RESUMEN
Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.
Asunto(s)
Proteínas Relacionadas con la Autofagia , Lipoilación , Proteínas Asociadas a Microtúbulos , Autofagosomas/genética , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Sistemas CRISPR-Cas , Células HCT116 , Células HEK293 , Humanos , Virus de la Influenza A/genética , Virus de la Influenza A/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Salmonella/genética , Salmonella/metabolismo , Proteínas de la Matriz Viral/genética , Proteínas de la Matriz Viral/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismoRESUMEN
KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.
Asunto(s)
Oncogenes , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Quinasas de la Proteína-Quinasa Activada por el AMP , Adenilato Quinasa/metabolismo , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Redes y Vías Metabólicas/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Pooled shRNA library is a powerful, rapid, and cost-effective technology to carry out functional genomic screens in mammalian cells. This approach has been applied extensively to identify genetic dependencies in cancer cells that might be exploited for therapeutic purposes. In this chapter we provide a detailed protocol for using the Hannon-Elledge miR30-based library to conduct dropout screens in cancer cell lines. This protocol is readily adaptable to other pooled shRNA libraries and should facilitate the functional annotation of the human genome.
