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1.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299206

RESUMO

Despite the intensive investigation of the molecular mechanism of skeletal muscle hypertrophy, the underlying signaling processes are not completely understood. Therefore, we used an overload model, in which the main synergist muscles (gastrocnemius, soleus) of the plantaris muscle were surgically removed, to cause a significant overload in the remaining plantaris muscle of 8-month-old Wistar male rats. SIRT1-associated pro-anabolic, pro-catabolic molecular signaling pathways, NAD and H2S levels of this overload-induced hypertrophy were studied. Fourteen days of overload resulted in a significant 43% (p < 0.01) increase in the mass of plantaris muscle compared to sham operated animals. Cystathionine-ß-synthase (CBS) activities and bioavailable H2S levels were not modified by overload. On the other hand, overload-induced hypertrophy of skeletal muscle was associated with increased SIRT1 (p < 0.01), Akt (p < 0.01), mTOR, S6 (p < 0.01) and suppressed sestrin 2 levels (p < 0.01), which are mostly responsible for anabolic signaling. Decreased FOXO1 and SIRT3 signaling (p < 0.01) suggest downregulation of protein breakdown and mitophagy. Decreased levels of NAD+, sestrin2, OGG1 (p < 0.01) indicate that the redox milieu of skeletal muscle after 14 days of overloading is reduced. The present investigation revealed novel cellular interactions that regulate anabolic and catabolic processes in the hypertrophy of skeletal muscle.


Assuntos
Cistationina beta-Sintase/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/patologia , Animais , Hipertrofia/genética , Hipertrofia/metabolismo , Hipertrofia/patologia , Masculino , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética , Sirtuínas/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
2.
J Cell Mol Med ; 25(7): 3460-3468, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33713531

RESUMO

Testosterone deficiency resulted in increased mortality in men. Our previous work found that hydrogen sulphide (H2 S) significantly alleviated the spermatogenesis disorder. To investigate whether H2 S could regulate testosterone synthesis and the relative signalling pathways. Disorder model of testosterone synthesis was constructed in vitro and in vivo. The cell viability was detected using CCK-8 method. The concentration of H2 S and testosterone were examined using ELISA kits. The relative mRNA and protein expression of CBS, PDE4A, PDE8A and proteins related to testosterone synthesis were detected by RT-qPCR and western blotting. PAS staining was used to detect the inflammatory status of testis. The sulfhydryl level of PDE4A and PDE8A was determined by Biotin Switch Technique. CBS overexpression inhibited while knockdown promoted LPS + H2 O2 induced injury in testosterone synthesis of MLTC-1 cells, though regulating the level of H2 S. The LPS + H2 O2 induced inhibition on cAMP and p-PKA was recovered by CBS overexpression, while addition of the specific inhibitor of PKA had opposite effects. CBS overexpression alleviated the inflammation status in testis and promoted the expression of StAR, P450scc, P450c17 and 3ß-HSD. CBS could also exhibit its protective role through promoting sulfhydrylation of PDE4A and PDE8A. H2 S catalysed by CBS could recover testosterone synthesis in vitro and in vivo through inhibiting PDE expression via sulfhydryl modification and activating cAMP/PKA pathway.


Assuntos
3',5'-AMP Cíclico Fosfodiesterases/metabolismo , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Testosterona/biossíntese , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Células HEK293 , Humanos , Sulfeto de Hidrogênio/farmacologia , Masculino , Camundongos , Transdução de Sinais , Testículo/metabolismo , Testosterona/deficiência
3.
Electron. j. biotechnol ; 50: 23-28, Mar. 2021. graf, ilus
Artigo em Inglês | LILACS | ID: biblio-1292311

RESUMO

BACKGROUND: H2S is proved to be functioning as a signaling molecule in an array of physiological processes in the plant and animal kingdom. However, the H2S synthesis pathway and the responses to cold conditions remain unclear in postharvest mushroom. RESULTS: The biosynthesis of H2S in the Agaricus bisporus mushroom tissues exhibited an increasing tendency during postharvest storage and was significantly triggered by cold treatment. The cystathionine clyase (AbCSE) and cystathionine b-synthase (AbCBS) genes were cloned and proved responsible for H2S biosynthesis. Furthermore, transcriptional and posttranscriptional regulation of AbCSE and AbCBS were crucial for the enzyme activities and subsequent H2S levels. However, the AbMST was not involved in this process. Moreover, the AbCSE and AbCBS genes displayed low identity to the characterized genes, but typical catalytic domains, activity sites, subunit interface sites, and cofactor binding sites were conserved in the respective protein sequences, as revealed by molecular modeling and docking study. The potential transcription factors responsible for the H2S biosynthesis in cold conditions were also provided. CONCLUSIONS: The H2S biosynthetic pathway in postharvest mushroom was unique and distinct to that of other horticultural products.


Assuntos
Agaricus/química , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/síntese química , Cultivos Agrícolas , Agaricus campestris , Temperatura Baixa , Armazenamento de Alimentos
4.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525421

RESUMO

The considerable post-traumatic brain recovery in fishes makes them a useful model for studying the mechanisms that provide reparative neurogenesis, which is poorly represented in mammals. After a mechanical injury to the telencephalon in adult fish, lost neurons are actively replaced due to the proliferative activity of neuroepithelial cells and radial glia in the neurogenic periventricular zone. However, it is not enough clear which signaling mechanisms are involved in the activation of adult neural stem cells (aNSC) after the injury (reactive proliferation) and in the production of new neurons (regenerative neurogenesis) from progenitor cells (NPC). In juvenile Pacific salmon, the predominant type of NSCs in the telencephalon are neuroepithelial cells corresponding to embryonic NSCs. Expression of glutamine synthetase (GS), a NSC molecular marker, was detected in the neuroepithelial cells of the pallium and subpallium of juvenile chum salmon, Oncorhynchus keta. At 3 days after a traumatic brain injury (TBI) in juvenile chum salmon, the GS expression was detected in the radial glia corresponding to aNSC in the pallium and subpallium. The maximum density of distribution of GS+ radial glia was found in the dorsal pallial region. Hydrogen sulfide (H2S) is a proneurogenic factor that reduces oxidative stress and excitotoxicity effects, along with the increased GS production in the brain cells of juvenile chum salmon. In the fish brain, H2S producing by cystathionine ß-synthase in neurogenic zones may be involved in maintaining the microenvironment that provides optimal conditions for the functioning of neurogenic niches during constitutive neurogenesis. After injury, H2S can determine cell survivability, providing a neuroprotective effect in the area of injury and reducing the process of glutamate excitotoxicity, acting as a signaling molecule involved in changing the neurogenic environment, which leads to the reactivation of neurogenic niches and cell regeneration programs. The results of studies on the control of the expression of regulatory Sonic Hedgehog genes (Shh) and the transcription factors Paired Box2 (Pax2) regulated by them are still insufficient. A comparative analysis of Pax2 expression in the telencephalon of intact chum salmon showed the presence of constitutive patterns of Pax2 expression in neurogenic areas and non-neurogenic parenchymal zones of the pallium and subpallium. After mechanical injury, the patterns of Pax2 expression changed, and the amount of Pax2+ decreased (p < 0.05) in lateral (Dl), medial (Dm) zones of the pallium, and the lateral zone (Vl) of the subpallium compared to the control. We believe that the decrease in the expression of Pax2 may be caused by the inhibitory effect of the Pax6 transcription factor, whose expression in the juvenile salmon brain increases upon injury.


Assuntos
Lesões Encefálicas/genética , Regeneração do Cérebro/genética , Cistationina beta-Sintase/genética , Proteínas de Peixes/genética , Glutamato-Amônia Ligase/genética , Fator de Transcrição PAX2/genética , Telencéfalo/metabolismo , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Lesões Encefálicas/metabolismo , Lesões Encefálicas/patologia , Diferenciação Celular , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica , Glutamato-Amônia Ligase/metabolismo , Ácido Glutâmico/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Sulfeto de Hidrogênio/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/citologia , Células Neuroepiteliais/metabolismo , Neurogênese/genética , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Oncorhynchus keta , Fator de Transcrição PAX2/metabolismo , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Telencéfalo/lesões , Telencéfalo/patologia
5.
Biol Pharm Bull ; 44(2): 162-168, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33518670

RESUMO

Folic acid (FA) affect human physiology and drug metabolism. Up to now, the effect of microgravity on the pharmacokinetics of FA remains unclear. The pharmacokinetics of FA in Sprague-Dawley (SD) rats are laying a foundation for safe medicine administration of astronauts. Proteins expression of such FA metabolic enzymes as Methyltetrahydrofolate reductase (MTHFR), Cystathionine beta synthase (CBS) and Methionine synthase (MS) in a variety of organs was analyzed with Western-Blot, and mRNA expression was detected by RT-PCR. The plasma concentration-time profile of FA in normal or tail-suspended SD rats was acquired by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after oral administration of FA. Area under curve (AUC) and Cmax of FA in SD rats decreased significantly with extending period of tail-suspension. In terms of expressed level of metabolic enzymes over four suspension terms, as well as the level of the corresponding mRNAs, the following regularities were found: an obvious sharp decline of MTHFR tissue in kidney, a time-dependent increase of CBS in liver tissue and duodenum tissues, the resemblance of MS fluctuation to that of CBS in tested tissues. A four-week simulated microgravity of SD rats exhibits an unequivocal diminish of bioavailability of FA, and simulated microgravity shows a varying effect on the expression of FA-metabolizing enzyme in a variety of tissues.


Assuntos
Ácido Fólico/farmacocinética , Fenômenos Fisiológicos da Nutrição , Voo Espacial , Vitaminas/farmacocinética , Simulação de Ausência de Peso , 5-Metiltetra-Hidrofolato-Homocisteína S-Metiltransferase/metabolismo , Animais , Cistationina beta-Sintase/metabolismo , Ácido Fólico/administração & dosagem , Masculino , Metilenotetra-Hidrofolato Redutase (NADPH2)/metabolismo , Modelos Animais , Ratos , Ratos Sprague-Dawley , Organismos Livres de Patógenos Específicos , Vitaminas/administração & dosagem
6.
Exp Eye Res ; 204: 108443, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33453277

RESUMO

Hydrogen sulfide (H2S) is an important gasotransmitter expressed in various tissues of the organism, including the eye. It is known that H2S is localized especially in the retina and corneal layers in bovine eye. The enzymes that mediate H2S synthesis are 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). Herein, we aimed to investigate the concentration levels and distribution profiles of these enzymes in bovine retina and retinal artery. Enzyme levels were measured by ELISA and distribution were determined by immunofluorescence microscopic analysis. Much higher concentrations of CBS and CSE have been detected in the retinal artery compared to the retina. In both tissues, particulary 3-MST was found at the lowest level while, CSE was determined to be the most abundant enzyme among the others. CBS distribution was shown in both endothelial and smooth muscle layers, while CSE was seen especially in the endothelial layer of the retinal artery. In the retina, CBS and CSE were expressed in cone-basil cells and retinal ganglion cells, while CSE was also present in bipolar cells. Our results indicated that H2S is synthesized endogenously in ocular tissues. The widespread expression of H2S synthesizing enzymes in the retina and retinal artery of the bovine eye, which has anatomical similarities with the human eye, may suggest a protective role for H2S against retinal vascular diseases as well as a regulatory role in the retinal vascular tone.


Assuntos
Cistationina gama-Liase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Retina/enzimologia , Artéria Retiniana/enzimologia , Animais , Bovinos , Cistationina beta-Sintase/metabolismo , Feminino , Masculino , Microscopia de Fluorescência , Sulfurtransferases/metabolismo
7.
Mol Genet Metab ; 132(2): 128-138, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33483253

RESUMO

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU. HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5'-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5'-methylthioadenosine. HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one­carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one­carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.


Assuntos
Cistationina beta-Sintase/genética , Homocistinúria/metabolismo , Fígado/metabolismo , Metionina/metabolismo , Adenosil-Homocisteinase/genética , Animais , Betaína/farmacologia , Cistationina beta-Sintase/metabolismo , Modelos Animais de Doenças , Ácido Fólico/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glicina Hidroximetiltransferase/genética , Homocisteína/sangue , Homocisteína/metabolismo , Homocistinúria/tratamento farmacológico , Homocistinúria/genética , Homocistinúria/patologia , Humanos , Fígado/enzimologia , Metionina/análogos & derivados , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Camundongos , Poliaminas/metabolismo
8.
Toxicology ; 451: 152685, 2021 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-33486070

RESUMO

Hydrogen sulfide (H2S) as the third gasotransmitter molecule serves various biological regulatory roles in health and disease. Acrylonitrile (AN) is a common occupational toxicant and environmental pollutant, causing brain and liver damage in mammals. The biotransformation of AN is dependent-upon reduced glutathione (GSH), cysteine and other sulfur-containing compounds. However, the effects of AN on the endogenous H2S biosynthesis pathway have yet to be determined. Herein, we demonstrated that a single exposure to AN (at 25, 50, or 75 mg/kg for 1, 6 or 24 h) decreased the endogenous H2S content and H2S-producing capacity in a dose-dependent manner, both in the cerebral cortex and liver of rats in vivo. In addition, the inhibitory effects of AN (1, 2.5, 5, 10 mM for 12 h) on the H2S content and/or the expression of H2S-producing enzymes were also found both in primary rat astrocytes and rat liver cell line (BRL cells). Impairment in the H2S biosynthesis pathway was also assessed in primary rat astrocytes treated with AN. It was found that inhibition of the cystathionine-ß-synthase (CBS)/3-mercaptopyruvate sulfurtransferase (3-MPST)-H2S pathway with the CBS inhibitor or 3-MPST-targeted siRNA significantly increased the AN-induced (5 mM for 12 h) cytotoxicity in astrocytes. In turn, CBS activation or 3-MPST overexpression as well as exogenous NaHS supplementation significantly attenuated AN-induced cytotoxicity. Taken together, endogenous H2S biosynthesis pathway was disrupted in rats acutely exposed to AN, which contributes to acute AN neurotoxicity in primary rat astrocytes.


Assuntos
Acrilonitrila/toxicidade , Astrócitos/metabolismo , Encéfalo/metabolismo , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Fígado/metabolismo , Sulfurtransferases/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Sulfeto de Hidrogênio/antagonistas & inibidores , Fígado/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
9.
FEBS J ; 288(2): 614-639, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32383312

RESUMO

Circadian disruption influences metabolic health. Metabolism modulates circadian function. However, the mechanisms coupling circadian rhythms and metabolism remain poorly understood. Here, we report that cystathionine ß-synthase (CBS), a central enzyme in one-carbon metabolism, functionally interacts with the core circadian protein cryptochrome 1 (CRY1). In cells, CBS augments CRY1-mediated repression of the CLOCK/BMAL1 complex and shortens circadian period. Notably, we find that mutant CBS-I278T protein, the most common cause of homocystinuria, does not bind CRY1 or regulate its repressor activity. Transgenic CbsZn/Zn  mice, while maintaining circadian locomotor activity period, exhibit reduced circadian power and increased expression of E-BOX outputs. CBS function is reciprocally influenced by CRY1 binding. CRY1 modulates enzymatic activity of the CBS. Liver extracts from Cry1-/- mice show reduced CBS activity that normalizes after the addition of exogenous wild-type (WT) CRY1. Metabolomic analysis of WT, CbsZn/Zn , Cry1-/- , and Cry2-/- samples highlights the metabolic importance of endogenous CRY1. We observed temporal variation in one-carbon and transsulfuration pathways attributable to CRY1-induced CBS activation. CBS-CRY1 binding provides a post-translational switch to modulate cellular circadian physiology and metabolic control.


Assuntos
Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Cistationina beta-Sintase/genética , Metaboloma/genética , Processamento de Proteína Pós-Traducional , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Sequência de Aminoácidos , Animais , Proteínas CLOCK/genética , Proteínas CLOCK/metabolismo , Criptocromos/deficiência , Cistationina beta-Sintase/metabolismo , Elementos E-Box , Feminino , Células HEK293 , Humanos , Masculino , Redes e Vias Metabólicas/genética , Camundongos , Camundongos Knockout , Mutação , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais
10.
Cell Prolif ; 54(1): e12950, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33179842

RESUMO

OBJECTIVES: Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS: We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine ß-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS: We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS: Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.


Assuntos
Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/metabolismo , Cistationina beta-Sintase/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Metionina Adenosiltransferase/metabolismo , Metionina/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Animais , Células Cultivadas , DNA/biossíntese , Feminino , Humanos , Metionina/análogos & derivados , Ratos , Ratos Sprague-Dawley
11.
Sci Total Environ ; 750: 141685, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32862004

RESUMO

Human exposure to bisphenol A (BPA) is unavoidable in daily life. Recently, research has showen that BPA could induce oxidative imbalance, thereby causing reproductive toxicity and liver dysfunction. Accumulated evidence has demonstrated that metformin possesses strong anti-oxidative properties. This study aimed to study the mechanism underlying the hepatic-protective effect of metformin on liver injury induced by BPA in rats via the UPLC-MS/MS metabolomics approach. Forty-two male rats were randomly divided into six groups (n = 7), namely the saline group (control), the corn oil group (vehicle), the metformin group (Met), the bisphenol A group (BPA), the bisphenol A and metformin group (BPA + Met), and the bisphenol A and diammonium glycyrrhizinate (positive control) group (BPA + DG). Serum was collected for biochemical analysis and metabolomics, and liver tissue was collected for histopathology and metabolomics in each group. We found that metformin could significantly reduce the levels of liver function enzymes (ALT, AST and GGT) and ameliorate inflammatory cell infiltration and hepatocyte necrosis induced by BPA. On the other hand, metformin could significantly enhance the total antioxidant capacity in BPA rats. Notably, metabolomics data indicated that the principal altered metabolic pathways based on the 26 differential metabolites in liver tissue, and 21 in serum among vehicle, BPA and BPA + Met groups, respectively, including cysteine and methionine metabolism, glutathione metabolism, and arginine biosynthesis and purine metabolism. Additionally, metformin significantly increased cystathionine ß synthase (CBS) and cystathionine γ lyase (CSE), thus reducing serum levels of homocysteine and increasing hepatic levels of cysteine and glutathione in BPA-treated rats. Overall, this study's results provided new insights into the role and mechanism of metformin in BPA-induced liver injury in rats.


Assuntos
Cistationina gama-Liase , Metformina , Animais , Compostos Benzidrílicos/toxicidade , Cromatografia Líquida , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Humanos , Fígado/metabolismo , Masculino , Metformina/toxicidade , Fenóis , Ratos , Espectrometria de Massas em Tandem , Regulação para Cima
12.
Expert Rev Proteomics ; 17(10): 751-765, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-33320032

RESUMO

Introduction: Homocystinuria due to cystathionine ß-synthase (CBS) deficiency, the most frequent inborn error of sulfur amino acid metabolism, is characterized biochemically by severely elevated homocysteine (Hcy) and related metabolites, such as Hcy-thiolactone and N-Hcy-protein. CBS deficiency reduces life span and causes pathological abnormalities affecting most organ systems in the human body, including the cardiovascular (thrombosis, stroke), skeletal/connective tissue (osteoporosis, thin/non-elastic skin, thin hair), and central nervous systems (mental retardation, seizures), as well as the liver (fatty changes), and the eye (ectopia lentis, myopia). Molecular basis of these abnormalities were largely unknown and available treatments remain ineffective. Areas covered: Proteomic and transcriptomic studies over the past decade or so, have significantly contributed to our understanding of mechanisms by which the CBS enzyme deficiency leads to clinical manifestations associated with it. Expert opinion: Recent findings, discussed in this review, highlight the involvement of dysregulated proteostasis in pathologies associated with CBS deficiency, including thromboembolism, stroke, neurologic impairment, connective tissue/collagen abnormalities, hair defects, and hepatic toxicity. To ameliorate these pathologies, pharmacological, enzyme replacement, and gene transfer therapies are being developed.


Assuntos
Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/metabolismo , Animais , Autofagia/fisiologia , Fibrinogênio/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteômica/métodos , Transcriptoma/genética , Transcriptoma/fisiologia
13.
PLoS One ; 15(11): e0241685, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33180827

RESUMO

The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-ß-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation.


Assuntos
Transdiferenciação Celular , Reprogramação Celular , Fibroblastos/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Compostos Alílicos/farmacologia , Células Cultivadas , Técnicas de Reprogramação Celular/métodos , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Ácido Láctico/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Sulfetos/farmacologia
14.
J Nutr ; 150(Suppl 1): 2494S-2505S, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33000151

RESUMO

Metabolism of excess methionine (Met) to homocysteine (Hcy) by transmethylation is facilitated by the expression of methionine adenosyltransferase (MAT) I/III and glycine N-methyltransferase (GNMT) in liver, and a lack of either enzyme results in hypermethioninemia despite normal concentrations of MATII and methyltransferases other than GNMT. The further metabolism of Hcy by the transsulfuration pathway is facilitated by activation of cystathionine ß-synthase (CBS) by S-adenosylmethionine (SAM) as well as the relatively high KM of CBS for Hcy. Transmethylation plus transsulfuration effects catabolism of the Met molecule along with transfer of the sulfur atom of Met to serine to synthesize cysteine (Cys). Oxidation and excretion of Met sulfur depend upon Cys catabolism and sulfur oxidation pathways. Excess Cys is oxidized by cysteine dioxygenase 1 (CDO1) and further metabolized to taurine or sulfate. Some Cys is normally metabolized by desulfhydration pathways, and the hydrogen sulfide (H2S) produced is further oxidized to sulfate. If Cys or Hcy concentrations are elevated, Cys or Hcy desulfhydration can result in excess H2S and thiosulfate production. Excess Cys or Met may also promote their limited metabolism by transamination pathways.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/metabolismo , Cisteína/metabolismo , Glicina N-Metiltransferase/deficiência , Homocisteína/metabolismo , Fígado/metabolismo , Metionina/metabolismo , Sulfetos/metabolismo , Enxofre/metabolismo , Aminoácidos/metabolismo , Animais , Cistationina beta-Sintase/metabolismo , Glicina N-Metiltransferase/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , S-Adenosilmetionina/metabolismo , Serina/metabolismo , Tiossulfatos/metabolismo
15.
Biochem Pharmacol ; 182: 114267, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33035509

RESUMO

BACKGROUND: Hydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. METHODS: H2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. RESULTS: While disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. CONCLUSIONS: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.


Assuntos
Inibidores de Acetaldeído Desidrogenases/farmacologia , Cobre/farmacologia , Cistationina beta-Sintase/antagonistas & inibidores , Dissulfiram/farmacologia , Ditiocarb/análogos & derivados , Compostos Organometálicos/farmacologia , Inibidores de Acetaldeído Desidrogenases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quelantes/metabolismo , Quelantes/farmacologia , Cobre/metabolismo , Cistationina beta-Sintase/metabolismo , Dissulfiram/metabolismo , Ditiocarb/metabolismo , Ditiocarb/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/metabolismo
16.
Commun Biol ; 3(1): 583, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067579

RESUMO

Hydrogen sulfide (H2S) is constitutively generated in the human body and works as a gasotransmitter in synaptic transmission. In this study, we aimed to evaluate the roles of endogenous H2S in generating eupnea at the respiratory center. We employed an in situ arterially perfused preparation of decerebrated rats and recorded the central respiratory outputs. When the H2S-producing enzyme cystathionine ß-synthase (CBS) was inhibited, respiration switched from the 3-phase eupneic pattern, which consists of inspiration, postinspiration, and expiration, to gasping-like respiration, which consists of inspiration only. On the other hand, when H2S synthesis was inhibited via cystathionine γ-lyase (CSE) or when H2S synthesis was activated via CBS, eupnea remained unchanged. These results suggest that H2S produced by CBS has crucial roles in maintaining the neuronal network to generate eupnea. The mechanism of respiratory pattern generation might be switched from a network-based system to a pacemaker cell-based system in low H2S conditions.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Centro Respiratório/irrigação sanguínea , Centro Respiratório/metabolismo , Animais , Seio Carotídeo/efeitos dos fármacos , Seio Carotídeo/inervação , Seio Carotídeo/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Denervação , Ratos , Respiração , Centro Respiratório/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismo
17.
Sci Rep ; 10(1): 14657, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887901

RESUMO

Cystathionine ß-synthase (CBS) catalyzes the condensation of serine and homocysteine to water and cystathionine, which is then hydrolyzed to cysteine, α-ketobutyrate and ammonia by cystathionine γ-lyase (CGL) in the reverse transsulfuration pathway. The protozoan parasite Toxoplasma gondii, the causative agent of toxoplasmosis, includes both CBS and CGL enzymes. We have recently reported that the putative T. gondii CGL gene encodes a functional enzyme. Herein, we cloned and biochemically characterized cDNA encoding CBS from T. gondii (TgCBS), which represents a first example of protozoan CBS that does not bind heme but possesses two C-terminal CBS domains. We demonstrated that TgCBS can use both serine and O-acetylserine to produce cystathionine, converting these substrates to an aminoacrylate intermediate as part of a PLP-catalyzed ß-replacement reaction. Besides a role in cysteine biosynthesis, TgCBS can also efficiently produce hydrogen sulfide, preferentially via condensation of cysteine and homocysteine. Unlike the human counterpart and similar to CBS enzymes from lower organisms, the TgCBS activity is not stimulated by S-adenosylmethionine. This study establishes the presence of an intact functional reverse transsulfuration pathway in T. gondii and demonstrates the crucial role of TgCBS in biogenesis of H2S.


Assuntos
Cistationina beta-Sintase/metabolismo , Cisteína/biossíntese , Sulfeto de Hidrogênio/metabolismo , Toxoplasma/enzimologia , Toxoplasma/genética , Biocatálise , Cistationina/biossíntese , Cistationina beta-Sintase/genética , Cistationina gama-Liase/metabolismo , DNA Complementar , Genes de Protozoários , Heme/metabolismo , Homocisteína/metabolismo , Cinética , Serina/análogos & derivados , Serina/metabolismo
18.
J Environ Pathol Toxicol Oncol ; 39(3): 281-290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865918

RESUMO

Objective-To investigate cystathionine ß synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine ß synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.


Assuntos
Apoptose , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mieloma Múltiplo/metabolismo , Adulto , Idoso , Alcinos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Transdução de Sinais
19.
Endocrinology ; 161(11)2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32987401

RESUMO

Angiogenesis is a physiological process for endometrial regeneration in the menstrual cycle and remodeling during pregnancy. Endogenous hydrogen sulfide (H2S), produced by cystathionine-ß synthase (CBS) and cystathionine-γ lyase (CSE), is a potent proangiogenic factor; yet, whether the H2S system is expressed in the endometrium and whether H2S plays a role in endometrial angiogenesis are unknown. This study was to test whether estrogens stimulate endometrial H2S biosynthesis to promote endometrial microvascular endothelial cell (EMEC) angiogenesis. CBS messenger RNA/protein and H2S production significantly differed among endometria from postmenopausal (POM), premenopausal secretory (sPRM), and proliferative (pPRM) nonpregnant (NP) and pregnant (Preg) women (P < .05) in a rank order of POM approximately equal to sPRM is less than pPRM is less than Preg, positively correlating with angiogenesis indices and endogenous estrogens and with no difference in CSE expression. CBS and CSE proteins were localized to stroma, glands, and vessels in endometrium, and greater stromal CBS protein was observed in the pPRM and Preg states. Estradiol-17ß (E2) (but not progesterone) stimulated CBS (but not CSE) expression and H2S production in pPRM endometrial stromal cells (ESCs) in vitro, which were attenuated by ICI 182 780. The H2S donor sodium hydrosulfide promoted in vitro EMEC angiogenesis. Co-culture with sPRM, pPRM, and Preg ESCs all stimulated EMEC migration with a rank order of sPRM less than pPRM approximately equal to Preg. CBS (but not CSE) inhibition attenuated ESC-stimulated EMEC migration. E2 did not affect EMEC migration but potentiated ESC-stimulated EMEC migration. Altogether, estrogens stimulate specific receptor-dependent stromal CBS-H2S production to promote endometrial EMEC angiogenesis in women.


Assuntos
Cistationina beta-Sintase/metabolismo , Endométrio/efeitos dos fármacos , Estradiol/farmacologia , Sulfeto de Hidrogênio/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Células Estromais/metabolismo , Adulto , Idoso , Indutores da Angiogênese/metabolismo , Células Cultivadas , Endométrio/irrigação sanguínea , Endométrio/citologia , Endométrio/metabolismo , Feminino , Humanos , Ciclo Menstrual/efeitos dos fármacos , Ciclo Menstrual/genética , Ciclo Menstrual/metabolismo , Pessoa de Meia-Idade , Neovascularização Fisiológica/genética , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/genética , Pós-Menopausa/metabolismo , Gravidez , Células Estromais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
20.
Molecules ; 25(16)2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32824311

RESUMO

Cystathionine ß-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine γ-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.


Assuntos
Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/análise , Pirazóis/farmacologia , Piridinas/farmacologia , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Redes Neurais de Computação , Pirazóis/química , Piridinas/química , S-Adenosilmetionina/química , Relação Estrutura-Atividade
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