RESUMO
Regulated necrosis (necroptosis) and apoptosis are crucially involved in severe cardiac pathological conditions, including myocardial infarction, ischemia-reperfusion injury and heart failure. Whereas apoptotic signaling is well defined, the mechanisms that underlie cardiomyocyte necroptosis remain elusive. Here we show that receptor-interacting protein 3 (RIP3) triggers myocardial necroptosis, in addition to apoptosis and inflammation, through activation of Ca(2+)-calmodulin-dependent protein kinase (CaMKII) rather than through the well-established RIP3 partners RIP1 and MLKL. In mice, RIP3 deficiency or CaMKII inhibition ameliorates myocardial necroptosis and heart failure induced by ischemia-reperfusion or by doxorubicin treatment. RIP3-induced activation of CaMKII, via phosphorylation or oxidation or both, triggers opening of the mitochondrial permeability transition pore and myocardial necroptosis. These findings identify CaMKII as a new RIP3 substrate and delineate a RIP3-CaMKII-mPTP myocardial necroptosis pathway, a promising target for the treatment of ischemia- and oxidative stress-induced myocardial damage and heart failure.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Insuficiência Cardíaca/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Isquemia Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/genética , Miócitos Cardíacos/metabolismo , Necrose/genética , Estresse Oxidativo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Apoptose/genética , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Sobrevivência Celular , Doxorrubicina/toxicidade , Insuficiência Cardíaca/induzido quimicamente , Insuficiência Cardíaca/patologia , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Poro de Transição de Permeabilidade Mitocondrial , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos Cardíacos/patologia , Necrose/etiologia , Necrose/patologia , Fosforilação , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase em Tempo Real , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismoRESUMO
SCOPE: Mushrooms are well known for their nutritional and medicinal value. Agrocybe aegerita has been used as a nutritious food around the world and for its herbal medicinal properties in Asia. In recent years, several antitumor proteins have been identified from A. aegerita. The objective of this study was to purify a novel antitumor protein from A. aegerita. METHODS AND RESULTS: A novel antitumor protein A. aegerita deoxyribonuclease (AAD) was purified through a two-step chromatographic procedure and was shown to possess antitumor activity against different cancer cell lines. Cells treated with AAD produced typical apoptotic morphological changes, which include chromatin condensation, the accumulation of sub-G1 cells and caspase-8 cleavage. Biochemical characterization of AAD showed that it was a member of the DNase I family and that it possessed divalent metal ion-dependent endonuclease activity. The optimal temperature for AAD activity was 50°C and its optimal pH was 8.5. The MS-identified peptides of AAD were found to match to Unigene3821, which has 97% homology with Aa-Pri1, in our A. aegerita transcriptome. CONCLUSION: We have identified a novel antitumor protein from A. aegerita. This fuller understanding of A. aegerita would help us to enhance its use in nutritional and medical applications.