RESUMEN
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Asunto(s)
Autofagia , Carcinoma Ductal Pancreático/metabolismo , Lisosomas/metabolismo , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Cromatina/genética , Cromatina/metabolismo , Regulación hacia Abajo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Antígenos de Histocompatibilidad/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Lisosomas/patología , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Nucleares/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Agregado de Proteínas , Unión Proteica , Proteolisis , Interferencia de ARN , Transducción de Señal , Sirtuina 1/genética , Sirtuina 1/metabolismo , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Factores de Transcripción/genética , TransfecciónRESUMEN
Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.
Asunto(s)
Actinas/metabolismo , Células Epiteliales/metabolismo , Mapas de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Animales , Células Cultivadas , Células Epiteliales/microbiología , Células HCT116 , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Immunoblotting , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteómica/métodos , Interferencia de ARN , Salmonella/fisiologíaRESUMEN
The hallmark of Salmonella Typhimurium infection is an acute intestinal inflammatory response, which is mediated through the action of secreted bacterial effector proteins. The pro-inflammatory Salmonella effector SopA is a HECT-like E3 ligase, which was previously proposed to activate host RING ligases TRIM56 and TRIM65. Here we elucidate an inhibitory mechanism of TRIM56 and TRIM65 targeting by SopA. We present the crystal structure of SopA in complex with the RING domain of human TRIM56, revealing the atomic details of their interaction and the basis for SopA selectivity towards TRIM56 and TRIM65. Structure-guided biochemical analysis shows that SopA inhibits TRIM56 E3 ligase activity by occluding the E2-interacting surface of TRIM56. We further demonstrate that SopA ubiquitinates TRIM56 and TRIM65, resulting in their proteasomal degradation during infection. Our results provide the basis for how a bacterial HECT ligase blocks host RING ligases and exemplifies the multivalent power of bacterial effectors during infection.