RESUMEN
Here, we show that the cellular DNA replication protein and ATR substrate SMARCAL1 is recruited to viral replication centers early during adenovirus infection and is then targeted in an E1B-55K/E4orf6- and cullin RING ligase-dependent manner for proteasomal degradation. In this regard, we have determined that SMARCAL1 is phosphorylated at S123, S129, and S173 early during infection in an ATR- and CDK-dependent manner, and that pharmacological inhibition of ATR and CDK activities attenuates SMARCAL1 degradation. SMARCAL1 recruitment to viral replication centers was shown to be largely dependent upon SMARCAL1 association with the RPA complex, while Ad-induced SMARCAL1 phosphorylation also contributed to SMARCAL1 recruitment to viral replication centers, albeit to a limited extent. SMARCAL1 was found associated with E1B-55K in adenovirus E1-transformed cells. Consistent with its ability to target SMARCAL1, we determined that E1B-55K modulates cellular DNA replication. As such, E1B-55K expression initially enhances cellular DNA replication fork speed but ultimately leads to increased replication fork stalling and the attenuation of cellular DNA replication. Therefore, we propose that adenovirus targets SMARCAL1 for degradation during infection to inhibit cellular DNA replication and promote viral replication.IMPORTANCE Viruses have evolved to inhibit cellular DNA damage response pathways that possess antiviral activities and utilize DNA damage response pathways that possess proviral activities. Adenovirus has evolved, primarily, to inhibit DNA damage response pathways by engaging with the ubiquitin-proteasome system and promoting the degradation of key cellular proteins. Adenovirus differentially regulates ATR DNA damage response signaling pathways during infection. The cellular adenovirus E1B-55K binding protein E1B-AP5 participates in ATR signaling pathways activated during infection, while adenovirus 12 E4orf6 negates Chk1 activation by promoting the proteasome-dependent degradation of the ATR activator TOPBP1. The studies detailed here indicate that adenovirus utilizes ATR kinase and CDKs during infection to promote the degradation of SMARCAL1 to attenuate normal cellular DNA replication. These studies further our understanding of the relationship between adenovirus and DNA damage and cell cycle signaling pathways during infection and establish new roles for E1B-55K in the modulation of cellular DNA replication.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Proteínas E1B de Adenovirus/metabolismo , Adenovirus Humanos/fisiología , ADN Helicasas/metabolismo , Replicación del ADN , Replicación Viral , Células A549 , Infecciones por Adenoviridae/virología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Transducción de Señal , Ubiquitina/metabolismoRESUMEN
DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades , Ribonucleoproteínas Nucleares Heterogéneas/fisiología , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Ácido Anhídrido Hidrolasas , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Adenovirus has evolved strategies to usurp host-cell factors and machinery to facilitate its life cycle, including cell entry, replication, assembly and egress. Adenovirus continues, therefore, to be an important model system for investigating fundamental cellular processes. The role of adenovirus E1B-55k in targeting host-cell proteins that possess antiviral activity for proteasomal degradation is now well established. To expand our understanding of E1B-55k in regulating the levels of host-cell proteins, we performed comparative proteome analysis of wild-type, and E1B-55k-deletion, adenovirus-infected cancer cells. As such we performed quantitative MS/MS analysis to monitor protein expression changes affected by viral E1B-55k. We identified 5937 proteins, and of these, 69 and 58 proteins were down-regulated during wild-type and E1B-55k (dl1520) adenovirus infection, respectively. This analysis revealed that there are many, previously unidentified, cellular proteins subjected to degradation by adenovirus utilizing pathways independent of E1B-55k expression. Moreover, we found that ALCAM, EPHA2 and PTPRF, three cellular proteins that function in the regulation of cell-cell contacts, appeared to be degraded by E1B-55k/E4orf3 and/or E1B-55k/E4orf6 complexes. These molecules, like integrin α3 (a known substrate of E1B-55k/E4orf6), are critical regulators of cell signalling, cell adhesion and cell surface modulation, and their degradation during infection is, potentially, pertinent to adenovirus propagation. The data presented in this study illustrate the broad nature of protein down-regulation mediated by adenovirus.
Asunto(s)
Infecciones por Adenoviridae/patología , Adenoviridae/crecimiento & desarrollo , Proteínas E1B de Adenovirus/genética , Eliminación de Gen , Interacciones Huésped-Patógeno , Proteoma/análisis , Adenoviridae/genética , Infecciones por Adenoviridae/virología , Línea Celular Tumoral , Humanos , Proteómica , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
The PTTG1-binding factor (PBF) is a transforming gene capable of eliciting tumor formation in xenograft models. However, the precise role of PBF in tumorigenesis and its prognostic value as a cancer biomarker remain largely uncharacterised, particularly in malignancies outside the thyroid. Here, we provide the first evidence that PBF represents a promising prognostic marker in colorectal cancer. Examination of a total of 39 patients demonstrated higher PBF expression at both the mRNA (P = 0.009) and protein (P < 0.0001) level in colorectal tumors compared to matched normal tissue. Critically, PBF was most abundant in colorectal tumors associated with Extramural Vascular Invasion (EMVI), increased genetic instability (GI) and somatic TP53 mutations, all features linked with recurrence and poorer patient survival. We further demonstrate by glutathione-S-transferase (GST) pull-down and coimmunoprecipitation that PBF binds to the tumor suppressor protein p53, as well as to p53 mutants (Δ126-132, M133K, V197E, G245D, I255F and R273C) identified in the colorectal tumors. Importantly, overexpression of PBF in colorectal HCT116 cells interfered with the transcriptional activity of p53-responsive genes such as mdm2, p21 and sfn. Diminished p53 stability (> 90%; P < 0.01) was also evident with a concurrent increase in ubiquitinated p53. Human colorectal tumors with wild-type TP53 and high PBF expression also had low p53 protein levels (P < 0.05), further emphasizing a putative interaction between these genes in vivo. Overall, these results demonstrate an emerging role for PBF in colorectal tumorigenesis through regulating p53 activity, with implications for PBF as a prognostic indicator for invasive tumors.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Proteínas de la Membrana/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Expresión Génica , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Invasividad Neoplásica , Pronóstico , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proto-Oncogenes Mas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ensayo de Tumor de Célula Madre , UbiquitinaciónRESUMEN
Down's syndrome (DS) is a genetic disorder caused by full or partial trisomy of human chromosome 21 and presents with many clinical phenotypes including a reduced incidence of solid tumours. Recent work with the Ts65Dn model of DS, which has orthologues of about 50% of the genes on chromosome 21 (Hsa21), has indicated that three copies of the ETS2 (ref. 3) or DS candidate region 1 (DSCR1) genes (a previously known suppressor of angiogenesis) is sufficient to inhibit tumour growth. Here we use the Tc1 transchromosomic mouse model of DS to dissect the contribution of extra copies of genes on Hsa21 to tumour angiogenesis. This mouse expresses roughly 81% of Hsa21 genes but not the human DSCR1 region. We transplanted B16F0 and Lewis lung carcinoma tumour cells into Tc1 mice and showed that growth of these tumours was substantially reduced compared with wild-type littermate controls. Furthermore, tumour angiogenesis was significantly repressed in Tc1 mice. In particular, in vitro and in vivo angiogenic responses to vascular endothelial growth factor (VEGF) were inhibited. Examination of the genes on the segment of Hsa21 in Tc1 mice identified putative anti-angiogenic genes (ADAMTS1and ERG) and novel endothelial cell-specific genes, never previously shown to be involved in angiogenesis (JAM-B and PTTG1IP), that, when overexpressed, are responsible for inhibiting angiogenic responses to VEGF. Three copies of these genes within the stromal compartment reduced tumour angiogenesis, explaining the reduced tumour growth in DS. Furthermore, we expect that, in addition to the candidate genes that we show to be involved in the repression of angiogenesis, the Tc1 mouse model of DS will permit the identification of other endothelium-specific anti-angiogenic targets relevant to a broad spectrum of cancer patients.
Asunto(s)
Carcinoma Pulmonar de Lewis/irrigación sanguínea , Modelos Animales de Enfermedad , Síndrome de Down/genética , Dosificación de Gen/genética , Melanoma Experimental/irrigación sanguínea , Neovascularización Patológica/genética , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Animales , Carcinoma Pulmonar de Lewis/complicaciones , Carcinoma Pulmonar de Lewis/genética , Carcinoma Pulmonar de Lewis/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Cromosomas de los Mamíferos/genética , Síndrome de Down/complicaciones , Síndrome de Down/fisiopatología , Femenino , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Masculino , Melanoma Experimental/complicaciones , Melanoma Experimental/genética , Melanoma Experimental/patología , Ratones , Trasplante de Neoplasias , Neovascularización Patológica/patología , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Proteína Proto-Oncogénica c-ets-2/genética , Proteína Proto-Oncogénica c-ets-2/metabolismo , Factores de Transcripción , Regulador Transcripcional ERG , Trisomía/genética , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Investigation of rare familial forms of renal cell carcinoma (RCC) has led to the identification of genes such as VHL and MET that are also implicated in the pathogenesis of sporadic RCC. In order to identify a novel candidate renal tumor suppressor gene, we characterized the breakpoints of a constitutional balanced translocation, t(5;19)(p15.3;q12), associated with familial RCC and found that a previously uncharacterized gene UBE2QL1 was disrupted by the chromosome 5 breakpoint. UBE2QL1 mRNA expression was downregulated in 78.6% of sporadic RCC and, although no intragenic mutations were detected, gene deletions and promoter region hypermethylation were detected in 17.3% and 20.3%, respectively, of sporadic RCC. Reexpression of UBE2QL1 in a deficient RCC cell line suppressed anchorage-independent growth. UBE2QL1 shows homology to the E2 class of ubiquitin conjugating enzymes and we found that (1) UBE2QL1 possesses an active-site cysteine (C88) that is monoubiquitinated in vivo, and (2) UBE2QL1 interacts with FBXW7 (an F box protein providing substrate recognition to the SCF E3 ubiquitin ligase) and facilitates the degradation of the known FBXW7 targets, CCNE1 and mTOR. These findings suggest UBE2QL1 as a novel candidate renal tumor suppressor gene.
Asunto(s)
Genes Supresores de Tumor , Predisposición Genética a la Enfermedad , Neoplasias Renales/genética , Translocación Genética , Enzimas Ubiquitina-Conjugadoras/genética , Adulto , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Puntos de Rotura del Cromosoma , Cromosomas Humanos Par 19 , Cromosomas Humanos Par 5 , Metilación de ADN , Epigénesis Genética , Proteínas F-Box/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Renales/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/genética , ARN Mensajero/química , ARN Viral/química , Adenosina Trifosfatasas/metabolismo , Proteínas E1A de Adenovirus/inmunología , Adenovirus Humanos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Línea Celular , Núcleo Celular/metabolismo , Inhibición de Contacto , Citoplasma/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Transcripción Genética , Replicación Viral/genéticaRESUMEN
The ability of adenovirus early region proteins, E1B-55K and E4orf6, to usurp control of cellular ubiquitin ligases and target proteins for proteasome-dependent degradation during infection is well established. Here we show that the E4 gene product, E4orf3 can, independently of E1B-55K and E4orf6, target the transcriptional corepressor transcriptional intermediary factor 1γ (TIF1γ) for proteasome-mediated degradation during infection. Initial mass spectrometric studies identified TIF1 family members-TIF1α, TIF1ß, and TIF1γ-as E1B-55K-binding proteins in both transformed and infected cells, but analyses revealed that, akin to TIF1α, TIF1γ is reorganized in an E4orf3-dependent manner to promyelocytic leukemia protein-containing nuclear tracks during infection. The use of a number of different adenovirus early region mutants identified the specific and sole requirement for E4orf3 in mediating TIF1γ degradation. Further analyses revealed that TIF1γ is targeted for degradation by a number of divergent human adenoviruses, suggesting that the ability of E4orf3 to regulate TIF1γ expression is evolutionarily conserved. We also determined that E4orf3 does not utilize the Cullin-based ubiquitin ligases, CRL2 and CRL5, or the TIF1α ubiquitin ligase in order to promote TIF1γ degradation. Further studies suggested that TIF1γ possesses antiviral activity and limits adenovirus early and late gene product expression during infection. Indeed, TIF1γ knockdown accelerates the adenovirus-mediated degradation of MRE11, while TIF1γ overexpression delays the adenovirus-mediated degradation of MRE11. Taken together, these studies have identified novel adenovirus targets and have established a new role for the E4orf3 protein during infection.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Proteínas E4 de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/metabolismo , Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/virología , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas E4 de Adenovirus/genética , Adenovirus Humanos/genética , Línea Celular , Humanos , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIbeta-binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Virales/metabolismo , Adenoviridae/genética , Adenoviridae/metabolismo , Adenoviridae/fisiología , Proteínas E1B de Adenovirus/genética , Proteínas E1B de Adenovirus/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Elonguina , Técnica del Anticuerpo Fluorescente , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Microscopía Confocal , Mutación , Proteínas Nucleares/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/genéticaRESUMEN
It is clear that a number of host-cell factors facilitate virus replication and, conversely, a number of other factors possess inherent antiviral activity. Research, particularly over the last decade or so, has revealed that there is a complex inter-relationship between viral infection and the host-cell DNA-damage response and repair pathways. There is now a realization that viruses can selectively activate and/or repress specific components of these host-cell pathways in a temporally coordinated manner, in order to promote virus replication. Thus, some viruses, such as simian virus 40, require active DNA-repair pathways for optimal virus replication, whereas others, such as adenovirus, go to considerable lengths to inactivate some pathways. Although there is ever-increasing molecular insight into how viruses interact with host-cell damage pathways, the precise molecular roles of these pathways in virus life cycles is not well understood. The object of this review is to consider how DNA viruses have evolved to manage the function of three principal DNA damage-response pathways controlled by the three phosphoinositide 3-kinase (PI3K)-related protein kinases ATM, ATR and DNA-PK and to explore further how virus interactions with these pathways promote virus replication.
Asunto(s)
Daño del ADN , Reparación del ADN , Virus ADN/genética , Transducción de Señal/genética , Replicación Viral/genética , Virus ADN/metabolismo , HumanosRESUMEN
Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.
Asunto(s)
Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/metabolismo , Adenoviridae/fisiología , Daño del ADN , Adenoviridae/clasificación , Adenoviridae/genética , Infecciones por Adenoviridae/enzimología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidad de la Especie , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Differentiated thyroid cancers and their metastases frequently exhibit reduced iodide uptake, impacting on the efficacy of radioiodine ablation therapy. PTTG binding factor (PBF) is a proto-oncogene implicated in the pathogenesis of thyroid cancer. We recently reported that PBF inhibits iodide uptake, and have now elucidated a mechanism by which PBF directly modulates sodium iodide symporter (NIS) activity in vitro. In subcellular localisation studies, PBF overexpression resulted in the redistribution of NIS from the plasma membrane into intracellular vesicles, where it colocalised with the tetraspanin CD63. Cell-surface biotinylation assays confirmed a reduction in plasma membrane NIS expression following PBF transfection compared with vector-only treatment. Coimmunoprecipitation and GST-pull-down experiments demonstrated a direct interaction between NIS and PBF, the functional consequence of which was assessed using iodide-uptake studies in rat thyroid FRTL-5 cells. PBF repressed iodide uptake, whereas three deletion mutants, which did not localise within intracellular vesicles, lost the ability to inhibit NIS activity. In summary, we present an entirely novel mechanism by which the proto-oncogene PBF binds NIS and alters its subcellular localisation, thereby regulating its ability to uptake iodide. Given that PBF is overexpressed in thyroid cancer, these findings have profound implications for thyroid cancer ablation using radioiodine.
Asunto(s)
Diferenciación Celular , Proteínas Represoras/metabolismo , Simportadores/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Animales , Antígenos CD/metabolismo , Caveolinas/metabolismo , Línea Celular , Eliminación de Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular , Yoduros/metabolismo , Proteínas de la Membrana/metabolismo , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Transporte de Proteínas , Proto-Oncogenes Mas , Ratas , Fracciones Subcelulares/metabolismo , Tetraspanina 30RESUMEN
The largest E1A isoform of human adenovirus (Ad) includes a C-4 zinc finger domain within conserved region 3 (CR3) that is largely responsible for activating transcription of the early viral genes. CR3 interacts with multiple cellular factors, but its mechanism of action is modeled primarily on the basis of the mechanism for the prototype E1A protein of human Ad type 5. We expanded this model to include a representative member from each of the six human Ad subgroups. All CR3 domains tested were capable of transactivation. However, there were dramatic differences in their levels of transcriptional activation. Despite these functional variations, the interactions of these representative CR3s with known cellular transcriptional regulators revealed only modest differences. Four common cellular targets of all representative CR3s were identified: the proteasome component human Sug1 (hSug1)/S8, the acetyltransferases p300/CREB binding protein (CBP), the mediator component mediator complex subunit 23 (MED23) protein, and TATA binding protein (TBP). The first three factors appear to be critical for CR3 function. RNA interference against human TBP showed no significant reduction in transactivation by any CR3 tested. These results indicate that the cellular factors previously shown to be important for transactivation by Ad5 CR3 are similarly bound by the E1A proteins of other types. This was confirmed experimentally using a transcriptional squelching assay, which demonstrated that the CR3 regions of each Ad type could compete with Ad5 CR3 for limiting factors. Interestingly, a mutant of Ad5 CR3 (V147L) was capable of squelching wild-type Ad5 CR3, despite its failure to bind TBP, MED23, p300/CBP-associated factor (pCAF), or p300/CBP, suggestive of the possibility that an additional as yet unidentified cellular factor is required for transactivation by E1A CR3.
Asunto(s)
Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/virología , Adenoviridae/clasificación , Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenoviridae/patogenicidad , Infecciones por Adenoviridae/metabolismo , Animales , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/virología , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Técnica del Anticuerpo Fluorescente , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Luciferasas/metabolismo , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a multicomponent E3 ubiquitin ligase that, by targeting protein substrates for 26S proteasome-mediated degradation through ubiquitination, coordinates the temporal progression of eukaryotic cells through mitosis and the subsequent G1 phase of the cell cycle. Other functions of the APC/C are, however, less well defined. Here we show that two APC/C components, APC5 and APC7, interact directly with the coactivators CBP and p300 through protein-protein interaction domains that are evolutionarily conserved in adenovirus E1A. This interaction stimulates intrinsic CBP/p300 acetyltransferase activity and potentiates CBP/p300-dependent transcription. We also show that APC5 and APC7 suppress E1A-mediated transformation in a CBP/p300-dependent manner, indicating that these components of the APC/C may be targeted during cellular transformation. Furthermore, we establish that CBP is required in APC/C function; specifically, gene ablation of CBP by RNA-mediated interference markedly reduces the E3 ubiquitin ligase activity of the APC/C and the progression of cells through mitosis. Taken together, our results define discrete roles for the APC/C-CBP/p300 complexes in growth regulation.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Ciclo Celular/fisiología , Regulación de la Expresión Génica , Transcripción Genética , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Secuencia de Aminoácidos , Ciclosoma-Complejo Promotor de la Anafase , Animales , Subunidad Apc5 del Ciclosoma-Complejo Promotor de la Anafase , Subunidad Apc7 del Ciclosoma-Complejo Promotor de la Anafase , Proteína de Unión a CREB/química , Proteína de Unión a CREB/genética , Línea Celular , Transformación Celular Neoplásica , Secuencia Conservada , Humanos , Mitosis , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Complejos de Ubiquitina-Proteína Ligasa/química , Complejos de Ubiquitina-Proteína Ligasa/genéticaRESUMEN
The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Activación Transcripcional , Factores de Transcripción p300-CBP/metabolismo , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E4 de Adenovirus/genética , Secuencia de Aminoácidos , Línea Celular , Secuencia Conservada , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Proteína p300 Asociada a E1A/genética , Humanos , Mutación , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053-32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.
Asunto(s)
Mitosis , Proteínas Nucleares/fisiología , Transactivadores/fisiología , Proteínas Adaptadoras Transductoras de Señales , Anafase , Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Immunoblotting/métodos , Metafase , Microscopía Fluorescente/métodos , Modelos Biológicos , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/química , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Viral genomes have high gene densities and complex transcription strategies rendering transcriptome analysis through short-read RNA-seq approaches problematic. Adenovirus transcription and splicing is especially complex. We used long-read direct RNA sequencing to study adenovirus transcription and splicing during infection. This revealed a previously unappreciated complexity of alternative splicing and potential for secondary initiating codon usage. Moreover, we find that most viral transcripts tend to shorten polyadenylation lengths as infection progresses. Development of an open reading frame centric bioinformatics analysis pipeline provided a deeper quantitative and qualitative understanding of adenovirus's genetic potential. Across the viral genome adenovirus makes multiple distinctly spliced transcripts that code for the same protein. Over 11,000 different splicing patterns were recorded across the viral genome, most occurring at low levels. This low-level use of alternative splicing patterns potentially enables the virus to maximise its coding potential over evolutionary timescales.
Asunto(s)
Adenovirus Humanos/genética , Empalme Alternativo/genética , Evolución Molecular , Transcriptoma , Secuencia de Bases , Línea Celular , Uso de Codones , Biología Computacional/métodos , Exones , Fibroblastos/virología , Perfilación de la Expresión Génica , Genoma Viral , Humanos , Poliadenilación , Regiones Promotoras Genéticas , ARN Viral/genética , RNA-SeqRESUMEN
The sodium iodide symporter (NIS) is required for iodide uptake, which facilitates thyroid hormone biosynthesis. NIS has been exploited for over 75 years in ablative radioiodine (RAI) treatment of thyroid cancer, where its ability to transport radioisotopes depends on its localization to the plasma membrane. The advent of NIS-based in vivo imaging and theranostic strategies in other malignancies and disease modalities has recently increased the clinical importance of NIS. However, NIS trafficking remains ill-defined. Here, we used tandem mass spectrometry followed by coimmunoprecipitation and proximity ligation assays to identify and validate two key nodes-ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP)-controlling NIS trafficking. Using cell-surface biotinylation assays and highly inclined and laminated optical sheet microscopy, we demonstrated that ARF4 enhanced NIS vesicular trafficking from the Golgi to the plasma membrane, whereas VCP-a principal component of endoplasmic reticulum (ER)-associated degradation-governed NIS proteolysis. Gene expression analysis indicated VCP expression was particularly induced in aggressive thyroid cancers and in patients who had poorer outcomes following RAI treatment. Two repurposed FDA-approved VCP inhibitors abrogated VCP-mediated repression of NIS function, resulting in significantly increased NIS at the cell-surface and markedly increased RAI uptake in mouse and human thyroid models. Collectively, these discoveries delineate NIS trafficking and highlight the new possibility of systemically enhancing RAI therapy in patients using FDA-approved drugs. SIGNIFICANCE: These findings show that ARF4 and VCP are involved in NIS trafficking to the plasma membrane and highlight the possible therapeutic role of VCP inhibitors in enhancing radioiodine effectiveness in radioiodine-refractory thyroid cancer.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Aparato de Golgi/metabolismo , Radioisótopos de Yodo/farmacología , Simportadores/metabolismo , Cáncer Papilar Tiroideo/terapia , Neoplasias de la Tiroides/terapia , Proteína que Contiene Valosina/metabolismo , Adulto , Animales , Mama/patología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimioradioterapia/métodos , Femenino , Perfilación de la Expresión Génica , Humanos , Radioisótopos de Yodo/uso terapéutico , Estimación de Kaplan-Meier , Masculino , Ratones , Persona de Mediana Edad , Cultivo Primario de Células , Pronóstico , Supervivencia sin Progresión , Proteolisis , Cáncer Papilar Tiroideo/mortalidad , Cáncer Papilar Tiroideo/patología , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/patología , Glándula Tiroides/efectos de la radiación , Neoplasias de la Tiroides/mortalidad , Neoplasias de la Tiroides/patología , Distribución Tisular , Proteína que Contiene Valosina/antagonistas & inhibidoresRESUMEN
C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.
Asunto(s)
Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Luciferasas/análisis , Neoplasias Pulmonares/metabolismo , Datos de Secuencia Molecular , Mutación , Osteosarcoma/metabolismo , Plásmidos , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.