RESUMEN
The Dot/Icm system of Legionella pneumophila is essential for virulence and delivers a large repertoire of effectors into infected host cells to create the Legionella containing vacuole. Since the secretion of effectors via the Dot/Icm system does not occur in the absence of host cells, we hypothesised that host factors actively participate in Dot/Icm effector translocation. Here we employed a high-throughput, genome-wide siRNA screen to systematically test the effect of silencing 18,120 human genes on translocation of the Dot/Icm effector, RalF, into HeLa cells. For the primary screen, we found that silencing of 119 genes led to increased translocation of RalF, while silencing of 321 genes resulted in decreased translocation. Following secondary screening, 70 genes were successfully validated as 'high confidence' targets. Gene set enrichment analysis of siRNAs leading to decreased RalF translocation, showed that ubiquitination was the most highly overrepresented category in the pathway analysis. We further showed that two host factors, the E2 ubiquitin-conjugating enzyme, UBE2E1, and the E3 ubiquitin ligase, CUL7, were important for supporting Dot/Icm translocation and L. pneumophila intracellular replication. In summary, we identified host ubiquitin pathways as important for the efficiency of Dot/Icm effector translocation by L. pneumophila, suggesting that host-derived ubiquitin-conjugating enzymes and ubiquitin ligases participate in the translocation of Legionella effector proteins and influence intracellular persistence and survival.
Asunto(s)
Legionella pneumophila , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células HeLa , Humanos , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Ubiquitinación , Vacuolas/metabolismoRESUMEN
The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.
Asunto(s)
Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Glándulas Mamarias Humanas/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Activación Transcripcional , Angiotensina II/metabolismo , Angiotensina II/farmacología , Línea Celular Transformada , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/genética , Colina Quinasa/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Receptores ErbB/genética , Femenino , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Glándulas Mamarias Humanas/citología , Glándulas Mamarias Humanas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor de Angiotensina Tipo 1/genética , Transducción de Señal , Trombina/metabolismo , Trombina/farmacologíaRESUMEN
The Victorian Centre for Functional Genomics (VCFG) is an RNAi screening facility housed at the Peter MacCallum Cancer Centre in Melbourne, Australia. The Peter Mac is Australia's largest dedicated Cancer Research Institute, home to a team of over 520 scientists that focus on understanding the genetic risk of cancer, the molecular events regulating cancer growth and dissemination and improving detection through new diagnostic tools (www.petermac.org). Peter Mac is a well recognised technology leader and established the VCFG with a view to enabling researchers Australia and New Zealand-wide access to cutting edge functional genomics technology, infrastructure and expertise. This review documents the technology platforms operated within the VCFG and provides insight into the workflows and analysis pipelines currently in operation.