RESUMO
Bcl-rambo, also known as BCL2L13, has been reported to regulate apoptosis, mitochondrial fragmentation, and mitophagy. However, the molecular mechanisms by which Bcl-rambo regulates these processes currently remain unclear. In the present study, we identified phosphoglycerate mutase member 5 (PGAM5) as an emerging partner interacting with Bcl-rambo through phenotypic Drosophila screening. The rough eye phenotype induced by human Bcl-rambo was partly rescued by the knockdown of pgam5-2, a mammalian ortholog of PGAM5. Bcl-rambo bound to PGAM5, and their interaction required the Bcl-rambo transmembrane domain. The co-expression of Bcl-rambo and PGAM5 promoted effector caspase activity in human embryonic kidney 293T cells. The transient overexpression of Bcl-rambo increased LC3B-II levels, which had been decreased by the co-expression of PGAM5. These results suggest that PGAM5 promotes Bcl-rambo-dependent apoptosis, but conversely interferes with Bcl-rambo-dependent mitophagy.
Assuntos
Mitofagia , Fosfoglicerato Mutase , Animais , Apoptose/genética , Caspases Efetoras/metabolismo , Drosophila/metabolismo , Humanos , Mamíferos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitofagia/genética , Fosfoglicerato Mutase/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismoRESUMO
We previously reported that the Bcl-2 family member human Bcl-rambo, also known as BCL2L13, induces apoptosis in human embryonic kidney 293T cells. Mouse Bcl-rambo has recently been reported to mediate mitochondrial fragmentation and mitophagy. In the present study, we showed that the transfection of human Bcl-rambo and its microtubule-associated protein light chain 3-interacting region motif mutant (W276A/I279A) caused mitochondrial fragmentation and the perinuclear accumulation of fragmented mitochondria in human lung adenocarcinoma A549â¯cells. In comprehensive screening using the Drosophila model in which human Bcl-rambo was ectopically expressed in eye imaginal discs, voltage-dependent anion channels (VDAC), also known as mitochondrial porin, were found to manifest a genetic interaction with human Bcl-rambo. In addition to human adenine nucleotide translocase (ANT) 1 and ANT2, the human Bcl-rambo protein bound to human VDAC1, albeit to a lesser extent than ANT2. Moreover, human VDAC1 and human VDAC2 in particular promoted the activation of effector caspases only when they were co-expressed with human Bcl-rambo in 293T cells. Bcl-rambo induced the perinuclear accumulation of fragmented mitochondria by the knockdown of VDAC1, VDAC2, and VDAC3 in A549â¯cells. Thus, the present study revealed that human Bcl-rambo and VDAC cooperatively promote the activation of effector caspases in human cultured cells.
Assuntos
Caspases Efetoras/metabolismo , Drosophila/genética , Epistasia Genética/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Canais de Ânion Dependentes de Voltagem/genética , Células A549 , Animais , Animais Geneticamente Modificados , Células Cultivadas , Ativação Enzimática/genética , Células HEK293 , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
Bcl-2 family proteins play a central role in regulating apoptosis. We previously reported that human Bcl-rambo, also termed BCL2L13, localized to mitochondria and induced apoptosis when overexpressed in human embryonic kidney 293T cells. However, the physiological function of Bcl-rambo currently remains unclear. In the present study, human Bcl-rambo was ectopically expressed in Drosophila melanogaster. Bcl-rambo mainly localized to the mitochondria of Drosophila Schneider 2 (S2) cells. The overexpression of Bcl-rambo, but not Bcl-rambo lacking a C-terminal transmembrane domain, induced apoptosis in S2 cells. Moreover, the ectopic expression of Bcl-rambo by a GAL4-UAS system induced aberrant morphological changes characterized by atrophied wing, split thorax, and rough eye phenotypes. Bcl-rambo induced the activation of effector caspases in eye imaginal discs. The rough eye phenotype induced by Bcl-rambo was partly rescued by the co-expression of p35, Diap1, and Diap2. By using this Drosophila model, we showed that human Bcl-rambo interacted genetically with Drosophila homologues of adenine nucleotide translocators and the autophagy-related 8 protein. The results of the present study demonstrated that human Bcl-rambo localized to mitochondria and at least regulated an apoptosis signaling pathway in Drosophila.