![]() ![]() NEM treatment increased the complex formation between Trx2 and Prx3. Direct modification of Trx2 was confirmed by mass spectrometry and redox Western analysis. In the present study, we showed that NEM caused higher cytotoxicity in cells overexpressing Trx2. How modification of a single cysteine affects the functions of this protein has not been well characterized. The oxidized cysteines often returned to the reduced state within relative short time after the initial oxidation ( 13). Under those conditions, a disulfide bond was reversibly formed between Cys90 and Cys93 in the active center. In our previous studies, we showed that Trx2 was susceptible to oxidation caused by various inducers of oxidative stress, such as peroxides ( 13), thiol cross-linker ( 14), heavy metal ( 15) and tumor necrosis factor-α ( 16). Trx2 interacts with Prx3, which is a major detoxification enzyme responsible for the clearance of mitochondrially generated hydrogen peroxide, and keeps it in the reduced and active state ( 12). The mitochondrially localized thioredoxin 2 (Trx2) is an indispensable component of the mitochondrial antioxidant system ( 8, 9) and shows anti-apoptotic functions against various stimuli ( 10, 11). In mammalian cells, the cytosolic thioredoxin 1 (Trx1) protein is implicated in stimulation of cell proliferation, regulation of transcription of cell survival genes and inhibition of apoptosis ( 7). The two cysteine residues can undergo reversible oxidation/reduction in the presence of thioredoxin reductase and NADPH. Trxs contain an invariant active center, -Cys-Gly-Pro-Cys. Among them, thioredoxins (Trxs) are a family of small pleiotropic proteins that are ubiquitously expressed and evolutionarily conserved from prokaryotes to mammals ( 6). There are a number of enzymatic systems that control the cellular redox status utilizing NAD(P)H as the ultimate electron donor ( 5). NEM also inhibited platelet-derived growth factor-induced Akt phosphorylation ( 3) but increased the activity of protein phosphatase 2A ( 4). Alkylation of Cys199 by NEM augmented dephosphorylation and inactivation of cAMP-dependent protein kinase A ( 2). For instance, exposure of endothelial cells to peroxynitrite caused glutathiolation of p21ras at Cys118, which subsequently led to its membrane translocation and increased binding with Raf-1 ( 1). Modifications of critical thiols of protein kinases and phosphatases can result in either loss or gain of function. ![]() The thiol/disulfide redox status is an important regulatory factor in controlling tissue and cell functions. ![]()
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