RESUMEN
Methionine sulfoxide reductase B3 (MsrB3) is a protein repair enzyme that specifically catalyzes the reduction of methionine-R-sulfoxide residues and has an antioxidant function. We have previously shown that depletion of MsrB3 suppresses the proliferation of normal mammalian cells by arresting cell cycle. In this study, we report the crucial role of MsrB3 in cancer cell death. Deficiency of MsrB3 induced cancer cell death, while MsrB3 overexpression stimulated cancer cell proliferation. MsrB3 depletion resulted in apoptotic cancer cell death through the activation of the intrinsic mitochondrial pathway. MsrB3 deficiency increased the levels of cellular reactive oxygen species (ROS) and led to redox imbalance, and also increased the Bax to Bcl-2 ratio and cytochrome c release, leading to caspase activation. Treatment of MsrB3-depleted cells with N-acetylcysteine, an ROS scavenger, prevented cell death, suggesting that MsrB3 deficiency-induced cell death is associated with increased ROS production. In addition, MsrB3 depletion activated poly(ADP ribose) polymerase-1 (PARP-1) and led to the translocation of apoptosis-inducing factor (AIF) to the nucleus. Taken together, our results suggest that MsrB3 plays an important role in cancer cell survival through the modulation of the intrinsic apoptosis pathway.
Asunto(s)
Metionina Sulfóxido Reductasas/metabolismo , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Células A549 , Acetilcisteína/farmacología , Apoptosis/fisiología , Factor Inductor de la Apoptosis/metabolismo , Núcleo Celular/metabolismo , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Humanos , Células MCF-7/efectos de los fármacos , Metionina Sulfóxido Reductasas/genética , Mitocondrias/genética , Transporte de ProteínasRESUMEN
Thioredoxin reductase 1 (TXNRD1) is associated with susceptibility to acetaminophen (APAP)-induced liver damage. Methionine sulfoxide reductase A (MsrA) is an antioxidant and protein repair enzyme that specifically catalyzes the reduction of methionine S-sulfoxide residues. We have previously shown that MsrA deficiency exacerbates acute liver injury induced by APAP. In this study, we used primary hepatocytes to investigate the underlying mechanism of the protective effect of MsrA against APAP-induced hepatotoxicity. MsrA gene-deleted (MsrA-/-) hepatocytes showed higher susceptibility to APAP-induced cytotoxicity than wild-type (MsrA+/+) cells, consistent with our previous in vivo results. MsrA deficiency increased APAP-induced glutathione depletion and reactive oxygen species production. APAP treatment increased Nrf2 activation more profoundly in MsrA-/- than in MsrA+/+ hepatocytes. Basal TXNRD1 levels were significantly higher in MsrA-/- than in MsrA+/+ hepatocytes, while TXNRD1 depletion in both MsrA-/- and MsrA+/+ cells resulted in increased resistance to APAP-induced cytotoxicity. In addition, APAP treatment significantly increased TXNRD1 expression in MsrA-/- hepatocytes, while no significant change was observed in MsrA+/+ cells. Overexpression of MsrA reduced APAP-induced cytotoxicity and TXNRD1 expression levels in APAP-treated MsrA-/- hepatocytes. Collectively, our results suggest that MsrA protects hepatocytes from APAP-induced cytotoxicity through the modulation of TXNRD1 expression.
Asunto(s)
Acetaminofén/efectos adversos , Supervivencia Celular/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/enzimología , Metionina Sulfóxido Reductasas/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Analgésicos no Narcóticos/efectos adversos , Animales , Apoptosis/efectos de los fármacos , Células Cultivadas , Citoprotección/fisiología , Relación Dosis-Respuesta a Droga , Hepatocitos/patología , Masculino , Metionina Sulfóxido Reductasas/genética , Ratones , Ratones NoqueadosRESUMEN
We have previously shown that down-regulation of methionine sulfoxide reductase B3 (MsrB3) induces cancer cell apoptosis through the activation of the intrinsic mitochondrial pathway. However, the mechanism through which MsrB3 deficiency results in cancer cell death is poorly understood. In this study, we investigated whether p53 and endoplasmic reticulum (ER) stress are involved in MsrB3 deficiency-induced cancer cell apoptosis using breast and colon cancer cells. MsrB3 depletion resulted in p53 down-regulation at the post-transcriptional level. MsrB3 deficiency induced cell death to a similar extent in both p53 wild-type (p53+/+) and null (p53-/-) cancer cells, suggesting that MsrB3 deficiency-induced apoptosis occurs independently of p53. MsrB3 deficiency significantly increased ER stress, which resulted in apoptosis. In addition, MsrB3 depletion activated the pro-apoptotic Bim molecule, which is essential for ER stress-induced apoptosis. MsrB3 deficiency increased cytosolic calcium levels, suggesting that MsrB3 down-regulation leads to a disturbance of calcium homeostasis in the ER, which consequently triggers ER stress. MsrB3 overexpression in MsrB3-depleted cells reduced ER stress, and was accompanied by at least partial recovery of cell viability. Taken together, our results suggest that MsrB3 plays a critical role in cancer cell apoptosis through the modulation of ER stress status.
Asunto(s)
Apoptosis , Estrés del Retículo Endoplásmico , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Proteína p53 Supresora de Tumor/metabolismo , Técnicas de Silenciamiento del Gen , Células HCT116 , Humanos , Células MCF-7 , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Acetaminophen (APAP) overdose induces acute liver damage and failure via reactive oxygen species production and glutathione (GSH) depletion. Methionine sulfoxide reductase B1 (MsrB1) is an antioxidant selenoenzyme that specifically catalyzes the reduction of methionine R-sulfoxide residues. In this study, we used MsrB1 gene-knockout mice and primary hepatocytes to investigate the effect of MsrB1 on APAP-induced hepatotoxicity. Analyses of histological alterations and serum indicators of liver damage showed that MsrB1-/- mice were more susceptible to APAP-induced acute liver injury than wild-type (MsrB1+/+) mice. Consistent with the in vivo results, primary MsrB1-/- hepatocytes displayed higher susceptibility to APAP-induced cytotoxicity than MsrB1+/+ cells. MsrB1 deficiency increased hepatic oxidative stress after APAP challenge such as hydrogen peroxide production, lipid peroxidation, and protein oxidation levels. Additionally, basal and APAP-induced ratios of reduced-to-oxidized GSH (GSH/GSSG) were significantly lower in MsrB1-/- than in MsrB1+/+ livers. Nrf2 nuclear accumulation and heme oxygenase-1 expression levels after APAP challenge were lower in MsrB1-/- than in MsrB1+/+ livers, suggesting that MsrB1 deficiency attenuates the APAP-induced activation of Nrf2. Collectively, the results of this study suggest that selenoprotein MsrB1 plays a protective role against APAP-induced hepatotoxicity via its antioxidative function.
Asunto(s)
Acetaminofén/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Metionina Sulfóxido Reductasas/deficiencia , Metionina Sulfóxido Reductasas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Acetaminofén/efectos adversos , Animales , Antioxidantes/metabolismo , Masculino , Metionina Sulfóxido Reductasas/genética , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Methionine sulfoxide reductase A (MsrA) is a major antioxidant enzyme that specifically catalyzes the reduction of methionine S-sulfoxide. In this study, we used MsrA gene-knockout (MsrA-/-) mice and bone marrow-derived macrophages (BMDMs) to investigate the role of MsrA in the regulation of inflammatory responses induced by lipopolysaccharide (LPS). MsrA-/- mice were more susceptible to LPS-induced lethal shock than wild-type (MsrA+/+) mice. Serum levels of the proinflammatory cytokines IL-6 and TNF-α induced by LPS were higher in MsrA-/- than in MsrA+/+ mice. MsrA deficiency in the BMDMs also increased the LPS-induced cytotoxicity as well as TNF-α level. Basal and LPS-induced reactive oxygen species (ROS) levels were higher in MsrA-/- than in MsrA+/+ BMDMs. Phosphorylation levels of p38, JNK, and ERK were higher in MsrA-/- than in MsrA+/+ BMDMs in response to LPS, suggesting that MsrA deficiency increases MAPK activation. Furthermore, MsrA deficiency increased the expression and nuclear translocation of NF-κB and the expression of inducible nitric oxide synthase, a target gene of NF-κB, in response to LPS. Taken together, our results suggest that MsrA protects against LPS-induced septic shock, and negatively regulates proinflammatory responses via inhibition of the ROS-MAPK-NF-κB signaling pathways.
Asunto(s)
Inflamación/inmunología , Lipopolisacáridos/inmunología , Metionina Sulfóxido Reductasas/inmunología , Choque Séptico/inmunología , Animales , Citocinas/inmunología , Femenino , Eliminación de Gen , Inflamación/complicaciones , Inflamación/genética , Mediadores de Inflamación/inmunología , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/inmunología , Especies Reactivas de Oxígeno/inmunología , Choque Séptico/complicaciones , Choque Séptico/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Many bacteria, particularly pathogens, possess methionine sulfoxide reductase A (MsrA) and B (MsrB) as a fusion form (MsrAB). However, it is not clear why they possess a fusion MsrAB form rather than the separate enzymes that exist in most organisms. In this study, we performed biochemical and kinetic analyses of MsrAB from Treponema denticola (TdMsrAB), single-domain forms (TdMsrA and TdMsrB), and catalytic Cys mutants (TdMsrAB(C11S) and TdMsrAB(C285S)). We found that the catalytic efficiency of both MsrA and MsrB increased after fusion of the domains and that the linker region (iloop) that connects TdMsrA and TdMsrB is required for the higher catalytic efficiency of TdMsrAB. We also determined the crystal structure of TdMsrAB at 2.3 Å, showing that the iloop mainly interacts with TdMsrB via hydrogen bonds. Further kinetic analysis using the iloop mutants revealed that the iloop-TdMsrB interactions are critical to MsrB and MsrA activities. We also report the structure in which an oxidized form of dithiothreitol, an in vitro reductant for MsrA and MsrB, is present in the active site of TdMsrA. Collectively, the results of this study reveal an essential role of the iloop in maintaining the higher catalytic efficiency of the MsrAB fusion enzyme and provide a better understanding of why the MsrAB enzyme exists as a fused form.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Biocatálisis , Dicroismo Circular , Proteínas Recombinantes de Fusión/química , Homología de Secuencia de AminoácidoRESUMEN
Methionine sulfoxide reductase B3 (MsrB3), which is primarily found in the endoplasmic reticulum (ER), is an important protein repair enzyme that stereospecifically reduces methionine-R-sulfoxide residues. We previously found that MsrB3 deficiency arrests the cell cycle at the G1/S stage through up-regulation of p21 and p27. In this study, we report a critical role of MsrB3 in gene expression of heme oxygenase-1 (HO-1), which has an anti-proliferative effect associated with p21 up-regulation. Depletion of MsrB3 elevated HO-1 expression in mammalian cells, whereas MsrB3 overexpression had no effect. MsrB3 deficiency increased cellular reactive oxygen species (ROS), particularly in the mitochondria. ER stress, which is associated with up-regulation of HO-1, was also induced by depletion of MsrB3. Treatment with N-acetylcysteine as an ROS scavenger reduced augmented HO-1 levels in MsrB3-depleted cells. MsrB3 deficiency activated Nrf2 transcription factor by enhancing its expression and nuclear import. The activation of Nrf2 induced by MsrB3 depletion was confirmed by increased expression levels of its other target genes, such as γ-glutamylcysteine ligase. Taken together, these data suggest that MsrB3 attenuates HO-1 induction by inhibiting ROS production, ER stress, and Nrf2 activation.
Asunto(s)
Hemo-Oxigenasa 1/metabolismo , Metionina Sulfóxido Reductasas/fisiología , Factor 2 Relacionado con NF-E2/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Células Cultivadas , Estrés del Retículo Endoplásmico , Hemo-Oxigenasa 1/biosíntesis , Humanos , Metionina Sulfóxido Reductasas/deficiencia , Ratones , Ratones Endogámicos C57BLRESUMEN
Methionine sulfoxide reductase B3 (MsrB3) is an oxidoreductase in the endoplasmic reticulum that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine. Here, we report the critical role and mechanisms of MsrB3 in cell proliferation. The deletion of MsrB3 led to a significant decrease in cell proliferation in mouse embryonic fibroblast (MEF) cells. MsrB3-knockout MEF cells showed increased p53 protein levels, compared to wild-type MEF cells, which subsequently elevated the protein level of cyclin-dependent kinase inhibitor p21. In addition, MsrB3 deficiency enhanced the protein level of p27, another cell cycle regulator, and caused cell cycle arrest at the G1 stage. The inhibitory effect of MsrB3 deficiency on cell proliferation through the activation of p53-p21 and p27 pathways was also confirmed in primary human dermal fibroblasts. Collectively, the data suggest that MsrB3 is a regulator of cell growth through the p53-p21 and p27 pathways.
Asunto(s)
Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/metabolismo , Metionina Sulfóxido Reductasas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ciclo Celular , Células Cultivadas , Dermis/citología , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Humanos , Metionina Sulfóxido Reductasas/metabolismo , Ratones , Transducción de SeñalRESUMEN
Free methionine-R-sulfoxide reductase (fRMsr) enzymes only reduce the free form of methionine-R-sulfoxide and can be grouped into two types with respect to the number of conserved Cys residues in the active sites. In this work, the crystal structures of type II fRMsr from Thermoplasma acidophilum (TafRMsr), which contains two conserved Cys, have been determined in native form and in a complex with the substrate. The overall structure of TafRMsr consists of a central ß-sheet encompassed by a two-α-helix bundle flanking on one side and one small α-helix on the other side. Based on biochemical and growth complementation assays, Cys(84) is demonstrated to be the catalytic Cys. The data also show that TafRMsr functions as an antioxidant protein. Structural analyses reveal insights into substrate recognition and orientation, conformational changes in the active site during substrate binding, and the role of active site residues in substrate binding. A model for the catalytic mechanism of type II TafRMsr is suggested, in which intramolecular disulfide bond formation is not involved. In addition, the biochemical, enzymatic, and structural properties of type II TafRMsr are compared with those of type I enzymes.
Asunto(s)
Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/metabolismo , Thermoplasma/enzimología , Secuencia de Aminoácidos , Biocatálisis , Dominio Catalítico , Disulfuros/química , Cinética , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Methionine sulfoxide reductase A (MsrA) reduces free and protein-based methionine-S-sulfoxide to methionine. Structures of 1-Cys MsrAs lacking a resolving Cys, which interacts with catalytic Cys, are unknown. In addition, no structural information on selenocysteine (Sec)-containing MsrA enzymes has been reported. In this work, we determined the crystal structures of 1-Cys type selenoprotein MsrA from Clostridium oremlandii at 1.6-1.8Å, including the reduced, oxidized (sulfenic acid), and substrate-bound forms. The overall structure of Clostridium MsrA, consisting of ten α-helices and six ß-strands, folds into a catalytic domain and a novel helical domain absent from other known MsrA structures. The helical domain, containing five helices, tightly interacts with the catalytic domain, and is likely critical for catalytic activity due to its association with organizing the active site. This helical domain is also conserved in several selenoprotein MsrAs. Our structural analysis reveals that the side chain length of Glu55 is critical for the proton donor function of this residue. Our structures also provide insights into the architecture of the 1-Cys MsrA active site and the roles of active site residues in substrate recognition and catalysis.
Asunto(s)
Clostridium/enzimología , Cisteína/química , Metionina Sulfóxido Reductasas/química , Sitios de Unión , Dominio Catalítico , Clostridium/química , Cristalografía por Rayos X , Cisteína/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
MsrA and MsrB catalyze the reduction of methionine-S-sulfoxide and methionine-R-sulfoxide, respectively, to methionine in different cellular compartments of mammalian cells. One of the three MsrBs, MsrB3, is an endoplasmic reticulum (ER)-type enzyme critical for stress resistance including oxidative and ER stresses. However, there is no evidence for the presence of an ER-type MsrA or the ER localization of MsrA. In this work, we developed an ER-targeted recombinant MsrA construct and investigated the potential effects of methionine-S-sulfoxide reduction in the ER on stress resistance. The ER-targeted MsrA construct contained the N-terminal ER-targeting signal peptide of human MsrB3A (MSPRRSLPRPLSLCLSLCLCLCLAAALGSAQ) and the C-terminal ER-retention signal sequence (KAEL). The over-expression of ER-targeted MsrA significantly increased cellular resistance to H2O2-induced oxidative stress. The ER-targeted MsrA over-expression also significantly enhanced resistance to dithiothreitol-induced ER stress; however, it had no positive effects on the resistance to ER stresses induced by tunicamycin and thapsigargin. Collectively, our data suggest that methionine-S-sulfoxide reduction in the ER compartment plays a protective role against oxidative and ER stresses.
Asunto(s)
Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Estrés Oxidativo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Metionina Sulfóxido Reductasas/química , Ratones , Datos de Secuencia MolecularRESUMEN
Methionine sulfoxide reductase B3A (MsrB3A), which catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine, is localized to the endoplasmic reticulum (ER). Here, we report a critical role of the ER-targeted MsrB3 in protection against ER stress in Drosophila and in mammalian cells. Flies overexpressing human MsrB3A exhibited significantly increased resistance to ER stress induced by dithiothreitol. These flies also showed slightly enhanced resistance to tunicamycin-induced ER stress. In addition, overexpression of MsrB3A in mammalian cells increased resistance to dithiothreitol- and thapsigargin-induced ER stresses. However, MsrB3A overexpression had no effect on the resistance to tunicamycin-induced ER stress. Knockdown of MsrB3A in mammalian cells led to a significant decrease in the resistance to thapsigargin-induced ER stress, but had no effects on the resistance to either dithiothreitol- or tunicamycin-induced ER stress. Collectively, our data provide evidence that the ER-type of MsrB3 plays an important role in protection against ER stress, suggesting that MsrB3 may be involved in the regulation of ER homeostasis.
Asunto(s)
Drosophila melanogaster/fisiología , Estrés del Retículo Endoplásmico/genética , Estrés del Retículo Endoplásmico/fisiología , Retículo Endoplásmico/enzimología , Metionina Sulfóxido Reductasas/fisiología , Estrés Oxidativo/genética , Estrés Oxidativo/fisiología , Animales , Animales Modificados Genéticamente , Línea Celular , Regulación hacia Abajo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Metionina Sulfóxido Reductasas/genética , Estrés Oxidativo/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
There have been insufficient kinetic data that enable a direct comparison between free and peptide methionine sulfoxide reductase activities of either MsrB or MsrA. In this study, we determined the kinetic parameters of mammalian and yeast MsrBs and MsrAs for the reduction of both free methionine sulfoxide (Met-O) and peptidyl Met-O under the same assay conditions. Catalytic efficiency of mammalian and yeast MsrBs towards free Met-O was >2000-fold lower than that of yeast fRMsr, which is specific for free Met-R-O. The ratio of free to peptide Msr activity in MsrBs was 1:20-40. In contrast, mammalian and yeast MsrAs reduced free Met-O much more efficiently than MsrBs. Their k(cat) values were 40-500-fold greater than those of the corresponding MsrBs. The ratio of free to peptide Msr activity was 1:0.8 in yeast MsrA, indicating that this enzyme can reduce free Met-O as efficiently as peptidyl Met-O. In addition, we analyzed the in vivo free Msr activities of MsrBs and MsrAs in yeast cells using a growth complementation assay. Mammalian and yeast MsrBs, as well as the corresponding MsrAs, had apparent in vivo free Msr activities. The in vivo free Msr activities of MsrBs and MsrAs agreed with their in vitro activities.
Asunto(s)
Metionina Sulfóxido Reductasas/metabolismo , Metionina/análogos & derivados , Péptidos/metabolismo , Animales , Eliminación de Gen , Humanos , Cinética , Metionina/metabolismo , Metionina Sulfóxido Reductasas/genética , Ratones , Oxidación-Reducción , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Free methionine-R-sulfoxide reductase (fRMsr) reduces free methionine R-sulfoxide back to methionine, but its catalytic mechanism is poorly understood. Here, we have determined the crystal structures of the reduced, substrate-bound, and oxidized forms of fRMsr from Staphylococcus aureus. Our structural and biochemical analyses suggest the catalytic mechanism of fRMsr in which Cys(102) functions as the catalytic residue and Cys(68) as the resolving Cys that forms a disulfide bond with Cys(102). Cys(78), previously thought to be a catalytic Cys, is a non-essential residue for catalytic function. Additionally, our structures provide insights into the enzyme-substrate interaction and the role of active site residues in substrate binding. Structural comparison reveals that conformational changes occur in the active site during catalysis, particularly in the loop of residues 97-106 containing the catalytic Cys(102). We have also crystallized a complex between fRMsr and isopropyl alcohol, which acts as a competitive inhibitor for the enzyme. This isopropyl alcohol-bound structure helps us to understand the inhibitory mechanism of fRMsr. Our structural and enzymatic analyses suggest that a branched methyl group in alcohol seems important for competitive inhibition of the fRMsr due to its ability to bind to the active site.
Asunto(s)
Metionina Sulfóxido Reductasas/química , Staphylococcus aureus/enzimología , Catálisis , Cisteína/química , Dimerización , Disulfuros/química , Cinética , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Mutación , Péptidos/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a ß-strand rich globular protein consisting of eight antiparallel ß-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.
Asunto(s)
Metionina Sulfóxido Reductasas/química , Metionina Sulfóxido Reductasas/metabolismo , Animales , Sitios de Unión , Catálisis , Dominio Catalítico , Concentración de Iones de Hidrógeno , Ratones , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de ProteínaRESUMEN
Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193-205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.
RESUMEN
Free methionine-R-sulfoxide reductase (fRMsr) is a new type of methionine sulfoxide reductase that catalyzes the reduction of free methionine-R-sulfoxide to methionine. This enzyme cannot reduce oxidized methionine residues in proteins. While three Cys residues, Cys-91, Cys-101 and Cys-125, have been demonstrated to be involved in the catalysis by Saccharomyces cerevisiae fRMsr, their specific functions have not been fully established. In this work, we performed in vivo growth complementation experiments using S. cerevisiae cells lacking all three known methionine sulfoxide reductases. Cells containing a C125S construct, in which Cys-125 in fRMsr was replaced with Ser, did not grow in methionine sulfoxide medium, whereas cells containing C91S, C101S, or C91/101S constructs could grow in this medium. In addition, when assayed with thioredoxin and glutaredoxin reduction systems, the C125S form was inactive, whereas C91S and C101S had 1-2% and 9-10%, respectively, of the activity of the wild-type fRMsr. These data show that Cys-125 is the catalytic residue in fRMsr.
Asunto(s)
Cisteína/metabolismo , Metionina Sulfóxido Reductasas/metabolismo , Saccharomyces cerevisiae/enzimología , Dominio Catalítico/genética , Cisteína/genética , Prueba de Complementación Genética , Metionina Sulfóxido Reductasas/genética , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Clostridium oremlandii MsrA (CoMsrA) is a natively selenocysteine-containing methionine-S-sulfoxide reductase and classified into a 1-Cys type MsrA. CoMsrA exists as a monomer in solution. Herein, we report evidence that CoMsrA can undergo homodimerization during catalysis. The monomeric CoMsrA dimerizes in the presence of its substrate methionine sulfoxide via an intermolecular disulfide bond between catalytic Cys16 residues. The dimeric CoMsrA is resolved by the reductant glutaredoxin, suggesting the relevance of dimerization in catalysis. The dimerization reaction occurs in a concentration- and time-dependent manner. In addition, the occurrence of homodimer formation in the native selenoprotein CoMsrA is confirmed. We also determine the crystal structure of the dimeric CoMsrA, having the dimer interface around the two catalytic Cys16 residues. A central cone-shaped hole is present in the surface model of dimeric structure, and the two Cys16 residues constitute the base of the hole. Collectively, our biochemical and structural analyses suggest a novel dimerization-mediated mechanism for CoMsrA catalysis that is additionally involved in CoMsrA regeneration by glutaredoxin.
Asunto(s)
Clostridium/enzimología , Metionina Sulfóxido Reductasas/metabolismo , Algoritmos , Catálisis , Cristalografía por Rayos X , Disulfuros/química , Glutarredoxinas/química , Espectrometría de Masas , Modelos Moleculares , Oxidación-Reducción , Multimerización de Proteína , Selenocisteína/metabolismo , Selenoproteínas/metabolismoRESUMEN
Human methionine sulfoxide reductase B3A (hMsrB3A) is an endoplasmic reticulum (ER) reductase that catalyzes the stereospecific reduction of methionine-R-sulfoxide to methionine in proteins. In this work, we identified an antimicrobial peptide from hMsrB3A protein. The N-terminal ER-targeting signal peptide (amino acids 1-31) conferred an antimicrobial effect in Escherichia coli cells. Sequence and structural analyses showed that the overall positively charged ER signal peptide had an Argand Pro-rich region and a potential hydrophobic α-helical segment that contains 4 cysteine residues. The potential α-helical region was essential for the antimicrobial activity within E. coli cells. A synthetic peptide, comprised of 2-26 amino acids of the signal peptide, was effective at killing Gram-negative E. coli, Klebsiella pneumoniae, and Salmonella paratyphi, but had no bactericidal activity against Gram-positive Staphylococcus aureus.
Asunto(s)
Antiinfecciosos , Péptidos Catiónicos Antimicrobianos , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Metionina Sulfóxido Reductasas/química , Señales de Clasificación de Proteína/genética , Antiinfecciosos/química , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Humanos , Metionina Sulfóxido Reductasas/genética , Metionina Sulfóxido Reductasas/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación ProteicaRESUMEN
Dimethyl sulfoxide (DMSO) can be reduced to dimethyl sulfide by MsrA, which stereospecifically catalyzes the reduction of methionine-S-sulfoxide to methionine. Our previous study showed that DMSO can competitively inhibit methionine sulfoxide reduction ability of yeast and mammalian MsrA in both in vitro and in vivo, and also act as a non-competitive inhibitor for mammalian MsrB2, specific for the reduction of methionine-R-sulfoxide, with lower inhibition effects. The present study investigated the effects of DMSO on the physiological antioxidant functions of methionine sulfoxide reductases. DMSO elevated hydrogen peroxide-mediated Saccharomyces cerevisiae cell death, whereas it protected human SK-Hep1 cells against oxidative stress. DMSO reduced the protein-carbonyl content in yeast cells in normal conditions, but markedly increased protein-carbonyl accumulation under oxidative stress. Using Msr deletion mutant yeast cells, we demonstrated the DMSO's selective inhibition of the antioxidant function of MsrA in S. cerevisiae, resulting in an increase in oxidative stress-induced cytotoxicity.