RESUMEN
The RAS pathway is among the most frequently activated signaling nodes in cancer. However, the mechanisms that alter RAS activity in human pathologies are not entirely understood. The most prevalent post-translational modification within the GTPase core domain of NRAS and KRAS is ubiquitination at lysine 128 (K128), which is significantly decreased in cancer samples compared to normal tissue. Here, we found that K128 ubiquitination creates an additional binding interface for RAS GTPase-activating proteins (GAPs), NF1 and RASA1, thus increasing RAS binding to GAP proteins and promoting GAP-mediated GTP hydrolysis. Stimulation of cultured cancer cells with growth factors or cytokines transiently induces K128 ubiquitination and restricts the extent of wild-type RAS activation in a GAP-dependent manner. In KRAS mutant cells, K128 ubiquitination limits tumor growth by restricting RAL/ TBK1 signaling and negatively regulating the autocrine circuit induced by mutant KRAS. Reduction of K128 ubiquitination activates both wild-type and mutant RAS signaling and elicits a senescence-associated secretory phenotype, promoting RAS-driven pancreatic tumorigenesis.
Asunto(s)
Unión Proteica , Proteínas Proto-Oncogénicas p21(ras) , Ubiquitinación , Humanos , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Animales , Proteína Activadora de GTPasa p120/metabolismo , Proteína Activadora de GTPasa p120/genética , Ratones , Línea Celular Tumoral , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Lisina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas ras/metabolismo , Proteínas ras/genética , Neurofibromina 1RESUMEN
RATIONALE: Noonan syndrome (NS) is one of the most frequent genetic disorders. Bleeding problems are among the most common, yet poorly defined complications associated with NS. A lack of consensus on the management of bleeding complications in patients with NS indicates an urgent need for new therapeutic approaches. OBJECTIVE: Bleeding disorders have recently been described in patients with NS harboring mutations of LZTR1 (leucine zipper-like transcription regulator 1), an adaptor for CUL3 (CULLIN3) ubiquitin ligase complex. Here, we assessed the pathobiology of LZTR1-mediated bleeding disorders. METHODS AND RESULTS: Whole-body and vascular specific knockout of Lztr1 results in perinatal lethality due to cardiovascular dysfunction. Lztr1 deletion in blood vessels of adult mice leads to abnormal vascular leakage. We found that defective adherent and tight junctions in Lztr1-depleted endothelial cells are caused by dysregulation of vesicular trafficking. LZTR1 affects the dynamics of fusion and fission of recycling endosomes by controlling ubiquitination of the ESCRT-III (endosomal sorting complex required for transport III) component CHMP1B (charged multivesicular protein 1B), whereas NS-associated LZTR1 mutations diminish CHMP1B ubiquitination. LZTR1-mediated dysregulation of CHMP1B ubiquitination triggers endosomal accumulation and subsequent activation of VEGFR2 (vascular endothelial growth factor receptor 2) and decreases blood levels of soluble VEGFR2 in Lztr1 haploinsufficient mice. Inhibition of VEGFR2 activity by cediranib rescues vascular abnormalities observed in Lztr1 knockout mice Conclusions: Lztr1 deletion phenotypically overlaps with bleeding diathesis observed in patients with NS. ELISA screening of soluble VEGFR2 in the blood of LZTR1-mutated patients with NS may predict both the severity of NS phenotypes and potential responders to anti-VEGF therapy. VEGFR inhibitors could be beneficial for the treatment of bleeding disorders in patients with NS.
Asunto(s)
Vasos Sanguíneos/metabolismo , Endosomas/metabolismo , Células Endoteliales/metabolismo , Hemorragia/metabolismo , Síndrome de Noonan/metabolismo , Factores de Transcripción/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Malformaciones Vasculares/metabolismo , Animales , Vasos Sanguíneos/anomalías , Vasos Sanguíneos/efectos de los fármacos , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patología , Modelos Animales de Enfermedad , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endosomas/genética , Endosomas/patología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Haploinsuficiencia , Células HeLa , Hemorragia/genética , Hemorragia/patología , Hemorragia/prevención & control , Humanos , Linfocinas/genética , Linfocinas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neovascularización Patológica , Síndrome de Noonan/tratamiento farmacológico , Síndrome de Noonan/genética , Síndrome de Noonan/patología , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Transporte de Proteínas , Quinazolinas/farmacología , Transducción de Señal , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Ubiquitinación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Malformaciones Vasculares/tratamiento farmacológico , Malformaciones Vasculares/genética , Malformaciones Vasculares/patologíaRESUMEN
Protein-protein interactions (PPIs) are central in cell metabolism but research tools for the structural and functional characterization of these PPIs are often missing. Here we introduce broadly applicable immunization (Cross-link PPIs and immunize llamas, ChILL) and selection strategies (Display and co-selection, DisCO) for the discovery of diverse nanobodies that either stabilize or disrupt PPIs in a single experiment. We apply ChILL and DisCO to identify competitive, connective, or fully allosteric nanobodies that inhibit or facilitate the formation of the SOS1â¢RAS complex and modulate the nucleotide exchange rate on this pivotal GTPase in vitro as well as RAS signalling in cellulo. One of these connective nanobodies fills a cavity that was previously identified as the binding pocket for a series of therapeutic lead compounds. The long complementarity-determining region (CDR3) that penetrates this binding pocket serves as pharmacophore for extending the repertoire of potential leads.
Asunto(s)
Unión Proteica , Proteína SOS1 , Anticuerpos de Dominio Único , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/metabolismo , Proteína SOS1/metabolismo , Proteína SOS1/química , Proteína SOS1/genética , Proteína SOS1/inmunología , Humanos , Animales , Regulación Alostérica , Proteínas ras/metabolismo , Proteínas ras/química , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/inmunología , Sitios de Unión , Camélidos del Nuevo Mundo/inmunología , Inmunización , Transducción de Señal , Modelos MolecularesRESUMEN
Aerobic glycolysis is a hallmark of cancer development, but this dogma has been challenged by reports showing a key role of oxidative phosphorylation (OXPHOS) in cancer cell survival. It has been proposed that increased levels of intramitochondrial proteins in cancer cells are associated with high OXPHOS activity and increased sensitivity to OXPHOS inhibitors. However, the molecular mechanisms leading to the high expression of OXPHOS proteins in cancer cells remain unknown. Multiple proteomics studies have detected the ubiquitination of intramitochondrial proteins, suggesting the contribution of the ubiquitin system to the proteostatic regulation of OXPHOS proteins. Here, we identified the ubiquitin hydrolase OTUB1 as a regulator of the mitochondrial metabolic machinery essential for lung cancer cell survival. Mitochondria-localized OTUB1 modulates respiration by inhibiting K48-linked ubiquitination and turnover of OXPHOS proteins. An increase in OTUB1 expression is commonly observed in one-third of non-small-cell lung carcinomas and is associated with high OXPHOS signatures. Moreover, OTUB1 expression highly correlates with the sensitivity of lung cancer cells to mitochondrial inhibitors.