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Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

ABSTRACT: Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the purpose of conserving persulfides under biologically relevant conditions. The resulting persulfide adducts (R-S-S-Alk) rapidly convert into the corresponding thioethers (R-S-Alk) by donating sulfur to ambient nucleophilic acceptors. In contrast, certain other alkylating agents, in particular monobromobimane and N-t-butyl-iodoacetamide, generate stable alkylated persulfides. We propose that the nature of the alkylating agent determines the ability of the disulfide bond (R-S-S-Alk) to tautomerize into the thiosulfoxide (R-(S=S)-Alk), and/or the ability of nucleophiles to remove the sulfane sulfur atom from the thiosulfoxide.

SUBMITTER: Schilling D

PROVIDER: S-EPMC9401046 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Schilling Danny D Barayeu Uladzimir U Steimbach Raphael R RR Talwar Deepti D Miller Aubry K AK Dick Tobias P TP

Angewandte Chemie (International ed. in English) 20220524 30

Protein persulfides (R-S-SH) have emerged as a common post-translational modification. Detection and quantitation of protein persulfides requires trapping with alkylating agents. Here we show that alkylating agents differ dramatically in their ability to conserve the persulfide's sulfur-sulfur bond for subsequent detection by mass spectrometry. The two alkylating agents most commonly used in cell biology and biochemistry, N-ethylmaleimide and iodoacetamide, are found to be unsuitable for the pur ...[more]

PMID: 35506673

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Project description:ObjectiveTo clarify the effects of smoking on the disposition of two commonly used antipsychotics, olanzapine and clozapine, and to create standards to adjust the doses of these drugs in clinical practice based on the smoking status.DesignA meta-analysis was conducted by searching MEDLINE, Scopus and the Cochrane Library for relevant prospective and retrospective studies.Included studiesWe included the studies that investigated the effects of smoking on the concentration to dose (C/D) ratio of olanzapine or clozapine.Primary outcome measureThe weighted mean difference was calculated using a DerSimonian-Laird random effects model, along with 95% CI.ResultsSeven association studies, comprising 1094 patients (652 smokers and 442 non-smokers) with schizophrenia or other psychiatric disorders, were included in the meta-analysis of olanzapine. The C/D ratio was significantly lower in smokers than in non-smokers (p<0.00001), and the mean difference was -0.75 (ng/mL)/(mg/day) (95% CI -0.89 to -0.61). Therefore, it was estimated that if 10 and 20 mg/day of olanzapine would be administered to smokers, about 7 and 14 mg/day, respectively, should be administered to non-smokers in order to obtain the equivalent olanzapine concentration. Four association studies of clozapine were included in the meta-analysis of clozapine, comprising 196 patients (120 smokers and 76 non-smokers) with schizophrenia or other psychiatric disorders. The C/D ratio was significantly lower in smokers than in non-smokers (p<0.00001), and the mean difference was -1.11 (ng/mL)/(mg/day) (95% CI -1.53 to -0.70). Therefore, it was estimated that if 200 and 400 mg/day of clozapine would be administered to smokers, about 100 and 200 mg/day, respectively, should be administered to non-smokers.ConclusionsWe suggest that the doses of olanzapine and clozapine should be reduced by 30% and 50%, respectively, in non-smokers compared with smokers in order to obtain an equivalent olanzapine or clozapine concentration.

Project description:Hydrogen sulfide signaling involves persulfide formation at specific protein Cys residues. However, overcoming current methodological challenges in persulfide detection and elucidation of Cys regeneration mechanisms from persulfides are prerequisites for constructing a bona fide signaling model. We here establish a novel, highly specific protein persulfide detection protocol, ProPerDP, with which we quantify 1.52 ± 0.6 and 11.6 ± 6.9 μg/mg protein steady-state protein persulfide concentrations in human embryonic kidney 293 (HEK293) cells and mouse liver, respectively. Upon treatment with polysulfides, HEK293 and A549 cells exhibited increased protein persulfidation. Deletion of the sulfide-producing cystathionine-γ-lyase or cystathionine-β-synthase enzymes in yeast diminished protein persulfide levels, thereby corroborating their involvement in protein persulfidation processes. We here establish that thioredoxin (Trx) and glutathione (GSH) systems can independently catalyze reductions of inorganic polysulfides and protein persulfides. Increased endogenous persulfide levels and protein persulfidation following polysulfide treatment in thioredoxin reductase-1 (TrxR1) or thioredoxin-related protein of 14 kDa (TRP14) knockdown HEK293 cells indicated that these enzymes constitute a potent regeneration system of Cys residues from persulfides in a cellular context. Furthermore, TrxR1-deficient cells were less viable upon treatment with toxic amounts of polysulfides compared to control cells. Emphasizing the dominant role of cytosolic disulfide reduction systems in maintaining sulfane sulfur homeostasis in vivo, protein persulfide levels were markedly elevated in mouse livers where hepatocytes lack both TrxR1 and glutathione reductase (TR/GR-null). The different persulfide patterns observed in wild-type, GR-null, and TR/GR-null livers suggest distinct roles for the Trx and GSH systems in regulating subsets of protein persulfides and thereby fine-tuning sulfide signaling pathways.

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Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

Publications

Commonly Used Alkylating Agents Limit Persulfide Detection by Converting Protein Persulfides into Thioethers.

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