RESUMO
Ischemic stroke represents a major cause of adult physical disability, which is triggered by cerebral artery occlusion induced blood flow blockage. MiR-874-3p has been reported to be down-regulated in the brain injury induced by ischemia-reperfusion (I/R), but the direct evidence associated with injury of I/R remains unknown. In this study, we found that miR-874-3p levels significantly decreased in rat I/R brain induced by middle cerebral artery occlusion/reperfusion (MCAO/R) and SH-SY5Y cells following oxygen-glucose deprivation and reperfusion (OGD/R) treatment. Upregulation of miR-874-3p reduced infarct volumes and cell apoptosis in the in vivo I/R stroke model using TTC and TUNEL staining, as well as increased proliferation and inhibited apoptosis in OGD/R induced SH-SY5Y cells by CCK-8, Edu staining and flow cytometry analysis. Mechanistically, bioinformatics analysis and luciferase reporter assay confirmed BCL-2-modifying factor (BMF) and Bcl-2 family protein Bcl-rambo (BCL2L13) were the direct targets of miR-874-3p. Furthermore, BMF or BCL2L13 knockdown also provided significant protection against OGD/R induced injury, while their overexpression reversed the protective effects of miR-874-3p on SH-SY5Y cells following OGD/R. In summary, our results suggest that miR-874-3p attenuated ischemic injury by negatively regulating BMF and BCL2L13, highlighting a novel therapeutic target for ischemic stroke.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Isquemia Encefálica/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Traumatismo por Reperfusão/genética , Regulação para Cima/genética , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose/genética , Sequência de Bases , Isquemia Encefálica/complicações , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo/genética , Glucose/deficiência , Humanos , Masculino , MicroRNAs/metabolismo , Oxigênio , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Traumatismo por Reperfusão/complicaçõesRESUMO
The import of proteins containing the peroxisomal targeting signal 1 (PTS1) into the Leishmania glycosome is dependent on the docking of the PTS1-loaded LdPEX5 cytosolic receptor with LdPEX14 on the glycosome surface. Here we show that, in the absence of PTS1, LdPEX5 is a tetramer that is stabilized by two distinct interaction domains; the first is a coiled-coil motif encompassing residues 277 to 310, whereas the second domain is localized to residues 1 to 202. By using microcalorimetry, surface plasmon resonance, and size exclusion chromatography techniques, we show that PTS1 peptide binding to LdPEX5 tetramers promotes their dissociation into dimeric structures, which are stabilized by a coiled-coil interaction. Moreover, we demonstrated that the resulting LdPEX5-PTS1 complex is remarkably stable and exhibits extremely slow dissociation kinetics. However, binding of LdPEX14 to LdPEX5 modulates the LdPEX5-PTS1 affinity as it decreases the thermodynamic dissociation constant for this latter complex by 10-fold. These changes in the oligomeric state of LdPEX5 and in its affinity for PTS1 ligand upon LdPEX14 binding may explain how, under physiological conditions, LdPEX5 can function to deliver and unload its cargo to the protein translocation machinery on the glycosomal membrane.
Assuntos
Leishmania donovani/metabolismo , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Quaternária de Proteína , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos , Ligação Proteica , Estrutura Secundária de Proteína , Transporte Proteico/fisiologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Several membrane proteins were previously shown to bind to the 5' leader of the chloroplast psbC mRNA in the unicellular eukaryotic alga Chlamydomonas reinhardtii. This study showed that these proteins have affinity for AU-rich RNAs, as determined by competition experiments. In addition, their binding activities are enhanced 13-15-fold by light, and a 46 kDa protein is activated within 1-10 min. This activation could be mediated by the modulation of ADP pools by the light-dependent reactions of photosynthesis and ATP synthase because (1) two inhibitors that block ATP synthesis also prevent this activation and (2) ADP inhibits the RNA-binding activity of this protein in vitro. An inhibitor of Photosystem II diminishes this induction, suggesting that reducing potential generated by the photosynthetic electron transport chain modulates this RNA-binding activity. The RNA-binding activities of two proteins (of 46 and 47 kDa) are inhibited by Mg-protoporphyrin IX methyl ester in vitro suggesting they could be regulated by these intermediates in the chlorophyll biosynthetic pathway.