RESUMO
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.
Assuntos
Proteínas Relacionadas à Autofagia , Lipoilação , Proteínas Associadas aos Microtúbulos , Autofagossomos/genética , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sistemas CRISPR-Cas , Células HCT116 , Células HEK293 , Humanos , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Salmonella/genética , Salmonella/metabolismo , Proteínas da Matriz Viral/genética , Proteínas da Matriz Viral/metabolismo , Proteínas Viroporinas/genética , Proteínas Viroporinas/metabolismoRESUMO
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.
Assuntos
Oncogenes , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Adenilato Quinase/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Modelos Biológicos , Mutação/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
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.
