RESUMO
Post-transcriptional modifications of tRNA nucleotide are important determinants in folding, structure and function. We have successfully identified and characterized a new modified base named 2-methylthio-methylenethio-N6 -(cis-4-hydroxyisopentenyl)adenosine, which is present at position 37 in some tRNAs. We also showed that this new modified adenosine is derived from the known 2-methylthio-methylenethio-N6 -(isopentenyl)adenosine nucleoside by a catalytic cycle of the tRNA-diiron monooxygenase, MiaE, present in Salmonella typhimurium.
Assuntos
Adenosina , Salmonella typhimurium , Salmonella typhimurium/genética , RNA de Transferência/química , Isopenteniladenosina/química , Oxigenases de Função Mista/químicaRESUMO
Hydrogen sulfide is synthesized by enzymes involved in sulfur metabolism and oxidized via a dedicated mitochondrial pathway that intersects with the electron transport chain at the level of complex III. Studies with H2S are challenging since it is volatile and also reacts with oxidized thiols in the culture medium, forming sulfane sulfur species. The half-life of exogenously added H2S to cultured cells is unknown. In this study, we first examined the half-life of exogenously added H2S to human colonic epithelial cells. In plate cultures, H2S disappeared with a t1/2 of 3 to 4 min at 37 °C with a small fraction being trapped as sulfane sulfur species. In suspension cultures, the rate of abiotic loss of H2S was slower, and we demonstrated that sulfide stimulated aerobic glycolysis, which was sensitive to the mitochondrial but not the cytoplasmic NADH pool. Oxidation of mitochondrial NADH using the genetically encoded mito-LbNOX tool blunted the cellular sensitivity to sulfide-stimulated aerobic glycolysis and enhanced its oxidation to thiosulfate. In contrast, sulfide did not affect flux through the oxidative pentose phosphate pathway or the TCA cycle. Knockdown of sulfide quinone oxidoreductase, which commits H2S to oxidation, sensitized cells to sulfide-stimulated aerobic glycolysis. Finally, we observed that sulfide decreased ATP levels in cells. The dual potential of H2S to activate oxidative phosphorylation at low concentrations, but inhibit it at high concentrations, suggests that it might play a role in tuning electron flux and, therefore, cellular energy metabolism, particularly during cell proliferation.
Assuntos
Glicólise , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Transdução de Sinais , Células HCT116 , Células HT29 , HumanosRESUMO
Mammalian cells synthesize H2S from sulfur-containing amino acids and are also exposed to exogenous sources of this signaling molecule, notably from gut microbes. As an inhibitor of complex IV in the electron transport chain, H2S can have a profound impact on metabolism, suggesting the hypothesis that metabolic reprogramming is a primary mechanism by which H2S signals. In this study, we report that H2S increases lipogenesis in many cell types, using carbon derived from glutamine rather than from glucose. H2S-stimulated lipid synthesis is sensitive to the mitochondrial NAD(P)H pools and is enabled by reductive carboxylation of α-ketoglutarate. Lipidomics analysis revealed that H2S elicits time-dependent changes across several lipid classes, e.g., upregulating triglycerides while downregulating phosphatidylcholine. Direct analysis of triglyceride concentration revealed that H2S induces a net increase in the size of this lipid pool. These results provide a mechanistic framework for understanding the effects of H2S on increasing lipid droplets in adipocytes and population studies that have pointed to a positive correlation between cysteine (a substrate for H2S synthesis) and fat mass.
Assuntos
Glutamina , Sulfeto de Hidrogênio , NAD , Metabolismo Energético , Lipogênese , Mitocôndrias/metabolismo , Transdução de SinaisRESUMO
Unlike most other tissues, the colon epithelium is exposed to high levels of H2S derived from gut microbial metabolism. H2S is a signaling molecule that modulates various physiological effects. It is also a respiratory toxin that inhibits complex IV in the electron transfer chain (ETC). Colon epithelial cells are adapted to high environmental H2S exposure as they harbor an efficient mitochondrial H2S oxidation pathway, which is dedicated to its disposal. Herein, we report that the sulfide oxidation pathway enzymes are apically localized in human colonic crypts at the host-microbiome interface, but that the normal apical-to-crypt gradient is lost in colorectal cancer epithelium. We found that sulfide quinone oxidoreductase (SQR), which catalyzes the committing step in the mitochondrial sulfide oxidation pathway and couples to complex III, is a critical respiratory shield against H2S poisoning. H2S at concentrations ≤20 µm stimulated the oxygen consumption rate in colon epithelial cells, but, when SQR expression was ablated, H2S concentrations as low as 5 µm poisoned cells. Mitochondrial H2S oxidation altered cellular bioenergetics, inducing a reductive shift in the NAD+/NADH redox couple. The consequent electron acceptor insufficiency caused uridine and aspartate deficiency and enhanced glutamine-dependent reductive carboxylation. The metabolomic signature of this H2S-induced stress response mapped, in part, to redox-sensitive nodes in central carbon metabolism. Colorectal cancer tissues and cell lines appeared to counter the growth-restricting effects of H2S by overexpressing sulfide oxidation pathway enzymes. Our findings reveal an alternative mechanism for H2S signaling, arising from alterations in mitochondrial bioenergetics that drive metabolic reprogramming.
Assuntos
Metabolismo Energético , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colo/citologia , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cisteína/química , Cisteína/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Sulfeto de Hidrogênio/química , Sulfeto de Hidrogênio/farmacologia , NAD/química , Oxirredução , Consumo de Oxigênio/efeitos dos fármacos , Quinona Redutases/antagonistas & inibidores , Quinona Redutases/genética , Quinona Redutases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Rhodanese domains are structural modules present in the sulfurtransferase superfamily. These domains can exist as single units, in tandem repeats, or fused to domains with other activities. Despite their prevalence across species, the specific physiological roles of most sulfurtransferases are not known. Mammalian rhodanese and mercaptopyruvate sulfurtransferase are perhaps the best-studied members of this protein superfamily and are involved in hydrogen sulfide metabolism. The relatively unstudied human thiosulfate sulfurtransferase-like domain-containing 1 (TSTD1) protein, a single-domain cytoplasmic sulfurtransferase, was also postulated to play a role in the sulfide oxidation pathway using thiosulfate to form glutathione persulfide, for subsequent processing in the mitochondrial matrix. Prior kinetic analysis of TSTD1 was performed at pH 9.2, raising questions about relevance and the proposed model for TSTD1 function. In this study, we report a 1.04 Å resolution crystal structure of human TSTD1, which displays an exposed active site that is distinct from that of rhodanese and mercaptopyruvate sulfurtransferase. Kinetic studies with a combination of sulfur donors and acceptors reveal that TSTD1 exhibits a low Km for thioredoxin as a sulfane sulfur acceptor and that it utilizes thiosulfate inefficiently as a sulfur donor. The active site exposure and its interaction with thioredoxin suggest that TSTD1 might play a role in sulfide-based signaling. The apical localization of TSTD1 in human colonic crypts, which interfaces with sulfide-releasing microbes, and the overexpression of TSTD1 in colon cancer provide potentially intriguing clues as to its role in sulfide metabolism.
Assuntos
Modelos Moleculares , NADP/metabolismo , Proteínas de Neoplasias/metabolismo , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Tiossulfato Sulfurtransferase/metabolismo , Substituição de Aminoácidos , Animais , Biocatálise , Domínio Catalítico , Colo/enzimologia , Colo/metabolismo , Colo/patologia , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Mucosa Intestinal/enzimologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Tiorredoxina Dissulfeto Redutase/química , Tiorredoxinas/química , Tiorredoxinas/genética , Tiossulfato Sulfurtransferase/química , Tiossulfato Sulfurtransferase/genéticaRESUMO
The chemical species involved in H2S signaling remain elusive despite the profound and pleiotropic physiological effects elicited by this molecule. The dominant candidate mechanism for sulfide signaling is persulfidation of target proteins. However, the relatively poor reactivity of H2S toward oxidized thiols, such as disulfides, the low concentration of disulfides in the reducing milieu of the cell and the low steady-state concentration of H2S raise questions about the plausibility of persulfide formation via reaction between an oxidized thiol and a sulfide anion or a reduced thiol and oxidized hydrogen disulfide. In contrast, sulfide oxidation pathways, considered to be primarily mechanisms for disposing of excess sulfide, generate a series of reactive sulfur species, including persulfides, polysulfides and thiosulfate, that could modify target proteins. We posit that sulfide oxidation pathways mediate sulfide signaling and that sulfurtransferases ensure target specificity.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Transdução de Sinais , Compostos de Sulfidrila/metabolismo , Sulfetos/metabolismo , Animais , Bivalves/metabolismo , Cisteína/metabolismo , Dissulfetos/metabolismo , Humanos , Metemoglobina/química , Metemoglobina/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Oxirredução , Poliquetos/metabolismo , Bactérias Redutoras de Enxofre/metabolismo , Sulfurtransferases/química , Sulfurtransferases/metabolismoRESUMO
Rhodanese is a component of the mitochondrial H2S oxidation pathway. Rhodanese catalyzes the transfer of sulfane sulfur from glutathione persulfide (GSSH) to sulfite generating thiosulfate and from thiosulfate to cyanide generating thiocyanate. Two polymorphic variations have been identified in the rhodanese coding sequence in the French Caucasian population. The first, 306AâC, has an allelic frequency of 1% and results in an E102D substitution in the encoded protein. The second polymorphism, 853CâG, has an allelic frequency of 5% and leads to a P285A substitution. In this study, we have examined differences in the stability between wild-type rhodanese and the E102D and P285A variants and in the kinetics of the sulfur transfer reactions. The Asp-102 and Ala-285 variants are more stable than wild-type rhodanese and exhibit kcat/Km,CN values that are 17- and 1.6-fold higher, respectively. All three rhodanese forms preferentially catalyze sulfur transfer from GSSH to sulfite, generating thiosulfate and glutathione. The kcat/Km,sulfite values for the variants in the sulfur transfer reaction from GSSH to sulfite were 1.6- (Asp-102) and 4-fold (Ala-285) lower than for wild-type rhodanese, whereas the kcat/Km,GSSH values were similar for all three enzymes. Thiosulfate-dependent H2S production in murine liver lysate is low, consistent with a role for rhodanese in sulfide oxidation. Our studies show that polymorphic variations that are distant from the active site differentially modulate the sulfurtransferase activity of human rhodanese to cyanide versus sulfite and might be important in differences in susceptibility to diseases where rhodanese dysfunction has been implicated, e.g. inflammatory bowel diseases.
Assuntos
Polimorfismo Genético , Tiossulfato Sulfurtransferase/metabolismo , Animais , Catálise , Estabilidade Enzimática , Temperatura Alta , Humanos , Sulfeto de Hidrogênio/metabolismo , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutagênese Sítio-Dirigida , Sulfurtransferases/metabolismo , Tiossulfato Sulfurtransferase/genéticaRESUMO
Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.
Assuntos
Sulfeto de Hidrogênio/química , Mitocôndrias/metabolismo , Quinona Redutases/química , Catálise , Cisteína/química , Citocromos c/química , Escherichia coli/enzimologia , Glutationa/química , Homeostase , Humanos , Concentração de Íons de Hidrogênio , Cinética , Oxirredução , Oxigênio/química , Espectrofotometria Ultravioleta , Sulfetos/química , Tiossulfato Sulfurtransferase/químicaRESUMO
A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys(118) is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.
Assuntos
Metionina Sulfóxido Redutases/química , Neisseria meningitidis/enzimologia , Alanina/química , Catálise , Cisteína/química , Dimerização , Dissulfetos/química , Genômica , Cinética , Modelos Químicos , Mutação , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Ácidos Sulfênicos/químicaRESUMO
Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.
Assuntos
Diabetes Mellitus/patologia , Dislipidemias/patologia , Gluconeogênese , Fígado/patologia , Sulfetos/metabolismo , Tiossulfato Sulfurtransferase/fisiologia , Animais , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Dislipidemias/etiologia , Dislipidemias/metabolismo , Glucose/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Proteoma/metabolismoRESUMO
Peroxiredoxins from the Prx1 subfamily (Prx) are moonlighting peroxidases that operate in peroxide signaling and are regulated by sulfinylation. Prxs offer a major model of protein-thiol oxidative modification. They react with H2O2 to form a sulfenic acid intermediate that either engages into a disulfide bond, committing the enzyme into its peroxidase cycle, or again reacts with peroxide to produce a sulfinic acid that inactivates the enzyme. Sensitivity to sulfinylation depends on the kinetics of these two competing reactions and is critically influenced by a structural transition from a fully folded (FF) to locally unfolded (LU) conformation. Analysis of the reaction of the Tsa1 Saccharomyces cerevisiae Prx with H2O2 by Trp fluorescence-based rapid kinetics revealed a process linked to the FF/LU transition that is kinetically distinct from disulfide formation and suggested that sulfenate formation facilitates local unfolding. Use of mutants of distinctive sensitivities and of different peroxide substrates showed that sulfinylation sensitivity is not coupled to the resolving step kinetics but depends only on the sulfenic acid oxidation and FF-to-LU transition rate constants. In addition, stabilization of the active site FF conformation, the determinant of sulfinylation kinetics, is only moderately influenced by the Prx C-terminal tail dynamics that determine the FF â LU kinetics. From these two parameters, the relative sensitivities of Prxs toward hyperoxidation with different substrates can be predicted, as confirmed by in vitro and in vivo patterns of sulfinylation.
RESUMO
Peroxiredoxins are H2O2 scavenging enzymes that also carry out H2O2 signaling and chaperone functions. In yeast, the major cytosolic peroxiredoxin, Tsa1 is required for both promoting resistance to H2O2 and extending lifespan upon caloric restriction. We show here that Tsa1 effects both these functions not by scavenging H2O2, but by repressing the nutrient signaling Ras-cAMP-PKA pathway at the level of the protein kinase A (PKA) enzyme. Tsa1 stimulates sulfenylation of cysteines in the PKA catalytic subunit by H2O2 and a significant proportion of the catalytic subunits are glutathionylated on two cysteine residues. Redox modification of the conserved Cys243 inhibits the phosphorylation of a conserved Thr241 in the kinase activation loop and enzyme activity, and preventing Thr241 phosphorylation can overcome the H2O2 sensitivity of Tsa1-deficient cells. Results support a model of aging where nutrient signaling pathways constitute hubs integrating information from multiple aging-related conduits, including a peroxiredoxin-dependent response to H2O2.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Longevidade , Oxirredução , Peroxidases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoAssuntos
Proteínas de Bactérias/química , Metionina Sulfóxido Redutases/química , Staphylococcus aureus/enzimologia , Catálise , Cisteína/química , Dimerização , Dissulfetos/química , Cinética , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Mutação , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de ProteínaRESUMO
H2S is produced from sulfur-containing amino acids, cysteine and homocysteine, or a catabolite, 3-mercaptopyruvate, by three known enzymes: cystathionine ß-synthase, γ-cystathionase, and 3-mercaptopyruvate sulfurtransferase. Of these, the first two enzymes reside in the cytoplasm and comprise the transsulfuration pathway, while the third enzyme is found both in the cytoplasm and in the mitochondrion. The following mitochondrial enzymes oxidize H2S: sulfide quinone oxidoreductase, sulfur dioxygenase, rhodanese, and sulfite oxidase. The products of the sulfide oxidation pathway are thiosulfate and sulfate. Assays for enzymes involved in the production and oxidative clearance of sulfide to thiosulfate are described in this chapter.