RESUMEN
Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.
Asunto(s)
Enfisema , Rarefacción Microvascular , Enfisema Pulmonar , Telomerasa , Ratones , Animales , Acortamiento del Telómero , Telomerasa/genéticaRESUMEN
DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.
Asunto(s)
Núcleo Celular , Daño del ADN , Molécula de Interacción Estromal 1 , Cromatina/genética , Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Mitomicina/farmacología , Proteómica , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Humanos , Núcleo Celular/metabolismo , Proteínas de Neoplasias/metabolismoRESUMEN
Chronic inflammatory diseases are associated with hematopoietic lineage bias, including neutrophilia and anemia. We have recently identified that the canonical inflammasome mediates the cleavage of the master erythroid transcription factor GATA1 in hematopoietic stem and progenitor cells (HSPCs). We report here that genetic inhibition of Nlrp1 resulted in reduced number of neutrophils and increased erythrocyte counts in zebrafish larvae. We also found that the NLRP1 inflammasome in human cells was inhibited by LRRFIP1 and FLII, independently of DPP9, and both inhibitors regulated hematopoiesis. Mechanistically, erythroid differentiation resulted in ribosomal stress-induced activation of the ZAKα/P38 kinase axis which, in turn, phosphorylated and promoted the assembly of NLRP1 in both zebrafish and human. Finally, inhibition of Zaka with the FDA/EMA-approved drug Nilotinib alleviated neutrophilia in a zebrafish model of neutrophilic inflammation and promoted erythroid differentiation and GATA1 accumulation in K562 cells. In conclusion, our results reveal that the NLRP1 inflammasome regulates hematopoiesis and pave the way to develop novel therapeutic strategies for the treatment of hematopoietic alterations associated with chronic inflammatory and rare diseases.
RESUMEN
Fanconi anemia (FA) is a genetic disorder associated with developmental defects, bone marrow failure and cancer. The FA pathway is crucial for the repair of DNA interstrand crosslinks (ICLs). In this study, we have developed and characterized a new tool to investigate ICL repair: a clickable version of the crosslinking agent melphalan which we name click-melphalan. Our results demonstrate that click-melphalan is as effective as its unmodified counterpart in generating ICLs and associated toxicity. The lesions induced by click-melphalan can be detected in cells by post-labelling with a fluorescent reporter and quantified using flow cytometry. Since click-melphalan induces both ICLs and monoadducts, we generated click-mono-melphalan, which only induces monoadducts, in order to distinguish between the two types of DNA repair. By using both molecules, we show that FANCD2 knock-out cells are deficient in removing click-melphalan-induced lesions. We also found that these cells display a delay in repairing click-mono-melphalan-induced monoadducts. Our data further revealed that the presence of unrepaired ICLs inhibits monoadduct repair. Finally, our study demonstrates that these clickable molecules can differentiate intrinsic DNA repair deficiencies in primary FA patient cells from those in primary xeroderma pigmentosum patient cells. As such, these molecules may have potential for developing diagnostic tests.
Asunto(s)
Anemia de Fanconi , Melfalán , Humanos , Melfalán/farmacología , Anemia de Fanconi/patología , Reparación del ADN , Daño del ADN , ADNRESUMEN
Fanconi anemia (FA) is a rare inherited disorder that mainly affects the bone marrow. This condition causes decreased production of all types of blood cells. FA is caused by a defective repair of DNA interstrand crosslinks and to date, mutations in over 20 genes have been linked to the disease. Advances in science and molecular biology have provided new insight between FA gene mutations and the severity of clinical manifestations. Here, we will highlight the current and promising therapeutic options for this rare disease. The current standard treatment for FA patients is hematopoietic stem cell transplantation, a treatment associated to exposure to radiation or chemotherapy, immunological complications, plus opportunistic infections from prolonged immune incompetence or increased risk of morbidity. New arising treatments include gene addition therapy, genome editing using CRISPR-Cas9 nuclease, and hematopoietic stem cell generation from induced pluripotent stem cells. Finally, we will also discuss the revolutionary developments in mRNA therapeutics as an opportunity for this disease.
Asunto(s)
Anemia de Fanconi , Humanos , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Médula Ósea/metabolismo , Terapia Genética , Células Madre Hematopoyéticas/metabolismo , Daño del ADNRESUMEN
Ubiquitin-fold modifier 1 (UFM1) is involved in neural and erythroid development, yet its biological roles in these processes are unknown. Here, we generated zebrafish models deficient in Ufm1 and Ufl1 that exhibited telomere shortening associated with developmental delay, impaired hematopoiesis and premature aging. We further report that HeLa cells lacking UFL1 have instability of telomeres replicated by leading-strand synthesis. We uncover that MRE11 UFMylation is necessary for the recruitment of the phosphatase PP1-α leading to dephosphorylation of NBS1. In the absence of UFMylation, NBS1 remains phosphorylated, thereby reducing MRN recruitment to telomeres. The absence of MRN at telomeres favors the formation of the TRF2-Apollo/SNM1 complex consistent with the loss of leading telomeres. These results suggest that MRE11-UFMylation may serve as module to recruit PP1-α. Last, zebrafish expressing Mre11 that cannot be UFMylated phenocopy Ufm1-deficient zebrafish, demonstrating that UFMylation of MRE11 is a previously undescribed evolutionarily conserved mechanisms regulating telomere length.
RESUMEN
Hematopoiesis is a complex process through which immature bone marrow precursor cells mature into all types of blood cells. Although the association of hematopoietic lineage bias (including anemia and neutrophilia) with chronic inflammatory diseases has long been appreciated, the causes involved are obscure. Recently, cytosolic multiprotein inflammasome complexes were shown to activate inflammatory and immune responses, and directly regulate hematopoiesis in zebrafish models; this was deemed to occur via cleavage and inactivation of the master erythroid transcription factor GATA1. Herein summarized are the zebrafish models that are currently available to study this unappreciated role of inflammasome-mediated regulation of hematopoiesis. Novel putative therapeutic strategies, for the treatment of hematopoietic alterations associated with chronic inflammatory diseases in humans, are also proposed.
Asunto(s)
Hematopoyesis , Inflamasomas , Modelos Animales , Pez Cebra , Animales , Hematopoyesis/genética , Hematopoyesis/inmunología , Humanos , Inflamasomas/metabolismo , Investigación/tendencias , Pez Cebra/genética , Pez Cebra/inmunología , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/inmunologíaRESUMEN
Chronic inflammatory diseases are associated with altered hematopoiesis that could result in neutrophilia and anemia. Here we report that genetic or chemical manipulation of different inflammasome components altered the differentiation of hematopoietic stem and progenitor cells (HSPC) in zebrafish. Although the inflammasome was dispensable for the emergence of HSPC, it was intrinsically required for their myeloid differentiation. In addition, Gata1 transcript and protein amounts increased in inflammasome-deficient larvae, enforcing erythropoiesis and inhibiting myelopoiesis. This mechanism is evolutionarily conserved, since pharmacological inhibition of the inflammasome altered erythroid differentiation of human erythroleukemic K562 cells. In addition, caspase-1 inhibition rapidly upregulated GATA1 protein in mouse HSPC promoting their erythroid differentiation. Importantly, pharmacological inhibition of the inflammasome rescued zebrafish disease models of neutrophilic inflammation and anemia. These results indicate that the inflammasome plays a major role in the pathogenesis of neutrophilia and anemia of chronic diseases and reveal druggable targets for therapeutic interventions.
Asunto(s)
Anemia/inmunología , Enfermedades de los Peces/inmunología , Factor de Transcripción GATA1/metabolismo , Inflamasomas/metabolismo , Inflamación/inmunología , Neutrófilos/inmunología , Proteínas de Pez Cebra/metabolismo , Pez Cebra/fisiología , Animales , Animales Modificados Genéticamente , Caspasa 1/genética , Caspasa 1/metabolismo , Diferenciación Celular , Células Eritroides/citología , Factor de Transcripción GATA1/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis , Humanos , Inflamasomas/genética , Células K562 , Masculino , Ratones , Ratones Endogámicos C57BL , Proteolisis , Proteínas de Pez Cebra/genéticaRESUMEN
Defects in the repair of DNA interstrand crosslinks (ICLs) are associated with the genome instability syndrome Fanconi anemia (FA). Here we report that cells with mutations in RFWD3, an E3 ubiquitin ligase that interacts with and ubiquitylates replication protein A (RPA), show profound defects in ICL repair. An amino acid substitution in the WD40 repeats of RFWD3 (I639K) found in a new FA subtype abolishes interaction of RFWD3 with RPA, thereby preventing RFWD3 recruitment to sites of ICL-induced replication fork stalling. Moreover, single point mutations in the RPA32 subunit of RPA that abolish interaction with RFWD3 also inhibit ICL repair, demonstrating that RPA-mediated RFWD3 recruitment to stalled replication forks is important for ICL repair. We also report that unloading of RPA from sites of ICL induction is perturbed in RFWD3-deficient cells. These data reveal important roles for RFWD3 localization in protecting genome stability and preserving human health.
Asunto(s)
Daño del ADN , Anemia de Fanconi/enzimología , Reparación del ADN por Recombinación , Origen de Réplica , Proteína de Replicación A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Anemia de Fanconi/genética , Células HeLa , Humanos , Mutación , Unión Proteica , Interferencia de ARN , Proteína de Replicación A/genética , Transfección , Ubiquitina-Proteína Ligasas/genéticaAsunto(s)
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Neoplasias de la Mama/fisiopatología , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Predisposición Genética a la Enfermedad , Mutación , Neoplasias Ováricas/fisiopatología , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/terapia , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Femenino , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapiaRESUMEN
The Fan1 endonuclease is required for repair of DNA interstrand cross-links (ICLs). Mutations in human Fan1 cause karyomegalic interstitial nephritis (KIN), but it is unclear whether defective ICL repair is responsible or whether Fan1 nuclease activity is relevant. We show that Fan1 nuclease-defective (Fan1(nd/nd)) mice develop a mild form of KIN. The karyomegalic nuclei from Fan1(nd/nd) kidneys are polyploid, and fibroblasts from Fan1(nd/nd) mice become polyploid upon ICL induction, suggesting that defective ICL repair causes karyomegaly. Thus, Fan1 nuclease activity promotes ICL repair in a manner that controls ploidy, a role that we show is not shared by the Fanconi anemia pathway or the Slx4-Slx1 nuclease also involved in ICL repair.
Asunto(s)
Daño del ADN/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Nefritis Intersticial/enzimología , Nefritis Intersticial/genética , Poliploidía , Animales , Células Cultivadas , Reparación del ADN/genética , Desoxirribonucleasas/metabolismo , Exodesoxirribonucleasas , Técnicas de Sustitución del Gen , Riñón/patología , Ratones , Enzimas Multifuncionales , Nefritis Intersticial/fisiopatologíaRESUMEN
Mono-ubiquitination of Fancd2 is essential for repairing DNA interstrand cross-links (ICLs), but the underlying mechanisms are unclear. The Fan1 nuclease, also required for ICL repair, is recruited to ICLs by ubiquitinated (Ub) Fancd2. This could in principle explain how Ub-Fancd2 promotes ICL repair, but we show that recruitment of Fan1 by Ub-Fancd2 is dispensable for ICL repair. Instead, Fan1 recruitment--and activity--restrains DNA replication fork progression and prevents chromosome abnormalities from occurring when DNA replication forks stall, even in the absence of ICLs. Accordingly, Fan1 nuclease-defective knockin mice are cancer-prone. Moreover, we show that a Fan1 variant in high-risk pancreatic cancers abolishes recruitment by Ub-Fancd2 and causes genetic instability without affecting ICL repair. Therefore, Fan1 recruitment enables processing of stalled forks that is essential for genome stability and health.
Asunto(s)
Aberraciones Cromosómicas , Replicación del ADN , Endodesoxirribonucleasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Inestabilidad Genómica/genética , Neoplasias Pancreáticas/genética , Ubiquitinación , Secuencia de Aminoácidos , Animales , Reparación del ADN , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Femenino , Técnicas de Sustitución del Gen , Predisposición Genética a la Enfermedad , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Linfoma/genética , Linfoma/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Enzimas MultifuncionalesRESUMEN
CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFß-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFß-responsive gene PAI-1. Using chemical and genetic regulators of TGFß signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.
Asunto(s)
Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genes Reporteros/genética , Transcripción Genética/genética , Línea Celular Tumoral , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , Inhibidor 1 de Activador Plasminogénico/genética , Regiones Promotoras Genéticas/genética , Factor de Crecimiento Transformador beta/genética , Transgenes/genética , Factores de Intercambio de Guanina Nucleótido rasRESUMEN
Reversible protein ubiquitylation plays important roles in various processes including DNA repair. Here, we identify the deubiquitylase USP45 as a critical DNA repair regulator. USP45 associates with ERCC1, a subunit of the DNA repair endonuclease XPF-ERCC1, via a short acidic motif outside of the USP45 catalytic domain. Wild-type USP45, but not a USP45 mutant defective in ERCC1 binding, efficiently deubiquitylates ERCC1 in vitro, and the levels of ubiquitylated ERCC1 are markedly enhanced in USP45 knockout cells. Cells lacking USP45 are hypersensitive specifically to UV irradiation and DNA interstrand cross-links, similar to cells lacking ERCC1. Furthermore, the repair of UV-induced DNA damage is markedly reduced in USP45-deficient cells. ERCC1 translocation to DNA damage-induced subnuclear foci is markedly impaired in USP45 knockout cells, possibly accounting for defective DNA repair. Finally, USP45 localises to sites of DNA damage in a manner dependent on its deubiquitylase activity, but independent of its ability to bind ERCC1-XPF. Together, these results establish USP45 as a new regulator of XPF-ERCC1 crucial for efficient DNA repair.
Asunto(s)
Núcleo Celular/metabolismo , Daño del ADN , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Endopeptidasas/metabolismo , Ubiquitinación , Transporte Activo de Núcleo Celular/genética , Transporte Activo de Núcleo Celular/efectos de la radiación , Animales , Línea Celular Tumoral , Núcleo Celular/genética , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Endopeptidasas/genética , Humanos , Ratones , Ratones Noqueados , Rayos Ultravioleta/efectos adversosRESUMEN
The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Marcación de Gen/métodos , Genoma , ARN Guía de Kinetoplastida/genética , Proteínas Asociadas a CRISPR/genética , Línea Celular , HumanosRESUMEN
Defects in SLX4, a scaffold for DNA repair nucleases, result in Fanconi anemia (FA), due to the defective repair of inter-strand DNA crosslinks (ICLs). Some FA patients have an SLX4 deletion removing two tandem UBZ4-type ubiquitin-binding domains that are implicated in protein recruitment to sites of DNA damage. Here, we show that human SLX4 is recruited to sites of ICL induction but that the UBZ-deleted form of SLX4 in cells from FA patients is not. SLX4 recruitment does not require either the ubiquitylation of FANCD2 or the E3 ligases RNF8, RAD18 and BRCA1. We show that the first (UBZ-1) but not the second UBZ domain of SLX4 binds to ubiquitin polymers, with a preference for K63-linked chains. Furthermore, UBZ-1 is required for SLX4 recruitment to ICL sites and for efficient ICL repair in murine fibroblasts. The SLX4 UBZ-2 domain does not bind to ubiquitin in vitro or contribute to ICL repair, but it is required for the resolution of Holliday junctions in vivo. These data shed light on SLX4 recruitment, and they point to the existence of currently unidentified ubiquitylated ligands and E3 ligases that are crucial for ICL repair.
Asunto(s)
Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Recombinasas/genética , Ubiquitina/metabolismo , Animales , Sitios de Unión , ADN/genética , ADN/metabolismo , Reparación del ADN , Humanos , Ratones , Estructura Terciaria de Proteína , Recombinasas/metabolismoRESUMEN
Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability.
Asunto(s)
Reparación del ADN , ADN Cruciforme , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Fibroblastos/citología , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Interferencia de ARN , Recombinasas/genética , Recombinasas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
While chitooligosaccharides (COs) derived from fungal chitin are potent elicitors of defense reactions, structurally related signals produced by certain bacteria and fungi, called lipo-chitooligosaccharides (LCOs), play important roles in the establishment of symbioses with plants. Understanding how plants distinguish between friend and foe through the perception of these signals is a major challenge. We report the synthesis of a range of COs and LCOs, including photoactivatable probes, to characterize a membrane protein from the legume Medicago truncatula. By coupling photoaffinity labeling experiments with proteomics and transcriptomics, we identified the likely LCO-binding protein as LYR3, a lysin motif receptor-like kinase (LysM-RLK). LYR3, expressed heterologously, exhibits high-affinity binding to LCOs but not COs. Homology modeling, based on the Arabidopsis CO-binding LysM-RLK AtCERK1, suggests that LYR3 could accommodate the LCO in a conserved binding site. The identification of LYR3 opens up ways for the molecular characterization of LCO/CO discrimination.
Asunto(s)
Quitina/análogos & derivados , Medicago truncatula/fisiología , Oligosacáridos/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Lípidos/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oligosacáridos/química , Proteínas de Plantas/química , Alineación de Secuencia , SimbiosisRESUMEN
Ubiquitin-binding domains (UBDs) are crucial for recruiting many proteins to sites of DNA damage. Here we characterize C1orf124 (Spartan; referred to as DVC1), which has an UBZ4-type UBD found predominantly in DNA repair proteins. DVC1 associates with DNA replication factories and localizes to sites of DNA damage in human cells, in a manner that requires the ability of the DVC1 UBZ domain to bind to ubiquitin polymers in vitro and a conserved PCNA-interacting motif. DVC1 interacts with the p97 protein 'segregase'. We show that DVC1 recruits p97 to sites of DNA damage, where we propose that p97 facilitates the extraction of the translesion synthesis (TLS) polymerase (Pol) η during DNA repair to prevent excessive TLS and limit the incidence of mutations induced by DNA damage. We introduce DVC1 as a regulator of cellular responses to DNA damage that prevents mutations when DNA damage occurs.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina/metabolismo , Daño del ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Immunoblotting , Inmunoprecipitación , ARN Interferente Pequeño/genética , Proteína que Contiene ValosinaRESUMEN
Sphinganine or dihydrosphingosine (d18:0, DHS), one of the most abundant free sphingoid Long Chain Base (LCB) in plants, is known to induce a calcium dependent programmed cell death (PCD) in tobacco BY-2 cells. In addition, we have recently shown that DHS triggers a production of H2O2, via the activation of NADPH oxidase(s). However, this production of H2O2 is not correlated with the DHS-induced cell death but would rather be associated with basal cell defense mechanisms. In the present study, we extend our current knowledge of the DHS signaling pathway, by demonstrating that DHS also promotes a production of nitric oxide (NO) in tobacco BY-2 cells. As for H2O2, this NO production is not necessary for cell death induction.