Search

Persulfides and polysulfides, collectively known as the sulfane sulfur pool along with hydrogen sulfide (H2S), play a central role in cellular physiology and disease. Exogenously enhancing these species in cells is an emerging therapeutic paradigm for mitigating oxidative stress and inflammation that are associated with several diseases. In this study, we present a unique approach of using the cell's own enzyme machinery coupled with an array of artificial substrates to enhance the cellular sulfane sulfur pool. We report the synthesis and validation of artificial/unnatural substrates specific for 3-mercaptopyruvate sulfurtransferase (3-MST), an important enzyme that contributes to sulfur trafficking in cells. We demonstrate that these artificial substrates generate persulfides in vitro as well as mediate sulfur transfer to low molecular weight thiols and to cysteine-containing proteins. A nearly 100-fold difference in the rates of H2S production for the various substrates is observed supporting the tunability of persulfide generation by the 3-MST enzyme/artificial substrate system. Next, we show that the substrate 1a permeates cells and is selectively turned over by 3-MST to generate 3-MST-persulfide, which protects against reactive oxygen species-induced lethality. Lastly, in a mouse model, 1a is found to significantly mitigate neuroinflammation in the brain tissue. Together, the approach that we have developed allows for the on-demand generation of persulfides in vitro and in vivo using a range of shelf-stable, artificial substrates of 3-MST, while opening up possibilities of harnessing these molecules for therapeutic applications., A persulfide/hydrogen sulfide generation strategy through artificial substrates for 3-mercaptopyruvate sulfurtransferase (3-MST) is reported, which enhances cellular persulfides, attenuates reactive oxygen species (ROS), and alleviates inflammation.

In cellular signaling, the diverse physiological actions of biological gases, including O2, CO, NO, and H2S, have attracted much interest. Hypoxia-inducible factors (HIFs), including HIF-1 and HIF-2, are transcription factors that respond to reduced intracellular O2 availability. Polysulfides are substances containing varying numbers of sulfur atoms (H2Sn) that are generated endogenously from H2S by 3-mercaptopyruvate sulfurtransferase in the presence of O2, and regulate ion channels, specific tumor suppressors, and protein kinases. However, the effect of polysulfides on HIF activation in hypoxic mammalian cells is largely unknown. Here, we have investigated the effect of polysulfide on cells in vitro. In established cell lines, polysulfide donors reversibly reduced cellular O2 consumption and inhibited hypoxia-induced HIF-1α protein accumulation and the expression of genes downstream of HIFs; however, these effects were not observed in anoxia. In Von Hippel-Lindau tumor suppressor (VHL)- and mitochondria-deficient cells, polysulfides did not affect HIF-1α protein synthesis but destabilized it in a VHL- and mitochondria-dependent manner. For the first time, we show that polysulfides modulate intracellular O2 homeostasis and regulate HIF activation and subsequent hypoxia-induced gene expression in a VHL- and mitochondria-dependent manner.

Graphical abstract, Background Over the last several decades, hydrogen sulfide (H2S) has been found to exert multiple physiological functions in mammal systems. The endogenous production of H2S is primarily mediated by cystathione β-synthase (CBS), cystathione γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST). These enzymes are widely expressed in the liver tissues and regulate hepatic functions by acting on various molecular targets. Aim of Review In the present review, we will highlight the recent advancements in the cellular events triggered by H2S under liver diseases. The therapeutic effects of H2S donors on hepatic diseases will also be discussed. Key Scientific Concepts of Review As a critical regulator of liver functions, H2S is critically involved in the etiology of various liver disorders, such as nonalcoholic steatohepatitis (NASH), hepatic fibrosis, hepatic ischemia/reperfusion (IR) injury, and liver cancer. Targeting H2S-producing enzymes may be a promising strategy for managing hepatic disorders.

3-Mercaptopyruvate sulfurtransferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate to generate an enzyme-bound hydropersulfide. Subsequently, MPST transfers the persulfide’s outer sulfur atom to proteins or small molecule acceptors. MPST activity is known to be involved in hydrogen sulfide generation, tRNA thiolation, protein urmylation and cyanide detoxification. Tissue-specific changes in MPST expression correlate with ageing and the development of metabolic disease. Deletion and overexpression experiments suggest that MPST contributes to oxidative stress resistance, mitochondrial respiratory function and the regulation of fatty acid metabolism. However, the role and regulation of MPST in the larger physiological context remain to be understood.

The amino acid biosynthetic pathway of invasive pathogenic fungi has been studied as a potential antifungal drug target. Studies of the disruption of genes involved in amino acid biosynthesis have demonstrated the importance of this pathway in the virulence of Cryptococcus neoformans. Here, we identified the MET5 (CNL05500) and MET10 (CNG03990) genes in this pathway, both encoding sulfite reductase, which catalyzes the reduction of sulfite to sulfide. The MET14 (CNE03880) gene was also identified, which is responsible for the conversion of sulfate to sulfite. The use of cysteine as a sulfur source led to the production of methionine via hydrogen sulfide synthesis mediated by CYS4 (CNA06170), CYS3 (CNN01730), and MST1 (CND03690). MST1 exhibited high homology with the TUM1 gene of Saccharomyces cerevisiae, which has functional similarity with the 3-mercaptopyruvate sulfurtransferase (3-MST) gene in humans. Although the hypothesis that hydrogen sulfide is produced from cysteine via CYS4, CYS3, and MST1 warrants further study, the new insight into the metabolic pathway of sulfur-containing amino acids in C. neoformans provided here indicates the usefulness of this system in the development of screening tools for antifungal drug agents.

The aim of the study was to determine the cytotoxic concentrations and incubation times of the commonly used dental adhesive system OptiBond Solo Plus in its non-polymerized form, and to test how it relates to oxidative stress by determining the reduced and oxidized glutathione (GSH and GSSG) levels as well as to study its influence on cell number and the expression of selected sulfur enzymes, with particular emphasis on cystathionine γ-lyase (CTH) and 3-mercaptopyruvate sulfurtransferase (MPST). All investigations were conducted on an in vitro model of human fibroblast cell line Hs27. Changes in cellular plasma membrane integrity were measured by the LDH test. The expression levels were determined by RT-PCR and Western blot protocols. Changes in cell number were visualized using crystal violet staining. The RP-HPLC method was used to determine the GSH and GSSG levels. Reduced cell number was shown for all tested concentrations and times. Changes in the expression on the mRNA and protein level were demonstrated for CTH and MPST enzymes upon exposure to the tested range of OptiBond concentrations. Levels of low-molecular sulfur compounds of reduced and oxidized glutathione were also established. Cytotoxic effect of OptiBond Solo Plus may be connected with the changes of MPST and CTH sulfur enzymes in the human fibroblast Hs27 cell line. The elevated levels of these enzymes could possibly show the antioxidant response to this dental adhesive system. OptiBond Solo Plus in vitro results should be taken into consideration for further in vivo tests.

Hydrogen sulfide (H(2)S) together with polysulfides (H(2)S(n), n > 2) are signalling molecules like NO with various physiological roles including regulation of neuronal transmission, vascular tone, inflammation and oxygen sensing. H(2)S and H(2)S(n) diffuse to the target proteins for S‐sulfurating their cysteine residues that induces the conformational changes to alter the activity. On the other hand, 3‐mercaptopyruvate sulfurtransferase transfers sulfur from a substrate 3‐mercaptopyruvate to the cysteine residues of acceptor proteins. A similar mechanism has also been identified in S‐nitrosylation. S‐sulfuration and S‐nitrosylation by enzymes proceed only inside the cell, while reactions induced by H(2)S, H(2)S(n) and NO even extend to the surrounding cells. Disturbance of signalling by these molecules as well as S‐sulfuration and S‐nitrosylation causes many nervous system diseases. This review focuses on the signalling by H(2)S and H(2)S(n) with S‐sulfuration comparing to that of NO with S‐nitrosylation and discusses on their roles in physiology and pathophysiology.

Background: 3-Mercaptopyruvate sulfurtransferase (3-MST) is a multifunctional, mitochondrial and cytoplasmic sulphurtransferase that catalyses the detoxification of cyanide to a less toxic thiocyanate. Limicolaria flammea feeds majorly on green leaves, plants and other cyanide containing foods. Methods: 3-MST from the hemolymph of Limicolaria flammae was purified by 70 % ammonium sulphate precipitation and ion exchange chromatography. The purified enzyme was characterized at different levels such as optimal activity, inhibitors, substrate preference, thermal stability and analysis of ki-netic parameters. Results: 3-MST from the hemolymph of Limicolaria flammae had a yield of 0.75 % with specific activity of 0.42 μ/mg/ml. The Km values for the substrates; KCN and 2-Mercaptoethanol were 1.09 and 2.83 mM, while the Vmax values were 3.08 μml/mol/min and 6.17 μml/mol/min respectively. The optimum pH and temperature of the enzyme were 5.0 and 60° C respectively. The metals (Al3+, Ca2+, and K+) demonstrated inhibitory activity in a concentration dependent manner. The substrate specificity study showed that sodium sulphite, ammonium per sulphate and ammonium sulphite showed enzymatic interference. Conclusion: This study affirmed the presence of 3-MST activity in the hemolymph of Limicolaria flammea, an indication that the enzyme possesses functional cyanide detoxification mechanism necessary for the survival of the animal in the environment.

The lack of the N-alpha-glucosaminidase (Naglu) is responsible for the incidence of a rare disease - mucopolysaccharidosis, type IIIB (MPS IIIB). To date, studies have been conducted based on cells derived from patients suffering from MPS or using in vivo MPS mouse models. These limitations have allowed for defining our research goal - to create and characterize the first in vitro murine cellular MPS IIIB model. In the current work we present a new, stable cell line with confirmed accumulation of glycosaminoglycans. The line stability was achieved by immortalization using a lentivirus carrying the T-antigens of SV40. The Naglu−/− cells were confirmed to produce no Naglu enzyme. To confirm the proper functioning of the in vitro MPS IIIB model, we determined the activity and expression of cystathionine γ-lyase, rhodanese and 3-mercaptopyruvate sulfurtransferase, as well as the level of low molecular-weight thiols (reduced and oxidized glutathione, cysteine and cystine). The results were referred to our earlier findings originating from the studies on the tissues of the Naglu−/− mice that were used to create the lines. The results obtained in the Naglu−/− cells were in accordance with the results found in the mouse model of MPS IIIB. It suggests that the presented murine Naglu−/− cell lines might be a convenient in vitro model of MPS IIIB.

The literature data and results of our research team concerning the physiological and pathological effects of hydrogen sulfide, a gas transmitter that has recently attracted significant and increasing attention of neurophysiologists, are analyzed. Hydrogen sulfide is a gaseous signaling molecule, the effects of which were discovered later than those of NO and CO; H2S was found to play great pathophysiological roles in various diseases. This compound is synthesized in the body by enzymatic and non-enzymatic pathways from cysteine, using the pyridoxal 5’-phosphate-dependent enzymes cystathionine β-synthase (CBS) or cystathionine γ-lyase (CSE); 3-mercaptopyruvate sulfurtransferase (3MST) plays a considerable role in H2S catabolism. Abnormal deviations in H2S metabolism are important factors involved in the development of a number of dangerous neurological pathologies, in particular of Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and Down’s syndrome (DS). This compound was also demonstrated to be involved in various biological and pathophysiological processes in the brain resulting from traumatic brain injury (TBI), stroke, oxidative stress, and cerebral edema. In the case of traumatic injury in the trout brain, the activation of CBS expression in the radial glia phenotypes occurs in the aNSC.

Made available in DSpace on 2020-12-12T02:04:13Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The pathogen Xylella fastidiosa belongs to the Xanthomonadaceae family, a large group of Gram-negative bacteria that cause diseases in many economically important crops. A predicted gene, annotated as glutaredoxin-like protein (glp), was found to be highly conserved among the genomes of different genera within this family and highly expressed in X. fastidiosa. Analysis of the GLP protein sequences revealed three protein domains: one similar to monothiol glutaredoxins (Grx), an Fe-S cluster and a thiosulfate sulfurtransferase/rhodanese domain (Tst/Rho), which is generally involved in sulfur metabolism and cyanide detoxification. To characterize the biochemical properties of GLP, we expressed and purified the X. fastidiosa recombinant GLP enzyme. Grx activity and Fe-S cluster formation were not observed, while an evaluation of Tst/Rho enzymatic activity revealed that GLP can detoxify cyanide and transfer inorganic sulfur to acceptor molecules in vitro. The biological activity of GLP relies on the cysteine residues in the Grx and Tst/Rho domains (Cys33 and Cys266, respectively), and structural analysis showed that GLP and GLPC266S were able to form high molecular weight oligomers (> 600 kDa), while replacement of Cys33 with Ser destabilized the quaternary structure. In vivo heterologous enzyme expression experiments in Escherichia coli revealed that GLP can protect bacteria against high concentrations of cyanide and hydrogen peroxide. Finally, phylogenetic analysis showed that homologous glp genes are distributed across Gram-negative bacterial families with conservation of the N- to C-domain order. However, no eukaryotic organism contains this enzyme. Altogether, these results suggest that GLP is an important enzyme with cyanide-decomposing and sulfurtransferase functions in bacteria, whose presence in eukaryotes we could not observe, representing a promising biological target for new pharmaceuticals. Laboratory of Structural Molecular Biology Biosciences Institute UNESP - São Paulo State University Laboratory of Biomarkers of Aquatic Contamination and Immunochemistry Department of Biochemistry Federal University of Santa Catarina Laboratory of Functional and Structural Characterization of Toxins of Venomous and Poisonous Animals Biosciences Institute UNESP - São Paulo State University Molecular Ecology Laboratory Biosciences Institute UNESP - São Paulo State University Laboratory of Structural Molecular Biology Biosciences Institute UNESP - São Paulo State University Laboratory of Functional and Structural Characterization of Toxins of Venomous and Poisonous Animals Biosciences Institute UNESP - São Paulo State University Molecular Ecology Laboratory Biosciences Institute UNESP - São Paulo State University FAPESP: 10/00172-8 FAPESP: 10/16827-3 FAPESP: 2007/50930-3 FAPESP: 2011/13500-6 FAPESP: 2013/16192-6 FAPESP: 2017/19942-7 FAPESP: 2017/20291-0

Transfer RNA (tRNA) is an adaptor molecule indispensable for assigning amino acids to codons on mRNA during protein synthesis. 2-thiouridine (s2U) derivatives in the anticodons (position 34) of tRNAs for glutamate, glutamine, and lysine are post-transcriptional modifications essential for precise and efficient codon recognition in all organisms. s2U34 is introduced either by (i) bacterial MnmA/eukaryote mitochondrial Mtu1 or (ii) eukaryote cytosolic Ncs6/archaeal NcsA, and the latter enzymes possess iron-sulfur (Fe–S) cluster. Here, we report the identification of novel-type MnmA homologs containing three conserved Cys residues, which could support Fe–S cluster binding and catalysis, in a broad range of bacteria, including thermophiles, Cyanobacteria, Mycobacteria, Actinomyces, Clostridium, and Helicobacter. Using EPR spectroscopy, we revealed that Thermus thermophilus MnmA (TtMnmA) contains an oxygen-sensitive [4Fe–4S]-type cluster. Efficient in vitro formation of s2U34 in tRNALys and tRNAGln by holo-TtMnmA occurred only under anaerobic conditions. Mutational analysis of TtMnmA suggested that the Fe–S cluster is coordinated by the three conserved Cys residues (Cys105, Cys108, and Cys200), and is essential for its activity. Evolutionary scenarios for the sulfurtransferases, including the Fe–S cluster containing Ncs6/NcsA s2U thiouridylases and several distantly related sulfurtransferases, are proposed.

Mitochondria are the key organelles of Fe–S cluster synthesis. They contain the enzyme cysteine desulfurase, a scaffold protein, iron and electron donors, and specific chaperons all required for the formation of Fe–S clusters. The newly formed cluster can be utilized by mitochondrial Fe–S protein synthesis or undergo further transformation. Mitochondrial Fe–S cluster biogenesis components are required in the cytosolic iron–sulfur cluster assembly machinery for cytosolic and nuclear cluster supplies. Clusters that are the key components of Fe–S proteins are vulnerable and prone to degradation whenever exposed to oxidative stress. However, once degraded, the Fe–S cluster can be resynthesized or repaired. It has been proposed that sulfurtransferases, rhodanese, and 3-mercaptopyruvate sulfurtransferase, responsible for sulfur transfer from donor to nucleophilic acceptor, are involved in the Fe–S cluster formation, maturation, or reconstitution. In the present paper, we attempt to sum up our knowledge on the involvement of sulfurtransferases not only in sulfur administration but also in the Fe–S cluster formation in mammals and yeasts, and on reconstitution-damaged cluster or restoration of enzyme’s attenuated activity.

BACKGROUND In the present work, we investigated the cardioprotective potential of pyridoxal-5-phosphate (PLP) in old rats as a cofactor of enzymes that synthesize hydrogen sulphide (H2 S). MATERIALS AND METHODS PLP was administered per os in a dose of 0.7 mg per kg daily for 2 weeks. Rats were divided into three groups (adult, old and old +PLP) of 20 animals. The cardiac mRNA levels of genes encoding H2 S-synthesizing enzymes cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), uncoupling proteins (UCP3), subunits of ATP-sensitive potassium (KATP ) channels were determined using real-time polymerase chain reaction analysis. We also studied the effect of PLP-administration on the content of H2 S, oxidative stress, the activities of inducible and constitutive NO-synthase (iNOS, cNOS), arginase and nitrate reductase in the heart homogenates as well as cardiac resistance to ischemia-reperfusion in Langendorff-isolated heart model. RESULTS It was shown that PLP restored mRNA levels of CSE, 3-MST and UCP3 genes, and H2 S content and also significantly increased the expression of SUR2 and Kir6.1 (2.2 and 3.3 times, respectively) in the heart of old rats. PLP significantly reduced the formation of superoxide, malondialdehyde, diene conjugates as well as the activity of iNOS and arginase. PLP significantly increased constitutive synthesis of NO and prevented reperfusion disturbances of the heart function after ischemia. CONCLUSIONS Thus, PLP-administration in old rats was associated with up-expression of CSE, 3-MST, UCP3 and SUR2 and Kir6.1 subunits of KATP channels, and also increased cNOS activity and reduced oxidative stress and prevented reperfusion dysfunction of the heart in ischemia-reperfusion.

Sulfides and persulfides/polysulfides (R-Sn-Rʹ, n > 2; R-Sn-H, n > 1) are endogenously produced metabolites that are abundant in mammalian and human cells and tissues. The most typical persulfides that are widely distributed among different organisms include various reactive persulfides—low-molecular-weight thiol compounds such as cysteine hydropersulfide, glutathione hydropersulfide, and glutathione trisulfide as well as protein-bound thiols. These species are generally more redox-active than are other simple thiols and disulfides. Although hydrogen sulfide (H2S) has been suggested for years to be a small signaling molecule, it is intimately linked biochemically to persulfides and may actually be more relevant as a marker of functionally active persulfides. Reactive persulfides can act as powerful antioxidants and redox signaling species and are involved in energy metabolism. Recent evidence revealed that cysteinyl-tRNA synthetases (CARSs) act as the principal cysteine persulfide synthases in mammals and contribute significantly to endogenous persulfide/polysulfide production, in addition to being associated with a battery of enzymes including cystathionine β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, which have been described as H2S-producing enzymes. The reactive sulfur metabolites including persulfides/polysulfides derived from CARS2, a mitochondrial isoform of CARS, also mediate not only mitochondrial biogenesis and bioenergetics but also anti-inflammatory and immunomodulatory functions. The physiological roles of persulfides, their biosynthetic pathways, and their pathophysiology in various diseases are not fully understood, however. Developing basic and high precision techniques and methods for the detection, characterization, and quantitation of sulfides and persulfides is therefore of great importance so as to thoroughly understand and clarify the exact functions and roles of these species in cells and in vivo.

The knowledge on sulfur incorporation mechanism involved in sulfur-containing molecule biosynthesis remains limited. Chuangxinmycin is a sulfur-containing antibiotic with a unique thiopyrano[4,3,2-cd]indole (TPI) skeleton and selective inhibitory activity against bacterial tryptophanyl-tRNA synthetase. Despite the previously reported biosynthetic gene clusters and the recent functional characterization of a P450 enzyme responsible for C-S bond formation, the enzymatic mechanism for sulfur incorporation remains unknown. Here, we resolve this central biosynthetic problem by in vitro biochemical characterization of the key enzymes and reconstitute the TPI skeleton in a one-pot enzymatic reaction. We reveal that the JAMM/MPN+ protein Cxm3 functions as a deubiquitinase-like sulfurtransferase to catalyze a non-classical sulfur-transfer reaction by interacting with the ubiquitin-like sulfur carrier protein Cxm4GG. This finding adds a new mechanism for sulfurtransferase in nature.

Hydrogen sulfide (H2S) has long been recognized as a putrid, toxic gas. However, as a result of intensive biochemical research in the past two decades, H2S is now considered to be the third gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO) in mammalian systems. H2S-producing enzymes are expressed in all organs, playing an important role in their physiology. In the kidney, H2S is a critical regulator of vascular and cellular function, although the mechanisms that affect (sub)cellular levels of H2S are not precisely understood. H2S modulates systemic and renal blood flow, glomerular filtration rate and the renin-angiotensin axis through direct inhibition of nitric oxide synthesis. Further, H2S affects cellular function by modulating protein activity via post-translational protein modification: a process termed persulfidation. Persulfidation modulates protein activity, protein localization and protein-protein interactions. Additionally, acute kidney injury (AKI) due to mitochondrial dysfunction, which occurs during hypoxia or ischemia-reperfusion (IR), is attenuated by H2S. H2S enhances ATP production, prevents damage due to free radicals and regulates endoplasmic reticulum stress during IR. In this review, we discuss current insights in the (sub)cellular regulation of H2S anabolism, retention and catabolism, with relevance to spatiotemporal regulation of renal H2S levels. Together, H2S is a versatile gasotransmitter with pleiotropic effects on renal function and offers protection against AKI. Unraveling the mechanisms that modulate (sub)cellular signaling of H2S not only expands fundamental insight in the regulation of functional effects mediated by H2S, but can also provide novel therapeutic targets to prevent kidney injury due to hypoxic or ischemic injury.

Hydrogen sulfide (H2S) has long been recognized as a putrid, toxic gas. However, as a result of intensive biochemical research in the past two decades, H2S is now considered to be the third gasotransmitter alongside nitric oxide (NO) and carbon monoxide (CO) in mammalian systems. H2S-producing enzymes are expressed in all organs, playing an important role in their physiology. In the kidney, H2S is a critical regulator of vascular and cellular function, although the mechanisms that affect (sub)cellular levels of H2S are not precisely understood. H2S modulates systemic and renal blood flow, glomerular filtration rate and the renin-angiotensin axis through direct inhibition of nitric oxide synthesis. Further, H2S affects cellular function by modulating protein activity via post-translational protein modification: a process termed persulfidation. Persulfidation modulates protein activity, protein localization and protein-protein interactions. Additionally, acute kidney injury (AKI) due to mitochondrial dysfunction, which occurs during hypoxia or ischemia-reperfusion (IR), is attenuated by H2S. H2S enhances ATP production, prevents damage due to free radicals and regulates endoplasmic reticulum stress during IR. In this review, we discuss current insights in the (sub)cellular regulation of H2S anabolism, retention and catabolism, with relevance to spatiotemporal regulation of renal H2S levels. Together, H2S is a versatile gasotransmitter with pleiotropic effects on renal function and offers protection against AKI. Unraveling the mechanisms that modulate (sub)cellular signaling of H2S not only expands fundamental insight in the regulation of functional effects mediated by H2S, but can also provide novel therapeutic targets to prevent kidney injury due to hypoxic or ischemic injury.

The authors have requested that this preprint be removed from Research Square.

Since the first description of hydrogen sulfide (H2S) as a toxic gas in 1713 by Bernardino Ramazzini, most studies on H2S have concentrated on its toxicity. In 1989, Warenycia et al. demonstrated the existence of endogenous H2S in the brain, suggesting that H2S may have physiological roles. In 1996, we demonstrated that hydrogen sulfide (H2S) is a potential signaling molecule, which can be produced by cystathionine β-synthase (CBS) to modify neurotransmission in the brain. Subsequently, we showed that H2S relaxes vascular smooth muscle in synergy with nitric oxide (NO) and that cystathionine γ-lyase (CSE) is another producing enzyme. This study also opened up a new research area of a crosstalk between H2S and NO. The cytoprotective effect, anti-inflammatory activity, energy formation, and oxygen sensing by H2S have been subsequently demonstrated. Two additional pathways for the production of H2S with 3-mercaptopyruvate sulfurtransferase (3MST) from l- and d-cysteine have been identified. We also discovered that hydrogen polysulfides (H2Sn, n ≥ 2) are potential signaling molecules produced by 3MST. H2Sn regulate the activity of ion channels and enzymes, as well as even the growth of tumors. S-Sulfuration (S-sulfhydration) proposed by Snyder is the main mechanism for H2S/H2Sn underlying regulation of the activity of target proteins. This mini review focuses on the key findings on H2S/H2Sn signaling during the first 25 years.

Hypertension and age are key risk factors for cardiovascular morbidity and mortality. Hydrogen sulfide (H2S), a gaseous transmitter, contributes significantly to regulating arterial blood pressure and aging processes. This study evaluated the effects of hypertension and aging on the hepatic metabolism of sulfur-containing compounds, the activity of the enzymes involved in sulfur homeostasis, and the liver’s ability to generate H2S. Livers isolated from 16- and 60-week-old normotensive Wistar Kyoto rats (WKY) and Spontaneously Hypertensive Rats (SHR) were used to evaluate gene expression using RT-PCR, and the activity of enzymes participating in H2S metabolism, including thiosulfate sulfurtransferase (rhodanese, TST), cystathionine gamma-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (MPST). The levels of cysteine, cystine, reduced and oxidized glutathione were measured using RP-HPLC. SHR livers from both age groups showed a higher capacity to generate H2S than livers from WKY. The gene expression and activity of enzymes involved in sulfur metabolism differed between WKY and SHR, and between the age groups. For example, 16-week-old SHR had significantly higher activity of TST than 16-week-old WKY. Furthermore, differences between younger and older WKY rats in the expression and/or activity of TST and MPST were present. In conclusion, our study shows that arterial hypertension and aging affect hepatic sulfur metabolism and H2S production in rats. These findings pave the way for interventional studies evaluating a potential causal relation between liver sulfur metabolism, hypertension and aging.

The enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is one of the more recently identified mammalian sources of H2S. A recent study identified several novel 3-MST inhibitors with micromolar potency. Among those, (2-[(4-hydroxy-6-methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one) or HMPSNE was found to be the most potent and selective. We now took the central core of this compound and modified the pyrimidone and the arylketone sides independently. A 63-compound library was synthesized; compounds were tested for H2S generation from recombinant 3-MST in vitro. Active compounds were subsequently tested to elucidate their potency and selectivity. Computer modeling studies have delineated some of the key structural features necessary for binding to the 3-MST's active site. Six novel 3-MST inhibitors were tested in cell-based assays: they exerted inhibitory effects in murine MC38 and CT26 colon cancer cell proliferation; the antiproliferative effect of the compound with the highest potency and best cell-based activity (1b) was also confirmed on the growth of MC38 tumors in mice.

While chemical protein synthesis (CPS) has granted access to challenging proteins, synthesis of longer proteins is often limited by low abundance or non-strategic placement of cysteine (Cys) residues, essential for native chemical ligations (NCL), as well as multiple purification and isolation steps. Selective deselenization and one-pot CPS serve as key technologies to circumvent these issues. Herein, we describe the one-pot total synthesis of human thiosulfate: glutathione sulfurtransferase (TSTD1), a 115-residue protein with a single Cys residue at its active site, and its seleno-analogue. WT-TSTD1 was synthesized in a C-to-N synthetic approach employing multiple NCL reactions, Cu(II)-mediated deprotection of selenazolidine (Sez), and chemoselective deselenization, all in one-pot. In addition, the protein’s seleno analogue (Se-TSTD1), in which the active site Cys is replaced with selenocysteine, was synthesized with a kinetically controlled ligation in a one-pot, N-to-C synthetic approach. TSTD1’s one-pot synthesis was made possible by the newly reported, rapid, and facile copper-mediated selenazolidine deprotection that can be accomplished in one minute. Finally, catalytic activity of the two proteins indicated that Se-TSTD1 possessed only four-fold lower activity than WT-TSTD1 as a thiosulfate: glutathione sulfurtransferase, suggesting that selenoproteins can have physiologically comparable sulfutransferase activity as their cysteine counterparts.

Prokaryotes and eukaryotes alike endogenously generate the gaseous molecule hydrogen sulfide (H2S). Bacterial H2S acts as a cytoprotectant against antibiotics-induced stress and promotes redox homeostasis. In E. coli, endogenous H2S production is primarily dependent on 3-mercaptopyruvate sulfurtransferase (3MST), encoded by mstA. Here, we show that cells lacking 3MST acquire a phenotypic suppressor mutation resulting in compensatory H2S production and tolerance to antibiotics and oxidative stress. Using whole genome sequencing, we identified a non-synonymous mutation within an uncharacterized LacI-type transcription factor, ycjW. We then mapped regulatory targets of YcjW and discovered it controls the expression of carbohydrate metabolic genes and thiosulfate sulfurtransferase PspE. Induction of pspE expression in the suppressor strain provides an alternative mechanism for H2S biosynthesis. Our results reveal a complex interaction between carbohydrate metabolism and H2S production in bacteria and the role, a hitherto uncharacterized transcription factor, YcjW, plays in linking the two., Hydrogen sulfide (H2S) production in Escherichia coli is controlled by the sulfurtransferase 3MST. Here, the authors describe an alternative mechanism for H2S biosynthesis via activation of the thiosulfate sulfurtransferase PspE, a process mediated by the transcription factor YcjW.

Hydrogen sulfide (H2S) is a signaling molecule used to modify neuronal transmission, regulate vascular tone, protect tissues from oxidative stress, sense oxygen, and generate ATP. Hydrogen polysulfides (H2Sn) have recently been identified as signaling molecules that mediate the activation of ion channels, regulation of tumor growth, and the transcriptional regulation of oxidative stress; some of which were previously ascribed to H2S. Cystathionine β-synthetase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are known as H2S-producing enzymes. 3MST also produces H2Sn and other persulfurated molecules such as cysteine persulfide, glutathione persulfide, and persulfurated proteins. The chemical interaction of H2S and nitric oxide (NO) also produces H2Sn, which may be the mechanism underlying the synergistic effect of H2S and NO that was initially reported on vascular relaxation. H2Sn and other persulfurated molecules elicit their effect via S-sulfuration (S-sulfhydration) of specific cysteine residues of the target proteins. This review article focuses on the production and roles of H2Sn as well as H2S in the central nervous system.

Hydrogen sulfide (H2S) has been reported to play an important role in biological systems. More recently, sulfane sulfur (sulfur with 0 or -1 charge) molecules have been also reported to be involved in various biological phenomena such as regulation of redox signaling and antioxidant functions. Fluorescent probes are one of the important chemical tools because it is easy to use and enable the real-time detection of the target molecules in living cells and tissues. We have successfully developed a highly selective H2S-detecting fluorescent probe, HSip-1. HSip-1 has been designed on the basis of the facts that the macrocyclic polyamine ligands form a stable complex with Cu2+, and Cu2+ also reacts with H2S and make a stable CuS complex. SSip-1 is a fluorescent probe for detecting sulfane sulfur and this fluorescent probe is designed on the basis of the unique feature of sulfane sulfur to bind reversibly to other sulfur atoms and the intramolecular spirocyclization reaction of xanthene dyes. SSip-1 is a highly selective fluorescent probe and can detect sulfane sulfur reversibly. Both HSip-1 and SSip-1 were able to be used for the live-cell fluorescence imaging. Further, we applied HSip-1 to the high-throughput screening (HTS) for the inhibitors of 3-mercaptopyruvate sulfurtransferase (3MST), one of the reactive sulfur species (RSS)-generating enzymes. We successfully found new 3MST inhibitors by screening of 174,118 compounds. We expect that these fluorescent probes and inhibitors would be useful to elucidate new functions of RSS and RSS-generating enzymes.

Many questions regarding proteins involved in microbial sulfur metabolism remain unsolved. For sulfur respiration at low pH, the terminal electron acceptor is still unclear. Desulfurella amilsii is a sulfur-reducing bacterium that respires elemental sulfur (S0 ) or thiosulfate, and grows by S0 disproportionation. Due to its versatility, comparative studies on D. amilsii may shed light on microbial sulfur metabolism. Requirement of physical contact between cells and S0 was analyzed. Sulfide production decreased by around 50% when S0 was trapped in dialysis membranes, suggesting that contact between cells and S0 is beneficial, but not strictly needed. Proteome analysis was performed under the aforementioned conditions. A Mo-oxidoreductase suggested from genome analysis to act as sulfur reductase was not detected in any growth condition. Thiosulfate and sulfite reductases showed increased abundance in thiosulfate-reducing cultures, while rhodanese-like sulfurtransferases were highly abundant in all conditions. DsrE and DsrL were abundantly detected during thiosulfate reduction, suggesting a modified mechanism of sulfite reduction. Proteogenomics suggest a different disproportionation pathway from what has been reported. This work points to an important role of rhodaneses in sulfur processes and these proteins should be considered in searches for sulfur metabolism in broader fields like meta-omics.

Mucoepidermoid carcinoma (MEC) is the most common malignant epithelial neoplasm of the salivary glands. MECs of the mouth floor are rare, with only a few cases reported. Here we report a MEC of the mouth floor in a 55-year-old woman. Since several studies have shown that hydrogen sulfide (H2S)-synthesizing enzymes are often increased in malignant tumors compared to benign counterpart tissues, we used western blotting to compare the protein levels of cystathionine-β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3-MST) in a mouth floor MEC to adjacent benign oral mucosae. We also used high-performance liquid chromatography to quantify possible differences in tissue sulfur fraction concentrations between the two biopsy types. Last, we used western blotting to examine nicotinamide phosphoribosyl transferase (Nampt), mitoNEET, and phospho-ser727-Stat3 levels in the biopsies. We found that all the proteins and phospho-ser727-Stat3 are increased in the MEC compared to benign mucosae. Interestingly, free H2S levels, acid-labile, and the sulfane sulfur factions were essentially the same between the MEC and benign tissue. Although limited to a single and unusual tumor type, to our knowledge this is only the third time H2S concentrations were directly quantified inside a human tumor. Last, our results replicate those of two previous studies where the H2S-synthesizing enzymes are increased in a malignant tumor, while free H2S is either not increased or only slightly increased, suggesting that malignant tumors rapidly metabolize H2S as part of tumor maintenance and growth.

Fluorescence lifetime and anisotropy has become a prevalent tool to detect the structure change and motility property of proteins. YgaP is the only membrane-integrated rhodanese in E. coli. The sulfur transfer process has been characterized by various studies. However, the mechanism of the outward transportation of SCN − remains unclear. In this work, we examined the fluorescence lifetime and anisotropy of site-specific incorporated unnatural amino acid 7−HC to study the conformational change of YgaP upon SCN − binding. We also compared the fluorescence changes between detergent-wrapped environment in DPC and intact native membrane environment in SMA. Our results suggested the presence of at least two different conformations in YgaP protein. Both the residues in the middle of TMH2 and the residues near extracellular side play important roles in the binding and/or output of SCN − . SMA is a good material to reflect the in situ conformation changes of protein than micelles.

Genetic deletion of 3-mercaptopyruvate sulfurtransferase (MST) is known to result in hypertension and cardiac hypertrophy in older mice, and is associated with increased anxiety-like behaviors. Endogenous hydrogen sulfide (H2S) produced by MST in the mitochondria is also known to be involved in physiological and cellular bioenergetics, and its dysfunction associated with depressive behavior and increased cardiovascular morbidity. Interestingly, early life stress has been shown to lead to a significant loss of cystathionine-γ-lyase (CSE) and oxytocin receptor (OTR) expression in the heart. Thus, we were interested in testing the hypothesis of whether genetic MST mutation (ΔMST) would affect cardiac CSE and OTR expression and affect the mitochondrial respiration in a clinically relevant, resuscitated, mouse model of trauma and hemorrhagic shock. In ΔMST mice, we found a reduction of CSE and OTR in both the naive as well as injured state, in contrast to the wild type (wt) controls. Interestingly, the ΔMST showed a different complex IV response to injury than the wt controls, although our claims are based on the non-demonstrated assumption that naive wt and naive ΔMST mice have comparable complex IV activity. Finally, hemorrhagic shock led to a reduction of CSE and OTR, confirming previous results in the injured mouse heart. To date, the exact mechanisms of the cardiac interaction between H2S and OT are not clear, but they point the way to potential cardioprotective therapies., publishedVersion

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form.

Hydrogen sulfide (H2S) has a long history as toxic gas and environmental hazard; inhibition of cytochrome c oxidase (mitochondrial Complex IV) is viewed as a primary mode of its cytotoxic action. However, studies conducted over the last two decades unveiled multiple biological regulatory roles of H2S as an endogenously produced mammalian gaseous transmitter. Cystathionine -lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) are currently viewed as the principal mammalian H2S-generating enzymes. In contrast to its inhibitory (toxicological) mitochondrial effects, at lower (physiological) concentrations, H2S serves as a stimulator of electron transport in mammalian mitochondria, by acting as an electron donor—with sulfide:quinone oxidoreductase (SQR) being the immediate electron acceptor. The mitochondrial roles of H2S are significant in various cancer cells, many of which exhibit high expression and partial mitochondrial localization of various H2S producing enzymes. In addition to the stimulation of mitochondrial ATP production, the roles of endogenous H2S in cancer cells include the maintenance of mitochondrial organization (protection against mitochondrial fission) and the maintenance of mitochondrial DNA repair (via the stimulation of the assembly of mitochondrial DNA repair complexes). The current article overviews the state-of-the-art knowledge regarding the mitochondrial functions of endogenously produced H2S in cancer cells.