Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase that can activate the c-Jun N-terminal kinase and the p38 signaling pathways. It plays a critical role in cytokine-and stressinduced apoptosis. To further characterize the mechanism of the regulation of the ASK1 signal, we searched for ASK1-interacting proteins employing the yeast twohybrid method. The yeast two-hybrid assay indicated that mouse glutathione S-transferase Mu 1-1 (mGSTM1-1), an enzyme involved in the metabolism of drugs and xenobiotics, interacted with ASK1. We subsequently confirmed that mGSTM1-1 physically associated with ASK1 both in vivo and in vitro. The in vitro binding assay indicated that the C-terminal portion of mGSTM1-1 and the N-terminal region of ASK1 were crucial for binding one another. Furthermore, mGSTM1-1 suppressed stress-stimulated ASK1 activity in cultured cells. mG-STM1-1 also blocked ASK1 oligomerization. The ASK1 inhibition by mGSTM1-1 occurred independently of the glutathione-conjugating activity of mGSTM1-1. Moreover, mGSTM1-1 repressed ASK1-dependent apoptotic cell death. Taken together, our findings suggest that mGSTM1-1 functions as an endogenous inhibitor of ASK1. This highlights a novel function for mGSTM1-1 insofar as mGSTM1-1 may modulate stress-mediated signals by repressing ASK1, and this activity occurs independently of its well-known catalytic activity in intracellular glutathione metabolism.
Hydrogen peroxide (H(2)O(2)) is an incompletely reduced metabolite of oxygen that has a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of its production. Characterization of the cellular functions of H(2)O(2) requires measurement of its concentration selectively in the presence of other oxygen metabolites and with spatial and temporal fidelity in live cells. For the measurement of H(2)O(2) in biological fluids, several sensitive methods based on horseradish peroxidase and artificial substrates (such as Amplex Red and 3,5,3'5'-tetramethylbenzidine) or on ferrous oxidation in the presence of xylenol orange (FOX) have been developed. For measurement of intracellular H(2)O(2), methods based on dihydro compounds such as 2',7'-dichlorodihydrofluorescein that fluoresce on oxidation are used widely because of their sensitivity and simplicity. However, such probes react with a variety of cellular oxidants including nitric oxide, peroxynitrite, and hypochloride in addition to H(2)O(2). Deprotection reaction-based probes (PG1 and PC1) that fluoresce on H(2)O(2)-specific removal of a boronate group rather than on nonspecific oxidation have recently been developed for selective measurement of H(2)O(2) in cells. Furthermore, a new class of organelle-targetable fluorescent probes has been devised by joining PG1 to a substrate of SNAP-tag. Given that SNAP-tag can be genetically targeted to various subcellular organelles, localized accumulation of H(2)O(2) can be monitored with the use of SNAP-tag bioconjugation chemistry. However, given that both dihydro- and deprotection-based probes react irreversibly with H(2)O(2), they cannot be used to monitor transient changes in H(2)O(2) concentration. This drawback has been overcome with the development of redox-sensitive green fluorescent protein (roGFP) probes, which are prepared by the introduction of two redox-sensitive cysteine residues into green fluorescent protein; the oxidation of these residues to form a disulfide results in a conformational change of the protein and altered fluorogenic properties. Such genetically encoded probes react reversibly with H(2)O(2) and can be targeted to various compartments of the cell, but they are not selective for H(2)O(2) because disulfide formation in roGFP is promoted by various cellular oxidants. A new type of H(2)O(2)-selective, genetically encoded, and reversible fluorescent probe, named HyPer, was recently prepared by insertion of a circularly permuted yellow fluorescent protein (cpYFP) into the bacterial peroxide sensor protein OxyR.
The predominant enzymes responsible for elimination of hydrogen peroxide (H(2)O(2)) in cells are peroxiredoxins (Prxs), catalase, and glutathione peroxidases (GPxs). Evidence suggests that catalytic activities of certain isoforms of these H(2)O(2)-eliminating enzymes are extensively regulated via posttranslational modification. Prx I and Prx II become inactivated when phosphorylated on Thr(90) by cyclin B-dependent kinase Cdc2. In addition, the active-site cysteine of Prx I-IV undergoes a reversible sulfinylation (oxidation to cysteine sulfinic acid) in cells. Desulfinylation (reduction to cysteine) is achieved by a novel enzyme named sulfiredoxin. c-Abl and Arg nonreceptor protein tyrosine kinases associate with catalase in cells treated with H(2)O(2) by mechanisms involving the SH3 domains of the kinases and the Pro(293)PheAsnPro motif of catalase and activate catalase by phosphorylating it on Tyr(231) and Tyr(386). Similarily, GPx1 is activated by c-Abl- and Arg-mediated phosphorylation. The tyrosine phosphorylation is critical for ubiquitination-dependent degradation of catalase.
H(2)O(2) is a reactive oxygen species that has drawn much interest because of its role as a second messenger in receptor-mediated signaling. Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H(2)O(2) generated in response to receptor stimulation. 2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H(2)O(2) reduction reaction in the presence of thioredoxin, thioredoxin reductase and NADPH. Several lines of evidence suggest that 2-Cys peroxiredoxins have dual roles as regulators of the H(2)O(2) signal and as defenders of oxidative stress. In particular, 2-Cys peroxiredoxin appears to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2-Cys peroxiredoxins is clear for diseases, such as cancer and cardiovascular diseases, that involve reactive oxygen species.
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