RESUMO
Mieap, a p53-inducible protein, controls mitochondrial quality by repairing unhealthy mitochondria. During repair, Mieap induces the accumulation of intramitochondrial lysosomal proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria) by interacting with NIX, leading to the elimination of oxidized mitochondrial proteins. Here, we report that an additional mitochondrial outer membrane protein, BNIP3, is also involved in MALM. BNIP3 interacts with Mieap in a reactive oxygen species (ROS)-dependent manner via the BH3 domain of BNIP3 and the coiled-coil domains of Mieap. The knockdown of endogenous BNIP3 expression severely inhibited MALM. Although the overexpression of either BNIP3 or NIX did not cause a remarkable change in the mitochondrial membrane potential (MMP), the co-expression of all three exogenous proteins, Mieap, BNIP3 and NIX, caused a dramatic reduction in MMP, implying that the physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may regulate the opening of a pore in the mitochondrial double membrane. This effect was not related to cell death. These results suggest that two mitochondrial outer membrane proteins, BNIP3 and NIX, mediate MALM in order to maintain mitochondrial integrity. The physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix.
Assuntos
Proteínas de Membrana/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Linhagem Celular Tumoral , Células HCT116 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Modelos Biológicos , Organelas/metabolismo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , Transporte Proteico/genética , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
NEEP21, also designated D4S234E or NSG1, is an endosomal protein expressed in neuronal cells under normal conditions. Here, we report that NEEP21 is a direct transcriptional target gene of the tumor suppressor p53. NEEP21 expression is inducible in non-neuronal human cancer cell lines by exposure to adriamycin, hydrogen peroxide, UV and γ-ray in a p53-dependent manner. Chromatin immunoprecipitation assay indicated that a potential p53-binding site (p53BS) is located in intron 1 of the NEEP21 gene. A reporter assay confirmed that p53BS has p53-responsive activity. The heterologous luciferase gene containing p53BS is also transactivated by p73-ß and p63-γ. The introduction of the NEEP21 gene into various cancer cell lines suppressed cell growth. Infection with an adenovirus vector containing NEEP21 induced apoptotic cell death via caspase-3 activation in many cancer cell lines. The expression of NEEP21 mRNA was remarkably induced by γ-ray irradiation in the spleen of p53+/+ mice but not in that of p53-/- mice. These results suggest that NEEP21 may play a critical role in apoptosis as a mediator of p53.
Assuntos
Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica/genética , Proteínas do Tecido Nervoso/genética , Ativação Transcricional/genética , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular Tumoral , Separação Celular , Imunoprecipitação da Cromatina , Citometria de Fluxo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The tumor suppressor p53 is a transcription factor that induces the transcription of various target genes in response to DNA damage and it protects the cells from malignant transformation. In this study, we performed cDNA microarray analysis and found that the transmembrane protein containing sushi domain (TMPS) gene, which encodes a putative type I transmembrane protein, is a novel p53-target gene. TMPS contains a sushi domain in the extracellular region, which is associated with protein-protein interaction. TMPS expression is induced by endogenous p53 under genotoxic stress in several cancer cell lines. Reporter assay revealed p53-dependent transactivation of the p53 binding-sites (BSs) located in the intron 1 of TMPS. Chromatin immunoprecipitation (ChIP) assay showed that p53 binds to these BSs in vivo. Overexpression of TMPS induced apoptosis through the activation of caspase-3, 8, and 9 in various cancer cell lines. Moreover, γ-irradiation induced the expression of TMPS mRNA in the spleen and colon of p53+/+ mice but not in those of p53-/- mice. These data indicate that TMPS may play a role in p53-dependent apoptosis under DNA damage condition.