RESUMEN
DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteínas de la Ataxia Telangiectasia Mutada/genética , Replicación del ADN/genética , Repeticiones de Microsatélite/genética , Animales , Proteínas de Ciclo Celular/genética , Cromatina/genética , Inmunoprecipitación de Cromatina/métodos , Roturas del ADN de Doble Cadena , Daño del ADN/genética , Femenino , Inestabilidad Genómica/genética , Humanos , Ratones , Proteína de Replicación A/genéticaRESUMEN
BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.
Asunto(s)
Citidina Desaminasa , Cambio de Clase de Inmunoglobulina , Estructuras R-Loop , Cambio de Clase de Inmunoglobulina/genética , Citidina Desaminasa/metabolismo , Citidina Desaminasa/genética , Citidina Desaminasa/química , Animales , Ratones , Metilación , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Pesadas de Inmunoglobulina/metabolismo , Recombinación Genética , ARN/metabolismo , ARN/genéticaRESUMEN
Familial neurodegenerative diseases commonly involve mutations that result in either aberrant proteins or dysfunctional components of the proteolytic machinery that act on aberrant proteins. UBQLN2 is a ubiquitin receptor of the UBL/UBA family that binds the proteasome through its ubiquitin-like domain and is thought to deliver ubiquitinated proteins to proteasomes for degradation. UBQLN2 mutations result in familial amyotrophic lateral sclerosis (ALS)/frontotemporal dementia in humans through an unknown mechanism. Quantitative multiplexed proteomics was used to provide for the first time an unbiased and global analysis of the role of Ubqln2 in controlling the composition of the proteome. We studied several murine models of Ubqln2-linked ALS and also generated Ubqln2 null mutant mice. We identified impacts of Ubqln2 on diverse physiological pathways, most notably serotonergic signaling. Interestingly, we observed an upregulation of proteasome subunits, suggesting a compensatory response to diminished proteasome output. Among the specific proteins whose abundance is linked to UBQLN2 function, the strongest hits were the ubiquitin ligase TRIM32 and two retroelement-derived proteins, PEG10 and CXX1B. Cycloheximide chase studies using induced human neurons and HEK293 cells suggested that PEG10 and TRIM32 are direct clients. Although UBQLN2 directs the degradation of multiple proteins via the proteasome, it surprisingly conferred strong protection from degradation on the Gag-like protein CXX1B, which is expressed from the same family of retroelement genes as PEG10. In summary, this study charts the proteomic landscape of ALS-related Ubqln2 mutants and identifies candidate client proteins that are altered in vivo in disease models and whose degradation is promoted by UBQLN2.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/genética , Proteínas Relacionadas con la Autofagia/genética , Demencia Frontotemporal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteómica/métodos , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia/deficiencia , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular , Cicloheximida/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Regulación de la Expresión Génica , Células HEK293 , Humanos , Masculino , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Serotonina/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
Asunto(s)
Antibacterianos/farmacología , Bacteriemia , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Espacio Intracelular/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Vancomicina/farmacología , Animales , Antibacterianos/uso terapéutico , Bacteriemia/tratamiento farmacológico , Bacteriemia/microbiología , Portador Sano/tratamiento farmacológico , Portador Sano/microbiología , Diseño de Fármacos , Femenino , Inmunoconjugados/química , Espacio Intracelular/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Fagosomas/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/patología , Staphylococcus aureus/patogenicidad , Vancomicina/uso terapéuticoRESUMEN
The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Ratones , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Recombinasas/metabolismo , Sumoilación , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , Quinasa Tipo Polo 1RESUMEN
In mammals, homologs that fail to synapse during meiosis are transcriptionally inactivated. This process, meiotic silencing, drives inactivation of the heterologous XY bivalent in male germ cells (meiotic sex chromosome inactivation [MSCI]) and is thought to act as a meiotic surveillance mechanism. The checkpoint protein ATM and Rad3-related (ATR) localizes to unsynapsed chromosomes, but its role in the initiation and maintenance of meiotic silencing is unknown. Here we show that ATR has multiple roles in silencing. ATR first regulates HORMA (Hop1, Rev7, and Mad2) domain protein HORMAD1/2 phosphorylation and localization of breast cancer I (BRCA1) and ATR cofactors ATR-interacting peptide (ATRIP)/topoisomerase 2-binding protein 1 (TOPBP1) at unsynapsed axes. Later, it acts as an adaptor, transducing signaling at unsynapsed axes into surrounding chromatin in a manner that requires interdependence with mediator of DNA damage checkpoint 1 (MDC1) and H2AFX. Finally, ATR catalyzes histone H2AFX phosphorylation, the epigenetic event leading to gene inactivation. Using a novel genetic strategy in which MSCI is used to silence a chosen gene in pachytene, we show that ATR depletion does not disrupt the maintenance of silencing and that silencing comprises two phases: The first is dynamic and reversible, and the second is stable and irreversible. Our work identifies a role for ATR in the epigenetic regulation of gene expression and presents a new technique for ablating gene function in the germline.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación de la Expresión Génica , Silenciador del Gen , Meiosis , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Cromosomas/metabolismo , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Ratones , Fosforilación , Transporte de Proteínas/genética , Proteínas Represoras/metabolismoRESUMEN
The type VII secretion system (T7SS) of Staphylococcus aureus is a multiprotein complex dedicated to the export of several virulence factors during host infection. This virulence pathway plays a key role in promoting bacterial survival and the long-term persistence of staphylococcal abscess communities. The expression of the T7SS is activated by bacterial interaction with host tissues including blood serum, nasal secretions, and pulmonary surfactant. In this work we identify the major stimulatory factors as host-specific cis-unsaturated fatty acids. Increased T7SS expression requires host fatty acid incorporation into bacterial biosynthetic pathways by the Saureus fatty acid kinase (FAK) complex, and FakA is required for virulence. The incorporated cis-unsaturated fatty acids decrease Saureus membrane fluidity, and these altered membrane dynamics are partially responsible for T7SS activation. These data define a molecular mechanism by which Saureus cells sense the host environment and implement appropriate virulence pathways.
Asunto(s)
Interacciones Huésped-Patógeno , Ácido Linoleico/metabolismo , Staphylococcus aureus/fisiología , Sistemas de Secreción Tipo VII/fisiología , Animales , Humanos , Ratones , Factores de Virulencia/metabolismoRESUMEN
The DNA damage response (DDR) orchestrates a network of cellular processes that integrates cell-cycle control and DNA repair or apoptosis, which serves to maintain genome stability. DNA-PKcs (the catalytic subunit of the DNA-dependent kinase, encoded by PRKDC), ATM (ataxia telangiectasia, mutated), and ATR (ATM and Rad3-related) are related PI3K-like protein kinases and central regulators of the DDR. Defects in these kinases have been linked to neurodegenerative or neurodevelopmental syndromes. In all cases, the key neuroprotective function of these kinases is uncertain. It also remains unclear how interactions between the three DNA damage-responsive kinases coordinate genome stability, particularly in a physiological context. Here, we used a genetic approach to identify the neural function of DNA-PKcs and the interplay between ATM and ATR during neurogenesis. We found that DNA-PKcs loss in the mouse sensitized neuronal progenitors to apoptosis after ionizing radiation because of excessive DNA damage. DNA-PKcs was also required to prevent endogenous DNA damage accumulation throughout the adult brain. In contrast, ATR coordinated the DDR during neurogenesis to direct apoptosis in cycling neural progenitors, whereas ATM regulated apoptosis in both proliferative and noncycling cells. We also found that ATR controls a DNA damage-induced G2/M checkpoint in cortical progenitors, independent of ATM and DNA-PKcs. These nonoverlapping roles were further confirmed via sustained murine embryonic or cortical development after all three kinases were simultaneously inactivated. Thus, our results illustrate how DNA-PKcs, ATM, and ATR have unique and essential roles during the DDR, collectively ensuring comprehensive genome maintenance in the nervous system. SIGNIFICANCE STATEMENT: The DNA damage response (DDR) is essential for prevention of a broad spectrum of different human neurologic diseases. However, a detailed understanding of the DDR at a physiological level is lacking. In contrast to many in vitro cellular studies, here we demonstrate independent biological roles for the DDR kinases DNA-PKcs, ATM, and ATR during neurogenesis. We show that DNA-PKcs is central to DNA repair in nonproliferating cells, and restricts DNA damage accumulation, whereas ATR controls damage-induced G2 checkpoint control and apoptosis in proliferating cells. Conversely, ATM is critical for controlling apoptosis in immature noncycling neural cells after DNA damage. These data demonstrate functionally distinct, but cooperative, roles for each kinase in preserving genome stability in the nervous system.
Asunto(s)
Daño del ADN/fisiología , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Neurogénesis/fisiología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/embriología , Corteza Cerebral/metabolismo , Proteína Quinasa Activada por ADN/genética , Proteínas de Unión al ADN/genética , Femenino , Genoma/fisiología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Nucleares/genéticaRESUMEN
The ATR (ATM (ataxia telangiectasia mutated) and rad3-related) checkpoint kinase is considered critical for signalling DNA replication stress and its dysfunction can lead to the neurodevelopmental disorder, ATR-Seckel syndrome. To understand how ATR functions during neurogenesis, we conditionally deleted Atr broadly throughout the murine nervous system, or in a restricted manner in the dorsal telencephalon. Unexpectedly, in both scenarios, Atr loss impacted neurogenesis relatively late during neural development involving only certain progenitor populations. Whereas the Atr-deficient embryonic cerebellar external germinal layer underwent p53- (and p16(Ink4a/Arf))-independent proliferation arrest, other brain regions suffered apoptosis that was partially p53 dependent. In contrast to other organs, in the nervous system, p53 loss did not worsen the outcome of Atr inactivation. Coincident inactivation of Atm also did not affect the phenotype after Atr deletion, supporting non-overlapping physiological roles for these related DNA damage-response kinases in the brain. Rather than an essential general role in preventing replication stress, our data indicate that ATR functions to monitor genomic integrity in a selective spatiotemporal manner during neurogenesis.
Asunto(s)
Encéfalo/embriología , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Células Madre/fisiología , Animales , Apoptosis , Proteínas de la Ataxia Telangiectasia Mutada , Encéfalo/patología , Proteínas de Ciclo Celular/deficiencia , Proliferación Celular , Histocitoquímica , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía , Proteínas Serina-Treonina Quinasas/deficienciaRESUMEN
Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S/fisiología , Alelos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Cromatina/metabolismo , Daño del ADN , Replicación del ADN/fisiología , Genes Dominantes/genética , Inestabilidad Genómica/fisiología , N-Metiltransferasa de Histona-Lisina , Histonas/química , Histonas/metabolismo , Humanos , Leucemia/genética , Lisina/metabolismo , Metilación , Ratones , Células Progenitoras Mieloides/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/deficiencia , Proteína de la Leucemia Mieloide-Linfoide/genética , Fosforilación , Fosfoserina/metabolismo , Unión Proteica , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Transducción de Señal , Translocación Genética/genéticaRESUMEN
Genome stability is essential for neural development and the prevention of neurological disease. Here we determined how DNA damage signaling from dysfunctional telomeres affects neurogenesis. We found that telomere uncapping by Pot1a inactivation resulted in an Atm-dependent loss of cerebellar interneurons and granule neuron precursors in the mouse nervous system. The activation of Atm by Pot1a loss occurred in an Atr-dependent manner, revealing an Atr to Atm signaling axis in the nervous system after telomere dysfunction. In contrast to telomere lesions, Brca2 inactivation in neural progenitors also led to ablation of cerebellar interneurons, but this did not require Atm. These data reveal that neural cell loss after DNA damage selectively engages Atm signaling, highlighting how specific DNA lesions can dictate neuropathology arising in human neurodegenerative syndromes.
Asunto(s)
Daño del ADN/fisiología , Proteínas de Unión al ADN/fisiología , Neuronas/fisiología , Telómero/metabolismo , Animales , Animales Recién Nacidos , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Encéfalo/citología , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/genética , Embrión de Mamíferos , Femenino , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Transgénicos , Nestina/genética , Complejo Shelterina , Proteínas de Unión a Telómeros , beta-Galactosidasa/metabolismoRESUMEN
Infection of host tissues by Staphylococcus aureus and S. epidermidis requires an unusual family of staphylococcal adhesive proteins that contain long stretches of serine-aspartate dipeptide-repeats (SDR). The prototype member of this family is clumping factor A (ClfA), a key virulence factor that mediates adhesion to host tissues by binding to extracellular matrix proteins such as fibrinogen. However, the biological siginificance of the SDR-domain and its implication for pathogenesis remain poorly understood. Here, we identified two novel bacterial glycosyltransferases, SdgA and SdgB, which modify all SDR-proteins in these two bacterial species. Genetic and biochemical data demonstrated that these two glycosyltransferases directly bind and covalently link N-acetylglucosamine (GlcNAc) moieties to the SDR-domain in a step-wise manner, with SdgB appending the sugar residues proximal to the target Ser-Asp repeats, followed by additional modification by SdgA. GlcNAc-modification of SDR-proteins by SdgB creates an immunodominant epitope for highly opsonic human antibodies, which represent up to 1% of total human IgG. Deletion of these glycosyltransferases renders SDR-proteins vulnerable to proteolysis by human neutrophil-derived cathepsin G. Thus, SdgA and SdgB glycosylate staphylococcal SDR-proteins, which protects them against host proteolytic activity, and yet generates major eptopes for the human anti-staphylococcal antibody response, which may represent an ongoing competition between host and pathogen.
Asunto(s)
Proteínas Bacterianas/inmunología , Glicosiltransferasas/inmunología , Interacciones Huésped-Patógeno/inmunología , Staphylococcus aureus Resistente a Meticilina/fisiología , Infecciones Estafilocócicas/inmunología , Staphylococcus epidermidis/fisiología , Factores de Virulencia/inmunología , Animales , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana/genética , Adhesión Bacteriana/inmunología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catepsina G/genética , Catepsina G/inmunología , Catepsina G/metabolismo , Línea Celular Tumoral , Pared Celular/enzimología , Pared Celular/genética , Pared Celular/inmunología , Epítopos/genética , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Glicosiltransferasas/genética , Glicosiltransferasas/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Masculino , Ratones , Secuencias Repetitivas de Aminoácido , Infecciones Estafilocócicas/enzimología , Infecciones Estafilocócicas/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Ubiquilins (Ubqlns)-a family of ubiquitin-binding proteins-are involved in several protein degradation pathways and have been implicated in various neurodegenerative diseases. Ubqln1 regulates autophagosome maturation during autophagy-mediated degradation. We now show that Ubqln4 mediates the interaction between Ubqln1 and the autophagy machinery by recruiting Ubqln1 to LC3. This targeting of Ubqln1 to autophagosomes requires the Ubqln4 UBL domain and the Ubqln1 UBA domain. This study identifies a new role for Ubqln4, expanding the role for Ubqlns in protein degradation.
Asunto(s)
Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/biosíntesis , Células HEK293 , Células HeLa , Humanos , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/biosíntesis , Microscopía Confocal , Microscopía Fluorescente , Proteínas Asociadas a Microtúbulos/metabolismo , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fagosomas/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , ARN Interferente Pequeño/genética , Proteína Fluorescente RojaRESUMEN
BACKGROUND: Detailed knowledge on protein repertoire of a pathogen during host infection is needed for both developing a better understanding of the pathogenesis and defining potential therapeutic targets. Such data, however, have been missing for Staphylococcus aureus, a major human pathogen. METHODS: We determined the surface proteome of methicillin-resistant S. aureus (MRSA) clone usa300 derived directly from murine systemic infectiON. RESULTS: The majority of the in vivo-expressed surface-associated proteins were lipoproteins involved in nutrient acquisition, especially uptake of metal ions. Enzyme-linked immunosorbent assay (ELISA) of convalescent human serum samples revealed that proteins that were highly produced during murine experimental infection were also produced during natural human infection. We found that among the 7 highly abundant lipoproteins only MntC, which is the manganese-binding protein of the MntABC system, was essential for MRSA virulence during murine systemic infection. Moreover, we show that MntA and MntB are equally important for MRSA virulence. CONCLUSIONS: Besides providing experimental evidence that MntABC might be a potential therapeutic target for the development of antibiotics, our in vivo proteomics data will serve as a valuable basis for defining potential antigen combinations for multicomponent vaccines.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/metabolismo , Proteómica , Animales , Proteínas Bacterianas/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Riñón/microbiología , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Suero/inmunología , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Vacunas Estafilocócicas/inmunología , VirulenciaRESUMEN
Little is known about the expression of methicillin-resistant Staphylococcus aureus (MRSA) genes during infection conditions. Here, we described the transcriptome of the clinical MRSA strain USA300 derived from human cutaneous abscesses, and compared it with USA300 bacteria derived from infected kidneys in a mouse model. Remarkable similarity between the transcriptomes allowed us to identify genes encoding multiple proteases and toxins, and iron- and peptide-transporter molecules, which are upregulated in both infections and are likely important for establishment of infection. We also showed that disruption of the global transcriptional regulators agr and sae prevents in vivo upregulation of many toxins and proteases, protecting mice from lethal infection dose, and hinting at the role of these transcriptional regulators in the pathology of MRSA infection.
Asunto(s)
Regulación Bacteriana de la Expresión Génica/fisiología , Staphylococcus aureus Resistente a Meticilina/metabolismo , Transcriptoma , Absceso/microbiología , Animales , Humanos , Staphylococcus aureus Resistente a Meticilina/clasificación , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Análisis por Matrices de Proteínas , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Enfermedades Cutáneas Bacterianas/microbiología , VirulenciaRESUMEN
Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.
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Replicación del ADN , Ubiquitinación , Humanos , Reparación del ADN , ADN/metabolismoRESUMEN
Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.
RESUMEN
A key event and potential therapeutic target in allergic and asthmatic diseases is signaling by the IgE receptor FcεRI, which depends on its interactions with Src family kinases (SFK). Here we tested the hypothesis that glycosylphosphatidylinositiol-anchored proteins (GPI-AP) are involved in FcεRI signaling, based on previous observations that GPI-AP colocalize with and mediate activation of SFK. We generated mice with a hematopoietic cell-specific GPI-AP deficiency by targeted disruption of the GPI biosynthesis gene PigA. In these mice, IgE-mediated passive cutaneous anaphylaxis was largely abolished. PigA-deficient mast cells cultured from these mice showed impaired degranulation in response to stimulation with IgE and antigen in vitro, despite normal IgE binding and antigen-induced FcεRI aggregation. On stimulation of these cells with IgE and antigen, coprecipitation of the FcεRI α-chain with the γ-chain and ß-chain was markedly reduced. As a result, IgE/antigen-induced FcεRI-Lyn association and γ-chain tyrosine phosphorylation were both impaired in PigA-deficient cells. These data provide genetic evidence for an unanticipated key role of GPI-AP in FcεRI interchain interactions and early FcεRI signaling events, necessary for antigen-induced mast cell degranulation.
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Glicosilfosfatidilinositoles/inmunología , Mastocitos/inmunología , Proteínas de la Membrana/genética , Receptores de IgE/inmunología , Anafilaxia/genética , Anafilaxia/inmunología , Animales , Degranulación de la Célula , Células Cultivadas , Eliminación de Gen , Inmunoglobulina E/inmunología , Masculino , Mastocitos/citología , Mastocitos/metabolismo , Proteínas de la Membrana/inmunología , Ratones , Fosforilación , Estabilidad Proteica , Transducción de SeñalRESUMEN
PURPOSE: Addition of ataxia telangiectasia and Rad3-related kinase inhibitors (ATRi) to PARP inhibitors (PARPi) overcomes PARPi resistance in high-grade serous ovarian cancer (HGSOC) cell and mouse models. We present the results of an investigator-initiated study of combination PARPi (olaparib) and ATRi (ceralasertib) in patients with acquired PARPi-resistant HGSOC. PATIENTS AND METHODS: Eligible patients had recurrent, platinum-sensitive BRCA1/2 mutated or homologous recombination (HR)-deficient (HRD) HGSOC and clinically benefited from PARPi (response by imaging/CA-125 or duration of maintenance therapy; > 12 months first-line or > 6 months ≥ second-line) before progression. No intervening chemotherapy was permitted. Patients received olaparib 300 mg twice daily and ceralasertib 160 mg daily on days 1 to 7 of a 28-day cycle. Primary objectives were safety and objective response rate (ORR). RESULTS: Thirteen patients enrolled were evaluable for safety and 12 for efficacy; 62% (n = 8) had germline BRCA1/2 mutations, 23% (n = 3) somatic BRCA1/2 mutations, and 15% (n = 2) tumors with positive HRD assay. Prior PARPi indication was treatment for recurrence (54%, n = 7), second-line maintenance (38%, n = 5) and first-line treatment with carboplatin/paclitaxel (8%, n = 1). There were 6 partial responses yielding an ORR of 50% (95% confidence interval, 0.15-0.72). Median treatment duration was 8 cycles (range 4-23+). Grade (G) 3/4 toxicities were 38% (n = 5); 15% (n = 2) G3 anemia, 23% (n = 3) G3 thrombocytopenia, 8% (n = 1) G4 neutropenia. Four patients required dose reductions. No patient discontinued treatment due to toxicity. CONCLUSIONS: Combination olaparib and ceralasertib is tolerable and shows activity in HR-deficient platinum-sensitive recurrent HGSOC that benefited and then progressed with PARPi as the penultimate regimen. These data suggest that ceralasertib resensitizes PARPi-resistant HGSOCs to olaparib, warranting further investigation.
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Antineoplásicos , Neoplasias Ováricas , Animales , Femenino , Humanos , Ratones , Antineoplásicos/uso terapéutico , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Recombinación Homóloga , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Ftalazinas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéuticoRESUMEN
ARID1A, an epigenetic tumor suppressor, is the most common gene mutation in clear-cell ovarian cancers (CCOCs). CCOCs are often resistant to standard chemotherapy and lack effective therapies. We hypothesized that ARID1A loss would increase CCOC cell dependency on chromatin remodeling and DNA repair pathways for survival. We demonstrate that combining BRD4 inhibitor (BRD4i) with DNA damage response inhibitors (ATR or WEE1 inhibitors; e.g. BRD4i-ATRi) was synergistic at low doses leading to decreased survival, and colony formation in CCOC in an ARID1A dependent manner. BRD4i-ATRi caused significant tumor regression and increased overall survival in ARID1AMUT but not ARID1AWT patient-derived xenografts. Combination BRD4i-ATRi significantly increased γH2AX, and decreased RAD51 foci and BRCA1 expression, suggesting decreased ability to repair DNA double-strand-breaks (DSBs) by homologous-recombination in ARID1AMUT cells, and these effects were greater than monotherapies. These studies demonstrate BRD4i-ATRi is an effective treatment strategy that capitalizes on synthetic lethality with ARID1A loss in CCOC.