RESUMEN
This study presents a global strategy for the transsulfuration of intracellular thiols (RSH) to persulfides (RSSH). Thiiranes comprising fluorenyl/diphenyl and malonate ester moieties directly convert intercellular RSH to low-molecular-weight RSSH in cells. The efficiency of transsulfuration is determined by counting the number of olefins produced as byproducts, providing ratiometric signals for the corresponding persulfide production. Specifically, the direct and rapid protein S-persulfidation by thiirane is validated. Thiiranes are expected to play a crucial role in the study of sulfur signaling.
RESUMEN
1,3,5-Trithiane functionalized with esterase-sensitive ester groups on the methylene linkers was developed as a class of enzymatic hydrolysis-based hydrogen sulfide (H2S) donors. The amount of H2S released from the donors was dependent on the number of ester bonds. The donors release H2S in a controllable manner in the presence of an enzyme.
RESUMEN
Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-ß-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and ß-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-ß-lactamase superfamily, but also provide detailed information on substrate binding by PDOs.
Asunto(s)
Proteínas Bacterianas/química , Dioxigenasas/química , Myxococcus xanthus/enzimología , Pseudomonas putida/enzimología , beta-Lactamasas/química , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , Glutatión , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Soluciones , Especificidad por SustratoRESUMEN
Hydrogen sulfide (H2S) is a critical signaling molecule that regulates many physiological and/or pathological processes. Modulation of H2S levels could have potential therapeutic value. In this work, we report the rational design, synthesis, and biological evaluation of a class of phosphonamidothioate-based H2S-releasing agents (i.e., H2S donors). A novel pH-dependent intramolecular cyclization was employed to promote H2S release from the donors. These water-soluble compounds showed slow, controllable, and pH-sensitive production of H2S in aqueous solutions. The donors also showed significant cytoprotective effects in cellular models of oxidative damage. Most importantly, the donors were found to exhibit potent cardioprotective effects in an in vivo murine model of myocardial ischemia-reperfusion (MI/R) injury through a H2S-related mechanism.
Asunto(s)
Modelos Animales de Enfermedad , Sulfuro de Hidrógeno/metabolismo , Daño por Reperfusión Miocárdica/metabolismo , Animales , Concentración de Iones de Hidrógeno , RatonesRESUMEN
Although non-alcoholic fatty liver disease has been reported as a cardiometabolic risk factor, the effect of non-alcoholic fatty liver is yet to be clarified on abdominal obesity. Therefore, this study was conducted to investigate the longitudinal relationship of non-alcoholic fatty liver on the development of abdominal obesity. The study participants were composed of 11,212 Korean men without abdominal obesity. They were followed up from 2005 to 2010 to be monitored for the development of abdominal obesity according to their degree of non-alcoholic fatty liver disease (normal, mild, and moderate to severe). Cox-proportional hazard model was used to calculate the hazard ratios for abdominal obesity according to the degree of non-alcoholic fatty liver disease. While the average incidence was 15.5%, the incidence of abdominal obesity increased according to the degree of non-alcoholic fatty liver (normal: 11.6%, mild: 25.2%, moderate to severe: 41.0%, P < 0.001). Multivariable-adjusted hazard ratios for abdominal obesity independently increased proportionally to the degree of NAFLD (mild [1.07; 0.94-1.23], moderate to severe [1.58; 1.11-2.26], P for trend < 0.001). The risk of abdominal obesity increased proportionally to the degree of non-alcoholic fatty liver disease. This finding guarantees further studies to reveal the incidental relationship of abdominal obesity with non-alcoholic fatty liver disease.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico/epidemiología , Obesidad Abdominal/complicaciones , Adulto , Pueblo Asiatico , Estudios de Cohortes , Demografía , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Obesidad Abdominal/epidemiología , Modelos de Riesgos Proporcionales , Estudios Prospectivos , República de Corea/epidemiología , Factores de Riesgo , Circunferencia de la CinturaRESUMEN
Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.
RESUMEN
Ascochyta rabiei and Alternaria solani, the causal agents of Ascochyta blight of chickpea (Cicer arietinum) and early blight of potato (Solanum tuberosum), respectively, produce a set of phytotoxic compounds including solanapyrones A, B, and C. Although both the phytotoxicity of solanapyrones and their universal production among field isolates have been documented, the role of solanapyrones in pathogenicity is not well understood. Here, we report the functional characterization of the sol5 gene, which encodes a Diels-Alderase that catalyzes the final step of solanapyrone biosynthesis. Deletion of sol5 in both Ascochyta rabiei and Alternaria solani completely prevented production of solanapyrones and led to accumulation of the immediate precursor compound, prosolanapyrone II-diol, which is not toxic to plants. Deletion of sol5 did not negatively affect growth rate or spore production in vitro, and led to overexpression of the other solanapyrone biosynthesis genes, suggesting a possible feedback regulation mechanism. Phytotoxicity tests showed that solanapyrone A is highly toxic to several legume species and Arabidopsis thaliana. Despite the apparent phytotoxicity of solanapyrone A, pathogenicity tests showed that solanapyrone-minus mutants of Ascochyta rabiei and Alternaria solani were equally virulent as their corresponding wild-type progenitors, suggesting that solanapyrones are not required for pathogenicity.
Asunto(s)
Alternaria/enzimología , Alternaria/patogenicidad , Ascomicetos/enzimología , Ascomicetos/patogenicidad , Proteínas Fúngicas/metabolismo , Micotoxinas/metabolismo , Alternaria/genética , Alternaria/metabolismo , Ascomicetos/genética , Ascomicetos/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Micotoxinas/genética , Naftalenos/metabolismo , Pironas/metabolismoRESUMEN
S-Nitrosothiols (SNO) and their biological implications as an important post-translational modification are under active investigation. In our work on bioorthogonal reactions of protein SNO we have uncovered chemistry of this functionality that shows synthetic promise. Herein we report a phosphine-mediated reaction between SNO and aldehydes to form thioimines. A simple synthesis of benzoisothiazole based on this reaction is presented.
RESUMEN
Protein S-sulfhydration (forming -S-SH adducts from cysteine residues) is a newly defined oxidative posttranslational modification and plays an important role in H2 S-mediated signaling pathways. In this study we report the first selective, "tag-switch" method which can directly label protein S-sulfhydrated residues by forming stable thioether conjugates. Furthermore we demonstrate that H2 S alone cannot lead to S-sulfhydration and that the two possible physiological mechanisms include reaction with protein sulfenic acids (P-SOH) or the involvement of metal centers which would facilitate the oxidation of H2 S to HS(.) .
Asunto(s)
Cisteína/química , Sulfuro de Hidrógeno/química , Procesamiento Proteico-Postraduccional , Compuestos de Sulfhidrilo/química , Sulfuros/química , Biotina/química , Glutatión Peroxidasa/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Jurkat , Microscopía Fluorescente , Albúmina Sérica Bovina/químicaRESUMEN
Hydrogen sulfide (H2S) has emerged as an endogenous signaling molecule that functions in many physiological and pathological processes of human cells in health and disease, including neuromodulation and neuroprotection, inflammation, angiogenesis, and vasorelaxation. The limited clinical applications of current H2S donors have led to the development of H2S donor hybrid compounds that combine current H2S donors with bioactive molecules. Finely tuned multi-targeting hybrid molecules have been shown to have complementary neuroprotective effects against reactive oxygen species (ROS)-induced oxidative stress. In this study, we developed hybrid molecules combining a dithiolethione-based slow-releasing H2S donor that exerts neuroprotective effects, with the tripeptides glycyl-L-histidyl-l-lysine (GHK) and L-alanyl-L-cystinyl-l-glutamine (ACQ), two natural products that exhibit powerful antioxidant effects. In particular, a hybrid combination of a dithiolethione-based slow-releasing H2S donor and ACQ exhibited significant neuroprotective effects against glutamate-induced oxidative damage in HT22 hippocampal neuronal cells. This hybrid remarkably suppressed Ca2+ accumulation and ROS production. Furthermore, it efficiently inhibited apoptotic neuronal cell death by blocking apoptosis-inducing factor release and its translocation to the nucleus. These results indicate that the hybrid efficiently inhibited apoptotic neuronal cell damage by complementary neuroprotective actions.
Asunto(s)
Sulfuro de Hidrógeno , Fármacos Neuroprotectores , Humanos , Fármacos Neuroprotectores/farmacología , Especies Reactivas de Oxígeno/metabolismo , Estrés Oxidativo , Péptidos/farmacología , Hipocampo/metabolismo , Sulfuro de Hidrógeno/metabolismoRESUMEN
A synthesis of aziridine-containing peptides via the Cu(II)-promoted coupling of unprotected peptide thioacids and N-H aziridine-2-carbonyl peptides is reported. The unique reactivity of the resulting N-acylated aziridine-2-carbonyl peptides facilitates their subsequent regioselective and stereoselective nucleophilic ring-opening to give unprotected peptides that are specifically modified at the ligation site. The aziridine-mediated peptide ligation concept is exemplified using H(2)O as the nucleophile, producing a Xaa-Thr linkage (where Xaa can be an epimerizable and hindered amino acid). The overall process is compatible with a variety of unprotected amino acid functionality, most notably the N-terminal and Lys side chain amines.
Asunto(s)
Aziridinas/química , Péptidos/químicaRESUMEN
The reactions between S-nitrosothiols and phosphite esters, including P(OPh)3, P(OBn)3, and P(OEt)3, were studied. Two different conjugated adducts, thiophosphoramidates and phosphorothioates, were formed, depending on the structures of the S-nitrosothiol substrate (e.g., primary vs tertiary). These reactions proceeded under mild conditions, and the reaction mechanisms were studied using experiments and calculations.
Asunto(s)
Ésteres/química , Fosfitos/química , S-Nitrosotioles/síntesis química , Estructura Molecular , S-Nitrosotioles/químicaRESUMEN
The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.
Asunto(s)
Descubrimiento de Drogas , Fenoles/química , Sulfonas/química , Tetrazoles/química , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Humanos , Espectrometría de Masas , Modelos Biológicos , Estructura Molecular , Reactivos de Sulfhidrilo/química , Reactivos de Sulfhidrilo/farmacocinética , Reactivos de Sulfhidrilo/farmacología , Sulfonas/farmacocinética , Sulfonas/farmacologíaRESUMEN
S-Nitrosothiols (RSNOs) have many biological implications but are rarely used in organic synthesis. In this work we report the development of proline-based phosphoramidite substrates that can effectively convert RSNOs to proline-based sulfenamides through a reductive ligation process. A unique property of this method is that the phosphine oxide moiety on the ligation products can be readily removed under acidic conditions. In conjugation with the facile preparation of RSNOs from the corresponding thiols (RSHs), this method provides a new way to prepare proline-based sulfenamides from simple thiol starting materials.
Asunto(s)
Compuestos Organofosforados/química , Prolina/química , S-Nitrosotioles/química , Óxido Nítrico/química , Oxidación-Reducción , Fosfinas/química , Sulfamerazina/químicaRESUMEN
The development of a functional disulfide, FmSSPy-A (Fm = 9-fluorenylmethyl; Py = pyridinyl), is reported. It can effectively convert small molecule and protein thiols (-SH) to form -S-SFm adducts under mild conditions. This method allows for a H2S-free and biomimetic protocol to generate highly reactive persulfides (in their anionic forms). The high nucleophilicity of persulfides toward a number of thiol-blocking reagents is also demonstrated. The method holds promise for further understanding the chemical biology of persulfides and S-sulfhydration.
Asunto(s)
Disulfuros/química , Fluorenos/química , Sulfuros/química , Biomimética , Estructura MolecularRESUMEN
Chemotaxonomy and the comparative analysis of metabolic features of fungi have the potential to provide valuable information relating to ecology and evolution, but have not been fully explored in fungal biology. Here, we investigated the chemical diversity of legume-associated Ascochyta and Phoma species and the possible use of a metabolomics approach using liquid chromatography-mass spectrometry for their classification. The metabolic features of 45 strains including 11 known species isolated from various legumes were extracted, and the datasets were analyzed using chemometrics methods such as principal component and hierarchical clustering analyses. We found a high degree of intra-species consistency in metabolic profiles, but inter-species diversity was high. Molecular phylogenies of the legume-associated Ascochyta/Phoma species were estimated using sequence data from three protein-coding genes and the five major chemical groups that were detected in the hierarchical clustering analysis were mapped to the phylogeny. Clusters based on similarity of metabolic features were largely congruent with the species phylogeny. These results indicated that evolutionarily distinct fungal lineages have diversified their metabolic capacities as they have evolved independently. This whole metabolomics approach may be an effective tool for chemotaxonomy of fungal taxa lacking information on their metabolic content.
Asunto(s)
Ascomicetos/metabolismo , Fabaceae/microbiología , Metabolómica , Ascomicetos/clasificación , Ascomicetos/aislamiento & purificación , Teorema de Bayes , Quitina Sintasa/genética , Cromatografía Líquida de Alta Presión , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/metabolismo , Proteínas Fúngicas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Metaboloma , Factor 1 de Elongación Peptídica/genética , Fragmentos de Péptidos/genética , Filogenia , Análisis de Componente Principal , Análisis de Secuencia de ADN , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
AIM: Oxidative stress is a key contributor to endothelial dysfunction and associated cardiovascular pathogenesis. Hydrogen sulfide (H2S) is an antioxidant gasotransmitter that protects endothelial cells against oxidative stress. Sirtuin3 (SIRT3), which belongs to the silent information regulator 2 (SIR2) family, is an important deacetylase under oxidative stress. H2S is able to regulate the activity of several sirtuins. The present study aims to investigate the role of SIRT3 in the antioxidant effect of H2S in endothelial cells. RESULTS: Cultured EA.hy926 endothelial cells were exposed to hydrogen peroxide (H2O2) as a model of oxidative stress-induced cell injury. GYY4137, a slow-releasing H2S donor, improved cell viability, reduced oxidative stress and apoptosis, and improved mitochondrial function following H2O2 treatment. H2S reversed the stimulation of MAPK phosphorylation, downregulation of SIRT3 mRNA and reduction of the superoxide dismutase 2 and isocitrate dehydrogenase 2 expression which were induced by H2O2. H2S also increased activator protein 1 (AP-1) binding activity with SIRT3 promoter and this effect was absent in the presence of the specific AP-1 inhibitor, SR11302 or curcumin. Paraquat administration to mice induced a defected endothelium-dependent aortic vasodilatation and increased oxidative stress in both mouse aorta and small mesenteric artery, which were alleviated by GYY4137 treatment. This vasoprotective effect of H2S was absent in SIRT3 knockout mice. INNOVATION: The present results highlight a novel role for SIRT3 in the protective effect of H2S against oxidant damage in the endothelium both in vitro and in vivo. CONCLUSION: H2S enhances AP-1 binding activity with the SIRT3 promoter, thereby upregulating SIRT3 expression and ultimately reducing oxidant-provoked vascular endothelial dysfunction. Antioxid. Redox Signal. 24, 329-343.
Asunto(s)
Antioxidantes/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Sulfuro de Hidrógeno/farmacología , Sirtuina 3/metabolismo , Animales , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/fisiología , Inmunoprecipitación de Cromatina , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Mutantes , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Sirtuina 3/genéticaRESUMEN
BACKGROUND: Hydrogen sulfide (H2S) is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel-like factor 5 (KLF5) exerts diverse functions in the cardiovascular system. Whether and how H2S regulates KLF5 in myocardial hypertrophy is unknown. METHODS AND RESULTS: In our study, hypertrophic myocardial samples in the clinic were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats and neonatal rat cardiomyocytes were studied for functional and signaling responses to GYY4137, an H2S-releasing compound. Expression of cystathionine γ-lyase, a principal enzyme for H2S generation in heart, decreased in human hypertrophic myocardium, whereas KLF5 expression increased. After GYY4137 administration for 4 weeks, myocardial hypertrophy was inhibited in spontaneously hypertensive rats, as demonstrated by improvement in cardiac structural parameters, heart mass, size of cardiac myocytes, and expression of atrial natriuretic peptide. H2S diminished expression of KLF5 in myocardium of spontaneously hypertensive rats and in hypertrophic cardiomyocytes. H2S also inhibits platelet-derived growth factor A promoter activity, decreased recruitment of KLF5 to the platelet-derived growth factor A promoter, and reduced atrial natriuretic peptide expression in angiotensin II-stimulated cardiomyocytes, and these effects are suppressed by KLF5 knockdown. KLF5 promoter activity and KLF5 expression was also reversed by H2S. H2S increased the S-sulfhydration on specificity protein 1 in cardiomyocytes. Moreover, H2S decreased KLF5 promoter activity; reduced KLF5 mRNA expression; attenuated specificity protein 1 binding activity with KLF5 promoter; and inhibited hypertrophy after specificity protein 1 mutated at Cys659, Cys689, and Cys692 but not Cys664 overexpression. CONCLUSIONS: These findings suggest that H2S regulates KLF5 transcription activity via specificity protein 1 S-sulfhydration at Cys664 to prevent myocardial hypertrophy.
Asunto(s)
Cardiomegalia/genética , Cistationina gamma-Liasa/metabolismo , Sulfuro de Hidrógeno/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , ARN Mensajero/metabolismo , Angiotensina II/sangre , Animales , Animales Recién Nacidos , Factor Natriurético Atrial/efectos de los fármacos , Factor Natriurético Atrial/metabolismo , Cardiomegalia/metabolismo , Estudios de Casos y Controles , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Factores de Transcripción de Tipo Kruppel/efectos de los fármacos , Masculino , Morfolinas/farmacología , Miocitos Cardíacos/efectos de los fármacos , Compuestos Organotiofosforados/farmacología , Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/metabolismo , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays were used to determine H2S release from the donors in both aqueous buffers and in cultured cells. These donors were found to be slow-release donors, much like the well-known GYY4137. These donors also showed some protective effects against hydrogen peroxide (H2O2)-induced oxidative damage in myocytes.
Asunto(s)
Sulfuro de Hidrógeno/química , Fosfatos/química , Animales , Calibración , Línea Celular , Sulfuro de Hidrógeno/metabolismo , Hidrólisis , Ratas , Estándares de Referencia , Espectrometría de Fluorescencia/normasRESUMEN
Protein S-sulfhydration (i.e., converting protein cysteines -SH to persulfides -SSH) is a redox-based posttranslational modification. This reaction plays an important role in signaling pathways mediated by hydrogen sulfide or other reactive sulfane sulfur species. Recently, our laboratories developed a "tag-switch" method which can be used to selectively label and detect protein S-sulfhydrated residues. In this chapter, we provide a comprehensive summary of this method, including the design of the method, preparation of the reagents, validation on small-molecule substrates, as well as applications in protein labeling. Experimental protocols for the use of the method are described in details.