RESUMEN
The ubiquitin system regulates the DNA damage response (DDR) by modifying histone H2A at Lys15 (H2AK15ub) and triggering downstream signaling events. Here, we find that phosphorylation of ubiquitin at Thr12 (pUbT12) controls the DDR by inhibiting the function of 53BP1, a key factor for DNA double-strand break repair by non-homologous end joining (NHEJ). Detectable as a chromatin modification on H2AK15ub, pUbT12 accumulates in nuclear foci and is increased upon DNA damage. Mutating Thr12 prevents the removal of ubiquitin from H2AK15ub by USP51 deubiquitinating enzyme, leading to a pronounced accumulation of ubiquitinated chromatin. Chromatin modified by pUbT12 is inaccessible to 53BP1 but permissive to the homologous recombination (HR) proteins RNF169, RAD51, and the BRCA1/BARD1 complex. Phosphorylation of ubiquitin at Thr12 in the chromatin context is a new histone mark, H2AK15pUbT12, that regulates the DDR by hampering the activity of 53BP1 at damaged chromosomes.
Asunto(s)
Daño del ADN/fisiología , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Cromatina/metabolismo , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Recombinación Homóloga/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Fosforilación , Transducción de Señal/genética , Treonina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/fisiología , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Chromatin ubiquitination by the ubiquitin ligase RNF168 is critical to regulate the DNA damage response (DDR). DDR deficiencies lead to cancer-prone syndromes, but whether this reflects DNA repair defects is still elusive. We identified key factors of the RNF168 pathway as essential mediators of efficient DNA replication in unperturbed S phase. We found that loss of RNF168 leads to reduced replication fork progression and to reversed fork accumulation, particularly evident at repetitive sequences stalling replication. Slow fork progression depends on MRE11-dependent degradation of reversed forks, implicating RNF168 in reversed fork protection and restart. Consistent with regular nucleosomal organization of reversed forks, the replication function of RNF168 requires H2A ubiquitination. As this novel function is shared with the key DDR players ATM, γH2A.X, RNF8, and 53BP1, we propose that double-stranded ends at reversed forks engage classical DDR factors, suggesting an alternative function of this pathway in preventing genome instability and human disease.
Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Histonas/metabolismo , Línea Celular , Roturas del ADN de Doble Cadena , Replicación del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Humanos , Fase S/fisiología , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiologíaRESUMEN
DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.
Asunto(s)
Daño del ADN , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Neoplasias/biosíntesis , Neoplasias/enzimología , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Origen de Réplica , Animales , Sistemas CRISPR-Cas , ADN Helicasas/genética , ADN de Neoplasias/genética , ADN de Neoplasias/ultraestructura , Células HCT116 , Células HEK293 , Humanos , Cinética , Ratones , Mutación , Neoplasias/genética , Neoplasias/ultraestructura , Antígeno Nuclear de Célula en Proliferación/genética , Interferencia de ARN , Transfección , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , UbiquitinaciónRESUMEN
Complex mechanisms are in place to maintain genome stability. Ubiquitination of chromatin plays a central role in these mechanisms. The ever-growing complexity of the ubiquitin (Ub) code and of chromatin modifications and dynamics challenges our ability to fully understand how histone ubiquitination regulates genome stability. Here we review the current knowledge on specific, low-abundant histone ubiquitination events that are highly regulated within the cellular DNA damage response (DDR), with particular emphasis on the latest discovery of Ub phosphorylation as a novel regulator of the DDR signaling pathway. We discuss players involved and potential implications of histone (phospho)ubiquitination on chromatin structure, and we highlight exciting open questions for future research.
Asunto(s)
Inestabilidad Genómica , Histonas/metabolismo , Ubiquitina/metabolismo , Animales , Daño del ADN , Reparación del ADN , Histonas/genética , Humanos , Metilación , Fosforilación , Ubiquitina/genética , UbiquitinaciónRESUMEN
BACKGROUND: Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins. METHODS: To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model. RESULTS: The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique. CONCLUSIONS: Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.
Asunto(s)
Roturas del ADN de Doble Cadena , Células Epiteliales/virología , Papillomavirus Humano 16/patogenicidad , Proteínas Oncogénicas Virales/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Proteínas Represoras/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Técnicas de Cultivo de Célula , Femenino , Humanos , Modelos Biológicos , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Infecciones por Papillomavirus , Unión Proteica , Proteínas Represoras/genéticaRESUMEN
Recent discoveries have highlighted the importance of Haspin kinase activity for the correct positioning of the kinase Aurora B at the centromere. Haspin phosphorylates Thr(3) of the histone H3 (H3), which provides a signal for Aurora B to localize to the centromere of mitotic chromosomes. To date, histone H3 is the only confirmed Haspin substrate. We used a combination of biochemical, pharmacological, and mass spectrometric approaches to study the consequences of Haspin inhibition in mitotic cells. We quantified 3964 phosphorylation sites on chromatin-associated proteins and identified a Haspin protein-protein interaction network. We determined the Haspin consensus motif and the co-crystal structure of the kinase with the histone H3 tail. The structure revealed a unique bent substrate binding mode positioning the histone H3 residues Arg(2) and Lys(4) adjacent to the Haspin phosphorylated threonine into acidic binding pockets. This unique conformation of the kinase-substrate complex explains the reported modulation of Haspin activity by methylation of Lys(4) of the histone H3. In addition, the identification of the structural basis of substrate recognition and the amino acid sequence preferences of Haspin aided the identification of novel candidate Haspin substrates. In particular, we validated the phosphorylation of Ser(137) of the histone variant macroH2A as a target of Haspin kinase activity. MacroH2A Ser(137) resides in a basic stretch of about 40 amino acids that is required to stabilize extranucleosomal DNA, suggesting that phosphorylation of Ser(137) might regulate the interactions of macroH2A and DNA. Overall, our data suggest that Haspin activity affects the phosphorylation state of proteins involved in gene expression regulation and splicing.
Asunto(s)
Aurora Quinasa B/metabolismo , Regulación de la Expresión Génica/genética , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Mitosis/genética , Mapas de Interacción de Proteínas/genética , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metilación , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Transcripción Genética/genéticaRESUMEN
DNA replication and repair defects or genotoxic treatments trigger interferon (IFN)-mediated inflammatory responses. However, whether and how IFN signaling in turn impacts the DNA replication process has remained elusive. Here we show that basal levels of the IFN-stimulated gene 15, ISG15, and its conjugation (ISGylation) are essential to protect nascent DNA from degradation. Moreover, IFNß treatment restores replication fork stability in BRCA1/2-deficient cells, which strictly depends on topoisomerase-1, and rescues lethality of BRCA2-deficient mouse embryonic stem cells. Although IFNß activates hundreds of genes, these effects are specifically mediated by ISG15 and ISGylation, as their inactivation suppresses the impact of IFNß on DNA replication. ISG15 depletion significantly reduces cell proliferation rates in human BRCA1-mutated triple-negative, whereas its upregulation results in increased resistance to the chemotherapeutic drug cisplatin in mouse BRCA2-deficient breast cancer cells, respectively. Accordingly, cells carrying BRCA1/2 defects consistently show increased ISG15 levels, which we propose as an in-built mechanism of drug resistance linked to BRCAness.
Asunto(s)
Proteína BRCA1 , Interferones , Animales , Humanos , Ratones , Proteína BRCA1/genética , Supervivencia Celular , Proteína BRCA2/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , Citocinas/metabolismoRESUMEN
DNA replication is highly regulated by the ubiquitin system, which plays key roles upon stress. The ubiquitin-like modifier ISG15 (interferon-stimulated gene 15) is induced by interferons, bacterial and viral infection, and DNA damage, but it is also constitutively expressed in many types of cancer, although its role in tumorigenesis is still largely elusive. Here, we show that ISG15 localizes at the replication forks, in complex with PCNA and the nascent DNA, where it regulates DNA synthesis. Indeed, high levels of ISG15, intrinsic or induced by interferon-ß, accelerate DNA replication fork progression, resulting in extensive DNA damage and chromosomal aberrations. This effect is largely independent of ISG15 conjugation and relies on ISG15 functional interaction with the DNA helicase RECQ1, which promotes restart of stalled replication forks. Additionally, elevated ISG15 levels sensitize cells to cancer chemotherapeutic treatments. We propose that ISG15 up-regulation exposes cells to replication stress, impacting genome stability and response to genotoxic drugs.
Asunto(s)
Neoplasias Óseas/metabolismo , Rotura Cromosómica , Citocinas/metabolismo , Replicación del ADN , ADN de Neoplasias/biosíntesis , Osteosarcoma/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/farmacología , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Citocinas/genética , Daño del ADN , ADN de Neoplasias/genética , Relación Dosis-Respuesta a Droga , Células HEK293 , Células HeLa , Humanos , Células MCF-7 , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/genética , Osteosarcoma/patología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Factores de Tiempo , Ubiquitinas/genéticaRESUMEN
Loss of function of BRCA1-associated protein 1 (BAP1) is observed in about 50% of malignant pleural mesothelioma (MPM) cases. The aim of this study was to investigate whether this aspect could be exploited for targeted therapy. A genetically engineered model was established expressing either functional or nonfunctional BAP1, and whole-genome siRNA synthetic lethality screens were performed assessing differentially impaired survival between the two cell lines. The whole-genome siRNA screen unexpectedly revealed 11 hits (FDR < 0.05) that were more cytotoxic to BAP1-proficient cells. Two actionable targets, ribonucleotide reductase (RNR) catalytic subunit M1 (RRM1) and RNR regulatory subunit M2 (RRM2), were validated. In line with the screen results, primary mesothelioma (BAP1 +/-) overexpressing BAP1 C91A (catalytically dead mutant) was more resistant to RNR inhibition, while BAP1 knockdown in the BAP1-proficient cell lines rescued the cells from their vulnerability to RNR depletion. Gemcitabine and hydroxyurea were more cytotoxic in BAP1-proficient cell line-derived spheroids compared with BAP1 deficient. Upregulation of RRM2 upon gemcitabine and hydroxyurea treatment was more profound in BAP1 mut/del cell lines. Increased lethality mediated by RNR inhibition was observed in NCI-H2452 cells reconstituted with BAP1-WT but not with BAP1 C91A. Upregulation of RRM2 in NCI-H2452-BAP1 WT spheroids was modest compared with control or C91A mutant. Together, we found that BAP1 is involved in the regulation of RNR levels during replication stress. Our observations reveal a potential clinical application where BAP1 status could serve as predictive or stratification biomarker for RNR inhibition-based therapy in MPM.
Asunto(s)
Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Neoplasias Pleurales/genética , Ribonucleósido Difosfato Reductasa/antagonistas & inhibidores , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Antimetabolitos Antineoplásicos/farmacología , Línea Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Genómica , Humanos , Hidroxiurea/farmacología , Mesotelioma/enzimología , Neoplasias Pleurales/tratamiento farmacológico , Neoplasias Pleurales/enzimología , Ribonucleósido Difosfato Reductasa/genética , Ribonucleósido Difosfato Reductasa/metabolismo , Transfección , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , GemcitabinaRESUMEN
BACKGROUND: Modulation of chromatin structure has emerged as a critical molecular device to control gene expression. Histones undergo different post-translational modifications that increase chromatin accessibility to a number of regulatory factors. Among them, histone ubiquitination appears relevant in nuclear processes that govern gene silencing, either by inhibiting or activating transcription, and maintain genome stability, acting as scaffold to properly organize the DNA damage response. Thus, it is of paramount importance the identification and the characterization of new ubiquitin ligases that address histones. RESULTS: We identified and characterized RNF168, a new chromatin-associated RING finger protein. We demonstrated that RNF168 is endowed with ubiquitin ligase activity both in vitro and in vivo, which targets histones H2A and H2AX, but not H2B, forming K63 polyubiquitin chains. We previously described the presence within RNF168 sequence of two MIU domains, responsible for the binding to ubiquitinated proteins. Here we showed that inactivation of the MIUs impairs ubiquitin binding ability in vitro and reduces chromatin association of RNF168 in vivo. Moreover, upon formation of DNA double strand breaks induced by chemical and physical agents, RNF168 is recruited to the DNA damage foci, where it co-localizes with gammaH2AX and 53BP1. The localization of RNF168 at the site of damage highly increases the local concentration of ubiquitinated proteins and determines the prolonged ubiquitination signal. CONCLUSION: The RING finger protein RNF168 is a new ubiquitin ligase that functions as chromatin modifier, through histone ubiquitination. We hypothesize a dual function for RNF168. In normal condition RNF168 modifies chromatin structure by modulating ubiquitination of histone H2A. Upon DNA lesions, RNF168 is recruited to DNA damage response foci where it contributes to increase the amount of ubiquitinated proteins, thereby facilitating the downstream signalling cascade.
Asunto(s)
Ensamble y Desensamble de Cromatina , Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Línea Celular , HumanosRESUMEN
Global-genome nucleotide excision repair (GG-NER) prevents ultraviolet (UV) light-induced skin cancer by removing mutagenic cyclobutane pyrimidine dimers (CPDs). These lesions are formed abundantly on DNA wrapped around histone octamers in nucleosomes, but a specialized damage sensor known as DDB2 ensures that they are accessed by the XPC initiator of GG-NER activity. We report that DDB2 promotes CPD excision by recruiting the histone methyltransferase ASH1L, which methylates lysine 4 of histone H3. In turn, methylated H3 facilitates the docking of the XPC complex to nucleosomal histone octamers. Consequently, DDB2, ASH1L and XPC proteins co-localize transiently on histone H3-methylated nucleosomes of UV-exposed cells. In the absence of ASH1L, the chromatin binding of XPC is impaired and its ability to recruit downstream GG-NER effectors diminished. Also, ASH1L depletion suppresses CPD excision and confers UV hypersensitivity. These findings show that ASH1L configures chromatin for the effective handoff between damage recognition factors during GG-NER activity.
Asunto(s)
Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Células HeLa , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Metilación , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Dímeros de Pirimidina/metabolismo , ARN Interferente Pequeño/genética , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Rayos Ultravioleta/efectos adversosRESUMEN
INTRODUCTION: BRCA1 associated protein1 (BAP1) is a tumor suppressor involved in multiple cellular processes such as transcriptional regulation, chromatin modification by deubiquitinating histone 2A, and DNA repair. BAP1 mutations are frequent in malignant pleural mesothelioma (MPM). Our aim was to functionally characterize a newly identified isoform of BAP1 and investigate the effects of its expression on drug sensitivity in MPM. METHODS: Expression of BAP1 isoforms was detected by quantitative polymerase chain reaction in MPM and normal mesothelium cell lines and tumor and nontumor samples. Histone H2A ubiquitination levels were analyzed by Western blot after acidic extraction of core histones. Subcellular localization of BAP1 isoforms was examined by immunofluorescence. MPM cell survival in response to poly(adenosine diphosphate-ribose) polymerase (PARP) and dual phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitors was analyzed by in vitro assays. RESULTS: We have identified a novel alternative splice isoform of BAP1 (BAP1Δ) that misses part of the catalytic domain. Cells transfected with BAP1Δ showed reduced deubiquitinating activity compared with full-length BAP1. The expression of BAP1Δ transcript is more abundant in nontumor than in tumor samples. MPM cell lines expressing more than 20% of BAP1Δ are more sensitive to olaparib (a PARP1 inhibitor) cytotoxicity, and this sensitivity is enhanced when olaparib treatment is combined with GDC0980 (a dual PI3K-mTOR inhibitor), which induces downregulation of BRCA1. CONCLUSIONS: These observations suggest that BAP1Δ does regulate DNA damage response and influences drug sensitivity. It might therefore be relevant to investigate whether patients with high expression of BAP1Δ may be responsive to PARP/PI3K-mTOR inhibitors.
Asunto(s)
Proteína BRCA1/metabolismo , Reparación del ADN , Mesotelioma/tratamiento farmacológico , Mesotelioma/genética , Proteínas Supresoras de Tumor/biosíntesis , Ubiquitina Tiolesterasa/biosíntesis , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteína BRCA1/genética , Compuestos Bicíclicos Heterocíclicos con Puentes/administración & dosificación , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Sinergismo Farmacológico , Humanos , Mesotelioma/metabolismo , Mesotelioma/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ftalazinas/administración & dosificación , Ftalazinas/farmacología , Piperazinas/administración & dosificación , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Isoformas de Proteínas , Pirimidinas/administración & dosificación , Pirimidinas/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
This corrects the article DOI: 10.1038/ncb3554.
RESUMEN
Naive pluripotency is established in preimplantation epiblast. Embryonic stem cells (ESCs) represent the immortalization of naive pluripotency. 2i culture has optimized this state, leading to a gene signature and DNA hypomethylation closely comparable to preimplantation epiblast, the developmental ground state. Here we show that Pramel7 (PRAME-like 7), a protein highly expressed in the inner cell mass (ICM) but expressed at low levels in ESCs, targets for proteasomal degradation UHRF1, a key factor for DNA methylation maintenance. Increasing Pramel7 expression in serum-cultured ESCs promotes a preimplantation epiblast-like gene signature, reduces UHRF1 levels and causes global DNA hypomethylation. Pramel7 is required for blastocyst formation and its forced expression locks ESCs in pluripotency. Pramel7/UHRF1 expression is mutually exclusive in ICMs whereas Pramel7-knockout embryos express high levels of UHRF1. Our data reveal an as-yet-unappreciated dynamic nature of DNA methylation through proteasome pathways and offer insights that might help to improve ESC culture to reproduce in vitro the in vivo ground-state pluripotency.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Blastocisto/enzimología , Células Madre Embrionarias/enzimología , Epigénesis Genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/enzimología , Complejo de la Endopetidasa Proteasomal/metabolismo , Animales , Antígenos de Neoplasias/genética , Blastocisto/citología , Proteínas Potenciadoras de Unión a CCAAT , Proteínas Cullin/metabolismo , Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fenotipo , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Proteolisis , Interferencia de ARN , Factores de Tiempo , Transcriptoma , Transfección , Ubiquitina-Proteína LigasasRESUMEN
Ron, the receptor tyrosine kinase (RTK) for the macrophage stimulating protein (MSP), activates multiple signaling pathways by recruiting several positive regulators to a multifunctional docking site. Here we show that stimulation by MSP also recruits a negative regulator, the c-Cbl ubiquitin ligase, to the multifunctional docking site as well as to a juxtamembrane tyrosine autophosphorylation site. c-Cbl recruitment to these two sites results in polyubiquitylation of Ron molecules, which are subsequently sorted for endocytosis and degradation. Both the phosphotyrosine binding domain of c-Cbl and its RING domain are essential for downregulation of Ron. Although Ron and c-Cbl are found also in physical complexes that include Grb2, these associations are insufficient for productive ubiquitylation of Ron. Our results shed light on the mechanism of receptor desensitization mediated by c-Cbl and its binding partner Grb2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Proto-Oncogénicas/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas , Endocitosis , Proteína Adaptadora GRB2 , Humanos , Proteínas/fisiología , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-cbl , Ubiquitina/metabolismo , Dominios Homologos srcRESUMEN
Ubiquitination regulates numerous cellular processes by generating a versatile communication system based on eight structurally and functionally different chains linked through distinct residues. Except for K48 and K63, the biological relevance of different linkages is largely unclear. Here, we show that RNF168 ubiquitin ligase promotes noncanonical K27-linked ubiquitination both in vivo and in vitro. We demonstrate that residue K27 of ubiquitin (UbK27) is required for RNF168-dependent chromatin ubiquitination, by targeting histones H2A/H2A.X, and that it is the major ubiquitin-based modification marking chromatin upon DNA damage. Indeed, UbK27 is strictly required for the proper activation of the DNA damage response (DDR) and is directly recognized by crucial DDR mediators, namely 53BP1, Rap80, RNF168, and RNF169. Mutation of UbK27 has dramatic consequences on DDR activation, preventing the recruitment of 53BP1 and BRCA1 to DDR foci. Similarly to the DDR, atypical ubiquitin chains could play unanticipated roles in other crucial ubiquitin-mediated biological processes.
Asunto(s)
Daño del ADN , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Aminoácidos , Proteína BRCA1/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Reparación del ADN , Células HEK293 , Histonas/química , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina/antagonistas & inhibidores , Ubiquitina/genética , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.
Asunto(s)
Citocinas/metabolismo , Melanoma/enzimología , Nicotinamida Fosforribosiltransferasa/metabolismo , Neoplasias Cutáneas/enzimología , Animales , Comunicación Autocrina/efectos de los fármacos , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Citocinas/sangre , Espacio Extracelular/enzimología , Humanos , Peróxido de Hidrógeno/farmacología , Melanoma/patología , Ratones Endogámicos C57BL , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Nicotinamida Fosforribosiltransferasa/sangre , Comunicación Paracrina/efectos de los fármacos , Vesículas Secretoras/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
Ubiquitination of histones plays a critical role in the regulation of several processes within the nucleus, including maintenance of genome stability and transcriptional regulation. The only known ubiquitination site on histones is represented by a conserved Lys residue located at the C terminus of the protein. Here, we describe a novel ubiquitin mark at the N-terminal tail of histone H2As consisting of two Lys residues at positions 13 and 15 (K13/K15). This "bidentate" site is a target of the DNA damage response (DDR) ubiquitin ligases RNF8 and RNF168. Histone mutants lacking the K13/K15 site impair RNF168- and DNA damage-dependent ubiquitination. Conversely, inactivation of the canonical C-terminal site prevents the constitutive monoubiquitination of histone H2As but does not abolish the ubiquitination induced by RNF168. A ubiquitination-defective mutant is obtained by inactivating both the N- and the C-terminal sites, suggesting that these are unique, non-redundant acceptors of ubiquitination on histone H2As. This unprecedented result implies that RNF168 generates a qualitatively different Ub mark on chromatin.
Asunto(s)
Histonas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Secuencia de Aminoácidos , Ensamble y Desensamble de Cromatina , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Células HEK293 , Histonas/química , Histonas/genética , Humanos , Datos de Secuencia Molecular , Mutación , Estructura Terciaria de ProteínaRESUMEN
Ubiquitination regulates important cellular processes, including the DNA damage response (DDR) and DNA repair. The complexity of the ubiquitin-mediated signals is decoded by ubiquitin receptors, which contain protein modules named ubiquitin binding domains (UBDs). We previously identified a new ubiquitin ligase, RNF168, involved in DDR and endowed with two UBDs named MIU (motif interacting with ubiquitin). Here we have provided the identification of a novel UBD, the UMI (UIM- and MIU-related UBD), present in RNF168, and characterized the interaction surface with ubiquitin, centered on two Leu residues. We have demonstrated that integrity of the UMI, in addition to the MIUs, is necessary for the proper localization of RNF168 and for ubiquitination of nuclear proteins, including histone H2A. Finally, we have shown that simultaneous inactivation of UMI and MIUs prevents the recruitment to DDR foci of the crucial downstream mediator 53BP1.
Asunto(s)
Daño del ADN/fisiología , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Línea Celular , Cartilla de ADN/genética , Reparación del ADN/fisiología , Histonas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucina/química , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Ubiquitin is a highly versatile post-translational modification that controls virtually all types of cellular events. Over the past ten years we have learned that diverse forms of ubiquitin modifications and of ubiquitin binding modules co-exist in the cell, giving rise to complex networks of protein:protein interactions. A central problem that continues to puzzle ubiquitinologists is how cells translate this myriad of stimuli into highly specific responses. This is a classical signalling problem. Here, we draw parallels with the phosphorylation signalling pathway and we discuss the expanding repertoire of ubiquitin signals, signal tranducers and signalling-regulated E3 enzymes. We examine recent advances in the field, including a new mechanism of regulation of E3 ligases that relies on ubiquitination.