Irreversible oxidation of Cys residues to sulfinic/sulfonic forms typically impairs protein function. We found that persulfidation (CysSSH) protects Cys from irreversible oxidative loss of function by the formation of CysSSO1-3H derivatives that can subsequently be reduced back to native thiols. Reductive reactivation of oxidized persulfides by the thioredoxin system was demonstrated in albumin, Prx2, and PTP1B. In cells, this mechanism protects and regulates key proteins of signaling pathways, including Prx2, PTEN, PTP1B, HSP90, and KEAP1. Using quantitative mass spectrometry, we show that (i) CysSSH and CysSSO3H species are abundant in mouse liver and enzymatically regulated by the glutathione and thioredoxin systems and (ii) deletion of the thioredoxin-related protein TRP14 in mice altered CysSSH levels on a subset of proteins, predicting a role for TRP14 in persulfide signaling. Furthermore, selenium supplementation, polysulfide treatment, or knockdown of TRP14 mediated cellular responses to EGF, suggesting a role for TrxR1/TRP14-regulated oxidative persulfidation in growth factor responsiveness.

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-Recently, we established a biotin-PEAC 5 -maleimide (BPM)-labeling assay, which can be used to determine the modification of electrophilic metals to proteins (Toyama et al., J. Toxicol. Sci., 38, 477-484, 2013). In the present study, we applied a BPM-labeling assay to detect protein S-modification by environmental organic electrophiles. After exposing A431 cells to 1,2-naphthoquinone (1,2-NQ) and 1,4-naphthoquinone (1,4-NQ), there was an inverse correlation between Western blot analysis with specific antibody against these electrophiles and that with BPM on the blot intensity to detect protein modification. Similar results were also observed using enzyme-linked immunosorbent assay (ELISA) with BPM. Modification of proteins in mouse liver cytosol by 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, 1,4-benzoquinone (1,4-BQ), tert-butyl-1,4-BQ, and N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen, was detected using ELISA, but not non-electrophilic quinones or hydroquinone. We also tested whether ELISA could be used to detect electrophiles contained in the vapor phase of ambient air samples collected in the midtown area of Los Angeles. Taken together, the results suggested that the ELI-SA, developed in this study, can detect the existence of electrophilic quinones that covalently modify cellular proteins, resulting in modulation of redox-signal transduction pathways or cell damage.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

We previously reported that 9,10-phenanthraquinone (9,10-PQ), an atmospheric electron acceptor, undergoes redox cycling with dithiols as electron donors, resulting in the formation of semiquinone radicals and monothiyl radicals; however, monothiols have little reactivity. Because persulfide and polysulfide species are highly reducing, we speculate that 9,10-PQ might undergo one-electron reduction with these reactive sulfides. In the present study, we explored the redox cycling capability of a variety of quinone-related electron acceptors, including 9,10-PQ, during interactions with the hydropersulfide Na 2 S 2 and its related polysulfides. No reaction occurred when 9,10-PQ was incubated with Na 2 S; however, when 5 μM 9,10-PQ was incubated with either 250 μM Na 2 S 2 or Na 2 S 4 , we detected extensive consumption of dissolved oxygen (84 μM). Under these conditions, both the semiquinone radicals of 9,10-PQ and their thiyl radical species were also detected using ESR, suggesting that a redox cycle reaction occurred utilizing one-electron reduction processes. Notably, the perthiyl radicals remained stable even under aerobic conditions. Similar phenomenon has also been observed with other electron acceptors, such as pyrroloquinoline quinone, vitamin K 3 , and coenzyme Q10. Our experiments with Nmethoxycarbonyl penicillamine persulfide (MCPSSH), a precursor for endogenous cysteine persulfide, suggested the possibility of a redox coupling reaction with 9,10-PQ inside cells. Our study indicates that hydropersulfide and its related polysulfides are efficient electron donors that interact with quinones. Redox coupling reactions between quinoid electron acceptors and such highly reactive thiols might occur in biological systems.