RESUMEN
Centrioles play important roles in the assembly of centrosomes and cilia. Centriole duplication occurs once per cell cycle and is dependent on polo-like kinase 4 (PLK4). To prevent centriole amplification, which is a hallmark of cancer, PLK4 protein levels need to be tightly regulated. Here, we show that the Cullin4A/B-DDB1-DCAF1, CRL4DCAF1, E3 ligase targets PLK4 for degradation in human cells. DCAF1 binds and ubiquitylates PLK4 in the G2 phase to prevent premature centriole duplication in mitosis. In contrast to the regulation of PLK4 by SCFß-TrCP, the interaction between PLK4 and DCAF1 is independent of PLK4 kinase activity and mediated by polo-boxes 1 and 2 of PLK4, suggesting that DCAF1 promotes PLK4 ubiquitylation independently of ß-TrCP. Thus, the SCFSlimb/ß-TrCP pathway, targeting PLK4 for ubiquitylation based on its phosphorylation state and CRL4DCAF1, which ubiquitylates PLK4 by binding to the conserved PB1-PB2 domain, appear to be complementary ways to control PLK4 abundance to prevent centriole overduplication.
Asunto(s)
Centriolos , Ubiquitina , Humanos , Centriolos/metabolismo , Ubiquitina/metabolismo , Proteínas con Repetición de beta-Transducina/genética , Proteínas con Repetición de beta-Transducina/metabolismo , Ciclo Celular , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Immune checkpoint inhibition represents an important therapeutic option for advanced melanoma patients. Results from clinical studies have shown that treatment with the PD-1 inhibitors Pembrolizumab and Nivolumab provides improved response and survival rates. Moreover, combining Nivolumab with the CTLA-4 inhibitor Ipilimumab is superior to the respective monotherapies. However, use of these immunotherapies frequently associated with, sometimes life-threatening, immune-related adverse events. Thus, more evidence-based studies are required to characterize the underlying mechanisms, towards more effective clinical management and treatment monitoring. Our study examines two sets of public adverse event data coming from FAERS and VigiBase, each with more than two thousand melanoma patients treated with Pembrolizumab. Standard disproportionality metrics are utilized to characterize the safety of Pembrolizumab and its reaction profile is compared to those of the widely used Ipilimumab and Nivolumab based on melanoma cases that report only one of them. Our results confirm known toxicological considerations for their related and distinct side-effect profiles and highlight specific immune-related adverse reactions. Our retrospective computational analysis includes more patients than examined in other studies and relies on evidence coming from public pharmacovigilance data that contain safety reports from clinical and controlled studies as well as reports of suspected adverse events coming from real-world post-marketing setting. Despite these informative insights, more prospective studies are necessary to fully characterize the efficacy of these agents.
Asunto(s)
Autofagia/efectos de los fármacos , Hidroxicloroquina/farmacología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Inhibidores de Proteínas Quinasas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Anciano , Anciano de 80 o más Años , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Médula Ósea/patología , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/tratamiento farmacológico , Recurrencia , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Infections with Epstein-Barr virus (EBV) are associated with cancer development, and EBV lytic replication (the process that generates virus progeny) is a strong risk factor for some cancer types. Here we report that EBV infection of B-lymphocytes (in vitro and in a mouse model) leads to an increased rate of centrosome amplification, associated with chromosomal instability. This effect can be reproduced with virus-like particles devoid of EBV DNA, but not with defective virus-like particles that cannot infect host cells. Viral protein BNRF1 induces centrosome amplification, and BNRF1-deficient viruses largely lose this property. These findings identify a new mechanism by which EBV particles can induce chromosomal instability without establishing a chronic infection, thereby conferring a risk for development of tumours that do not necessarily carry the viral genome.
Asunto(s)
Centrosoma/virología , Inestabilidad Cromosómica , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Animales , Linfocitos B/metabolismo , Linfocitos B/virología , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica , Centrosoma/metabolismo , Infecciones por Virus de Epstein-Barr/genética , Células HEK293 , Células HeLa , Herpesvirus Humano 4/genética , Humanos , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Virión/genética , Virión/fisiologíaRESUMEN
Topoisomerase IIα is an essential enzyme that resolves topological constraints in genomic DNA. It functions in disentangling intertwined chromosomes during anaphase leading to chromosome segregation thus preserving genomic stability. Here we describe a previously unrecognized mechanism regulating topoisomerase IIα activity that is dependent on the F-box protein Fbxo28. We find that Fbxo28, an evolutionarily conserved protein, is required for proper mitotic progression. Interfering with Fbxo28 function leads to a delay in metaphase-to-anaphase progression resulting in mitotic defects as lagging chromosomes, multipolar spindles and multinucleation. Furthermore, we find that Fbxo28 interacts and colocalizes with topoisomerase IIα throughout the cell cycle. Depletion of Fbxo28 results in an increase in topoisomerase IIα-dependent DNA decatenation activity. Interestingly, blocking the interaction between Fbxo28 and topoisomerase IIα also results in multinucleated cells. Our findings suggest that Fbxo28 regulates topoisomerase IIα decatenation activity and plays an important role in maintaining genomic stability.
Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Mitosis , Proteínas Ligasas SKP Cullina F-box/metabolismo , Secuencia de Aminoácidos , Cromosomas Humanos/metabolismo , Secuencia Conservada , Regulación hacia Abajo , Evolución Molecular , Eliminación de Gen , Células HEK293 , Células HeLa , Humanos , Unión Proteica , Proteínas Ligasas SKP Cullina F-box/químicaRESUMEN
Centrioles are core components of centrosomes, the major microtubule-organizing centers of animal cells, and act as basal bodies for cilia formation. Control of centriole number is therefore crucial for genome stability and embryogenesis. Centriole duplication requires the serine/threonine protein kinase Plk4. Here, we identify Cep78 as a human centrosomal protein and a new interaction partner of Plk4. Cep78 is mainly a centriolar protein that localizes to the centriolar wall. Furthermore, we find that Plk4 binds to Cep78 through its N-terminal domain but that Cep78 is not an in vitro Plk4 substrate. Cep78 colocalizes with Plk4 at centrioles and is required for Plk4-induced centriole overduplication. Interestingly, upon depletion of Cep78, newly synthesized Plk4 is not localized to centrosomes. Our results suggest that the interaction between Cep78 and the N-terminal catalytic domain of Plk4 is a new and important element in the centrosome overduplication process.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Centriolos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células HeLa , Humanos , Interfase , Unión Proteica , Transporte de ProteínasRESUMEN
BACKGROUND: The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. METHODS: Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. RESULTS: More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. CONCLUSIONS: Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.
Asunto(s)
Regulación de la Expresión Génica/genética , Oryzias/genética , Proteínas Proto-Oncogénicas c-mdm2/genética , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/genética , Animales , Línea Celular Tumoral , Evolución Molecular , Biblioteca de Genes , HumanosRESUMEN
Duplication of centrioles, namely the formation of a procentriole next to the parental centriole, is regulated by the polo-like kinase Plk4. Only a few other proteins, including STIL (SCL/TAL1 interrupting locus, SIL) and Sas-6, are required for the early step of centriole biogenesis. Following Plk4 activation, STIL and Sas-6 accumulate at the cartwheel structure at the initial stage of the centriole assembly process. Here, we show that STIL interacts with Plk4 in vivo. A STIL fragment harboring both the coiled-coil domain and the STAN motif shows the strongest binding affinity to Plk4. Furthermore, we find that STIL is phosphorylated by Plk4. We identified Plk4-specific phosphorylation sites within the C-terminal domain of STIL and show that phosphorylation of STIL by Plk4 is required to trigger centriole duplication.
RESUMEN
The impact of self-efficacy on pain-related disability in multimorbid elderly patients in primary care is not known. The aim of our study was to analyze the influence of self-efficacy on the relation between pain intensity and pain-related disability, controlled for age and disease count, in aged multimorbid primary care patients with osteoarthritis and chronic pain. Patients were recruited in the German MultiCare study (trial registration: ISRCTN89818205). Pain was assessed using the Graded Chronic Pain Scale, and self-efficacy using the General Self-Efficacy Scale. We employed SPSS for statistical analysis. One thousand eighteen primary care patients were included in the study. Correlation analyses showed significant correlations between pain intensity and pain-related disability (r = 0.591, p < 0.001), pain intensity and general self-efficacy (r = 0.078, p < 0.05), and between general self-efficacy and pain-related disability (r = 0.153, p < 0.001). Multiple mediator analysis gives indications that self-efficacy partially mediates the relation between pain intensity and pain-related disability. In our results, we found little evidence that self-efficacy partially mediates the relation between pain intensity and pain-related disability in aged multimorbid primary care patients with osteoarthritis and chronic pain. Further research is necessary to prove the effect.
Asunto(s)
Comorbilidad , Osteoartritis/complicaciones , Osteoartritis/terapia , Manejo del Dolor/métodos , Dolor/diagnóstico , Autoeficacia , Anciano , Anciano de 80 o más Años , Estudios Transversales , Personas con Discapacidad , Femenino , Geriatría , Humanos , Masculino , Dimensión del Dolor , Atención Primaria de Salud/organización & administración , Índice de Severidad de la Enfermedad , Encuestas y CuestionariosRESUMEN
Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many-but not all-herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.
Asunto(s)
Glicoproteínas/metabolismo , Pulmón/virología , Células Mieloides/virología , Rhadinovirus/fisiología , Proteínas del Envoltorio Viral/metabolismo , Animales , Anticuerpos Neutralizantes/inmunología , Secuencia de Bases , Western Blotting , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/virología , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Células Epiteliales/virología , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Furina/metabolismo , Glicoproteínas/genética , Pulmón/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mutación/genética , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Proteínas del Envoltorio Viral/genética , Virión , Replicación ViralRESUMEN
Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4) entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.
Asunto(s)
Antígenos Virales/inmunología , Fusión de Membrana/inmunología , Rhadinovirus/inmunología , Proteínas Virales de Fusión/inmunología , Cloruro de Amonio/farmacología , Animales , Línea Celular , Endosomas/efectos de los fármacos , Endosomas/metabolismo , Ensayo de Inmunoadsorción Enzimática , Epítopos/inmunología , Humanos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Cinética , Macrólidos/farmacología , Fusión de Membrana/efectos de los fármacos , Pruebas de Neutralización , Rhadinovirus/efectos de los fármacos , Virión/inmunología , Internalización del Virus/efectos de los fármacosRESUMEN
Glycoprotein B (gB) is a conserved, essential component of gammaherpes virions and so potentially vulnerable to neutralization. However, few good gB-specific neutralizing antibodies have been identified. Here, we show that murid herpesvirus 4 is strongly neutralized by mAbs that recognize an epitope close to one of the gB fusion loops. Antibody binding did not stop gB interacting with its cellular ligands or initiating its fusion-associated conformation change, but did stop gB resolving stably to its post-fusion form, and so blocked membrane fusion to leave virions stranded in late endosomes. The conservation of gB makes this mechanism a possible general route to gammaherpesvirus neutralization.