RESUMO
Oogenesis is essential for female gamete production in mammals. The total number of ovarian follicles is determined early in life and production of ovarian oocytes is thought to stop during the lifetime. However, the molecular mechanisms underling oogenesis, particularly autophagy regulation in the ovary, remain largely unknown. Here, we reveal an important MYBL2-VDAC2-BECN1-BCL2L1 pathway linking autophagy suppression in the developing ovary. The transcription factors GATA1 and MYBL2 can bind to and activate the Vdac2 promoter. MYBL2 regulates the spatiotemporal expression of VDAC2 in the developing ovary. Strikingly, in the VDAC2 transgenic pigs (Sus scrofa/Ss), VDAC2 exerts its function by inhibiting autophagy in the ovary. In contrast, Vdac2 knockout promotes autophagy. Moreover, VDAC2-mediated autophagy suppression is dependent on its interactions with both BECN1 and BCL2L1 to stabilize the BECN1 and BCL2L1 complex, suggesting VDAC2 as an autophagy suppressor in the pathway. Our findings provide a functional connection among the VDAC2, MYBL2, the BECN1-BCL2L1 pathway and autophagy suppression in the developing ovary, which is implicated in improving female fecundity.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Mamíferos/metabolismo , Ovário/crescimento & desenvolvimento , Transativadores/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Proteína bcl-X/metabolismo , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Sequência de Bases , Imunoprecipitação da Cromatina , Análise Mutacional de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Fator de Transcrição GATA1/metabolismo , Camundongos , Dados de Sequência Molecular , Ovário/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Sus scrofa , Canal de Ânion 2 Dependente de Voltagem/deficiência , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
Protein modification by O-linked ß-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2(-/-) cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.
Assuntos
Mitocôndrias/metabolismo , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Acetilglucosamina/metabolismo , Animais , Linhagem Celular , Eletroforese em Gel de Campo Pulsado , Corantes Fluorescentes/química , Glicoproteínas/metabolismo , Glicosilação , Células HEK293 , Humanos , Células Jurkat , Camundongos , Proteômica , Especificidade por Substrato , Canal de Ânion 2 Dependente de Voltagem/deficiência , Canal de Ânion 2 Dependente de Voltagem/genéticaRESUMO
The proapoptotic proteins BAX and BAK constitute the mitochondrial apoptotic gateway that executes cellular demise after integrating death signals. The lethal BAK is kept in check by voltage-dependent anion channel 2 (VDAC2), a mammalian-restricted VDAC isoform. Here, we provide evidence showing a critical role for the VADC2-BAK complex in determining thymocyte survival in vivo. Genetic depletion of Vdac2 in the thymus resulted in excessive cell death and hypersensitivity to diverse death stimuli including engagement of the T cell receptor. These phenotypes were completely rescued by the concurrent deletion of Bak but not that of Bax. Thus, the VDAC2-BAK axis provides a mechanism that governs the homeostasis of thymocytes. Our study reveals a sophisticated built-in rheostat that likely fine-tunes immune competence to balance autoimmunity and immunodeficiency.
