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Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

ABSTRACT: Hydrogen peroxide (H2O2) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H2O2-reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion models of H2O2 signaling. The results show that at H2O2 supply rates commensurate with mitogenic signaling a H2O2 concentration gradient with a length scale of a few tenths of ?m is established. Even near the supply sites H2O2 concentrations are far too low to oxidize typical targets in an early mitogenic signaling time frame. Furthermore, any inhibition of the peroxiredoxin or increase in H2O2 supply able to drastically increase the local H2O2 concentration would collapse the concentration gradient and/or cause an extensive oxidation of the peroxiredoxins I and II, inconsistent with experimental observations. In turn, the local concentrations of peroxiredoxin sulfenate and disulfide forms exceed those of H2O2 by several orders of magnitude. Redox targets reacting with these forms at rate constants much lower than that for, say, thioredoxin could be oxidized within seconds. Moreover, the spatial distribution of the concentrations of these peroxiredoxin forms allows them to reach targets within 1 ?m from the H2O2 sites while maintaining signaling localized. The recruitment of peroxiredoxins to specific sites such as caveolae can dramatically increase the local concentrations of the sulfenic and disulfide forms, thus further helping these species to outcompete H2O2 for the oxidation of redox targets. Altogether, these results suggest that H2O2 signaling is mediated by localized redox relays whereby peroxiredoxins are oxidized to sulfenate and disulfide forms at H2O2 supply sites and these forms in turn oxidize the redox targets near these sites.

SUBMITTER: Travasso RDM

PROVIDER: S-EPMC5339411 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

Travasso Rui D M RDM Sampaio Dos Aidos Fernando F Bayani Anahita A Abranches Pedro P Salvador Armindo A

Redox biology 20170106

Hydrogen peroxide (H2O2) is a key signaling agent. Its best characterized signaling actions in mammalian cells involve the early oxidation of thiols in cytoplasmic phosphatases, kinases and transcription factors. However, these redox targets are orders of magnitude less H2O2-reactive and abundant than cytoplasmic peroxiredoxins. How can they be oxidized in a signaling time frame? Here we investigate this question using computational reaction-diffusion ...[more]

PMID: 28279943

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Project description:Preexposure to a low concentration of H2O2 significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H2O2 However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H2O2 and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn2+-coordinated Cys139 and Cys142 mutations eliminated the H2O2 oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn2+ in H2O2-treated PerR, demonstrating that cysteine oxidation results in Zn2+ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn2+-bound PerR protein (PerR:Zn,Mn) was abolished by H2O2 treatment but was restored by dithiothreitol reduction, verifying that H2O2 inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H2O2-concentration-induced adaptation of S. oligofermentans to a higher H2O2 concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H2O2 concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H2O2 defense strategy employed by catalase-negative streptococci in Mn2+-rich cellular environments.IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H2O2 This work reports the molecular mechanisms of low-H2O2-concentration-induced adaptation to higher H2O2 stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H2O2 through histidine oxidation by the Fe2+-triggered Fenton reaction, the streptococcal PerR is inactivated by H2O2 oxidation of the structural Zn2+ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.

Project description:The regulatory mechanisms by which hydrogen peroxide (H2O2) modulates the activity of transcription factors in bacteria (OxyR and PerR), lower eukaryotes (Yap1, Maf1, Hsf1 and Msn2/4) and mammalian cells (AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1) are reviewed. The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm-nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M(-1) s(-1) and ≥1.3 × 10(3) M(-1) s(-1) were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.

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Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

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Localized redox relays as a privileged mode of cytoplasmic hydrogen peroxide signaling.

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