Biophysical characterization of hydrogen sulfide: A fundamental exploration in understanding significance in cell signaling.

Hydrogen sulfide (H2S) has emerged as a significant signaling molecule involved in various physiological processes, including vasodilation, neurotransmission, and cytoprotection. Its interactions with biomolecules are critical to understand its roles in health and disease. Recent advances in biophysical characterization techniques have shed light on the complex interactions of H2S with proteins, nucleic acids, and lipids. Proteins are primary targets for H2S, which can modify cysteine residues through S-sulfhydration, impacting protein function and signaling pathways. Advanced spectroscopic techniques, such as mass spectrometry and NMR, have enabled the identification of specific sulfhydrated sites and provided insights into the structural and functional consequences of these modifications. Nucleic acids also interact with H2S, although this area is less explored compared to proteins. Recent studies have demonstrated that H2S can induce modifications in nucleic acids, affecting gene expression and stability. Techniques like gel electrophoresis and fluorescence spectroscopy have been utilized to investigate these interactions, revealing that H2S can protect DNA from oxidative damage and modulate RNA stability and function. Lipids, being integral components of cell membranes, interact with H2S, influencing membrane fluidity and signaling. Biophysical techniques such as electron paramagnetic resonance (EPR) and fluorescence microscopy have elucidated the effects of H2S on lipid membranes. These studies have shown that H2S can alter lipid packing and dynamics, which may impact membrane-associated signaling pathways and cellular responses to stress. In the current work we have integrated this with key scientific explainations to provide a comprehensive review.

Neuroprotective signaling by hydrogen sulfide and its dysregulation in Alzheimer's disease.

The ancient messenger molecule hydrogen sulfide (H2S) modulates myriad signaling cascades and has been conserved across evolutionary boundaries. Although traditionally known as an environmental toxin, H2S is also synthesized endogenously to exert modulatory and homeostatic effects in a broad array of physiologic functions. Notably, H2S levels are tightly physiologically regulated, as both its excess and paucity can be toxic. Accumulating evidence has revealed pivotal roles for H2S in neuroprotection and normal cognitive function, and H2S homeostasis is dysregulated in neurodegenerative conditions. Here, we review the normal neuroprotective roles of H2S that go awry in Alzheimer's disease, the most common form of neurodegenerative disease.

SIRT3 sulfhydration using hydrogen sulfide inhibited angiotensin II-induced atrial fibrosis and vulnerability to atrial fibrillation via suppression of the TGF-ß1/smad2/3 signalling pathway.

Atrial fibrosis is associated with the occurrence of atrial fibrillation (AF) and regulated by the transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signalling pathway. Unfortunately, the mechanisms of regulation of TGF-ß1/Smad2/3-induced atrial fibrosis and vulnerability to AF remain still unknown. Previous studies have shown that sirtuin3 (SIRT3) sulfhydration has strong anti-fibrotic effects. We hypothesised that SIRT3 sulfhydration inhibits angiotensin II (Ang-II)-induced atrial fibrosis via blocking the TGF-ß1/Smad2/3 signalling pathway. In this study, we found that SIRT3 expression was decreased in the left atrium of patients with AF compared to that in those with sinus rhythm (SR). In vitro, SIRT3 knockdown by small interfering RNA significantly expanded Ang-II-induced atrial fibrosis and TGF-ß1/Smad2/3 signalling pathway activation, whereas supplementation with Sodium Hydrosulfide (NaHS, exogenous hydrogen sulfide donor and sulfhydration agonist) and SIRT3 overexpression using adenovirus ameliorated Ang-II-induced atrial fibrosis. Moreover, we observed suppression of the TGF-ß1/Smad2/3 pathway when Ang-II was combined with NaHS treatment, and the effect of this co-treatment was consistent with that of Ang-II combined with LY3200882 (Smad pathway inhibitor) on reducing atrial fibroblast proliferation and cell migration in vitro. Supplementation with dithiothreitol (DTT, a sulfhydration inhibitor) and adenovirus SIRT3 shRNA blocked the ameliorating effect of NaHS and AngII co-treatment on atrial fibrosis in vitro. Finally, continued treatment with NaHS in rats ameliorated atrial fibrosis and remodelling, and further improved AF vulnerability induced by Ang-II, which was reversed by DTT and adenovirus SIRT3 shRNA, suggesting that SIRT3 sulfhydration might be a potential therapeutic target in atrial fibrosis and AF.

Hydrogen sulfide mitigates ox­LDL­induced NLRP3/caspase­1/GSDMD dependent macrophage pyroptosis by S­sulfhydrating caspase­1.

Macrophage pyroptosis mediates vascular inflammation and atherosclerosis (AS). Hydrogen sulfide (H2S) exerts a protective role in preventing inflammation and AS. However, its molecular mechanisms of regulating the pyroptosis signaling pathway and inhibiting macrophage pyroptosis remain unexplored. The present study aimed to determine whether H2S mitigates macrophage pyroptosis by downregulating the pyroptosis signaling pathway and S­sulfhydrating caspase­1 under the stimulation of oxidized low­density lipoprotein (ox­LDL), a pro­atherosclerotic factor. Macrophages derived from THP­1 monocytes were pre­treated using exogenous H2S donors sodium hydrosulfide (NaHS) and D,L­propargylglycine (PAG), a pharmacological inhibitor of endogenous H2S­producing enzymes, alone or in combination. Subsequently, cells were stimulated with ox­LDL or the desulfhydration reagent dithiothreitol (DTT) in the presence or absence of NaHS and/or PAG. Following treatment, the levels of H2S in THP­1 derived macrophages were measured by a methylene blue colorimetric assay. The pyroptotic phenotype of THP­1 cells was observed and evaluated by light microscopy, Hoechst 33342/propidium iodide fluorescent staining and lactate dehydrogenase (LDH) release assay. Caspase­1 activity in THP­1 cells was assayed by caspase­1 activity assay kit. Immunofluorescence staining was used to assess the accumulation of active caspase­1. Western blotting and ELISA were performed to determine the expression of pyroptosis­specific markers (NLRP3, pro­caspase­1, caspase­1, GSDMD and GSDMD­N) in cells and the secretion of pyroptosis­related cytokines [interleukin (IL)­1ß and IL­18] in the cell­free media, respectively. The S­sulfhydration of pro­caspase­1 in cells was assessed using a biotin switch assay. ox­LDL significantly induced macrophage pyroptosis by activating the pyroptosis signaling pathway. Inhibition of endogenous H2S synthesis by PAG augmented the pro­pyroptotic effects of ox­LDL. Conversely, exogenous H2S (NaHS) ameliorated ox­LDL­and ox­LDL + PAG­induced macrophage pyroptosis by suppressing the activation of the pyroptosis signaling pathway. Mechanistically, ox­LDL and the DTT increased caspase­1 activity and downstream events (IL­1ß and IL­18 secretion) of the caspase­1­dependent pyroptosis pathway by reducing S­sulfhydration of pro­caspase­1. Conversely, NaHS increased S­sulfhydration of pro­caspase­1, reducing caspase­1 activity and caspase­1­dependent macrophage pyroptosis. The present study demonstrated the molecular mechanism by which H2S ameliorates macrophage pyroptosis by suppressing the pyroptosis signaling pathway and S­sulfhydration of pro­caspase­1, thereby suppressing the generation of active caspase-1 and activity of caspase-1.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

Allicin Promotes Glucose Uptake by Activating AMPK through CSE/H2S-Induced S-Sulfhydration in a Muscle-Fiber Dependent Way in Broiler Chickens.

SCOPE: Allicin, a product of enzymatic reaction when garlic is injured, plays an important role in maintaining glucose homeostasis in mammals. However, the effect of allicin on glucose homeostasis in the state of insulin resistance remains to be elucidated. This study investigates the effect of allicin on glucose metabolism using different muscle fibers in a chicken model. METHODS AND RESULTS: Day-old male Arbor Acres broilers are randomly divided into three groups and fed a basal diet supplemented with 0, 150, or 300 mg kg-1 allicin for 42 days. Results show that allicin improves the zootechnical performance of broilers at the finishing stage. The glucose loading test (2 g kg-1 body mass) indicates the regulatory role of allicin on glucose homeostasis. In vitro results demonstrate allicin increases glutathione (GSH) level and the expression of cystathionine γ lyase (CSE), leading to endogenous hydrogen sulfide (H2S) production in M. pectoralis major (PM) muscle-derived myotubes. Allicin stimulates adenosine monophosphate-activated protein kinase (AMPK) S-sulfhydration and AMPK phosphorylation to promote glucose uptake, which is suppressed in the presence of d,l-propargylglycine (PAG, a CSE inhibitor). CONCLUSION: This study demonstrates that allicin induces AMPK S-sulfhydration and AMPK phosphorylation to promote glucose uptake via the CSE/H2S system in a muscle fiber-dependent manner.

Investigating the impact of protein S-sulfhydration modification on vascular diseases: A comprehensive review.

The post-translational modification of cysteine through redox reactions, especially S-sulfhydration, plays a critical role in regulating protein activity, interactions, and spatial arrangement. This review focuses on the impact of protein S-sulfhydration on vascular function and its implications in vascular diseases. Dysregulated S-sulfhydration has been linked to the development of vascular pathologies, including aortic aneurysms and dissections, atherosclerosis, and thrombotic diseases. The H2S signaling pathway and the enzyme cystathionine γ-lyase (CSE), which is responsible for H2S generation, are identified as key regulators of vascular function. Additionally, potential therapeutic targets for the treatment of vascular diseases, such as the H2S donor GYY4137 and the HDAC inhibitor entinostat, are discussed. The review also emphasizes the antithrombotic effects of H2S in regulating platelet aggregation and thrombosis. The aim of this review is to enhance our understanding of the function and mechanism of protein S-sulfhydration modification in vascular diseases, and to provide new insights into the clinical application of this modification.

Redox Regulation of Xenobiotics by Reactive Sulfur and Supersulfide Species.

Significance: Routine exposure to xenobiotics is unavoidable during our lifetimes. Certain xenobiotics are hazardous to human health, and are metabolized in the body to render them less toxic. During this process, several detoxification enzymes cooperatively metabolize xenobiotics. Glutathione (GSH) conjugation plays an important role in the metabolism of electrophilic xenobiotics. Recent Advances: Recent advances in reactive sulfur and supersulfide (RSS) analyses showed that persulfides and polysulfides bound to low-molecular-weight thiols, such as GSH, and to protein thiols are abundant in both eukaryotes and prokaryotes. The highly nucleophilic nature of hydropersulfides and hydropolysulfides contributes to cell protection against oxidative stress and electrophilic stress. Critical Issues: In contrast to GSH conjugation to electrophiles that is aided by glutathione S-transferase (GST), persulfides and polysulfides can directly form conjugates with electrophiles without the catalytic actions of GST. The polysulfur bonds in the conjugates are further reduced by perthioanions and polythioanions derived from RSS to form sulfhydrated metabolites that are no longer electrophilic but rather nucleophilic, and differ from metabolites that are formed via GSH conjugation. Future Directions: In view of the abundance of RSS in cells and tissues, metabolism of xenobiotics that is mediated by RSS warrants additional investigations, such as studies of the impact of microbiota-derived RSS on xenobiotic metabolism. Metabolites formed from reactions between electrophiles and RSS may be potential biomarkers for monitoring exposure to electrophiles and for studying their metabolism by RSS. Antioxid. Redox Signal. 40, 679-690.

Sulfur signaling pathway in cardiovascular disease.

Hydrogen sulfide (H2S) and sulfur dioxide (SO2), recognized as endogenous sulfur-containing gas signaling molecules, were the third and fourth molecules to be identified subsequent to nitric oxide and carbon monoxide (CO), and exerted diverse biological effects on the cardiovascular system. However, the exact mechanisms underlying the actions of H2S and SO2 have remained elusive until now. Recently, novel post-translational modifications known as S-sulfhydration and S-sulfenylation, induced by H2S and SO2 respectively, have been proposed. These modifications involve the chemical alteration of specific cysteine residues in target proteins through S-sulfhydration and S-sulfenylation, respectively. H2S induced S-sulfhydrylation can have a significant impact on various cellular processes such as cell survival, apoptosis, cell proliferation, metabolism, mitochondrial function, endoplasmic reticulum stress, vasodilation, anti-inflammatory response and oxidative stress in the cardiovascular system. Alternatively, S-sulfenylation caused by SO2 serves primarily to maintain vascular homeostasis. Additional research is warranted to explore the physiological function of proteins with specific cysteine sites, despite the considerable advancements in comprehending the role of H2S-induced S-sulfhydration and SO2-induced S-sulfenylation in the cardiovascular system. The primary objective of this review is to present a comprehensive examination of the function and potential mechanism of S-sulfhydration and S-sulfenylation in the cardiovascular system. Proteins that undergo S-sulfhydration and S-sulfenylation may serve as promising targets for therapeutic intervention and drug development in the cardiovascular system. This could potentially expedite the future development and utilization of drugs related to H2S and SO2.

Dissecting molecular mechanisms underlying ferroptosis in human umbilical cord mesenchymal stem cells: Role of cystathionine γ-lyase/hydrogen sulfide pathway.

BACKGROUND: Ferroptosis can induce low retention and engraftment after mesenchymal stem cell (MSC) delivery, which is considered a major challenge to the effectiveness of MSC-based pulmonary arterial hypertension (PAH) therapy. Interestingly, the cystathionine γ-lyase (CSE)/hydrogen sulfide (H2S) pathway may contribute to mediating ferroptosis. However, the influence of the CSE/H2S pathway on ferroptosis in human umbilical cord MSCs (HUCMSCs) remains unclear. AIM: To clarify whether the effect of HUCMSCs on vascular remodelling in PAH mice is affected by CSE/H2S pathway-mediated ferroptosis, and to investigate the functions of the CSE/H2S pathway in ferroptosis in HUCMSCs and the underlying mechanisms. METHODS: Erastin and ferrostatin-1 (Fer-1) were used to induce and inhibit ferroptosis, respectively. HUCMSCs were transfected with a vector to overexpress or inhibit expression of CSE. A PAH mouse model was established using 4-wk-old male BALB/c nude mice under hypoxic conditions, and pulmonary pressure and vascular remodelling were measured. The survival of HUCMSCs after delivery was observed by in vivo bioluminescence imaging. Cell viability, iron accumulation, reactive oxygen species production, cystine uptake, and lipid peroxidation in HUCMSCs were tested. Ferroptosis-related proteins and S-sulfhydrated Kelch-like ECH-associating protein 1 (Keap1) were detected by western blot analysis. RESULTS: In vivo, CSE overexpression improved cell survival after erastin-treated HUCMSC delivery in mice with hypoxia-induced PAH. In vitro, CSE overexpression improved H2S production and ferroptosis-related indexes, such as cell viability, iron level, reactive oxygen species production, cystine uptake, lipid peroxidation, mitochondrial membrane density, and ferroptosis-related protein expression, in erastin-treated HUCMSCs. In contrast, in vivo, CSE inhibition decreased cell survival after Fer-1-treated HUCMSC delivery and aggravated vascular remodelling in PAH mice. In vitro, CSE inhibition decreased H2S levels and restored ferroptosis in Fer-1-treated HUCMSCs. Interestingly, upregulation of the CSE/H2S pathway induced Keap1 S-sulfhydration, which contributed to the inhibition of ferroptosis. CONCLUSION: Regulation of the CSE/H2S pathway in HUCMSCs contributes to the inhibition of ferroptosis and improves the suppressive effect on vascular remodelling in mice with hypoxia-induced PAH. Moreover, the protective effect of the CSE/H2S pathway against ferroptosis in HUCMSCs is mediated via S-sulfhydrated Keap1/nuclear factor erythroid 2-related factor 2 signalling. The present study may provide a novel therapeutic avenue for improving the protective capacity of transplanted MSCs in PAH.

Activating transcription factor (ATF) 6 upregulates cystathionine ß synthetase (CBS) expression and hydrogen sulfide (H2S) synthesis to ameliorate liver metabolic damage.

Activating transcription factor 6 (ATF6) is an endoplasmic reticulum stress responsive gene. We previously reported that conditional knockout of hepatic ATF6 exacerbated liver metabolic damage by repressing autophagy through mTOR pathway. However, the mechanism by which ATF6 influence liver metabolism has not been well established. Hydrogen sulfide (H2S) is a gaseous signaling molecule that plays an important role in regulating inflammation, and suppress nonalcoholic fatty liver in mice. Based on the previous study, we assumed that ATF6 may regulate H2S production to participate in liver metabolism. In order to clarify the mechanism by which ATF6 regulates H2S synthesis to ameliorate liver steatosis and inflammatory environment, we conducted the present study. We used the liver specific ATF6 knockout mice and fed on high-fat-diet, and found that H2S level was significantly downregulated in hepatic ATF6 knockout mice. Restoring H2S by the administration of slow H2S releasing agent GYY4137 ameliorated the hepatic steatosis and glucose tolerance. ATF6 directly binds to the promoter of cystathionine ß synthetase (CBS), an important enzyme in H2S synthesis. Thus, ATF6 could upregulate H2S production through CBS. Sulfhydrated Sirtuin-1 (SIRT1) was downregulated in ATF6 knockout mice. The expression of pro-inflammatory factor IL-17A was upregulated and anti-inflammatory factor IL-10 was downregulated in ATF6 knockout mice. Our results suggest that ATF6 can transcriptionally enhance CBS expression as well as H2S synthesis. ATF6 increases SIRT1 sulfhydration and ameliorates lipogenesis and inflammation in the fatty liver. Therefore, ATF6 could be a novel therapeutic strategy for high-fat diet induced fatty liver metabolic abnormalities.

Hydrogen Sulfide Regulates Glucose Uptake in Skeletal Muscles via S-Sulfhydration of AMPK in Muscle Fiber Type-Dependent Way.

BACKGROUND: The effect of hydrogen sulfide (H2S) on glucose homeostasis remains to be elucidated, especially in the state of insulin resistance. OBJECTIVES: In the present study, we aimed to investigate H2S-regulated glucose uptake in the M. pectoralis major (PM) muscle (which mainly consists of fast-twitch glycolytic fibers) and M. biceps femoris (BF) muscle (which mainly consists of slow-twitch oxidative fibers) of the chicken, a potential model of insulin resistance. METHODS: Chicks were subjected to intraperitoneal injection of sodium hydrosulfide (NaHS, 50 µmol/kg body mass/day) twice a day to explore glucose homeostasis. In vitro, myoblasts from PM and BF muscles were used to detect glucose uptake and utilization. Effects of AMP-activated protein kinase (AMPK) phosphorylation, AMPK S-sulfhydration, and mitogen-activated protein kinase (MAPK) pathway induction by NaHS were detected. RESULTS: NaHS enhanced glucose uptake and utilization in chicks (P < 0.05). In myoblasts from PM muscle, NaHS (100 µM) increased glucose uptake by activating AMPK S-sulfhydration, AMPK phosphorylation, and the AMPK/p38 MAPK pathway (P < 0.05). However, NaHS decreased glucose uptake in myoblasts from BF muscle by suppressing the p38 MAPK pathway (P < 0.05). Moreover, NaHS increased S-sulfhydration and, in turn, the phosphorylation of AMPK (P < 0.05). CONCLUSIONS: This study reveals the role of H2S in enhancing glucose uptake and utilization in chicks. The results suggest that NaHS is involved in glucose uptake in skeletal muscle in a fiber type-dependent way. The AMPK/p38 pathway and protein S-sulfhydration promote glucose uptake in fast-twitch glycolytic muscle fibers, which provides a muscle fiber-specific potential therapeutic target to ameliorate glucose metabolism.

Hydrogen sulfide-induced post-translational modification as a potential drug target.

Hydrogen sulfide (H2S) is one of the three known gas signal transducers, and since its potential physiological role was reported, the literature on H2S has been increasing. H2S is involved in processes such as vasodilation, neurotransmission, angiogenesis, inflammation, and the prevention of ischemia-reperfusion injury, and its mechanism remains to be further studied. At present, the role of post-translational processing of proteins has been considered as a possible mechanism for the involvement of H2S in a variety of physiological processes. Current studies have shown that H2S is involved in S-sulfhydration, phosphorylation, and S-nitrosylation of proteins, etc. This paper focuses on the effects of protein modification involving H2S on physiological and pathological processes, looking forward to providing guidance for subsequent research.

Mapping Pregnancy-dependent Sulfhydrome Unfolds Diverse Functions of Protein Sulfhydration in Human Uterine Artery.

Uterine artery (UA) hydrogen sulfide (H2S) production is augmented in pregnancy and, on stimulation by systemic/local vasodilators, contributes to pregnancy-dependent uterine vasodilation; however, how H2S exploits this role is largely unknown. S-sulfhydration converts free thiols to persulfides at reactive cysteine(s) on targeted proteins to affect the entire proteome posttranslationally, representing the main route for H2S to elicit its function. Here, we used Tag-Switch to quantify changes in sulfhydrated (SSH-) proteins (ie, sulfhydrome) in H2S-treated nonpregnant and pregnant human UA. We further used the low-pH quantitative thiol reactivity profiling platform by which paired sulfhydromes were subjected to liquid chromatography tandem mass spectrometry-based peptide sequencing to generate site (cysteine)-specific pregnancy-dependent H2S-responsive human UA sulfhydrome. Total levels of sulfhydrated proteins were significantly greater in pregnant vs nonpregnant human UA and further stimulated by treatment with sodium hydrosulfide. We identified a total of 360 and 1671 SSH-peptides from 480 and 1186 SSH-proteins in untreated and sodium hydrosulfide-treated human UA, respectively. Bioinformatics analyses identified pregnancy-dependent H2S-responsive human UA SSH peptides/proteins, which were categorized to various molecular functions, pathways, and biological processes, especially vascular smooth muscle contraction/relaxation. Pregnancy-dependent changes in these proteins were rectified by immunoblotting of the Tag-Switch labeled SSH proteins. Low-pH quantitative thiol reactivity profiling failed to identify low abundance SSH proteins such as KATP channels in human UA; however, immunoblotting of Tag-Switch-labeled SSH proteins identified pregnancy-dependent upregulation of SSH-KATP channels without altering their total proteins. Thus, comprehensive analyses of human UA sulfhydromes influenced by endogenous and exogenous H2S inform novel roles of protein sulfhydration in uterine hemodynamics regulation.

Cysteine and methionine oxidation in thrombotic disorders.

Thrombosis is the leading cause of death in many diseased conditions. Oxidative stress is characteristic of these conditions. Yet, the mechanisms through which oxidants become prothrombotic are unclear. Recent evidence suggests protein cysteine and methionine oxidation as prothrombotic regulators. These oxidative post-translational modifications occur on proteins that participate in the thrombotic process, including Src family kinases, protein disulfide isomerase, ß2 glycoprotein I, von Willebrand factor, and fibrinogen. New chemical tools to identify oxidized cysteine and methionine proteins in thrombosis and hemostasis, including carbon nucleophiles for cysteine sulfenylation and oxaziridines for methionine, are critical to understanding why clots occur during oxidative stress. These mechanisms will identify alternative or novel therapeutic approaches to treat thrombotic disorders in diseased conditions.

Pioglitazone treatment increases the cellular acid-labile and protein-bound sulfane sulfur fractions.

BACKGROUND: Iron-sulfur clusters play a central role in cellular function and are regulated by the ATM protein. Iron-sulfur clusters are part of the cellular sulfide pool, which functions to maintain cardiovascular health, and consists of free hydrogen sulfide, iron-sulfur clusters, protein bound sulfides, which constitute the total cellular sulfide fraction. ATM protein signaling and the drug pioglitazone share some cellular effects, which led us to examine the effects of this drug on cellular iron-sulfur cluster formation. Additionally, as ATM functions in the cardiovasculature and its signaling may be diminished in cardiovascular disease, we examined pioglitazone in the same cell type, with and without ATM protein expression. METHODS: We examined the effects of pioglitazone treatment on the total cellular sulfide profile, the glutathione redox state, cystathionine gamma-lyase enzymatic activity, and on double-stranded DNA break formation in cells with and without ATM protein expression. RESULTS: Pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions and reduced cystathionine gamma-lyase enzymatic activity in cells with and without ATM protein expression. Interestingly, pioglitazone also increased reduced glutathione and lowered DNA damage in cells without ATM protein expression, but not in ATM wild-type cells. These results are interesting as the acid-labile (iron-sulfur cluster), bound sulfur cellular fractions, and reduced glutathione are low in cardiovascular disease. CONCLUSION: Here we found that pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions, impinges on hydrogen sulfide synthesis, and exerts beneficial effect on cells with deficient ATM protein signaling. Thus, we show a novel pharmacologic action for pioglitazone.

Hydrogen sulfide signalling in neurodegenerative diseases.

The gaseous neurotransmitter hydrogen sulfide (H2 S) exerts neuroprotective efficacy in the brain via post-translational modification of cysteine residues by sulfhydration, also known as persulfidation. This process is comparable in biological impact to phosphorylation and mediates a variety of signalling events. Unlike conventional neurotransmitters, H2 S cannot be stored in vesicles due to its gaseous nature. Instead, it is either locally synthesized or released from endogenous stores. Sulfhydration affords both specific and general neuroprotective effects and is critically diminished in several neurodegenerative disorders. Conversely, some forms of neurodegenerative disease are linked to excessive cellular H2 S. Here, we review the signalling roles of H2 S across the spectrum of neurodegenerative diseases, including Huntington's disease, Parkinson's disease, Alzheimer's disease, Down syndrome, traumatic brain injury, the ataxias, and amyotrophic lateral sclerosis, as well as neurodegeneration generally associated with ageing.

Hydrogen sulfide attenuates TMAO­induced macrophage inflammation through increased SIRT1 sulfhydration.

Chronic inflammation is a key factor that accelerates the progression of inflammatory vascular disease. Hydrogen sulfide (H2S) has potent anti­inflammatory effects; however, its underlying mechanism of action has not been fully elucidated. The present study aimed to investigate the potential effect of H2S on sirtuin 1 (SIRT1) sulfhydration in trimethylamine N­oxide (TMAO)­induced macrophage inflammation, and its underlying mechanism. Pro­inflammatory M1 cytokines (MCP­1, IL­1ß, and IL­6) and anti­inflammatory M2 cytokines (IL­4 and IL­10) were detected by RT­qPCR. CSE, p65 NF­κB, p­p65 NF­κB, IL­1ß, IL­6 and TNF­α levels were measured by Western blot. The results revealed that cystathionine γ­lyase protein expression was negatively associated with TMAO­induced inflammation. Sodium hydrosulfide (a donor of H2S) increased SIRT1 expression and inhibited the expression of inflammatory cytokines in TMAO­stimulated macrophages. Furthermore, nicotinamide, a SIRT1 inhibitor, antagonized the protective effect of H2S, which contributed to P65 NF­κB phosphorylation and upregulated the expression of inflammatory factors in macrophages. H2S ameliorated TMAO­induced activation of the NF­κB signaling pathway via SIRT1 sulfhydration. Moreover, the antagonistic effect of H2S on inflammatory activation was largely eliminated by the desulfhydration reagent dithiothreitol. These results indicated that H2S may prevent TMAO­induced macrophage inflammation by reducing P65 NF­κB phosphorylation via the upregulation and sulfhydration of SIRT1, suggesting that H2S may be used to treat inflammatory vascular diseases.

Endothelial-dependent S-Sulfhydration of tissue factor pathway inhibitor regulates blood coagulation.

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. Here we investigated whether endothelial cell-driven oxidative post-translational modifications could have an impact on TFPI activity. We focused on S-sulfhydration, which is a hydrogen sulfide-dependent post-translational modification that, in endothelial cells, is regulated by the enzyme cystathionine γ-lyase (CSE). The study made use of human primary endothelial cells and blood from healthy individuals or subjects with atherosclerosis as well as from mice lacking endothelial CSE. TFPI was S-sulfhydrated in endothelial cells from healthy individuals and mice, while the loss of endothelial CSE expression/activity reduced its modification. Non-S-sulfhydrated TFPI was no longer able to interact with factor Xa, which facilitated the activation of tissue factor. Similarly, non-S-sulfhydratable TFPI mutants bound less protein S, while supplementation with hydrogen sulfide donors, preserved TFPI activity. Phenotypically, loss of TFPI S-sulfhydration increased clot retraction, suggesting that this post-translational modification is a new endothelial cell-dependent mechanism that contributes to the regulation of blood coagulation.

The biological functions of protein S-sulfhydration in eukaryotes and the ever-increasing understanding of its effects on bacteria.

As a critical endogenous signaling molecule, hydrogen sulfide may induce reversible post-translational modifications on cysteine residues of proteins, generating a persulfide bond known as S-sulfhydration. A systemic overview of the biofunctions of S-sulfhydration will equip us better to characterize its regulatory roles in antioxidant defense, inflammatory response, and cell fate, as well as its pathological mechanisms related to cardiovascular, neurological, and multiple organ diseases, etc. Nevertheless, the understanding of S-sulfhydration is mostly built on mammalian cells and animal models. We subsequently summarized the mediation effects of this specific post-transcriptional modification on physiological processes and virulence in bacteria. The high-sensitivity and high-throughput detection technologies are required for studying the signal transduction mechanism of H2S and protein S-sulfhydration modification. Herein, we reviewed the establishment and development of different approaches to assess S-sulfhydration, including the biotin-switch method, modified biotin-switch method, alkylation-based cysteine-labelled assay, and Tag-switch method. Finally, we discussed the limitations of the impacts of S-sulfhydration in pathogens-host interactions and envisaged the challenges to design drugs and antibiotics targeting the S-sulfhydrated proteins in the host or pathogens.