conditions where active site cysteines were oxidized (54,59,166). A recent study, using C32S and C35S mutants, demonstrated that Trx S-nitrosylated in Cys73 is able to transnitrosylate many cellular targets including caspase-3 (112,166,168). Interestingly, when Trx is S-nitrosylated in Cys73, the disulfide bond in the active site C32-C35 is not reduced by Trx reductase; both enzymes are then uncoupled, which can be a mechanism to prevent the denitrosylase activity of Trx while promoting its transnitrosylase activity (59).…”

“…Benhar et al used a quantitative LC-MS/MS approach to identify proteins that were denitrosylated when they added Trx/TR to cell extracts that had been nitrosylated in vitro (18). Interestingly, Trx targets of transnitrosylase activity could be differentiated from those of denitrosylation, providing specificity to the search of Trx substrates in the context of S-nitrosylation (168).…”

S-nitrosylation: integrator of cardiovascular performance and oxygen delivery

“…27-29, 34, 35, and Figure 2). Members of the TRX enzyme family (TRX1 and TRX2) mediate the denitrosylation of multiple SNO-protein substrates in the cytosol and mitochondria (25,36).…”