RESUMEN
NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1ß secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/metabolismo , Proteínas de Unión al Calcio/metabolismo , Inflamasomas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Adaptadoras de Señalización CARD/química , Proteínas Adaptadoras de Señalización CARD/deficiencia , Proteínas Adaptadoras de Señalización CARD/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Caspasa 1/metabolismo , Activación Enzimática , Técnicas de Sustitución del Gen , Humanos , Inmunidad Innata/inmunología , Interleucina-1beta/inmunología , Interleucina-1beta/metabolismo , Macrófagos/inmunología , Ratones , Datos de Secuencia Molecular , Fosforilación , Conformación Proteica , Proteína Quinasa C-delta/deficiencia , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/metabolismo , Salmonella typhimurium/inmunología , Alineación de SecuenciaRESUMEN
Apoptotic cell death is important for embryonic development, immune cell homeostasis, and pathogen elimination. Innate immune cells also undergo a very rapid form of cell death termed pyroptosis after activating the protease caspase-1. The hemichannel pannexin-1 has been implicated in both processes. In this study, we describe the characterization of pannexin-1-deficient mice. LPS-primed bone marrow-derived macrophages lacking pannexin-1 activated caspase-1 and secreted its substrates IL-1ß and IL-18 normally after stimulation with ATP, nigericin, alum, silica, flagellin, or cytoplasmic DNA, indicating that pannexin-1 is dispensable for assembly of caspase-1-activating inflammasome complexes. Instead, thymocytes lacking pannexin-1, but not the P2X7R purinergic receptor, were defective in their uptake of the nucleic acid dye YO-PRO-1 during early apoptosis. Cell death was not delayed but, unlike their wild-type counterparts, Panx1(-/-) thymocytes failed to recruit wild-type peritoneal macrophages in a Transwell migration assay. These data are consistent with pannexin-1 liberating ATP and other yet to be defined "find me" signals necessary for macrophage recruitment to apoptotic cells.
Asunto(s)
Adenosina Trifosfato/metabolismo , Apoptosis , Conexinas/metabolismo , Inflamasomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adenosina Trifosfato/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzoxazoles/metabolismo , Benzoxazoles/farmacocinética , Western Blotting , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Caspasa 1/metabolismo , Movimiento Celular , Supervivencia Celular , Células Cultivadas , Conexinas/genética , Proteínas de Unión al ADN , Citometría de Flujo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Macrófagos Peritoneales/citología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Compuestos de Quinolinio/metabolismo , Compuestos de Quinolinio/farmacocinética , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 - 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry-based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines.
Asunto(s)
Células CHO/citología , Técnicas de Cultivo de Célula/métodos , Separación Celular/métodos , Análisis de la Célula Individual , Animales , Supervivencia Celular/genética , Cricetinae , Cricetulus , Citometría de Flujo/métodos , HumanosRESUMEN
BACKGROUND: Oncogenic RAS is a highly validated cancer target. Attempts at targeting RAS directly have so far not succeeded in the clinic. Understanding downstream RAS-effectors that mediate oncogenesis in a RAS mutant setting will help tailor treatments that use RAS-effector inhibitors either alone or in combination to target RAS-driven tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have investigated the sufficiency of targeting RAS-effectors, RAF, MEK and PI3-Kinase either alone or in combination in RAS mutant lines, using an inducible shRNA in vivo mouse model system. We find that in colon cancer cells harboring a KRAS(G13D) mutant allele, knocking down KRAS alone or the RAFs in combination or the RAF effectors, MEK1 and MEK2, together is effective in delaying tumor growth in vivo. In melanoma cells harboring an NRAS(Q61L) or NRAS(Q61K) mutant allele, we find that targeting NRAS alone or both BRAF and CRAF in combination or both BRAF and PIK3CA together showed efficacy. CONCLUSION/SIGNIFICANCE: Our data indicates that targeting oncogenic NRAS-driven melanomas require decrease in both pERK and pAKT downstream of RAS-effectors for efficacy. This can be achieved by either targeting both BRAF and CRAF or BRAF and PIK3CA simultaneously in NRAS mutant tumor cells.