RESUMEN
Western equine encephalitis virus (WEEV) is an arthropod-borne virus (arbovirus) that frequently caused major outbreaks of encephalitis in humans and horses in the early twentieth century, but the frequency of outbreaks has since decreased markedly, and strains of this alphavirus isolated in the past two decades are less virulent in mammals than strains isolated in the 1930s and 1940s1-3. The basis for this phenotypic change in WEEV strains and coincident decrease in epizootic activity (known as viral submergence3) is unclear, as is the possibility of re-emergence of highly virulent strains. Here we identify protocadherin 10 (PCDH10) as a cellular receptor for WEEV. We show that multiple highly virulent ancestral WEEV strains isolated in the 1930s and 1940s, in addition to binding human PCDH10, could also bind very low-density lipoprotein receptor (VLDLR) and apolipoprotein E receptor 2 (ApoER2), which are recognized by another encephalitic alphavirus as receptors4. However, whereas most of the WEEV strains that we examined bind to PCDH10, a contemporary strain has lost the ability to recognize mammalian PCDH10 while retaining the ability to bind avian receptors, suggesting WEEV adaptation to a main reservoir host during enzootic circulation. PCDH10 supports WEEV E2-E1 glycoprotein-mediated infection of primary mouse cortical neurons, and administration of a soluble form of PCDH10 protects mice from lethal WEEV challenge. Our results have implications for the development of medical countermeasures and for risk assessment for re-emerging WEEV strains.
Asunto(s)
Cadherinas , Virus de la Encefalitis Equina del Oeste , Receptores Virales , Animales , Ratones , Humanos , Receptores Virales/metabolismo , Cadherinas/metabolismo , Femenino , Receptores de LDL/metabolismo , Receptores de LDL/genética , Masculino , Encefalomielitis Equina/virología , Encefalomielitis Equina/transmisión , Encefalomielitis Equina/veterinariaRESUMEN
PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
Asunto(s)
Complejo CD3 , Endopeptidasas , Proteínas Ligadas a GPI , Inmunoterapia Adoptiva , Mesotelina , Neoplasias Pancreáticas , Receptores Quiméricos de Antígenos , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Humanos , Animales , Ratones , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/inmunología , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Receptores Quiméricos de Antígenos/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Línea Celular Tumoral , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Fibroblastos Asociados al Cáncer/metabolismo , Fibroblastos Asociados al Cáncer/inmunología , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Adenocarcinoma/inmunología , Adenocarcinoma/terapia , Adenocarcinoma/patologíaRESUMEN
Annotation of immunologic gene function in vivo typically requires the generation of knockout mice, which is time consuming and low throughput. We previously developed CHimeric IMmune Editing (CHIME), a CRISPR-Cas9 bone marrow delivery system for constitutive, ubiquitous deletion of single genes. Here we describe X-CHIME, four new CHIME-based systems for modular and rapid interrogation of gene function combinatorially (C-CHIME), inducibly (I-CHIME), lineage-specifically (L-CHIME) or sequentially (S-CHIME). We use C-CHIME and S-CHIME to assess the consequences of combined deletion of Ptpn1 and Ptpn2, an embryonic lethal gene pair, in adult mice. We find that constitutive deletion of both PTPN1 and PTPN2 leads to bone marrow hypoplasia and lethality, while inducible deletion after immune development leads to enteritis and lethality. These findings demonstrate that X-CHIME can be used for rapid mechanistic evaluation of genes in distinct in vivo contexts and that PTPN1 and PTPN2 have some functional redundancy important for viability in adult mice.
Asunto(s)
Sistemas CRISPR-Cas , Proteína Tirosina Fosfatasa no Receptora Tipo 2 , Ratones , Animales , Sistemas CRISPR-Cas/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 2/genética , Ratones Noqueados , Sistema Inmunológico , Edición GénicaRESUMEN
NUT carcinoma is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of progrowth genes. BET bromodomain inhibitors (BETi) are a promising treatment for NUT carcinoma that can impede BRD4-NUT's ability to activate genes, but the efficacy of BETi as monotherapy is limited. Here, we demonstrated that enhancer of zeste homolog 2 (EZH2), which silences genes through establishment of repressive chromatin, is a dependency in NUT carcinoma. Inhibition of EZH2 with the clinical compound tazemetostat potently blocked growth of NUT carcinoma cells. Epigenetic and transcriptomic analysis revealed that tazemetostat reversed the EZH2-specific H3K27me3 silencing mark and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4-NUT-regulated genes. Indeed, H3K27me3 and H3K27ac domains were found to be mutually exclusive in NUT carcinoma cells. CDKN2A was identified as the only gene among all tazemetostat-derepressed genes to confer resistance to tazemetostat in a CRISPR-Cas9 screen. Combined inhibition of EZH2 and BET synergized to downregulate cell proliferation genes, resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In preclinical models, combined tazemetostat and BETi synergistically blocked tumor growth and prolonged survival of NUT carcinoma-xenografted mice, with complete remission without relapse in one cohort. Identification of EZH2 as a dependency in NUT carcinoma substantiates the reliance of NUT carcinoma tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary, chromatin regulatory pathways to maintain NUT carcinoma growth. SIGNIFICANCE: Repression of tumor suppressor genes, including CDKN2A, by EZH2 provides a mechanistic rationale for combining EZH2 and BET inhibitors for the clinical treatment of NUT carcinoma. See related commentary by Kazansky and Kentsis, p. 3827.
Asunto(s)
Carcinoma , Proteínas Nucleares , Animales , Humanos , Ratones , Carcinoma/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Cromatina , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Genes Supresores de Tumor , Histonas/metabolismo , Recurrencia Local de Neoplasia/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
NUT carcinoma (NC) is an aggressive carcinoma driven by the BRD4-NUT fusion oncoprotein, which activates chromatin to promote expression of pro-growth genes. BET bromodomain inhibitors (BETi) impede BRD4-NUT's ability to activate genes and are thus a promising treatment but limited as monotherapy. The role of gene repression in NC is unknown. Here, we demonstrate that EZH2, which silences genes through establishment of repressive chromatin, is a dependency in NC. Inhibition of EZH2 with the clinical compound tazemetostat (taz) potently blocked growth of NC cells. Epigenetic and transcriptomic analysis revealed that taz reversed the EZH2-specific H3K27me3 silencing mark, and restored expression of multiple tumor suppressor genes while having no effect on key oncogenic BRD4- NUT-regulated genes. CDKN2A was identified as the only gene amongst all taz-derepressed genes to confer resistance to taz in a CRISPR-Cas9 screen. Combined EZH2 inhibition and BET inhibition synergized to downregulate cell proliferation genes resulting in more pronounced growth arrest and differentiation than either inhibitor alone. In pre-clinical models, combined taz and BETi synergistically blocked growth and prolonged survival of NC-xenografted mice, with all mice cured in one cohort. STATEMENT OF SIGNIFICANCE: Identification of EZH2 as a dependency in NC substantiates the reliance of NC tumor cells on epigenetic dysregulation of functionally opposite, yet highly complementary chromatin regulatory pathways to maintain NC growth. In particular, repression of CDKN2A expression by EZH2 provides a mechanistic rationale for combining EZH2i with BETi for the clinical treatment of NC.
RESUMEN
Neuroepithelial crosstalk is critical for gut physiology. However, the mechanisms by which sensory neurons communicate with epithelial cells to mediate gut barrier protection at homeostasis and during inflammation are not well understood. Here, we find that Nav1.8+CGRP+ nociceptor neurons are juxtaposed with and signal to intestinal goblet cells to drive mucus secretion and gut protection. Nociceptor ablation led to decreased mucus thickness and dysbiosis, while chemogenetic nociceptor activation or capsaicin treatment induced mucus growth. Mouse and human goblet cells expressed Ramp1, receptor for the neuropeptide CGRP. Nociceptors signal via the CGRP-Ramp1 pathway to induce rapid goblet cell emptying and mucus secretion. Notably, commensal microbes activated nociceptors to control homeostatic CGRP release. In the absence of nociceptors or epithelial Ramp1, mice showed increased epithelial stress and susceptibility to colitis. Conversely, CGRP administration protected nociceptor-ablated mice against colitis. Our findings demonstrate a neuron-goblet cell axis that orchestrates gut mucosal barrier protection.
Asunto(s)
Colitis , Células Caliciformes , Ratones , Humanos , Animales , Células Caliciformes/metabolismo , Nociceptores/metabolismo , Péptido Relacionado con Gen de Calcitonina/metabolismo , Colitis/metabolismo , Moco/metabolismo , Proteína 1 Modificadora de la Actividad de Receptores/metabolismoRESUMEN
Chimeric antigen receptor (CAR) therapy has had a transformative effect on the treatment of haematologic malignancies1-6, but it has shown limited efficacy against solid tumours. Solid tumours may have cell-intrinsic resistance mechanisms to CAR T cell cytotoxicity. Here, to systematically identify potential resistance pathways in an unbiased manner, we conducted a genome-wide CRISPR knockout screen in glioblastoma, a disease in which CAR T cells have had limited efficacy7,8. We found that the loss of genes in the interferon-γ receptor (IFNγR) signalling pathway (IFNGR1, JAK1 or JAK2) rendered glioblastoma and other solid tumours more resistant to killing by CAR T cells both in vitro and in vivo. However, loss of this pathway did not render leukaemia or lymphoma cell lines insensitive to CAR T cells. Using transcriptional profiling, we determined that glioblastoma cells lacking IFNγR1 had lower upregulation of cell-adhesion pathways after exposure to CAR T cells. We found that loss of IFNγR1 in glioblastoma cells reduced overall CAR T cell binding duration and avidity. The critical role of IFNγR signalling in susceptibility of solid tumours to CAR T cells is surprising, given that CAR T cells do not require traditional antigen-presentation pathways. Instead, in glioblastoma tumours, IFNγR signalling was required for sufficient adhesion of CAR T cells to mediate productive cytotoxicity. Our work demonstrates that liquid and solid tumours differ in their interactions with CAR T cells and suggests that enhancing binding interactions between T cells and tumour cells may yield improved responses in solid tumours.
Asunto(s)
Glioblastoma , Receptores Quiméricos de Antígenos , Muerte Celular , Glioblastoma/genética , Glioblastoma/terapia , Humanos , Inmunoterapia Adoptiva , Linfocitos T/patologíaRESUMEN
Traditional neuroanatomy immunohistology studies involve low-content analyses of a few antibodies of interest, typically applied and compared across sequential tissue sections. The efficiency, consistency, and ultimate insights of these studies can be substantially improved using high-plex immunofluorescence labelling on a single tissue section to allow direct comparison of many markers. Here we present an expanded and efficient multiplexed fluorescence-based immunohistochemistry (MP-IHC) approach that improves throughput with sequential labelling of up to 10 antibodies per cycle, with no limitation on the number of cycles, and maintains versatility and accessibility by using readily available commercial reagents and standard epifluorescence microscopy imaging. We demonstrate this approach by cumulatively screening up to 100 markers on formalin-fixed paraffin-embedded sections of human olfactory bulb sourced from neurologically normal (no significant pathology), Alzheimer's (AD), and Parkinson's disease (PD) patients. This brain region is involved early in the symptomology and pathophysiology of AD and PD. We also developed a spatial pixel bin analysis approach for unsupervised analysis of the high-content anatomical information from large tissue sections. Here, we present a comprehensive immunohistological characterisation of human olfactory bulb anatomy and a summary of differentially expressed biomarkers in AD and PD using the MP-IHC labelling and spatial protein analysis pipeline.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Inmunohistoquímica/métodos , Bulbo Olfatorio/química , Enfermedad de Parkinson/metabolismo , Estudios de Casos y Controles , Humanos , Bulbo Olfatorio/metabolismo , Bulbo Olfatorio/patología , Adhesión en ParafinaRESUMEN
BACKGROUND AND PURPOSE: Inhibitors of DNA-dependent protein kinase (DNA-PK) are effective radiation sensitisers in preclinical tumours, but little is known about risks of normal tissue radiosensitisation. Here, we evaluate radiosensitisation of head and neck squamous cell carcinoma (HNSCC) cells by DNA-PK inhibitor AZD7648 under oxia and anoxia in vitro, and tumour (SCCVII), oral mucosa and small intestine in mice. MATERIALS AND METHODS: Radiosensitisation of human (UT-SCC-54C) and murine (SCCVII) HNSCC cells by AZD7648 under oxia and anoxia was evaluated by clonogenic assay. Radiosensitisation of SCCVII tumours in C3H mice by oral AZD7648 (75 mg/kg) was determined by ex vivo clonogenic assay 3.5 days post-irradiation, with evaluation of normal tissue surrogate endpoints using 5-ethynyl-2'-deoxyuridine to facilitate detection of regenerating crypts in the ileum and repopulating S-phase cells in the ileum and oral mucosa of the same animals. RESULTS: AZD7648 potently radiosensitised both cell lines, with similar sensitiser enhancement ratios for 10% survival (SER10) under oxia and anoxia. AZD7648 diffused rapidly through multicellular layers, suggesting rapid equilibration between plasma and hypoxic zones in tumours. SCCVII tumours were radiosensitised by AZD7648 (SER10 2.5). AZD7648 also enhanced radiation-induced body weight loss and suppressed regenerating intestinal crypts and repopulating S-phase cells in the ileum and tongue epithelium with SER values similar to SCCVII tumours. CONCLUSION: AZD7648 is a potent radiation sensitiser of both oxic and anoxic tumour cells, but also markedly radiosensitises stem cells in the small intestine and oral mucosa.
Asunto(s)
Proteína Quinasa Activada por ADN , Neoplasias de Cabeza y Cuello , Animales , ADN , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Hipoxia , Ratones , Ratones Endogámicos C3H , Purinas , Piranos , Carcinoma de Células Escamosas de Cabeza y Cuello/radioterapia , TriazolesRESUMEN
Previous commercial studies carried out in New Zealand showed that mechanical shaking significantly reduced the incidence of Botrytis cinerea infection in wine grapes. However, the reasons behind this reduction are not well understood. Here, we employed a metabolomics approach to gain insights into the biochemical changes that occur in grape berries due to mechanical shaking. Berry samples were analyzed using three different analytical approaches including gas chromatography and mass spectrometry (MS), liquid chromatography and MS, and imaging mass spectrometry (IMS). Combined data provided a comprehensive overview of metabolic changes in grape berry, indicating the initiation of different stress mitigation strategies to overcome the effect of mechanical shaking. Berry primary metabolism was distinctly altered in the green berries in response to mechanical shaking, while secondary metabolism significantly changed in berries collected after veraison. Pathway analysis showed upregulation of metabolites related to nitrogen and lipid metabolism in the berries from shaken vines when compared with controls. From IMS data, we observed an accumulation of different groups of metabolites including phenolic compounds and amino and fatty acids in the areas near to the skin of berries from shaken vines. This observation suggests that mechanical shaking caused an accumulation of these metabolites, which may be associated with the formation of a protective barrier, leading to the reduction in B. cinerea infection in berries from mechanically shaken vines.
Asunto(s)
Frutas , Vitis , Botrytis , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas , Metabolómica , Nueva ZelandaRESUMEN
In situ hybridization (ISH) is a powerful tool that can be used to localize mRNA expression in tissue samples. Combining ISH with immunohistochemistry (IHC) to determine cell type provides cellular context of mRNA expression, which cannot be achieved with gene microarray or polymerase chain reaction. To study mRNA and protein expression on the same section we investigated the use of RNAscope® ISH in combination with fluorescent IHC on paraffin-embedded human brain tissue. We first developed a high-throughput, automated image analysis workflow for quantifying RNA puncta across the total cell population and within neurons identified by NeuN+ immunoreactivity. We then applied this automated analysis to tissue microarray (TMA) sections of middle temporal gyrus tissue (MTG) from neurologically normal and Alzheimer's Disease (AD) cases to determine the suitability of three commonly used housekeeping genes: ubiquitin C (UBC), peptidyl-prolyl cis-trans isomerase B (PPIB) and DNA-directed RNA polymerase II subunit RPB1 (POLR2A). Overall, we saw a significant decrease in total and neuronal UBC expression in AD cases compared to normal cases. Total expression results were validated with RT-qPCR using fresh frozen tissue from 5 normal and 5 AD cases. We conclude that this technique combined with our novel automated analysis pipeline provides a suitable platform to study changes in gene expression in diseased human brain tissue with cellular and anatomical context. Furthermore, our results suggest that UBC is not a suitable housekeeping gene in the study of post-mortem AD brain tissue.
Asunto(s)
Enfermedad de Alzheimer , Perfilación de la Expresión Génica/métodos , Genes Esenciales , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Anciano , Anciano de 80 o más Años , Ciclofilinas/análisis , ARN Polimerasas Dirigidas por ADN/análisis , Femenino , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/análisis , Transcriptoma , Ubiquitina C/análisis , Flujo de TrabajoRESUMEN
Olfactory dysfunction is an early and prevalent symptom of Alzheimer's disease (AD) and the olfactory bulb is a nexus of beta-amyloid plaque and tau neurofibrillary tangle (NFT) pathology during early AD progression. To mitigate the accumulation of misfolded proteins, an endoplasmic reticulum stress response called the unfolded protein response (UPR) occurs in the AD hippocampus. However, chronic UPR activation can lead to apoptosis and the upregulation of beta-amyloid and tau production. Therefore, UPR activation in the olfactory system could be one of the first changes in AD. In this study, we investigated whether two proteins that signal UPR activation are expressed in the olfactory system of AD cases with low or high amounts of aggregate pathology. We used immunohistochemistry to label two markers of UPR activation (p-PERK and p-eIF2α) concomitantly with neuronal markers (NeuN and PGP9.5) and pathology markers (beta-amyloid and tau) in the olfactory bulb, piriform cortex, entorhinal cortex and the CA1 region of the hippocampus in AD and normal cases. We show that UPR activation, as indicated by p-PERK and p-eIF2α expression, is significantly increased throughout the olfactory system in AD cases with low (Braak stage III-IV) and high-level (Braak stage V-VI) pathology. We further show that UPR activation occurs in the mitral cells and in the anterior olfactory nucleus of the olfactory bulb where tau and amyloid pathology is abundant. However, UPR activation is not present in neurons when they contain NFTs and only rarely occurs in neurons containing diffuse tau aggregates. We conclude that UPR activation is prevalent in all regions of the olfactory system and support previous findings suggesting that UPR activation likely precedes NFT formation. Our data indicate that chronic UPR activation in the olfactory system might contribute to the olfactory dysfunction that occurs early in the pathogenesis of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Neuronas/metabolismo , Vías Olfatorias/metabolismo , Respuesta de Proteína Desplegada/fisiología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Factor 2 Eucariótico de Iniciación/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ovillos Neurofibrilares/patología , Neuronas/patología , Vías Olfatorias/patología , eIF-2 Quinasa/análisis , Proteínas tau/metabolismoRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disorder for which no cognition-restoring therapies exist. Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. Increasing evidence suggests a remodeling of the GABAergic system in AD, which might represent an important therapeutic target. An inverse agonist of 5 subunit-containing GABAA receptors (α5GABAARs), 3-(5-Methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3-a]phthalazine (5IA) has cognition-enhancing properties. This study aimed to characterize the effects of 5IA on amyloid beta (A1-42)-induced molecular and cellular changes. Mouse primary hippocampal cultures were exposed to either A1-42 alone, or 5IA alone, 5IA with A1-42 or vehicle alone, and changes in cell viability and mRNA expression of several GABAergic signaling components were assessed. Treatment with 100 nM of 5IA reduced A1-42-induced cell loss by 23.8% (p < 0.0001) after 6 h and by 17.3% after 5 days of treatment (p < 0.0001). Furthermore, we observed an A1-42-induced increase in ambient GABA levels, as well as upregulated mRNA expression of the GABAAR α2,α5,2/3 subunits and the GABABR R1 and R2 subunits. Such changes in GABARs expression could potentially disrupt inhibitory neurotransmission and normal network activity. Treatment with 5IA restored A1-42-induced changes in the expression of α5GABAARs. In summary, this compound might hold neuroprotective potential and represent a new therapeutic avenue for AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Agonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Fragmentos de Péptidos/toxicidad , Ftalazinas/farmacología , Triazoles/farmacología , Animales , Muerte Celular , Células Cultivadas , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Hipocampo/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transmisión SinápticaRESUMEN
In the preterm brain, accumulating evidence suggests toll-like receptors (TLRs) are key mediators of the downstream inflammatory pathways triggered by hypoxia-ischemia (HI), which have the potential to exacerbate or ameliorate injury. Recently we demonstrated that central acute administration of the TLR7 agonist Gardiquimod (GDQ) confers neuroprotection in the preterm fetal sheep at 3 days post-asphyxial recovery. However, it is unknown whether GDQ can afford long-term protection. To address this, we examined the long-term effects of GDQ. Briefly, fetal sheep (0.7 gestation) received sham asphyxia or asphyxia induced by umbilical cord occlusion, and were studied for 7 days recovery. Intracerebroventricular (ICV) infusion of GDQ (total dose 3.34 mg) or vehicle was performed from 1-4 hours after asphyxia. GDQ was associated with a robust increase in concentration of tumor necrosis factor-(TNF)-α in the fetal plasma, and interleukin-(IL)-10 in both the fetal plasma and cerebrospinal fluid. GDQ did not significantly change the number of total and immature/mature oligodendrocytes within the periventricular and intragyral white matter. No changes were observed in astroglial and microglial numbers and proliferating cells in both white matter regions. GDQ increased neuronal survival in the CA4 region of the hippocampus, but was associated with exacerbated neuronal injury within the caudate nucleus. In conclusion, our data suggest delayed acute ICV administration of GDQ after severe HI in the developing brain may not support long-term neuroprotection.
Asunto(s)
Aminoquinolinas/administración & dosificación , Aminoquinolinas/uso terapéutico , Asfixia/embriología , Encéfalo/patología , Feto/patología , Imidazoles/administración & dosificación , Imidazoles/uso terapéutico , Nacimiento Prematuro/tratamiento farmacológico , Receptor Toll-Like 7/agonistas , Aminoquinolinas/farmacología , Animales , Apoptosis/efectos de los fármacos , Presión Arterial/efectos de los fármacos , Asfixia/sangre , Asfixia/líquido cefalorraquídeo , Asfixia/fisiopatología , Análisis de los Gases de la Sangre , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Caspasa 3/metabolismo , Polaridad Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Citocinas/sangre , Citocinas/líquido cefalorraquídeo , Femenino , Feto/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Concentración de Iones de Hidrógeno , Imidazoles/farmacología , Inyecciones Intraventriculares , Masculino , Metaboloma/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Oligodendroglía/patología , Tamaño de los Órganos/efectos de los fármacos , Nacimiento Prematuro/sangre , Nacimiento Prematuro/líquido cefalorraquídeo , Nacimiento Prematuro/fisiopatología , Ovinos , Factores de Tiempo , Cordón Umbilical/patologíaRESUMEN
PAK4 is a metazoan-specific kinase acting downstream of Cdc42. Here we describe the structure of human PAK4 in complex with Inka1, a potent endogenous kinase inhibitor. Using single mammalian cells containing crystals 50 µm in length, we have determined the in cellulo crystal structure at 2.95 Å resolution, which reveals the details of how the PAK4 catalytic domain binds cellular ATP and the Inka1 inhibitor. The crystal lattice consists only of PAK4-PAK4 contacts, which form a hexagonal array with channels of 80 Å in diameter that run the length of the crystal. The crystal accommodates a variety of other proteins when fused to the kinase inhibitor. Inka1-GFP was used to monitor the process crystal formation in living cells. Similar derivatives of Inka1 will allow us to study the effects of PAK4 inhibition in cells and model organisms, to allow better validation of therapeutic agents targeting PAK4.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasas p21 Activadas/metabolismo , Adenosina Trifosfato , Animales , Células COS , Dominio Catalítico , Línea Celular Tumoral , Chlorocebus aethiops , Cristalización , Cristalografía por Rayos X , Escherichia coli , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intracelular/química , Microscopía Confocal , Unión Proteica , Estructura Terciaria de Proteína , Quinasas p21 Activadas/químicaRESUMEN
The leading edge of migrating cells contains rapidly translocating activated integrins associated with growing actin filaments that form 'sticky fingers' to sense extracellular matrix and guide cell migration. Here we utilized indirect bimolecular fluorescence complementation to visualize a molecular complex containing a Mig-10/RIAM/lamellipodin (MRL) protein (Rap1-GTP-interacting adaptor molecule (RIAM) or lamellipodin), talin and activated integrins in living cells. This complex localizes at the tips of growing actin filaments in lamellipodial and filopodial protrusions, thus corresponding to the tips of the 'sticky fingers.' Formation of the complex requires talin to form a bridge between the MRL protein and the integrins. Moreover, disruption of the MRL protein-integrin-talin (MIT) complex markedly impairs cell protrusion. These data reveal the molecular basis of the formation of 'sticky fingers' at the leading edge of migrating cells and show that an MIT complex drives these protrusions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Movimiento Celular , Células/metabolismo , Integrinas/metabolismo , Proteínas de la Membrana/metabolismo , Talina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Portadoras/genética , Células/citología , Humanos , Integrinas/genética , Proteínas de la Membrana/genética , Unión Proteica , Talina/genéticaRESUMEN
Cell migration requires dynamic remodeling of the actomyosin network. We report here that an adapter protein, ArgBP2, is a component of α-actinin containing stress fibers and inhibits migration. ArgBP2 is undetectable in many commonly studied cancer-derived cell lines. COS-7 and HeLa cells express ArgBP2 (by Western analysis), but expression was detectable only in approximately half the cells by immunofluorescence. Short term clonal analysis demonstrated 0.2-0.3% of cells switch ArgBP2 expression (on or off) per cell division. ArgBP2 can have a fundamental impact on the actomyosin network: ArgBP2 positive COS-7 cells, for example, are clearly distinguishable by their denser actomyosin (stress fiber) network. ArgBP2γ binding to α-actinin appears to underlie its ability to localize to stress fibers and decrease cell migration. We map a small α-actinin binding region in ArgBP2 (residues 192-228) that is essential for these effects. Protein kinase A phosphorylation of ArgBP2γ at neighboring Ser-259 and consequent 14-3-3 binding blocks its interaction with α-actinin. ArgBP2 is known to be down-regulated in some aggressively metastatic cancers. Our work provides a biochemical explanation for the anti-migratory effect of ArgBP2.
Asunto(s)
Actinina/metabolismo , Actinas/metabolismo , Movimiento Celular , Regulación Enzimológica de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas 14-3-3/metabolismo , Actomiosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Colforsina/química , Citoesqueleto/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Metástasis de la Neoplasia , Fosforilación , Unión Proteica , Proteínas de Unión al ARN , TransfecciónRESUMEN
Increased ligand binding to cellular integrins ("activation") plays important roles in processes such as development, cell migration, extracellular matrix assembly, tumor metastasis, hemostasis, and thrombosis. Integrin activation encompasses both increased integrin monomer affinity and increased receptor clustering and depends on integrin-talin interactions. Loss of kindlins results in reduced activation of integrins. Kindlins might promote talin binding to integrins through a cooperative mechanism; however, kindlins do not increase talin association with integrins. Here, we report that, unlike talin head domain (THD), kindlin-3 has little effect on the affinity of purified monomeric αIIbß3, and it does not enhance activation by THD. Furthermore, studies with ligands of varying valency show that kindlins primarily increase cellular αIIbß3 avidity rather than monomer affinity. In platelets or nucleated cells, loss of kindlins markedly reduces αIIbß3 binding to multivalent but not monovalent ligands. Finally, silencing of kindlins reduces the clustering of ligand-occupied αIIbß3 as revealed by total internal reflection fluorescence and electron microscopy. Thus, in contrast to talins, kindlins have little primary effect on integrin αIIbß3 affinity for monovalent ligands and increase multivalent ligand binding by promoting the clustering of talin-activated integrins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Animales , Plaquetas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Humanos , Ligandos , Proteínas de la Membrana/genética , Ratones , Proteínas de Neoplasias/genética , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Talin-mediated integrin activation drives integrin-based adhesions. Here we examine the roles of two proteins that induce talin-integrin interactions--vinculin and Rap1-GTP-interacting adaptor molecule (RIAM)--in the formation and maturation of integrin-based adhesions. RIAM-containing adhesions are primarily in the lamellipodium; RIAM is subsequently reduced in mature focal adhesions due to direct competition with vinculin for talin-binding sites. We show that vinculin binding to talin induces Rap1-independent association of talin with integrins and resulting integrin activation, in sharp contrast to Rap1-dependent RIAM-induced activation. Vinculin stabilizes adhesions, increasing their ability to transmit force, whereas RIAM played a critical role in lamellipodial protrusion. Thus displacement of RIAM by vinculin acts as a molecular switch that mediates the transition of integrin-based adhesions from drivers of lamellipodial protrusion to stable, force-bearing adhesions. Consequently changes in the abundance of two multiprotein modules within maturing adhesions, one regulated by Rap1 and one by tension, result in the temporal evolution of adhesion functions.