RESUMO
The small heat shock protein αB-Crystallin (CryAB, HspB5) and SH2 domain-containing tyrosine phosphatase 2 (Shp2) are important molecules in heart response to pathophysiological stress. Here we show that CryAB interacts with and potentially regulates Shp2 catalytic activity in stretched cardiomyocytes. Such an interaction requires CryAB oligomer to attenuate Shp2 activation. Stretched cardiomyocytes show a robust CryAB/Shp2 association accompanied by a reduction in the Shp2 phosphatase activity. Accordingly, CryAB knock-down in cardiomyocytes enhances Shp2 activity induced by mechanical stress. These results revealed a new role for CryAB, as a modulator of Shp2 phosphatase activity during a functionally relevant stimulus in cardiomyocytes.
Assuntos
Cristalinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Miócitos Cardíacos/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Estresse Fisiológico/fisiologia , Animais , Células Cultivadas , Cristalinas/genética , Ativação Enzimática/fisiologia , Técnicas de Silenciamento de Genes , Proteínas Associadas aos Microtúbulos/genética , Miócitos Cardíacos/citologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Ratos , Ratos Wistar , Estresse MecânicoRESUMO
Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that αB-crystallin interacts with and confers protection to FAK against calpain-mediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the ß4-ß8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. αB-crystallin silencing results in calpain-dependent FAK depletion and in the increased apoptosis of cardiomyocytes in response to mechanical stress. FAK overexpression protects cardiomyocytes depleted of αB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of αB-crystallin by RNA interference. These studies define a role for αB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.
Assuntos
Calpaína/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Regulação Enzimológica da Expressão Gênica , Miócitos Cardíacos/citologia , Cadeia B de alfa-Cristalina/química , Animais , Aorta/metabolismo , Apoptose , Sobrevivência Celular , Transferência Ressonante de Energia de Fluorescência , Inativação Gênica , Homeostase , Masculino , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Modelos Moleculares , Miocárdio/metabolismo , Fosforilação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Estresse Mecânico , Quinases da Família src/metabolismoRESUMO
The main goal of this work is to evaluate some differential protein species in transgenic (T) and nontransgenic (NT) Arabidopsis thaliana plants after their cultivation in the presence or absence of sodium selenite. The transgenic line was obtained through insertion of CaMV 35S controlling nptII gene. Comparative proteomics through 2D-DIGE is carried out in four different groups (NT × T; NT × Se-NT (where Se is selenium); Se-NT × Se-T, and T × Se-T). Although no differential proteins are achieved in the T × Se-T group, for the others, 68 differential proteins (by applying a regulation factor ≥1.5) are achieved, and 27 of them accurately characterized by ESI-MS/MS. These proteins are classified into metabolism, energy, signal transduction, disease/defense categories, and some of them are involved in the glycolysis pathway-Photosystems I and II and ROS combat. Additionally, laser ablation imaging is used for evaluating the Se and sulfur distribution in leaves of different groups, corroborating some results obtained and related to proteins involved in the glycolysis pathway. From these results, it is possible to conclude that the genetic modification also confers to the plant resistance to oxidative stress.
Assuntos
Arabidopsis/genética , Folhas de Planta/genética , Proteômica , Selenito de Sódio/administração & dosagem , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional/métodos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Lasers , Imagem Molecular/métodos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/biossíntese , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
A large majority of the 1000-1500 proteins in the mitochondria are encoded by the nuclear genome, and therefore, they are translated in the cytosol in the form and contain signals to enable the import of proteins into the organelle. The TOM complex is the major translocase of the outer membrane responsible for preprotein translocation. It consists of a general import pore complex and two membrane import receptors, Tom20 and Tom70. Tom70 contains a characteristic TPR domain, which is a docking site for the Hsp70 and Hsp90 chaperones. These chaperones are involved in protecting cytosolic preproteins from aggregation and then in delivering them to the TOM complex. Although highly significant, many aspects of the interaction between Tom70 and Hsp90 are still uncertain. Thus, we used biophysical tools to study the interaction between the C-terminal domain of Hsp90 (C-Hsp90), which contains the EEVD motif that binds to TPR domains, and the cytosolic fragment of Tom70. The results indicate a stoichiometry of binding of one monomer of Tom70 per dimer of C-Hsp90 with a K(D) of 360±30nM, and the stoichiometry and thermodynamic parameters obtained suggested that Tom70 presents a different mechanism of interaction with Hsp90 when compared with other TPR proteins investigated.
Assuntos
Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Sequência de Aminoácidos , Fenômenos Biofísicos , Dimerização , Proteínas de Choque Térmico HSP90/genética , Humanos , Técnicas In Vitro , Cinética , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
Hop is a tetratricopeptide repeat domain (TPR)-containing co-chaperone that is able to directly associate with both Hsp70 and Hsp90. Previous data showed that the TPR2A-domain is the primary site for dimerization and that the TPR2B-domain may also play a role in dimerization. We present Hop-D456G, a mutant within the TPR2B-domain, that is a mixture of monomeric and dimeric species.