Biosynthesis and Reactivity of Cysteine Persulfides in Signaling.
ABSTRACT: Hydrogen sulfide (H2S) elicits pleiotropic physiological effects ranging from modulation of cardiovascular to CNS functions. A dominant method for transmission of sulfide-based signals is via posttranslational modification of reactive cysteine thiols to persulfides. However, the source of the persulfide donor and whether its relationship to H2S is as a product or precursor is controversial. The transsulfuration pathway enzymes can synthesize cysteine persulfide (Cys-SSH) from cystine and H2S from cysteine and/or homocysteine. Recently, Cys-SSH was proposed as the primary product of the transsulfuration pathway with H2S representing a decomposition product of Cys-SSH. Our detailed kinetic analyses demonstrate a robust capacity for Cys-SSH production by the human transsulfuration pathway enzymes, cystathionine beta-synthase and ?-cystathionase (CSE) and for homocysteine persulfide synthesis from homocystine by CSE only. However, in the reducing cytoplasmic milieu where the concentration of reduced thiols is significantly higher than of disulfides, substrate level regulation favors the synthesis of H2S over persulfides. Mathematical modeling at physiologically relevant hepatic substrate concentrations predicts that H2S rather than Cys-SSH is the primary product of the transsulfuration enzymes with CSE being the dominant producer. The half-life of the metastable Cys-SSH product is short and decomposition leads to a mixture of polysulfides (Cys-S-(S)n-S-Cys). These in vitro data, together with the intrinsic reactivity of Cys-SSH for cysteinyl versus sulfur transfer, are consistent with the absence of an observable increase in protein persulfidation in cells in response to exogenous cystine and evidence for the formation of polysulfides under these conditions.
Project description:Hydrogen sulfide has emerged as an important gaseous signaling molecule and a regulator of critical biological processes. However, the physiological significance of hydrogen sulfide metabolites such as persulfides, polysulfides, and other reactive sulfur species (RSS) has only recently been appreciated. Emerging evidence suggests that these RSS molecules may have similar or divergent regulatory roles compared with hydrogen sulfide in various biological activities. However, the chemical nature of persulfides and polysulfides is complex and remains poorly understood within cardiovascular and other pathophysiological conditions. Recent reports suggest that RSS can be produced endogenously, with different forms having unique chemical properties and biological implications involving diverse cellular responses such as protein biosynthesis, cell-cell barrier functions, and mitochondrial bioenergetics. Enzymes of the transsulfuration pathway, CBS (cystathionine beta-synthase) and CSE (cystathionine gamma-lyase), may also produce RSS metabolites besides hydrogen sulfide. Moreover, CARSs (cysteinyl-tRNA synthetase) are also able to generate protein persulfides via cysteine persulfide (CysSSH) incorporation into nascently formed polypeptides suggesting a new biologically relevant amino acid. This brief review discusses the biochemical nature and potential roles of RSS, associated oxidative stress redox signaling, and future research opportunities in cardiovascular disease.
Project description:Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine ?-synthase and cystathionine ?-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 ?M) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H2O2 and a recently described biologically generated electrophile 8-nitroguanosine 3',5'-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.
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 ?g/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-?-lyase or cystathionine-?-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.
Project description:Many physiological functions of hydrogen sulfide (H2S) have been reported in mammalian cells over the last 20 years. These physiological effects have been ascertained through in vitro treatment of cells with Na2S or NaHS, both of which are precursors of H2S. Since H2S exists as HS- in a neutral solution, a disulfide compound such as cystine could react with HS- in culture medium as well as in the cell. This study demonstrated that after the addition of Na2S solution into culture medium, HS- was transiently generated and disappeared immediately through the reaction between HS- and cystine to form cysteine persulfides and polysulfides in the culture medium (bound sulfur mixture: BS-Mix). Furthermore, we found that the addition of Na2S solution resulted in an increase of intracellular cysteine persulfide levels in SH-SY5Y cells. This alteration in intracellular persulfide was also observed in cystine-free medium. Considering this reaction of HS- as a precursor of BS-Mix, we highlighted the cytoprotective effect of Na2S on human neuroblastoma SH-SY5Y cells against methylglyoxal (MG)-induced toxicity. BS-Mix produced with Na2S in cystine-containing medium provided SH-SY5Y cells significant protective effect against MG-induced toxicity. However, the protective effect was attenuated in cystine-free medium. Moreover, we observed that Na2S or BS-Mix activated the Keap1/Nrf2 system and increased glutathione (GSH) levels in the cell. In addition, the activation of Nrf2 is significantly attenuated in cystine-free medium. These results suggested that Na2S protects SH-SY5Y cells from MG cytotoxicity through the activation of Nrf2, mediated by cysteine persulfides and polysulfides that were generated by Na2S addition.
Project description:H2S is an important signalling molecule involved in diverse biological processes. It mediates the formation of cysteine persulfides (R-S-SH), which affect the activity of target proteins. Like thiols, persulfides show reactivity towards electrophiles and behave similarly to other cysteine modifications in a biotin switch assay. In this manuscript, we report on qPerS-SID a mass spectrometry-based method allowing the isolation of persulfide containing peptides in the mammalian proteome. With this method, we demonstrated that H2S donors differ in their efficacy to induce persulfides in HEK293 cells. Furthermore, data analysis revealed that persulfide formation affects all subcellular compartments and various cellular processes. Negatively charged amino acids appeared more frequently adjacent to cysteines forming persulfides. We confirmed our proteomic data using pyruvate kinase M2 as a model protein and showed that several cysteine residues are prone to persulfide formation finally leading to its inactivation. Taken together, the site-specific identification of persulfides on a proteome scale can help to identify target proteins involved in H2S signalling and enlightens the biology of H2S and its releasing agents.
Project description:Mercaptopyruvate sulfurtransferase (MST) is a source of endogenous H2S, a gaseous signaling molecule implicated in a wide range of physiological processes. The contribution of MST versus the other two H2S generators, cystathionine β-synthase and γ-cystathionase, has been difficult to evaluate because many studies on MST have been conducted at high pH and have used varied reaction conditions. In this study, we have expressed, purified, and crystallized human MST in the presence of the substrate 3-mercaptopyruvate (3-MP). The kinetics of H2S production by MST from 3-MP was studied at pH 7.4 in the presence of various physiological persulfide acceptors: cysteine, dihydrolipoic acid, glutathione, homocysteine, and thioredoxin, and in the presence of cyanide. The crystal structure of MST reveals a mixture of the product complex containing pyruvate and an active site cysteine persulfide (Cys(248)-SSH) and a nonproductive intermediate in which 3-MP is covalently linked via a disulfide bond to an active site cysteine. The crystal structure analysis allows us to propose a detailed mechanism for MST in which an Asp-His-Ser catalytic triad is positioned to activate the nucleophilic cysteine residue and participate in general acid-base chemistry, whereas our kinetic analysis indicates that thioredoxin is likely to be the major physiological persulfide acceptor for MST.
Project description:Hydrogen sulfide (H2S) participates in prokaryotic metabolism and is associated with several physiological functions in mammals. H2S reacts with oxidized thiol derivatives (i.e. disulfides and sulfenic acids) and thereby forms persulfides, which are plausible transducers of the H2S-mediated signaling effects. The one-cysteine peroxiredoxin alkyl hydroperoxide reductase E from Mycobacterium tuberculosis (MtAhpE-SH) reacts fast with hydroperoxides, forming a stable sulfenic acid (MtAhpE-SOH), which we chose here as a model to study the interactions between H2S and peroxiredoxins (Prx). MtAhpE-SOH reacted with H2S, forming a persulfide (MtAhpE-SSH) detectable by mass spectrometry. The rate constant for this reaction was (1.4 ± 0.2) × 103 m-1 s-1 (pH 7.4, 25 °C), six times higher than that reported for the reaction with the main low-molecular-weight thiol in M. tuberculosis, mycothiol. H2S was able to complete the catalytic cycle of MtAhpE and, according to kinetic considerations, it could represent an alternative substrate in M. tuberculosis. MtAhpE-SSH reacted 43 times faster than did MtAhpE-SH with the unspecific electrophile 4,4'-dithiodipyridine, a disulfide that exhibits no preferential reactivity with peroxidatic cysteines, but MtAhpE-SSH was less reactive toward specific Prx substrates such as hydrogen peroxide and peroxynitrite. According to molecular dynamics simulations, this loss of specific reactivity could be explained by alterations in the MtAhpE active site. MtAhpE-SSH could transfer its sulfane sulfur to a low-molecular-weight thiol, a process likely facilitated by the low pKa of the leaving thiol MtAhpE-SH, highlighting the possibility that Prx participates in transpersulfidation. The findings of our study contribute to the understanding of persulfide formation and reactivity.
Project description:Substrate ambiguity and relaxed reaction specificity underlie the diversity of reactions catalyzed by the transsulfuration pathway enzymes, cystathionine ?-synthase (CBS) and ?-cystathionase (CSE). These enzymes either commit sulfur metabolism to cysteine synthesis from homocysteine or utilize cysteine and/or homocysteine for synthesis of H2S, a signaling molecule. We demonstrate that a kinetically controlled heme-dependent metabolite switch in CBS regulates these competing reactions where by cystathionine, the product of CBS, inhibits H2S synthesis by the second enzyme, CSE. Under endoplasmic reticulum stress conditions, induction of CSE and up-regulation of the CBS inhibitor, CO, a product of heme oxygenase-1, flip the operating preference of CSE from cystathionine to cysteine, transiently stimulating H2S production. In contrast, genetic deficiency of CBS leads to chronic stimulation of H2S production. This metabolite switch from cystathionine to cysteine and/or homocysteine renders H2S synthesis by CSE responsive to the known modulators of CBS: S-adenosylmethionine, NO, and CO. Used acutely, it regulates H2S synthesis; used chronically, it might contribute to disease pathology.
Project description:Hydrogen sulfide (H2S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or S-sulfhydration) has been proposed as the main pathway for H2S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H2S release suggesting that thioredoxin could be an important regulator of H2S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H2S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role in vivo.
Project description:Sulfane sulfur is common inside cells, playing both regulatory and antioxidant roles. However, there are unresolved issues about its chemistry and biochemistry. We report the discovery that reactive sulfane sulfur such as polysulfides and persulfides could be detected by using resonance synchronous spectroscopy (RS2). With RS2, we showed that inorganic polysulfides at low concentrations were unstable with a half-life about 1?min under physiological conditions due to reacting with glutathione. The protonated form of glutathione persulfide (GSSH) was electrophilic and had RS2 signal. GSS- was nucleophilic, prone to oxidation, but had no RS2 signal. Using this phenomenon, pKa of GSSH was determined as 6.9. GSSH/GSS- was 50-fold more reactive than H2S/HS- towards H2O2 at pH 7.4, supporting reactive sulfane sulfur species like GSSH/GSS- may act as antioxidants inside cells. Further, protein persulfides were shown to be in two forms: at pH 7.4 the deprotonated form (R-SS-) without RS2 signal was not reactive toward sulfite, and the protonated form (R-SSH) in the active site of a rhodanese had RS2 signal and readily reacted with sulfite to produce thiosulfate. These data suggest that RS2 of sulfane sulfur is likely associated with its electrophilicity. Sulfane sulfur showed species-specific RS2 spectra and intensities at physiological pH, which may reveal the relative abundance of a reactive sulfane sulfur species inside cells.