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Role of thiosulfate on hydrogen sulfide-mediated redox signaling in endothelial cells
Abstracts/Nitric Oxide 47 (2015) S5–S13
Our research focuses on the molecular basis of oxidant sensing and signalling in the heart and in blood vessels. Reactive protein thiols (which are typically ionised and known as thiolates) can react with a variety of oxidant or electrophilic species generated within biological systems. These reactions can result in the oxidative posttranslational modifications of proteins, which potentially can alter the activity of these proteins and so modulate cell function to provide homeostasis or adaptive signalling. Using such mechanisms, oxidants may serve as signalling molecules via their ability to structurally modify proteins with a coupled functional alteration. By identifying cardiovascular redox sensor proteins susceptible to oxidant-induced alterations in structure and function, we can perhaps better understand the significance of these modifications during health and disease. Redox proteomic approaches have allowed us to identify a number of proteins regulated by the oxidation of their cysteinyl thiols. These include protein kinase A, protein kinase G and soluble epoxide hydrolase, which are of note because we have subsequently engineered ‘redox dead’ knock-in mice for each of these proteins. These ‘redox dead’ knock-in mice have the reactive thiol replaced with an oxidant-unreactive serine. Comparing these knockin mice with wild-type littermates during interventions of interest allows the importance of each redox sensing mechanisms to be defined in isolation. In this way we have found that C42S PKG knockin mice are resistant to blood pressure-lowering induced by hydrogen sulfide. Biochemical studies showed that hydrogen sulfide has a propensity to form persulfides which can then react with PKG to induce its oxidation, which activates the kinase to lower blood pressure. The deficit in the ability of the C42S PKG knock-in mice (and mesenteric vessels isolated from them) to undergo vasodilation is explained by the kinase lacking the oxidant sensor that transduces the persulfide signals. This provides a mechanism that contributes to hydrogen sulfide-induced vasodilation and blood pressure lowering. http://dx.doi.org/10.1016/j.niox.2015.02.013
OP8 Role of thiosulfate on hydrogen sulfide-mediated redox signaling in endothelial cells Xinggui Shen, Anna Leskova, Sibile Pardue, John D Glawe, Christopher G. Kevil LSU Health Sciences Center, Shreveport, USA Recent reports have revealed that hydrogen sulfide (H2S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate (S2O32−) is one of the products formed during oxidative H2S metabolism, and thiosulfate has been used extensively and safely to treat calcific uremic arteriopathy (a disease, in part due to calcification of small arteries) in dialysis patients. It increases the solubility of calcium by up to 100,000 fold and is also a potent antioxidant. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In this study, we examined the effect of thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, we observed a decreased ratio of GSH:GSSG at 0.5 h and 4 h, unchanged thiosulfate levels at 0.5 h, increased thiosulfate at 4 h, as well as decreased free H2S and acid-labile sulfide, and increased bound sulfide at all time points. Treatment with thiosulfate greatly decreased the ratio of GSH:GSSG under hypoxia by more than what was observed due to hypoxia alone. Treatment with thiosulfate also decreased bound sulfane sulfur under hypoxia. This response showed that thiosulfate has a different role at low and high concentrations or oxygen tension. Interestingly, treatment with thiosulfate also
S7
reduced proliferation in vascular endothelial growth factor (VEGF)induced HUVECs under normoxic and hypoxic conditions. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions through non-enzymatic and enzymatic pathways and that this is not only a ready source of H2S production but also serves as a means of recycling sulfur and thereby conserving biologically relevant thiols. http://dx.doi.org/10.1016/j.niox.2015.02.014
OP9 CBS update: Structure, CBS replacement therapy, and H 2 S production Tomas Majtan a, Erez Bublil a, InSun Park a, Richard Carrillo a, June Ereño-Orbea b, Luis Alfonso Martínez-Cruz b, Helena Hu˚lková c, Jakub Krijt c, Viktor Kožich c, Warren Kruger d, Jan P. Kraus a a University of Colorado School of Medicine, Aurora, CO, USA b Structural Biology Unit, Center for Cooperative Research in Biosciences, Derio, Bizkaia, Spain c Institute of Inherited Metabolic Disorders, Charles University, First Faculty of Medicine and General University Hospital, Czech Republic d Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA Cystathionine beta-synthase (CBS), a pivotal enzyme in the transsulfuration pathway, binds three cofactors, heme, whose function remains an enigma, pyridoxal 5′-phosphate (PLP), part of the catalytic site where homocysteine and serine are condensed to cystathionine, and S-adenosylmethionine (SAM), which upon binding greatly stimulates CBS activity. Deficiency of CBS results in recessively inherited metabolic disease, homocystinuria, characterized by high concentrations of homocysteine, methionine and S-adenosylhomocysteine in body fluids and greatly decreased cysteine and cystathionine. The X-ray structure of CBS in its basal conformation having low activity and the access to the catalytic sites partially obstructed will be compared to its activated structure with SAM bound, the catalytic sites uncovered, and high CBS activity. We will also show that a pegylated CBS truncate can be efficiently used to normalize the sulfur amino acid metabolites in plasma and improve or prevent a number of abnormal pathologies in several mouse models of the disease. Lastly, the potential alternative role of CBS in H2S metabolism will be discussed. http://dx.doi.org/10.1016/j.niox.2015.02.015
OP10 Therapeutic potential of mitochondria-targeted hydrogen sulphide donors Matthew Whiteman University of Exeter Medical School, Exeter, UK Very recently several mitochondrial functions have been shown to be regulated by hydrogen sulphide (H2S) e.g. mitochondrial respiration and ATP synthesis, apoptosis, inflammation etc. Mitochondria are also capable of generating H2S. Endogenous H2S is produced within mitochondria from mercaptopyruvate via 3-mercaptopyruvate sulphurtransferase (3-MST) and cytosolic H 2 S from cysteine/ homocysteine via cystathionine-β-synthase (CBS) and cystathionineγ-lyase (CSE). Oxidative stress results in the inhibition of mitochondrial H2S synthesis and/or depletion of mitochondrial H2S and the translocation of CSE and CBS to mitochondria resulting in mitochondrial
Our research focuses on the molecular basis of oxidant sensing and signalling in the heart and in blood vessels. Reactive protein thiols (which are typically ionised and known as thiolates) can react with a variety of oxidant or electrophilic species generated within biological systems. These reactions can result in the oxidative posttranslational modifications of proteins, which potentially can alter the activity of these proteins and so modulate cell function to provide homeostasis or adaptive signalling. Using such mechanisms, oxidants may serve as signalling molecules via their ability to structurally modify proteins with a coupled functional alteration. By identifying cardiovascular redox sensor proteins susceptible to oxidant-induced alterations in structure and function, we can perhaps better understand the significance of these modifications during health and disease. Redox proteomic approaches have allowed us to identify a number of proteins regulated by the oxidation of their cysteinyl thiols. These include protein kinase A, protein kinase G and soluble epoxide hydrolase, which are of note because we have subsequently engineered ‘redox dead’ knock-in mice for each of these proteins. These ‘redox dead’ knock-in mice have the reactive thiol replaced with an oxidant-unreactive serine. Comparing these knockin mice with wild-type littermates during interventions of interest allows the importance of each redox sensing mechanisms to be defined in isolation. In this way we have found that C42S PKG knockin mice are resistant to blood pressure-lowering induced by hydrogen sulfide. Biochemical studies showed that hydrogen sulfide has a propensity to form persulfides which can then react with PKG to induce its oxidation, which activates the kinase to lower blood pressure. The deficit in the ability of the C42S PKG knock-in mice (and mesenteric vessels isolated from them) to undergo vasodilation is explained by the kinase lacking the oxidant sensor that transduces the persulfide signals. This provides a mechanism that contributes to hydrogen sulfide-induced vasodilation and blood pressure lowering. http://dx.doi.org/10.1016/j.niox.2015.02.013
OP8 Role of thiosulfate on hydrogen sulfide-mediated redox signaling in endothelial cells Xinggui Shen, Anna Leskova, Sibile Pardue, John D Glawe, Christopher G. Kevil LSU Health Sciences Center, Shreveport, USA Recent reports have revealed that hydrogen sulfide (H2S) exerts critical actions to promote cardiovascular homeostasis and health. Thiosulfate (S2O32−) is one of the products formed during oxidative H2S metabolism, and thiosulfate has been used extensively and safely to treat calcific uremic arteriopathy (a disease, in part due to calcification of small arteries) in dialysis patients. It increases the solubility of calcium by up to 100,000 fold and is also a potent antioxidant. Yet despite its significance, fundamental questions regarding how thiosulfate and H2S interact during redox signaling remain unanswered. In this study, we examined the effect of thiosulfate on hypoxia-induced H2S metabolite bioavailability in human umbilical vein endothelial cells (HUVECs). Under hypoxic conditions, we observed a decreased ratio of GSH:GSSG at 0.5 h and 4 h, unchanged thiosulfate levels at 0.5 h, increased thiosulfate at 4 h, as well as decreased free H2S and acid-labile sulfide, and increased bound sulfide at all time points. Treatment with thiosulfate greatly decreased the ratio of GSH:GSSG under hypoxia by more than what was observed due to hypoxia alone. Treatment with thiosulfate also decreased bound sulfane sulfur under hypoxia. This response showed that thiosulfate has a different role at low and high concentrations or oxygen tension. Interestingly, treatment with thiosulfate also
S7
reduced proliferation in vascular endothelial growth factor (VEGF)induced HUVECs under normoxic and hypoxic conditions. These results indicate that thiosulfate can contribute to H2S signaling under hypoxic conditions through non-enzymatic and enzymatic pathways and that this is not only a ready source of H2S production but also serves as a means of recycling sulfur and thereby conserving biologically relevant thiols. http://dx.doi.org/10.1016/j.niox.2015.02.014
OP9 CBS update: Structure, CBS replacement therapy, and H 2 S production Tomas Majtan a, Erez Bublil a, InSun Park a, Richard Carrillo a, June Ereño-Orbea b, Luis Alfonso Martínez-Cruz b, Helena Hu˚lková c, Jakub Krijt c, Viktor Kožich c, Warren Kruger d, Jan P. Kraus a a University of Colorado School of Medicine, Aurora, CO, USA b Structural Biology Unit, Center for Cooperative Research in Biosciences, Derio, Bizkaia, Spain c Institute of Inherited Metabolic Disorders, Charles University, First Faculty of Medicine and General University Hospital, Czech Republic d Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA, USA Cystathionine beta-synthase (CBS), a pivotal enzyme in the transsulfuration pathway, binds three cofactors, heme, whose function remains an enigma, pyridoxal 5′-phosphate (PLP), part of the catalytic site where homocysteine and serine are condensed to cystathionine, and S-adenosylmethionine (SAM), which upon binding greatly stimulates CBS activity. Deficiency of CBS results in recessively inherited metabolic disease, homocystinuria, characterized by high concentrations of homocysteine, methionine and S-adenosylhomocysteine in body fluids and greatly decreased cysteine and cystathionine. The X-ray structure of CBS in its basal conformation having low activity and the access to the catalytic sites partially obstructed will be compared to its activated structure with SAM bound, the catalytic sites uncovered, and high CBS activity. We will also show that a pegylated CBS truncate can be efficiently used to normalize the sulfur amino acid metabolites in plasma and improve or prevent a number of abnormal pathologies in several mouse models of the disease. Lastly, the potential alternative role of CBS in H2S metabolism will be discussed. http://dx.doi.org/10.1016/j.niox.2015.02.015
OP10 Therapeutic potential of mitochondria-targeted hydrogen sulphide donors Matthew Whiteman University of Exeter Medical School, Exeter, UK Very recently several mitochondrial functions have been shown to be regulated by hydrogen sulphide (H2S) e.g. mitochondrial respiration and ATP synthesis, apoptosis, inflammation etc. Mitochondria are also capable of generating H2S. Endogenous H2S is produced within mitochondria from mercaptopyruvate via 3-mercaptopyruvate sulphurtransferase (3-MST) and cytosolic H 2 S from cysteine/ homocysteine via cystathionine-β-synthase (CBS) and cystathionineγ-lyase (CSE). Oxidative stress results in the inhibition of mitochondrial H2S synthesis and/or depletion of mitochondrial H2S and the translocation of CSE and CBS to mitochondria resulting in mitochondrial