RESUMEN
UV-induced DNA damage, a major risk factor for skin cancers, is primarily repaired by nucleotide excision repair (NER). UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy. It was initially isolated as a cDNA partially complementing UV sensitivity in xeroderma pigmentosum (XP), but this was not explored further. Here we show that UVRAG plays an integral role in UV-induced DNA damage repair. It localizes to photolesions and associates with DDB1 to promote the assembly and activity of the DDB2-DDB1-Cul4A-Roc1 (CRL4(DDB2)) ubiquitin ligase complex, leading to efficient XPC recruitment and global genomic NER. UVRAG depletion decreased substrate handover to XPC and conferred UV-damage hypersensitivity. We confirmed the importance of UVRAG for UV-damage tolerance using a Drosophila model. Furthermore, increased UV-signature mutations in melanoma correlate with reduced expression of UVRAG. Our results identify UVRAG as a regulator of CRL4(DDB2)-mediated NER and suggest that its expression levels may influence melanoma predisposition.
Asunto(s)
Autofagia/efectos de la radiación , Daño del ADN , Reparación del ADN/efectos de la radiación , Proteínas de Unión al ADN/metabolismo , Melanoma Experimental/enzimología , Neoplasias Cutáneas/enzimología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Rayos Ultravioleta , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/efectos de la radiación , Activación Enzimática , Células HEK293 , Células HeLa , Humanos , Melanoma Experimental/genética , Melanoma Experimental/patología , Proteolisis , Interferencia de ARN , Retina/enzimología , Retina/efectos de la radiación , Transducción de Señal/efectos de la radiación , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transfección , Proteínas Supresoras de Tumor/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Enveloped viruses exploit the endomembrane system to enter host cells. Through a cascade of membrane-trafficking events, virus-bearing vesicles fuse with acidic endosomes and/or lysosomes mediated by SNAREs triggering viral fusion. However, the molecular mechanisms underlying this process remain elusive. Here, we found that UV-radiation resistance-associated gene (UVRAG), an autophagic tumor suppressor, is required for the entry of the prototypic negative-strand RNA virus, including influenza A virus and vesicular stomatitis virus, by a mechanism independent of IFN and autophagy. UVRAG mediates viral endocytic transport and membrane penetration through interactions with the class C vacuolar protein sorting (C-Vps) tethering complex and endosomal glutamine-containing SNAREs [syntaxin 7 (STX7), STX8, and vesicle transport through t-SNARE homolog 1B (Vti1b)], leading to the assembly of a fusogenic trans-SNARE complex involving vesicle-associated membrane protein (VAMP8), but not VAMP7. Indeed, UVRAG stimulates VAMP8 translocation to virus-bearing endosomes. Inhibition of VAMP8, but not VAMP7, significantly reduces viral entry. Our data indicate that UVRAG, in concert with C-Vps, regulates viral entry by assembling a specific fusogenic SNARE complex. Thus, UVRAG governs downstream viral entry, highlighting an important pathway capable of potential antiviral therapeutics.
Asunto(s)
Proteínas R-SNARE/metabolismo , Virus ARN/fisiología , Proteínas Supresoras de Tumor/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Internalización del Virus , Análisis de Varianza , Animales , Western Blotting , Chlorocebus aethiops , Cricetinae , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Inmunoprecipitación , Virus de la Influenza A/fisiología , Ratones , Microscopía Confocal , Microscopía Fluorescente , Células 3T3 NIH , Plásmidos/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Vero , Vesiculovirus/fisiologíaRESUMEN
Autophagy-related factors are implicated in metabolic adaptation and cancer metastasis. However, the role of autophagy factors in cancer progression and their effect in treatment response remain largely elusive. Recent studies have shown that UVRAG, a key autophagic tumour suppressor, is mutated in common human cancers. Here we demonstrate that the cancer-related UVRAG frameshift (FS), which does not result in a null mutation, is expressed as a truncated UVRAG(FS) in colorectal cancer (CRC) with microsatellite instability (MSI), and promotes tumorigenesis. UVRAG(FS) abrogates the normal functions of UVRAG, including autophagy, in a dominant-negative manner. Furthermore, expression of UVRAG(FS) can trigger CRC metastatic spread through Rac1 activation and epithelial-to-mesenchymal transition, independently of autophagy. Interestingly, UVRAG(FS) expression renders cells more sensitive to standard chemotherapy regimen due to a DNA repair defect. These results identify UVRAG as a new MSI target gene and provide a mechanism for UVRAG participation in CRC pathogenesis and treatment response.
Asunto(s)
Carcinogénesis/genética , Neoplasias Colorrectales/genética , Resistencia a Antineoplásicos/genética , Transición Epitelial-Mesenquimal/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Animales , Antimetabolitos Antineoplásicos/farmacología , Autofagia/genética , Línea Celular Tumoral , Neoplasias Colorrectales/patología , Reparación del ADN/genética , Femenino , Fluorouracilo/farmacología , Mutación del Sistema de Lectura , Predisposición Genética a la Enfermedad , Células HCT116 , Células HEK293 , Células HT29 , Células HeLa , Humanos , Inmunohistoquímica , Masculino , Ratones , Inestabilidad de Microsatélites , Microscopía Confocal , Persona de Mediana Edad , Células Madre Embrionarias de Ratones , Células 3T3 NIH , Invasividad Neoplásica/genética , Metástasis de la Neoplasia/genética , Trasplante de Neoplasias , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína de Unión al GTP rac1/metabolismoRESUMEN
While the cell imposes multiple barriers to virus entry, enveloped viruses are remarkably still able to gain entry to their cellular hosts by hitchhiking and remodeling the endomembrane system to traffic within, and eventually escape from, endosomal organelles for their genome release. Elucidating viral entry mechanisms and their interaction with the host trafficking network is necessary for antiviral therapy. Here, we focus on the use of host autophagy molecular factors during the entry of prototypic negative-stranded RNA viruses, and highlight recent progress in our understanding of the role of one such factor, UVRAG, in both viral and cellular endocytic membrane trafficking and fusion events.
Asunto(s)
Autofagia , Proteínas Supresoras de Tumor/metabolismo , Internalización del Virus , Virus/metabolismo , Endosomas/metabolismo , Humanos , Proteínas SNARE/metabolismoRESUMEN
Endoplasmic reticulum (ER)-Golgi membrane transport and autophagy are intersecting trafficking pathways that are tightly regulated and crucial for homeostasis, development and disease. Here, we identify UVRAG, a beclin-1-binding autophagic factor, as a phosphatidylinositol-3-phosphate (PtdIns(3)P)-binding protein that depends on PtdIns(3)P for its ER localization. We further show that UVRAG interacts with RINT-1, and acts as an integral component of the RINT-1-containing ER tethering complex, which couples phosphoinositide metabolism to COPI-vesicle tethering. Displacement or knockdown of UVRAG profoundly disrupted COPI cargo transfer to the ER and Golgi integrity. Intriguingly, autophagy caused the dissociation of UVRAG from the ER tether, which in turn worked in concert with the Bif-1-beclin-1-PI(3)KC3 complex to mobilize Atg9 translocation for autophagosome formation. These findings identify a regulatory mechanism that coordinates Golgi-ER retrograde and autophagy-related vesicular trafficking events through physical and functional interactions between UVRAG, phosphoinositide and their regulatory factors, thereby ensuring spatiotemporal fidelity of membrane trafficking and maintenance of organelle homeostasis.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Secuencia de Aminoácidos , Animales , Autofagia , Proteínas Relacionadas con la Autofagia , Beclina-1 , Transporte Biológico , Células COS , Línea Celular , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Proteínas de Transporte VesicularRESUMEN
UVRAG is a promoter of the autophagy pathway, and its deficiency may fuel the development of cancers. Intriguingly, our recent study has demonstrated that this protein also mediates the repair of damaged DNA and patrols centrosome stability, mechanisms that commonly prevent cancer progression, in a manner independent of its role in autophagy signaling. Given the central role of UVRAG in genomic stability and autophagic cleaning, it is speculated that UVRAG is a bona fide genome protector and that the decrease in UVRAG seen in some cancers may render these cells vulnerable to chromosomal damage, making UVRAG an appealing target for cancer therapy.
Asunto(s)
Autofagia/genética , Inestabilidad Genómica/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Centrosoma/metabolismo , Daño del ADN , Reparación del ADN , RatonesRESUMEN
Autophagy defects have recently been associated with chromosomal instability, a hallmark of human cancer. However, the functional specificity and mechanism of action of autophagy-related factors in genome stability remain elusive. Here we report that UVRAG, an autophagic tumor suppressor, plays a dual role in chromosomal stability, surprisingly independent of autophagy. We establish that UVRAG promotes DNA double-strand-break repair by directly binding and activating DNA-PK in nonhomologous end joining. Disruption of UVRAG increases genetic instability and sensitivity of cells to irradiation. Furthermore, UVRAG was also found to be localized at centrosomes and physically associated with CEP63, an integral component of centrosomes. Disruption of the association of UVRAG with centrosomes causes centrosome instability and aneuploidy. UVRAG thus represents an autophagy-related molecular factor that also has a convergent role in patrolling both the structural integrity and proper segregation of chromosomes, which may confer autophagy-independent tumor suppressor activity.
Asunto(s)
Inestabilidad Cromosómica/genética , Reparación del ADN/fisiología , Proteína Quinasa Activada por ADN/metabolismo , ADN/metabolismo , Neoplasias/genética , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Aneuploidia , Autofagia/genética , Proteínas de Ciclo Celular , Centrosoma/metabolismo , Segregación Cromosómica/genética , Roturas del ADN de Doble Cadena/efectos de la radiación , Células HEK293 , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Bcl-2, originally identified as a universal inhibitor of apoptotic cell death, has since been implicated in suppressing autophagy, the cell's quality control mechanism. Our recent study demonstrates that the anti-autophagic aspect of Bcl-2 can function as a promoter of oncogenic growth, independently of its role in apoptosis signaling. It is likely that the increase in Bcl-2 often seen in breast and other cancers might render cells error-prone by blunting autophagy, while concomitantly keeping damaged cells alive. The outcome of such a 'double hit' of Bcl-2 may synergistically promote tumor growth and increase the chance of cancer development and drug resistance.
Asunto(s)
Autofagia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Apoptosis , Supervivencia Celular , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Resistencia a Antineoplásicos , HumanosRESUMEN
γ-Herpesviruses (γ-HVs) are notable for their ability to establish latent infections of lymphoid cells(1). The narrow host range of human γ-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine γ-herpesvirus 68 (γHV68) shares extensive genetic and biological similarities with human γ-HVs and is a natural pathogen of murid rodents(2). As such, evaluation of γHV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during γ-HVs infection. Upon intranasal inoculation, γHV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host(3,4). In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of γHV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.