RESUMEN
The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.
Asunto(s)
Puntos de Control de la Fase G2 del Ciclo Celular , Infecciones por VIH/patología , VIH-1/metabolismo , Inmunidad Innata , Complejos Multiproteicos/metabolismo , Recombinasas/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Células HEK293 , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Células HeLa , Humanos , Interferón gamma/metabolismoRESUMEN
Activating mutations in NOTCH1, an essential regulator of T cell development, are frequently found in human T cell acute lymphoblastic leukemia (T-ALL). Despite important advances in our understanding of Notch signal transduction, the regulation of Notch functions in the nucleus remains unclear. Using immunoaffinity purification, we identified NOTCH1 nuclear partners in T-ALL cells and showed that, beyond the well-characterized core activation complex (ICN1-CSL-MAML1), NOTCH1 assembles a multifunctional complex containing the transcription coactivator AF4p12, the PBAF nucleosome remodeling complex, and the histone demethylases LSD1 and PHF8 acting through their demethylase activity to promote epigenetic modifications at Notch-target genes. Remarkably, LSD1 functions as a corepressor when associated with CSL-repressor complex and as a NOTCH1 coactivator upon Notch activation. Our work provides new insights into the molecular mechanisms that govern Notch transcriptional activity and represents glimpse into NOTCH1 interaction landscape, which will help in deciphering mechanisms of NOTCH1 functions and regulation.
Asunto(s)
Proteínas Oncogénicas/metabolismo , Receptor Notch1/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Regulación Leucémica de la Expresión Génica , Células HEK293 , Células HeLa , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Immunoblotting , Ratones , Ratones SCID , Modelos Genéticos , Proteínas Oncogénicas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patología , Unión Proteica , Interferencia de ARN , Receptor Notch1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Trasplante HeterólogoRESUMEN
In response to DNA double-strand breaks (DSBs), BRCA1 forms biochemically distinct complexes with certain other DNA damage response proteins. These structures, some of which are required for homologous recombination (HR)-type DSB repair, concentrate at distinct nuclear foci that demarcate sites of genome breakage. Polyubiquitin binding by one of these structures, the RAP80/BRCA1 complex, is required for efficient BRCA1 focal recruitment, but the relationship of this process to the execution of HR has been unclear. We found that this complex actively suppresses otherwise exaggerated, BRCA1-driven HR. By controlling the kinetics by which other BRCA1-interacting proteins that promote HR concentrate together with BRCA1 in nuclear foci, RAP80/BRCA1 complexes suppress excessive DSB end processing, HR-type DSB repair, and overt chromosomal instability. Since chromosomal instability emerges when BRCA1 HR function is either unbridled or absent, active tuning of BRCA1 activity, executed in nuclear foci, is important to genome integrity maintenance.
Asunto(s)
Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Reparación del ADN , Proteínas Nucleares/metabolismo , Radiación Ionizante , Recombinación Genética , Proteínas Portadoras/genética , Línea Celular Tumoral , Inestabilidad Cromosómica , Cromosomas/química , Cromosomas/genética , Cromosomas/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN , Células HEK293 , Células HeLa , Chaperonas de Histonas , Humanos , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genéticaRESUMEN
HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.
Asunto(s)
Núcleo Celular/metabolismo , VIH-1/genética , ARN Viral/biosíntesis , Ribonucleoproteínas Nucleares Pequeñas/metabolismo , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Sitios de Unión , Línea Celular , Quinasa 9 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH-1/metabolismo , Células HeLa , N-Metiltransferasa de Histona-Lisina , Humanos , Complejos Multiproteicos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/genética , Estrés Fisiológico , Factores de Transcripción , Factores de Elongación Transcripcional/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.
Asunto(s)
Células Dendríticas/metabolismo , VIH-1/fisiología , Proteínas de Unión al GTP Monoméricas/metabolismo , Células Mieloides/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Línea Celular , ADN Viral/metabolismo , Células Dendríticas/virología , Silenciador del Gen , Infecciones por VIH/metabolismo , Células HeLa , Humanos , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína 1 que Contiene Dominios SAM y HD , Células U937 , Replicación ViralRESUMEN
The hepatitis B virus X protein (HBx) is essential for virus replication and has been implicated in the development of liver cancer. HBx is recruited to viral and cellular promoters and activates transcription by interacting with transcription factors and coactivators. Here, we purified HBx-associated factors in nuclear extracts from HepG2 hepatoma cells and identified protein arginine methyltransferase 1 (PRMT1) as a novel HBx-interacting protein. We showed that PRMT1 overexpression reduced the transcription of hepatitis B virus (HBV), and this inhibition was dependent on the methyltransferase function of PRMT1. Conversely, depletion of PRMT1 correlated with increased HBV transcription. Using a quantitative chromatin immunoprecipitation assay, we found that PRMT1 is recruited to HBV DNA, suggesting a direct effect of PRMT1 on the regulation of HBV transcription. Finally, we showed that HBx expression inhibited PRMT1-mediated protein methylation. Downregulation of PRMT1 activity was further observed in HBV-replicating cells in an in vivo animal model. Altogether, our results support the notion that the binding of HBx to PRMT1 might benefit viral replication by relieving the inhibitory activity of PRMT1 on HBV transcription.
Asunto(s)
Virus de la Hepatitis B/patogenicidad , Interacciones Huésped-Patógeno , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Replicación Viral , Línea Celular , Inmunoprecipitación de Cromatina , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hepatocitos/virología , Humanos , Evasión Inmune , Unión Proteica , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Helicasas/metabolismo , ADN/metabolismo , Proteómica/métodos , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Western Blotting , Línea Celular Tumoral , Nucléolo Celular/metabolismo , ADN/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HeLa , Humanos , Autoantígeno Ku , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Unión Proteica/genética , Transporte de Proteínas/efectos de la radiación , Proteoma/clasificación , Proteoma/genética , Proteoma/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Factores de Tiempo , Rayos UltravioletaRESUMEN
The Super Elongation Complex (SEC), containing transcription elongation activators/coactivators P-TEFb, ELL2, AFF4/1, ENL, and AF9, is recruited by HIV-1 Tat and mixed lineage leukemia (MLL) proteins to activate the expression of HIV-1 and MLL-target genes, respectively. In the absence of Tat and MLL, however, it is unclear how SEC is targeted to RNA polymerase (Pol) II to stimulate elongation in general. Furthermore, although ENL and AF9 can bind the H3K79 methyltransferase Dot1L, it is unclear whether these bindings are required for SEC-mediated transcription. Here, we show that the homologous ENL and AF9 exist in separate SECs with similar but nonidentical functions. ENL/AF9 contacts the scaffolding protein AFF4 that uses separate domains to recruit different subunits into SEC. ENL/AF9 also exists outside SEC when bound to Dot1L, which is found to inhibit SEC function. The YEATS domain of ENL/AF9 targets SEC to Pol II on chromatin through contacting the human Polymerase-Associated Factor complex (PAFc) complex. This finding explains the YEATS domain's dispensability for leukemogenesis when ENL/AF9 is translocated to MLL, whose interactions with PAFc and DNA likely substitute for the PAFc/chromatin-targeting function of the YEATS domain.
Asunto(s)
Cromatina/metabolismo , Complejos Multiproteicos/metabolismo , ARN Polimerasa II/metabolismo , Factores de Elongación Transcripcional/metabolismo , Cromatina/genética , Regulación Viral de la Expresión Génica/fisiología , VIH-1/genética , VIH-1/metabolismo , Células HeLa , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Complejos Multiproteicos/genética , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Elongación Transcripcional/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.
Asunto(s)
Biosíntesis de Proteínas , Ribonucleasas , Regulación de la Expresión Génica , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleasas/genética , Ribonucleasas/metabolismoRESUMEN
Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.
Asunto(s)
Ácido Graso Desaturasas , Síndrome Metabólico , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Humanos , Inflamación , Metabolismo de los LípidosRESUMEN
BACKGROUND: Platinum-containing chemotherapy produces specific DNA damage and is used to treat several human solid tumors. Tumors initially sensitive to platinum-based drugs frequently become resistant. Inhibition of DNA repair is a potential strategy to enhance cisplatin effectiveness. After cisplatin treatment, a balance between repair and apoptosis determines whether cancer cells proliferate or die. DNA-dependent protein kinase (DNA-PK) binds to DNA double strand breaks (DSBs) through its Ku subunits and initiates non-homologous end joining. Inhibition of DNA-PK sensitizes cancer cells to cisplatin killing. The goal of this study is to elucidate the mechanism underlying the effects of DNA-PK on cisplatin sensitivity. RESULTS: Silencing the expression of the catalytic subunit of DNA-PK (DNA-PKcs) increased sensitivity to cisplatin and decreased the appearance of γH2AX after cisplatin treatment. We purified DNA-PK by its Ku86 subunit and identified interactors by tandem mass spectrometry before and after cisplatin treatment. The structure specific recognition protein 1 (SSRP1), Spt16 and γH2AX appeared in the Ku86 complex 5 hours after cisplatin treatment. SSRP1 and Spt16 form the facilitator of chromatin transcription (FACT). The cisplatin-induced association of FACT with Ku86 and γH2AX was abrogated by DNase treatment. In living cells, SSRP1 and Ku86 were recruited at sites of DSBs induced by laser beams. Silencing SSRP1 expression increased sensitivity to cisplatin and decreased γH2AX appearance. However, while silencing SSRP1 in cisplatin-treated cells increased both apoptosis and necrosis, DNA-PKcs silencing, in contrast, favored necrosis over apoptosis. CONCLUSIONS: DNA-PK and FACT both play roles in DNA repair. Therefore both are putative targets for therapeutic inhibition. Since DNA-PK regulates apoptosis, silencing DNA-PKcs redirects cells treated with cisplatin toward necrosis. Silencing FACT however, allows both apoptosis and necrosis. Targeting DNA repair in cancer patients may have different therapeutic effects depending upon the roles played by factors targeted.
Asunto(s)
Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Reparación del ADN/efectos de los fármacos , Proteína Quinasa Activada por ADN/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas del Grupo de Alta Movilidad/fisiología , Factores de Elongación Transcripcional/fisiología , Antineoplásicos/farmacología , Apoptosis/genética , Línea Celular Tumoral , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Reparación del ADN/genética , Proteína Quinasa Activada por ADN/genética , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Evaluación Preclínica de Medicamentos , Células HEK293 , Células HeLa , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Necrosis/inducido químicamente , Necrosis/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
PURPOSE: BRCA1-interacting protein 1 (BRIP1; FANCJ/BACH1), which encodes a DNA helicase that interacts with BRCA1, has been suggested to be a low-penetrance breast cancer predisposing gene. We aimed to assess whether BRIP1 mutations contribute to breast cancer susceptibility in our population and, if so, to investigate the effect of such mutation(s) on BRIP1 function. EXPERIMENTAL DESIGN: A series of 49 breast/ovarian cancer families, devoid of a BRCA1/BRCA2 mutation, were screened for BRIP1 mutations. Functional analyses, including coimmunoprecipitation and stability assays, were employed to further characterize a previously unreported variant. RESULTS: Five sequence alterations were identified, of which four had been already described. Herein, we report a novel BRIP1 germ-line mutation identified in a woman with early-onset breast cancer. The mutation consists of a 4-nucleotide deletion (c.2992-2995delAAGA) in BRIP1 exon 20 that causes a shift in the reading frame, disrupts the BRCA1-binding domain of BRIP1, and creates a premature stop codon. Functional analysis of the recombinant mutant protein in transfected cells showed that the truncation interferes with the stability of the protein and with its ability to interact with BRCA1. Loss of the wild-type BRIP1 allele with retention of the mutated one was observed in the patient's breast tumor tissue. CONCLUSIONS: These results, by showing that the newly identified BRIP1 c.2992-2995delAAGA mutation is associated with instability and functional impairment of the encoded protein, provide further evidence of a breast cancer-related role for BRIP1.
Asunto(s)
Neoplasias de la Mama/genética , Análisis Mutacional de ADN , Proteínas de Unión al ADN/genética , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/genética , ARN Helicasas/genética , Adulto , Anciano , Secuencia de Bases , Western Blotting , Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi , Femenino , Humanos , Inmunoprecipitación , Pérdida de Heterocigocidad , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Mutación , Neoplasias Ováricas/genética , Linaje , ARN Helicasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
RNA polymerase II (RNAPII) pausing/termination shortly after initiation is a hallmark of gene regulation. Here, we show that negative elongation factor (NELF) interacts with Integrator complex subunits (INTScom), RNAPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both human immunodeficiency virus type 1 promoter and genome-wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated INTScom subunits. Interestingly, in addition to controlling RNAPII pause-release INTS11 catalytic subunit of the INTScom is required for RNAPII processivity. Finally, INTScom target genes are enriched in human immunodeficiency virus type 1 transactivation response element/NELF binding element and in a 3' box sequence required for small nuclear RNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pause-release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/metabolismo , Endorribonucleasas , Regulación de la Expresión Génica , VIH-1/genética , Humanos , Fosforilación , Regiones Promotoras Genéticas , Subunidades de Proteína/metabolismo , Proteínas Represoras/metabolismoRESUMEN
SAMHD1 has recently been identified as an HIV-1 restriction factor operating in myeloid cells. As a countermeasure, the Vpx accessory protein from HIV-2 and certain lineages of SIV have evolved to antagonize SAMHD1 by inducing its ubiquitin-proteasome-dependent degradation. Here, we show that SAMHD1 experienced strong positive selection episodes during primate evolution that occurred in the Catarrhini ancestral branch prior to the separation between hominoids (gibbons and great apes) and Old World monkeys. The identification of SAMHD1 residues under positive selection led to mapping the Vpx-interaction domain of SAMHD1 to its C-terminal region. Importantly, we found that while SAMHD1 restriction activity toward HIV-1 is evolutionarily maintained, antagonism of SAMHD1 by Vpx is species-specific. The distinct evolutionary signature of SAMHD1 sheds light on the development of its antiviral specificity.
Asunto(s)
Evolución Molecular , VIH-2/patogenicidad , Interacciones Huésped-Patógeno , Proteínas de Unión al GTP Monoméricas/genética , Proteínas de Unión al GTP Monoméricas/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Sitios de Unión , Análisis por Conglomerados , VIH-1/inmunología , VIH-1/patogenicidad , VIH-2/inmunología , Humanos , Filogenia , Primates , Unión Proteica , Mapeo de Interacción de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
PALB2/FANCN is mutated in breast and pancreatic cancers and Fanconi anemia (FA). It controls the intranuclear localization, stability, and DNA repair function of BRCA2 and links BRCA1 and BRCA2 in DNA homologous recombination repair and breast cancer suppression. Here, we show that PALB2 directly interacts with KEAP1, an oxidative stress sensor that binds and represses the master antioxidant transcription factor NRF2. PALB2 shares with NRF2 a highly conserved ETGE-type KEAP1 binding motif and can effectively compete with NRF2 for KEAP1 binding. PALB2 promotes NRF2 accumulation and function in the nucleus and lowers the cellular reactive oxygen species (ROS) level. In addition, PALB2 also regulates the rate of NRF2 export from the nucleus following induction. Our findings identify PALB2 as a regulator of cellular redox homeostasis and provide a new link between oxidative stress and the development of cancer and FA.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Línea Celular Tumoral , Núcleo Celular/metabolismo , Transformación Celular Neoplásica , Reparación del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Humanos , Proteína 1 Asociada A ECH Tipo Kelch , Neoplasias/patología , Oxidación-Reducción , Estrés Oxidativo , Unión Proteica , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Mutations affecting the BRCT domains of the breast cancer-associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.
Asunto(s)
Proteína BRCA1/metabolismo , Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , ADN/metabolismo , Proteínas Nucleares/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Reparación del ADN/fisiología , Proteínas de Unión al ADN , Células HeLa , Chaperonas de Histonas , Humanos , Ratones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Estructura Terciaria de Proteína , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Many cancer-associated genes remain to be identified to clarify the underlying molecular mechanisms of cancer susceptibility and progression. Better understanding is also required of how mutations in cancer genes affect their products in the context of complex cellular networks. Here we have used a network modeling strategy to identify genes potentially associated with higher risk of breast cancer. Starting with four known genes encoding tumor suppressors of breast cancer, we combined gene expression profiling with functional genomic and proteomic (or 'omic') data from various species to generate a network containing 118 genes linked by 866 potential functional associations. This network shows higher connectivity than expected by chance, suggesting that its components function in biologically related pathways. One of the components of the network is HMMR, encoding a centrosome subunit, for which we demonstrate previously unknown functional associations with the breast cancer-associated gene BRCA1. Two case-control studies of incident breast cancer indicate that the HMMR locus is associated with higher risk of breast cancer in humans. Our network modeling strategy should be useful for the discovery of additional cancer-associated genes.
Asunto(s)
Neoplasias de la Mama/genética , Centrosoma/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Redes Reguladoras de Genes , Receptores de Hialuranos/metabolismo , Redes Neurales de la Computación , Aurora Quinasas , Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/antagonistas & inhibidores , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Estudios de Casos y Controles , Biología Computacional , Proteínas de la Matriz Extracelular/antagonistas & inhibidores , Proteínas de la Matriz Extracelular/genética , Femenino , Perfilación de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Receptores de Hialuranos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Mapeo de Interacción de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/farmacología , Ubiquitina/metabolismoRESUMEN
The BRCA1 gene product and its stoichiometric binding partner, BARD1, play a vital role in the cellular response to DNA damage. However, how they acquire specific biochemical functions after DNA damage is poorly understood. Following exposure to genotoxic stress, DNA damage-specific interactions were observed between BRCA1/BARD1 and the DNA damage-response proteins, TopBP1 and Mre11/Rad50/NBS1. Two distinct DNA damage-dependent super complexes emerged; their activation was dependent, in part, on the actions of specific checkpoint kinases, and each super complex contributed to a distinctive aspect of the DNA damage response. The results support a new, multifactorial model that describes how genotoxic stress enables BRCA1 to execute a diverse set of DNA damage-response functions.