RESUMO
The role and responses of the dimeric DJ-1 protein to cardiac oxidative stress is incompletely understood. H2O2 induces a 50-kDa DJ-1 interprotein homodimer disulfide, known to form between Cys-53 on each subunit. A trimeric 75-kDa DJ-1 complex that mass spectrometry shows contained 2-Cys peroxiredoxin also formed and precedes the appearance of the disulfide dimer. These observations may represent peroxiredoxin sensing and transducing the oxidant signal to DJ-1. The dimeric disulfide DJ-1 complex was stabilized by auranofin, suggesting that thioredoxin recycles it in cells. Higher concentrations of H2O2 concomitantly induce DJ-1 Cys-106 hyperoxidation (sulfination or sulfonation) in myocytes, perfused heart, or HEK cells. An oxidation-resistant C53A DJ-1 shows potentiated H2O2-induced Cys-106 hyperoxidation. DJ-1 also forms multiple disulfides with unknown target proteins during H2O2 treatment, the formation of which is also potentiated in cells expressing the C53A mutant. This suggests that the intersubunit disulfide induces a conformational change that limits Cys-106 forming heterodisulfide protein complexes or from hyperoxidizing. High concentrations of H2O2 also induce cell death, with DJ-1 Cys-106 sulfonation appearing causal in these events, as expressionof C53A DJ-1 enhanced both Cys-106 sulfonation and cell death. Nonetheless, expression of the DJ-1 C106A mutant, which fully prevents hyperoxidation, also showed exacerbated cell death responses to H2O2 A rational explanation for these findings is that DJ-1 Cys-106 forms disulfides with target proteins to limit oxidant-induced cell death. However, when Cys-106 is hyperoxidized, formation of these potentially protective heterodimeric disulfide complexes is limited, and so cell death is exacerbated.
Assuntos
Dissulfetos/química , Peróxido de Hidrogênio/farmacologia , Proteínas Associadas aos Microtúbulos/metabolismo , Oxidantes/farmacologia , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , Animais , Western Blotting , Células Cultivadas , Imunofluorescência , Células HEK293 , Humanos , Imunoprecipitação , Masculino , Mutação/genética , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxirredução , Peroxirredoxinas/genética , Proteína Desglicase DJ-1 , Proteômica , Ratos , Ratos WistarRESUMO
Cardiac myosin-binding protein C (cMyBP-C) regulates actin-myosin interaction and thereby cardiac myocyte contraction and relaxation. This physiologic function is regulated by cMyBP-C phosphorylation. In our study, reduced site-specific cMyBP-C phosphorylation coincided with increased S-glutathiolation in ventricular tissue from patients with dilated or ischemic cardiomyopathy compared to nonfailing donors. We used redox proteomics, to identify constitutive and disease-specific S-glutathiolation sites in cMyBP-C in donor and patient samples, respectively. Among those, a cysteine cluster in the vicinity of the regulatory phosphorylation sites within the myosin S2 interaction domain C1-M-C2 was identified and showed enhanced S-glutathiolation in patients. In vitro S-glutathiolation of recombinant cMyBP-C C1-M-C2 occurred predominantly at Cys(249), which attenuated phosphorylation by protein kinases. Exposure to glutathione disulfide induced cMyBP-C S-glutathiolation, which functionally decelerated the kinetics of Ca(2+)-activated force development in ventricular myocytes from wild-type, but not those from Mybpc3-targeted knockout mice. These oxidation events abrogate protein kinase-mediated phosphorylation of cMyBP-C and therefore potentially contribute to the reduction of its phosphorylation and the contractile dysfunction observed in human heart failure.-Stathopoulou, K., Wittig, I., Heidler, J., Piasecki, A., Richter, F., Diering, S., van der Velden, J., Buck, F., Donzelli, S., Schröder, E., Wijnker, P. J. M., Voigt, N., Dobrev, D., Sadayappan, S., Eschenhagen, T., Carrier, L., Eaton, P., Cuello, F. S-glutathiolation impairs phosphoregulation and function of cardiac myosin-binding protein C in human heart failure.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Glutationa/metabolismo , Insuficiência Cardíaca/metabolismo , Adulto , Animais , Fármacos Cardiovasculares/uso terapêutico , Proteínas de Transporte/genética , Feminino , Insuficiência Cardíaca/tratamento farmacológico , Ventrículos do Coração/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Oxirredução , Fosforilação , Adulto JovemRESUMO
The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a ß-bulge at the C-terminus of ß-strand 3, which is a feature observed in many proteins of this superfamily.
Assuntos
Proteínas de Bactérias/química , Oxigenases/química , Pseudomonas putida/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Cristalografia por Raios X , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oxigenases/genética , Oxigenases/metabolismo , Plasmídeos/genética , Conformação Proteica , Dobramento de Proteína , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Alinhamento de SequênciaRESUMO
Exogenous H(2)O(2) is widely applied to cardiovascular tissues in order to elicit oxidant-dependent responses relevant to signalling and disease. Lower levels of endogenous H(2)O(2) are essential for normal physiological functioning and signalling, whereas higher levels are associated with disease. Within diseased tissues, concentrations in excess of 100 microM have been measured, though 1-15 microM appears to be the upper limit of the healthy physiological range. Analysing the kinetic constants and abundance of peroxidases suggests that they may, on occasion, encounter tissue H(2)O(2) concentrations as high as 1 mM. Extracellular application of 0.01-1 mM peroxide appears to be directly relevant to biology and broadly mimics the release of H(2)O(2) endogenously by growth factors and other effectors. However, the intracellular H(2)O(2) may only ever reach 1-15% of the applied exogenous concentration.
Assuntos
Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Oxidantes/farmacologia , Animais , Doenças Cardiovasculares/fisiopatologia , Relação Dose-Resposta a Droga , Humanos , Peróxido de Hidrogênio/administração & dosagem , Peróxido de Hidrogênio/metabolismo , Modelos Biológicos , Oxidantes/administração & dosagem , Oxidantes/metabolismo , Transdução de SinaisRESUMO
Peroxiredoxins (Prxs) are a ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels which mediate signal transduction in mammalian cells. Prxs can be regulated by changes to phosphorylation, redox and possibly oligomerization states. Prxs are divided into three classes: typical 2-Cys Prxs; atypical 2-Cys Prxs; and 1-Cys Prxs. All Prxs share the same basic catalytic mechanism, in which an active-site cysteine (the peroxidatic cysteine) is oxidized to a sulfenic acid by the peroxide substrate. The recycling of the sulfenic acid back to a thiol is what distinguishes the three enzyme classes. Using crystal structures, a detailed catalytic cycle has been derived for typical 2-Cys Prxs, including a model for the redox-regulated oligomeric state proposed to control enzyme activity.
Assuntos
Peroxidases/metabolismo , Sítios de Ligação , Catálise , Dimerização , Oxirredução , Peroxidases/química , Peroxirredoxinas , Conformação ProteicaRESUMO
A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].
RESUMO
The production of a higher-order assembly of peroxiredoxin-2 (Prx-2) from human erythrocytes has been achieved during specimen preparation on holey carbon support films, in the presence of ammonium molybdate and polyethylene glycol. TEM study suggested that this assembly is a regular dodecahedron, containing 12 Prx-2 decamers (Mr 2.62 MDa, external diameter approximately 20 nm). This interpretation has been supported by production of a approximately 1.6 nm 3D reconstruction from the negative stain TEM data, with automated docking of the available X-ray data of the Prx-2 decamer. Comparison with other known protein dodecahedral and viral icosahedral structures indicates that this arrangement of protein molecules is one of the fundamental macromolecular higher-order assemblies found in biology. Widespread biotechnological interest in macromolecular "cage" structures is relevant to the production of the Prx-2 dodecahedron.
Assuntos
Eritrócitos/química , Peroxidases/química , Estrutura Quaternária de Proteína , Humanos , Processamento de Imagem Assistida por Computador , Substâncias Macromoleculares , Microscopia Eletrônica de Transmissão , Modelos Moleculares , PeroxirredoxinasRESUMO
Protein sulfenic acids (SOHs) are the principal oxidation products formed when redox active proteins interact with peroxide molecules. We have developed a new antibody reagent that detects protein SOHs derivatized with dimedone. Using this new antibody, we found that glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is the predominant protein sulfenate present in isolated rat ventricular myocytes under basal conditions. During oxidative stress with hydrogen peroxide (H(2)O(2)), GAPDH SOH labeling is lost, but a number of secondary dimedone-reactive protein sulfenates then appear. As the sulfenate labeling is lost, the Cys-149 sulfinic/sulfonic acid oxidation states of GAPDH appear. This hyperoxidized GAPDH is associated with both the inhibition of glycolysis and its ability to reduce H(2)O(2). We examined whether inactivation of GAPDH was causative in the generation of secondary protein sulfenates that coincide with its hyperoxidation. The selective GAPDH inhibitor koningic acid (which functions by forming a covalent adduct at Cys-149) fully prevented basal SOH labeling, as well as subsequent peroxide-induced hyperoxidation. However, koningic acid-mediated inhibition of GAPDH alone did not induce the formation of intracellular H(2)O(2) or secondary protein sulfenates and also failed to potentiate their peroxide-induced formation. Overall, GAPDH appears to have peroxidase-like properties, but its inhibition failed to impact on downstream oxidant signaling involving secondary protein sulfenation.
Assuntos
Anticorpos/imunologia , Cicloexanonas/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Peróxido de Hidrogênio/metabolismo , Transdução de Sinais , Ácidos Sulfênicos/imunologia , Animais , Anticorpos/análise , Cicloexanonas/análise , Cicloexanonas/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/enzimologia , Ventrículos do Coração/metabolismo , Masculino , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Ácidos Sulfênicos/análise , Ácidos Sulfênicos/metabolismoRESUMO
MRCKα and MRCKß (myotonic dystrophy kinase-related Cdc42-binding kinases) belong to a subfamily of Rho GTPase activated serine/threonine kinases within the AGC-family that regulate the actomyosin cytoskeleton. Reflecting their roles in myosin light chain (MLC) phosphorylation, MRCKα and MRCKß influence cell shape and motility. We report further evidence for MRCKα and MRCKß contributions to the invasion of cancer cells in 3-dimensional matrix invasion assays. In particular, our results indicate that the combined inhibition of MRCKα and MRCKß together with inhibition of ROCK kinases results in significantly greater effects on reducing cancer cell invasion than blocking either MRCK or ROCK kinases alone. To probe the kinase ligand pocket, we screened 159 kinase inhibitors in an in vitro MRCKß kinase assay and found 11 compounds that inhibited enzyme activity >80% at 3 µM. Further analysis of three hits, Y-27632, Fasudil and TPCA-1, revealed low micromolar IC(50) values for MRCKα and MRCKß. We also describe the crystal structure of MRCKß in complex with inhibitors Fasudil and TPCA-1 bound to the active site of the kinase. These high-resolution structures reveal a highly conserved AGC kinase fold in a typical dimeric arrangement. The kinase domain is in an active conformation with a fully-ordered and correctly positioned αC helix and catalytic residues in a conformation competent for catalysis. Together, these results provide further validation for MRCK involvement in regulation of cancer cell invasion and present a valuable starting point for future structure-based drug discovery efforts.
Assuntos
Invasividade Neoplásica/patologia , Inibidores de Proteínas Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/química , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Amidas/química , Amidas/farmacologia , Domínio Catalítico , Linhagem Celular Tumoral , Colágeno/metabolismo , Cristalografia por Raios X , Combinação de Medicamentos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Concentração Inibidora 50 , Laminina/metabolismo , Modelos Moleculares , Miotonina Proteína Quinase , Inibidores de Proteínas Quinases/análise , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica/efeitos dos fármacos , Proteoglicanas/metabolismo , Piridinas/química , Piridinas/farmacologia , Tiofenos/química , Tiofenos/farmacologia , Quinases Associadas a rho/antagonistas & inibidores , Quinases Associadas a rho/metabolismoRESUMO
Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. ß-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters.
Assuntos
Actomiosina/fisiologia , Epiderme/patologia , Neoplasias Cutâneas/etiologia , beta Catenina/fisiologia , Animais , Fenômenos Biomecânicos , Proliferação de Células , Células Cultivadas , Humanos , Hiperplasia , Camundongos , Papiloma/etiologia , Transdução de Sinais , Quinases Associadas a rho/análise , Quinases Associadas a rho/genética , Quinases Associadas a rho/fisiologiaRESUMO
Peroxiredoxins (Prdxs), a family of antioxidant and redox-signaling proteins, are plentiful within the heart; however, their cardiac functions are poorly understood. These studies were designed to characterize the complex changes in Prdxs induced by oxidant stress in rat myocardium. Hydrogen peroxide, a Prdx substrate, was used as the model oxidant pertinent to redox signaling during health and to injury at higher concentrations. Rat hearts were aerobically perfused with a broad concentration range of hydrogen peroxide by the Langendorff method, homogenized, and analyzed by immunoblotting. Heart extracts were also analyzed by size-exclusion chromatography under nondenaturing conditions. Hydrogen peroxide-induced changes in disulfide bond formation, nonreversible oxidation of cysteine (hyperoxidation), and subcellular localization were determined. Hydrogen peroxide induced an array of changes in the myocardium, including formation of disulfide bonds that were intermolecular for Prdx1, Prdx2, and Prdx3 but intramolecular within Prdx5. For Prdx1, Prdx2, and Prdx5, disulfide bond formation can be approximated to an EC(50) of 10-100, 1-10, and 100-1,000 microM peroxide, respectively. Hydrogen peroxide induced hyperoxidation, not just within monomeric Prdx (by SDS-PAGE), but also within Prdx disulfide dimers, and reflects a flexibility within the dimeric unit. Prdx oxidation was also associated with movement from the cytosolic to the membrane and myofilament-enriched fractions. In summary, Prdxs undergo a complex series of redox-dependent structural changes in the heart in response to oxidant challenge with its substrate hydrogen peroxide.
Assuntos
Peróxido de Hidrogênio/metabolismo , Miocárdio/enzimologia , Oxidantes/metabolismo , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Animais , Cromatografia em Gel , Cisteína/metabolismo , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Peróxido de Hidrogênio/toxicidade , Immunoblotting , Técnicas In Vitro , Masculino , Oxidantes/toxicidade , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Perfusão , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
Protein sulfenic acids are reactive intermediates in the catalytic cycles of many enzymes as well as the in formation of other redox states. Sulfenic acid formation is a reversible post-translational modification with potential for protein regulation. Dimedone (5,5-dimethyl-1,3-cyclohexanedione) is commonly used in vitro to study sulfenation of purified proteins, selectively "tagging" them, allowing monitoring by mass spectrometry. However dimedone is of little use in complex protein mixtures because selective monitoring of labeling is not possible. To address this issue, we synthesized a novel biotinylated derivative of dimedone, keeping the dione cassette required for sulfenate reactivity but adding the functionality of a biotin tag. Biotin-amido(5-methyl-5-carboxamidocyclohexane 1,3-dione) tetragol (biotin dimedone) was prepared in six steps, combining 3,5-dimethoxybenzoic acid (Birch reduction, ultimately leading to the dimedone unit with a carboxylate functionality), 1-amino-11-azido-3,6,9-trioxaundecane (a differentially substituted tetragol spacer), and biotin. We loaded biotin dimedone (0.1 mm, 30 min) into rat ventricular myocytes, treated them with H(2)O(2) (0.1-10,000 microm, 5 min), and monitored derivatization on Western blots using streptavidin-horseradish peroxidase. There was a dose-dependent increase in labeling of multiple proteins that was maximal at 0.1 or 1 mm H(2)O(2) and declined sharply below basal with 10 mm treatment. Cell-wide labeling was observed in fixed cells probed with avidin-FITC using a confocal fluorescence microscope. Similar H(2)O(2)-induced labeling was observed in isolated rat hearts. Hearts loaded and subjected to hypoxia showed a striking loss of labeling, which returned when oxygen was resupplied, highlighting the protein sulfenates as oxygen sensors. Cardiac proteins that were sulfenated during oxidative stress were purified with avidin-agarose and identified by separation of tryptic digests by liquid chromatography with on-line analysis by mass spectrometry.
Assuntos
Cicloexanonas/farmacologia , Proteômica/instrumentação , Ácidos Sulfênicos/química , Animais , Biotina/química , Cromatografia Líquida , Cicloexanonas/química , Peroxidase do Rábano Silvestre/metabolismo , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/farmacologia , Modelos Químicos , Células Musculares/metabolismo , Oxirredução , Estresse Oxidativo , Oxigênio/metabolismo , Proteínas/química , Proteômica/métodos , Ratos , Tripsina/químicaRESUMO
Changes in the concentration of oxidants in cells can regulate biochemical signaling mechanisms that control cell function. We have found that guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) functions directly as a redox sensor. The Ialpha isoform, PKGIalpha, formed an interprotein disulfide linking its two subunits in cells exposed to exogenous hydrogen peroxide. This oxidation directly activated the kinase in vitro, and in rat cells and tissues. The affinity of the kinase for substrates it phosphorylates was enhanced by disulfide formation. This oxidation-induced activation represents an alternate mechanism for regulation along with the classical activation involving nitric oxide and cGMP. This mechanism underlies cGMP-independent vasorelaxation in response to oxidants in the cardiovascular system and provides a molecular explantion for how hydrogen peroxide can operate as an endothelium-derived hyperpolarizing factor.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Cisteína/metabolismo , Oxidantes/metabolismo , Animais , Aorta , Linhagem Celular , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I , Proteínas Quinases Dependentes de GMP Cíclico/genética , Dissulfetos/metabolismo , Ativação Enzimática , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Óxido Nítrico/metabolismo , Oxirredução , Estresse Oxidativo , Ratos , Ratos Wistar , Transdução de Sinais , Técnicas de Cultura de Tecidos , Transfecção , Vasodilatação/fisiologiaRESUMO
Here we demonstrate that type I protein kinase A is redoxactive, forming an interprotein disulfide bond between its two regulatory RI subunits in response to cellular hydrogen peroxide. This oxidative disulfide formation causes a subcellular translocation and activation of the kinase, resulting in phosphorylation of established substrate proteins. The translocation is mediated at least in part by the oxidized form of the kinase having an enhanced affinity for alpha-myosin heavy chain, which serves as a protein kinase A (PKA) anchor protein and localizes the PKA to its myofilament substrates troponin I and myosin binding protein C. The functional consequence of these events in cardiac myocytes is that hydrogen peroxide increases contractility independently of beta-adrenergic stimulation and elevations of cAMP. The oxidant-induced phosphorylation of substrate proteins and increased contractility is blocked by the kinase inhibitor H89, indicating that these events involve PKA activation. In essence, type I PKA contains protein thiols that operate as redox sensors, and their oxidation by hydrogen peroxide directly activates the kinase.