Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here we show that aB-crystallin interacts with and confers protection to FAK against calpainmediated proteolysis in cardiomyocytes. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the b4-b8 groove of aB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation. aB-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 aB-crystallin against the stretch-induced apoptosis. Consistently, load-induced apoptosis is blunted in the hearts from cardiac-specific FAK transgenic mice transiently depleted of aB-crystallin by RNA interference. These studies define a role for aB-crystallin in controlling FAK function and cardiomyocyte survival through the prevention of calpain-mediated degradation of FAK.
Focal adhesion kinase (FAK) regulates cellular processes that affect several aspects of development and disease. The FAK N-terminal FERM (4.1 protein-ezrin-radixin-moesin homology) domain, a compact clover-leaf structure, binds partner proteins and mediates intramolecular regulatory interactions. Combined chemical cross-linking coupled to MS, small-angle X-ray scattering, computational docking and mutational analyses showed that the FAK FERM domain has a molecular cleft (~998 Å(2)) that interacts with sarcomeric myosin, resulting in FAK inhibition. Accordingly, mutations in a unique short amino acid sequence of the FERM myosin cleft, FP-1, impaired the interaction with myosin and enhanced FAK activity in cardiomyocytes. An FP-1 decoy peptide selectively inhibited myosin interaction and increased FAK activity, promoting cardiomyocyte hypertrophy through activation of the AKT-mammalian target of rapamycin pathway. Our findings uncover an inhibitory interaction between the FAK FERM domain and sarcomeric myosin that presents potential opportunities to modulate the cardiac hypertrophic response through changes in FAK activity.
The small heat shock protein aB-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.
Focal adhesion kinase (FAK) contributes to cellular homeostasis under stress conditions. Here, we show that αB-crystallin confers protection to FAK against calpain-mediated proteolysis under mechanical stress in cardiomyocytes. Biochemical assays, chemical cross-linking coupled to mass spectrometry experiments, mutational analyses and Förster resonance energy transfer (FRET) were combined to investigate the basis of FAK and αB-crystallin interaction. A hydrophobic patch mapped between helices 1 and 4 of the FAK FAT domain was found to bind to the β4-β8 groove of αB-crystallin. Such an interaction requires FAK tyrosine 925 and is enhanced following its phosphorylation by Src, which occurs upon FAK stimulation by mechanical stress. αB-crystallin silencing results in calpain-dependent FAK depletion and in increased apoptosis of cardiomyocytes. The overexpression of a myc-FAK construct or treatment with a calpain inhibitor (E64) restored the survival of cardiomyocytes depleted of αB-crystallin. The association between FAK and αB-crystallin was also demonstrated to occur in response to pressure overload in rat left ventricle. The myocardial depletion of αB-crystallin by siRNA results in enhanced apoptosis of cardiomyocytes and myocardial fibrosis in overloaded hearts. These alterations were markedly attenuated in the overloaded left ventricles of transgenic mice with cardiac specific FAK expression. These findings define a novel mechanism by which αB-crystallin controls FAK function, with impact on cardiomyocyte survival and cardiac remodelling in response to stress.
AimFocal adhesion kinase (FAK) is activated and plays a role in cardiomyocyte hypertrophy induced by mechanical stress. FAK activation may depend on cooperation with protein partners. We investigated whether interaction with alpha‐B crystalline (αB Crys) regulates FAK activation by mechanical stress in cardiomyocytes.MethodsαB Crys and FAK expression and phosphorylation at Tyr397 (pFAK) were quantified in neonatal rat ventricular myocytes (NRVMs). FAK/αB Crys interaction was investigated by co‐immunoprecipitation and pulldown assays. αB Crys depletion by RNAi was used to examine its effect on FAK activation.ResultsCyclic stretch (10%, 2 hs) increased pFAK (2‐fold) and enhanced αB Crys expression (4‐fold) and phosphorylation at Ser59 (3‐fold). Stretch also induced FAK/αB Crys association. Pulldown assays with recombinant FAK domains indicated that the interaction with αB Crys is mediated by FAK C‐terminal domain. FAK immunostaining was coincident with myofilaments in non‐stretched while in stretched cells it was predominantly located in the nuclei. αB Crys was predominantly located at the nuclei of stretched NRVMs, coincident with FAK staining. Depletion of αB Crys markedly reduced FAK activation and re‐location to the nucleus.ConclusionThis study demonstrates that αB Crys associates and regulates FAK activation in cardiomyocytes in response to mechanical stress.
e4506067Active learning occurs when the student interacts with the subject under study by listening, speaking, doing and teaching. Thus, the student is encouraged to build knowledge instead of passively receiving it from the teacher. In this sense, the construction of educational games encourages active learning, then students are challenged to seek knowledge and practice it to win. The general objective of this articlo, which used a case study methodology, is to present whether the systematization of a game created and evaluated for the study can contribute to increase the knowledge in genetics of the students involved. This methodology was used in two schools in the Vale do Aço / MG region, involving 364 students, in the year 2019. Students were organized into groups and encouraged to build genotypes that condition predetermined phenotypes, using available alleles. The winning group would be the one that achieved the greatest number of genetic constitutions in the established time. Students evaluated, in a questionnaire, the contribution of the game as being positive in learning genetics. There was significant variation in student performance on an issue directly linked to gambling that was tested at the study. The game stimulated the development of important skills and competences that cannot be measured and/or quantified through the test performed by the students. The study allowed us to conclude that the use of teaching support tools by teachers can make teaching more attractive and interesting for students, causing them to build and reconstruct new concepts in a participatory way.ResumoA aprendizagem ativa ocorre quando o aluno interage com o assunto em estudo, ouvindo, falando, fazendo e ensinando. Assim, o aluno é estimulado a construir o conhecimento ao invés de recebê-lo passivamente do professor. Nesse sentido, a construção de jogos educativos estimula a aprendizagem ativa, pois os alunos são desafiados a buscar o conhecimento e praticá-lo para vencer. O objetivo geral deste artigo, que utilizou metodologia do tipo de estudo de caso é apresentar se a sistematização de um jogo, criado e avaliado para o estudo, pode contribuir para aumentar o conhecimento em genética dos estudantes envolvidos. Esta investigação foi desenvolvida em duas escolas da região do Vale do Aço/MG, envolvendo 364 estudantes, no ano 2019. Os estudantes foram organizados em grupos e estimulados a construir genótipos que condicionam fenótipos pré-determinados, utilizando alelos disponíveis. O grupo vencedor seria aquele que conseguisse maior número de constituições genéticas no tempo estabelecido. Os alunos avaliaram, em um questionário, a contribuição do jogo como sendo positiva na aprendizagem em genética. Houve variação significativa no desempenho dos estudantes em uma questão diretamente ligada ao jogo, que foi testada no estudo. Como resultado, foi percebido que o jogo estimulou o desenvolvimento de habilidades e competências importantes que não podem ser medidas e/ou quantificadas por meio do teste realizado pelos estudantes. O estudo permitiu concluir que a utilização de ferramentas de apoio ao ensino pelos professores pode tornar o ensino mais atrativo e interessante para os estudantes, provocando-lhes a construir e reconstruir novos conceitos de forma participativa.Palavras-chave: Educação significativa, Ensino cooperativo, Educação lúdica, Aprendizagem Associativa.Keywords: Significant education, Cooperative teaching, Playful education, Associative Learning.ReferencesALVES, Luciana; BIANCHIN, Maysa Alahmar: O jogo como instrumento de aprendizagem. Revista Psicopedagogia, São Paulo, vol. 27, n.83, 282-287, Agosto/2010.ANASTASIOU, Léa das Graças Camargos; ALVES, Leonir Pessati. Processos de ensinagem na universidade: pressupostos para as estratégias de trabalho em aula. 3ª edição / 2ª tiragem. Joinville, SC: UNIVILLE, 2004, 155p.ARAUJO, Adriano Bruno; GUSMÃO, Fabio Alexandre Ferreira. As principais dificuldades encontradas no ensino de genética na educação básica brasileira. 10º Encontro Internacional de Formação de Professores, 2017, ISSN: 2179-0663CATARINACHO, Renata Lobato. O Ensino de Genética com Super-Heróis: Uma Abordagem Mutante na Sala de Aula. Monografia – Universidade Presbiteriana Mackenzie, São Paulo, 2011, 32p.DIESEL, Aline; BALDEZ, Alda Leila Santos; MARTINS, Silvana Neumann. Os princípios das metodologias ativas de ensino: uma abordagem teórica. Thema, Pelotas, V.14, Nº 1, 268-288, Fevereiro/2017.DOUGHERTY, Michael; PLEASANTS, Chad; SOLOW, Lee; ZHANG, Huiping. A Comprehensive Analysis of High School Genetics Standards: Are States Keeping Pace with Modern Genetics? Life Sciences Education, V. 10, 318–327, Abril/2011MALAFAIA, Guilherme; RODRIGUES, Aline Sueli de Lima. Uma reflexão sobre o ensino de Ciências no nível fundamental da educação. Ciência Ensino, Campinas, V. 2, nº 2, p. 1-9, Junho/2008.ANDRÉ, Marli. O que é um estudo de caso qualitativo em Educação? Revista da FAEEBA – Educação e Contemporaneidade, Salvador, v. 22, n. 40, p. 95-103, jul./dez. 2013MESQUITA, Kelly Marinho Vieira; CARDOSO, Josiane Barbosa; VIGARIO, Ana Flávia. O uso de jogos didáticos no processo de ensino e aprendizagem de biologia. II Congresso Nacional de Ensino de Ciências e Formação de Professores. V. 02/2019. Disponível em http://cecifop.sistemasph.com.br. Acesso em 30 julho 2019.NASCIMENTO, Tuliana Euzébio; COUTINHO, Cadidja. Metodologias ativas de aprendizagem e o ensino de Ciências. Multiciência Online, Santiago, V.2, nº 3 p.1-20, Abril/2017SILVA, Ana Carolina Rosa da; SANTOS, Ludmylla Ribeiro dos; SILVA, Fabiana Mota da; COSTA, Livia Ribeiro da; LACERDA, Paloma Lopes de; CLEOPHAS, Maria das Graças. Importância da Aplicação de Atividades Lúdicas no Ensino de Ciências para Crianças. Revista Brasileira de ensino de CT, Curitiba, V.8, nº 3, p. 84-103, Mai-Agosto/2015 TOLMAN, Richard R. Difficulties in genetics problem solving. American Biology Teacher, V. 44, nº 9, p. 525-527, Dezembro/1982.ZUANON, Átima Clemente Alves; DINIZ, Renato Eugênio da Silva. Aulas de Biologia e a participação dos alunos: conhecendo como um grupo de estudantes do ensino médio avalia uma experiência. IV Encontro Nacional de Pesquisa em Educação em Ciências, Novembro/2003 Disponível em: http://fep.if.usp.br/~profis/arquivos/ivenpec/Arquivos/Orais/ORAL045.pdf Acesso em 10 julho 2019ZUANON, Átima Clemente Alves; DINIZ, Renato Eugênio da Silva; NASCIMENTO, Luiziane Helena. Construção de jogos didáticos para o ensino de Biologia: um recurso para integração dos alunos à prática Docente. Revista Brasileira de Ensino de Ciência e Tecnologia. Curitiba, V.3, nº 3 Janeiro/2010
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