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1.
Genes Dev ; 37(19-20): 913-928, 2023 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-37932011

RESUMEN

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.


Asunto(s)
Replicación del ADN , Neoplasias , Humanos , Replicación del ADN/genética , ADN Helicasas/metabolismo , Repeticiones de Microsatélite , Daño del ADN , Neoplasias/tratamiento farmacológico , Neoplasias/genética , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Helicasa del Síndrome de Werner/genética , Helicasa del Síndrome de Werner/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo
2.
Nat Immunol ; 19(8): 898, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29959442

RESUMEN

In the version of this article initially published, in second paragraph of the second subsection of Results ('Peripheral licensing of CD4+ TH17 cells in Tbx21-/- hosts'), the figure citation ('Fig. 1c') in the sentence that begins "In addition to" was incorrect. The correct citation is 'Fig. 1d'.

3.
Nat Immunol ; 18(10): 1117-1127, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28805812

RESUMEN

The transcription factor T-bet has been associated with increased susceptibility to systemic and organ-specific autoimmunity, but the mechanism by which T-bet expression promotes neuroinflammation remains unknown. In this study, we demonstrate a cardinal role of T-bet-dependent NKp46+ innate lymphoid cells (ILCs) in the initiation of CD4+ TH17-mediated neuroinflammation. Loss of T-bet specifically in NKp46+ ILCs profoundly impaired the ability of myelin-reactive TH17 cells to invade central nervous system (CNS) tissue and protected the mice from autoimmunity. T-bet-dependent NKp46+ ILCs localized in the meninges and acted as chief coordinators of meningeal inflammation by inducing the expression of proinflammatory cytokines, chemokines and matrix metalloproteinases, which together facilitated T cell entry into CNS parenchyma. Our findings uncover a detrimental role of T-bet-dependent NKp46+ ILCs in the development of CNS autoimmune disease.


Asunto(s)
Inmunidad Innata , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , Animales , Biomarcadores , Movimiento Celular/genética , Movimiento Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Expresión Génica , Inmunofenotipificación , Ratones , Ratones Noqueados , Vaina de Mielina/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Proteínas de Dominio T Box , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo
4.
Cell ; 153(6): 1266-80, 2013 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-23727112

RESUMEN

The DNA damage response (DDR) protein 53BP1 protects DNA ends from excessive resection in G1, and thereby favors repair by nonhomologous end-joining (NHEJ) as opposed to homologous recombination (HR). During S phase, BRCA1 antagonizes 53BP1 to promote HR. The pro-NHEJ and antirecombinase functions of 53BP1 are mediated in part by RIF1, the only known factor that requires 53BP1 phosphorylation for its recruitment to double-strand breaks (DSBs). Here, we show that a 53BP1 phosphomutant, 53BP18A, comprising alanine substitutions of the eight most N-terminal S/TQ phosphorylation sites, mimics 53BP1 deficiency by restoring genome stability in BRCA1-deficient cells yet behaves like wild-type 53BP1 with respect to immunoglobulin class switch recombination (CSR). 53BP18A recruits RIF1 but fails to recruit the DDR protein PTIP to DSBs, and disruption of PTIP phenocopies 53BP18A. We conclude that 53BP1 promotes productive CSR and suppresses mutagenic DNA repair through distinct phosphodependent interactions with RIF1 and PTIP.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Reparación del ADN por Unión de Extremidades , Proteínas de Unión al ADN/metabolismo , Cambio de Clase de Inmunoglobulina , Proteínas Nucleares/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Animales , Linfocitos B/metabolismo , Proteína BRCA1/metabolismo , Proteínas Cromosómicas no Histona/genética , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Inestabilidad Genómica , Ratones , Mutación , Proteína 1 de Unión al Supresor Tumoral P53
5.
Mol Cell ; 77(1): 26-38.e7, 2020 01 02.
Artículo en Inglés | MEDLINE | ID: mdl-31653568

RESUMEN

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.


Asunto(s)
Recombinación Homóloga/genética , Proteína 1 de Unión al Supresor Tumoral P53/genética , Envejecimiento/efectos de los fármacos , Envejecimiento/genética , Animales , Proteína BRCA1/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Inestabilidad Genómica/efectos de los fármacos , Inestabilidad Genómica/genética , Recombinación Homóloga/efectos de los fármacos , Ratones , Mutación/efectos de los fármacos , Mutación/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Ubiquitina-Proteína Ligasas/genética
6.
Mol Cell ; 73(6): 1267-1281.e7, 2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-30704900

RESUMEN

BRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.


Asunto(s)
Proteína BRCA1/genética , Cromatina/enzimología , Fibroblastos/enzimología , Haploinsuficiencia , Neoplasias/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Animales , Proteína BRCA2/genética , Línea Celular Tumoral , Cromatina/genética , Daño del ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Reparación del ADN por Recombinación , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genética
7.
Mol Cell ; 73(6): 1174-1190.e12, 2019 03 21.
Artículo en Inglés | MEDLINE | ID: mdl-30745086

RESUMEN

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.


Asunto(s)
Transformación Celular Neoplásica/genética , Cromatina/genética , Linfoma de Células B/genética , Oncogenes , Receptores Notch/genética , Neoplasias de la Mama Triple Negativas/genética , Sitios de Unión , Linaje de la Célula/genética , Proliferación Celular/genética , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Ciclina D1/genética , Ciclina D1/metabolismo , Elementos de Facilitación Genéticos , Regulación Neoplásica de la Expresión Génica , Redes Reguladoras de Genes , Células HEK293 , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Mutación , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología
8.
Nucleic Acids Res ; 51(17): 9337-9355, 2023 09 22.
Artículo en Inglés | MEDLINE | ID: mdl-37427791

RESUMEN

Two prominent cytoplasmic RNA granules, ubiquitous RNA-processing bodies (PB) and inducible stress granules (SG), regulate mRNA translation and are intimately related. In this study, we found that arsenite (ARS)-induced SG formed in a stepwise process is topologically and mechanically linked to PB. Two essential PB components, GW182 and DDX6, are repurposed under stress to play direct but distinguishable roles in SG biogenesis. By providing scaffolding activities, GW182 promotes the aggregation of SG components to form SG bodies. DEAD-box helicase DDX6 is also essential for the proper assembly and separation of PB from SG. DDX6 deficiency results in the formation of irregularly shaped 'hybrid' PB/SG granules with accumulated components of both PB and SG. Wild-type DDX6, but not its helicase mutant E247A, can rescue the separation of PB from SG in DDX6KO cells, indicating a requirement of DDX6 helicase activity for this process. DDX6 activity in biogenesis of both PB and SG in the cells under stress is further modulated by its interaction with two protein partners, CNOT1 and 4E-T, of which knockdown affects the formation of both PB and also SG. Together, these data highlight a new functional paradigm between PB and SG biogenesis during the stress.


Asunto(s)
Cuerpos de Procesamiento , Gránulos de Estrés , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Humanos , Línea Celular
9.
Nature ; 560(7718): 387-391, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29925955

RESUMEN

B cell receptor (BCR) signalling has emerged as a therapeutic target in B cell lymphomas, but inhibiting this pathway in diffuse large B cell lymphoma (DLBCL) has benefited only a subset of patients1. Gene expression profiling identified two major subtypes of DLBCL, known as germinal centre B cell-like and activated B cell-like (ABC)2,3, that show poor outcomes after immunochemotherapy in ABC. Autoantigens drive BCR-dependent activation of NF-κB in ABC DLBCL through a kinase signalling cascade of SYK, BTK and PKCß to promote the assembly of the CARD11-BCL10-MALT1 adaptor complex, which recruits and activates IκB kinase4-6. Genome sequencing revealed gain-of-function mutations that target the CD79A and CD79B BCR subunits and the Toll-like receptor signalling adaptor MYD885,7, with MYD88(L265P) being the most prevalent isoform. In a clinical trial, the BTK inhibitor ibrutinib produced responses in 37% of cases of ABC1. The most striking response rate (80%) was observed in tumours with both CD79B and MYD88(L265P) mutations, but how these mutations cooperate to promote dependence on BCR signalling remains unclear. Here we used genome-wide CRISPR-Cas9 screening and functional proteomics to determine the molecular basis of exceptional clinical responses to ibrutinib. We discovered a new mode of oncogenic BCR signalling in ibrutinib-responsive cell lines and biopsies, coordinated by a multiprotein supercomplex formed by MYD88, TLR9 and the BCR (hereafter termed the My-T-BCR supercomplex). The My-T-BCR supercomplex co-localizes with mTOR on endolysosomes, where it drives pro-survival NF-κB and mTOR signalling. Inhibitors of BCR and mTOR signalling cooperatively decreased the formation and function of the My-T-BCR supercomplex, providing mechanistic insight into their synergistic toxicity for My-T-BCR+ DLBCL cells. My-T-BCR supercomplexes characterized ibrutinib-responsive malignancies and distinguished ibrutinib responders from non-responders. Our data provide a framework for the rational design of oncogenic signalling inhibitors in molecularly defined subsets of DLBCL.


Asunto(s)
Carcinogénesis , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Complejos Multiproteicos/metabolismo , Transducción de Señal , Adenina/análogos & derivados , Animales , Biopsia , Sistemas CRISPR-Cas/genética , Carcinogénesis/genética , Diseño de Fármacos , Femenino , Humanos , Linfoma de Células B Grandes Difuso/genética , Ratones , Complejos Multiproteicos/química , Mutación , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Piperidinas , Proteómica , Pirazoles/farmacología , Pirazoles/uso terapéutico , Pirimidinas/farmacología , Pirimidinas/uso terapéutico , Receptores de Antígenos de Linfocitos B/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos B/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
10.
Biochem Biophys Res Commun ; 663: 171-178, 2023 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-37121127

RESUMEN

Zinc finger transcription factor CASZ1b is essential for nervous system development and suppresses neuroblastoma growth. Our previous study showed that CASZ1b interacts with DNA repair proteins, however, whether CASZ1b is involved in the DNA damage response remains unclear. In this study, we investigated the kinetic recruitment of CASZ1b to sites of DNA damage upon induction by laser microirradiation. We find that CASZ1b is transiently recruited to sites of DNA damage in multiple cell lines. Mutagenesis of either the poly-(ADP-ribose) (PAR) binding motif or NuRD complex binding region in CASZ1b significantly reduces the recruitment of CASZ1b to these sites of DNA damage (∼65% and ∼30%, respectively). In addition, treatment of cells with a poly-(ADP-ribose) polymerase (PARP) inhibitor significantly attenuates the recruitment of CASZ1b to these DNA damaged sites. Loss of CASZ1 increases cell sensitivity to DNA damage induced by gamma irradiation as shown by decreased colony formation. Our studies reveal that CASZ1b is transiently recruited to DNA damage sites mainly in a PARP-dependent way and regulates cell sensitivity to DNA damage. Our results suggest that CASZ1b has a role, although perhaps a minor one, in the DNA damage response and ultimately regulating the efficiency of DNA repair during normal development and tumorigenesis.


Asunto(s)
Inhibidores de Poli(ADP-Ribosa) Polimerasas , Factores de Transcripción , Factores de Transcripción/metabolismo , Dedos de Zinc , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Reparación del ADN , Poli(ADP-Ribosa) Polimerasas/metabolismo , Daño del ADN , Poli Adenosina Difosfato Ribosa/metabolismo
11.
Nucleic Acids Res ; 47(17): 9368-9385, 2019 09 26.
Artículo en Inglés | MEDLINE | ID: mdl-31400113

RESUMEN

Cellular non-membranous RNA-granules, P-bodies (RNA processing bodies, PB) and stress granules (SG), are important components of the innate immune response to virus invasion. Mechanisms governing how a virus modulates PB formation remain elusive. Here, we report the important roles of GW182 and DDX6, but not Dicer, Ago2 and DCP1A, in PB formation, and that Kaposi's sarcoma-associated herpesvirus (KSHV) lytic infection reduces PB formation through several specific interactions with viral RNA-binding protein ORF57. The wild-type ORF57, but not its N-terminal dysfunctional mutant, inhibits PB formation by interacting with the N-terminal GW-domain of GW182 and the N-terminal domain of Ago2, two major components of PB. KSHV ORF57 also induces nuclear Ago2 speckles. Homologous HSV-1 ICP27, but not EBV EB2, shares this conserved inhibitory function with KSHV ORF57. By using time-lapse confocal microscopy of HeLa cells co-expressing GFP-tagged GW182, we demonstrated that viral ORF57 inhibits primarily the scaffolding of GW182 at the initial stage of PB formation. Consistently, KSHV-infected iSLK/Bac16 cells with reduced GW182 expression produced far fewer PB and SG, but 100-fold higher titer of infectious KSHV virions when compared to cells with normal GW182 expression. Altogether, our data provide the first evidence that a DNA virus evades host innate immunity by encoding an RNA-binding protein that promotes its replication by blocking PB formation.


Asunto(s)
Autoantígenos/genética , ARN Helicasas DEAD-box/genética , Herpesvirus Humano 8/genética , Proteínas Proto-Oncogénicas/genética , Proteínas de Unión al ARN/genética , Proteínas Reguladoras y Accesorias Virales/genética , Proteínas Argonautas/genética , Regulación Viral de la Expresión Génica/genética , Células HeLa , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/virología , Herpesvirus Humano 8/patogenicidad , Interacciones Huésped-Patógeno/genética , Humanos , ARN Viral/genética , Replicación Viral/genética
12.
PLoS Pathog ; 13(10): e1006677, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-29084250

RESUMEN

TIA-1 positive stress granules (SG) represent the storage sites of stalled mRNAs and are often associated with the cellular antiviral response. In this report, we provide evidence that Kaposi's sarcoma-associated herpesvirus (KSHV) overcomes the host antiviral response by inhibition of SG formation via a viral lytic protein ORF57. By immunofluorescence analysis, we found that B lymphocytes with KSHV lytic infection are refractory to SG induction. KSHV ORF57, an essential post-transcriptional regulator of viral gene expression and the production of new viral progeny, inhibits SG formation induced experimentally by arsenite and poly I:C, but not by heat stress. KSHV ORF37 (vSOX) bearing intrinsic endoribonuclease activity also inhibits arsenite-induced SG formation, but KSHV RTA, vIRF-2, ORF45, ORF59 and LANA exert no such function. ORF57 binds both PKR-activating protein (PACT) and protein kinase R (PKR) through their RNA-binding motifs and prevents PACT-PKR interaction in the PKR pathway which inhibits KSHV production. Consistently, knocking down PKR expression significantly promotes KSHV virion production. ORF57 interacts with PKR to inhibit PKR binding dsRNA and its autophosphorylation, leading to inhibition of eIF2α phosphorylation and SG formation. Homologous protein HSV-1 ICP27, but not EBV EB2, resembles KSHV ORF57 in the ability to block the PKR/eIF2α/SG pathway. In addition, KSHV ORF57 inhibits poly I:C-induced TLR3 phosphorylation. Altogether, our data provide the first evidence that KSHV ORF57 plays a role in modulating PKR/eIF2α/SG axis and enhances virus production during virus lytic infection.


Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Infecciones por Herpesviridae/metabolismo , Herpesvirus Humano 8/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , eIF-2 Quinasa/metabolismo , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/patología , Gránulos Citoplasmáticos/virología , Activación Enzimática/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Células HeLa , Infecciones por Herpesviridae/genética , Infecciones por Herpesviridae/patología , Herpesvirus Humano 8/genética , Humanos , Poli I-C/farmacología , Antígeno Intracelular 1 de las Células T/genética , Antígeno Intracelular 1 de las Células T/metabolismo , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Virión/genética , Virión/metabolismo , eIF-2 Quinasa/genética
13.
J Biol Chem ; 291(4): 1789-1802, 2016 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-26559976

RESUMEN

Chromatin undergoes a rapid ATP-dependent, ATM and H2AX-independent decondensation when DNA damage is introduced by laser microirradiation. Although the detailed mechanism of this decondensation remains to be determined, the kinetics of decondensation are similar to the kinetics of poly(ADP-ribosyl)ation. We used laser microirradiation to introduce DNA strand breaks into living cells expressing a photoactivatable GFP-tagged histone H2B. We find that poly(ADP-ribosyl)ation mediated primarily by poly(ADP-ribose) polymerase 1 (PARP1) is responsible for the rapid decondensation of chromatin at sites of DNA damage. This decondensation of chromatin correlates temporally with the displacement of histones, which is sensitive to PARP inhibition and is transient in nature. Contrary to the predictions of the histone shuttle hypothesis, we did not find that histone H1 accumulated on poly(ADP-ribose) (PAR) in vivo. Rather, histone H1, and to a lessor extent, histones H2A and H2B were rapidly depleted from the sites of PAR accumulation. However, histone H1 returns to chromatin and the chromatin recondenses. Thus, the PARP-dependent relaxation of chromatin closely correlates with histone displacement.


Asunto(s)
Ensamble y Desensamble de Cromatina/efectos de la radiación , Cromatina/metabolismo , Cromatina/efectos de la radiación , Histonas/metabolismo , Animales , Línea Celular , Daño del ADN/efectos de la radiación , Reparación del ADN , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Humanos , Rayos Láser , Ratones , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo
14.
J Immunol ; 192(2): 630-40, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24337745

RESUMEN

A critical process during thymic development of the T cell repertoire is the induction of self-tolerance. Tolerance in developing T cells is highly dependent on medullary thymic epithelial cells (mTEC), and mTEC development in turn requires signals from mature single-positive thymocytes, a bidirectional relationship termed thymus crosstalk. We show that CD28-CD80/86 and CD40-CD40L costimulatory interactions, which mediate negative selection and self-tolerance, upregulate expression of LTα, LTß, and receptor activator for NF-κB in the thymus and are necessary for medullary development. Combined absence of CD28-CD80/86 and CD40-CD40L results in profound deficiency in mTEC development comparable to that observed in the absence of single-positive thymocytes. This requirement for costimulatory signaling is maintained even in a TCR transgenic model of high-affinity TCR-ligand interactions. CD4 thymocytes maturing in the altered thymic epithelial environment of CD40/CD80/86 knockout mice are highly autoreactive in vitro and are lethal in congenic adoptive transfer in vivo, demonstrating a critical role for these costimulatory pathways in self-tolerance as well as thymic epithelial development. These findings demonstrate that cooperativity between CD28-CD80/86 and CD40-CD40L pathways is required for normal medullary epithelium and for maintenance of self-tolerance in thymocyte development.


Asunto(s)
Antígeno B7-1/inmunología , Antígeno B7-2/inmunología , Antígenos CD28/inmunología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Epitelio/inmunología , Autotolerancia/inmunología , Timocitos/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Células Epiteliales/inmunología , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , FN-kappa B/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T Reguladores/inmunología , Regulación hacia Arriba/inmunología
15.
Blood ; 119(2): 445-53, 2012 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-22106344

RESUMEN

ERM (ezrin, radixin moesin) proteins in lymphocytes link cortical actin to plasma membrane, which is regulated in part by ERM protein phosphorylation. To assess whether phosphorylation of ERM proteins regulates lymphocyte migration and membrane tension, we generated transgenic mice whose T-lymphocytes express low levels of ezrin phosphomimetic protein (T567E). In these mice, T-cell number in lymph nodes was reduced by 27%. Lymphocyte migration rate in vitro and in vivo in lymph nodes decreased by 18% to 47%. Lymphocyte membrane tension increased by 71%. Investigations of other possible underlying mechanisms revealed impaired chemokine-induced shape change/lamellipod extension and increased integrin-mediated adhesion. Notably, lymphocyte homing to lymph nodes was decreased by 30%. Unlike most described homing defects, there was not impaired rolling or sticking to lymph node vascular endothelium but rather decreased migration across that endothelium. Moreover, decreased numbers of transgenic T cells in efferent lymph suggested defective egress. These studies confirm the critical role of ERM dephosphorylation in regulating lymphocyte migration and transmigration. Of particular note, they identify phospho-ERM as the first described regulator of lymphocyte membrane tension, whose increase probably contributes to the multiple defects observed in the ezrin T567E transgenic mice.


Asunto(s)
Membrana Celular/patología , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/fisiología , Ganglios Linfáticos/patología , Mutación/genética , Linfocitos T/patología , Migración Transendotelial y Transepitelial/fisiología , Animales , Membrana Celular/metabolismo , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosfoproteínas/metabolismo , Fosforilación , Linfocitos T/metabolismo
16.
Circ Res ; 110(9): 1202-10, 2012 Apr 27.
Artículo en Inglés | MEDLINE | ID: mdl-22456181

RESUMEN

RATIONALE: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. OBJECTIVE: We addressed the role of platelets in mediating CNS inflammation in EAE. METHODS AND RESULTS: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ibα (GPIbα) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIbα attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice. CONCLUSIONS: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.


Asunto(s)
Plaquetas/metabolismo , Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/sangre , Leucocitos/inmunología , Animales , Antiinflamatorios/farmacología , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Células Cultivadas , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos/efectos de los fármacos , Glicoproteínas de Membrana/antagonistas & inhibidores , Glicoproteínas de Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Adhesividad Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Complejo GPIb-IX de Glicoproteína Plaquetaria/antagonistas & inhibidores , Complejo GPIb-IX de Glicoproteína Plaquetaria/metabolismo , Factores de Tiempo
17.
Nature ; 456(7221): 529-33, 2008 Nov 27.
Artículo en Inglés | MEDLINE | ID: mdl-18931658

RESUMEN

Variable, diversity and joining (V(D)J) recombination and class-switch recombination use overlapping but distinct non-homologous end joining pathways to repair DNA double-strand-break intermediates. 53BP1 is a DNA-damage-response protein that is rapidly recruited to sites of chromosomal double-strand breaks, where it seems to function in a subset of ataxia telangiectasia mutated (ATM) kinase-, H2A histone family member X (H2AX, also known as H2AFX)- and mediator of DNA damage checkpoint 1 (MDC1)-dependent events. A 53BP1-dependent end-joining pathway has been described that is dispensable for V(D)J recombination but essential for class-switch recombination. Here we report a previously unrecognized defect in the joining phase of V(D)J recombination in 53BP1-deficient lymphocytes that is distinct from that found in classical non-homologous-end-joining-, H2ax-, Mdc1- and Atm-deficient mice. Absence of 53BP1 leads to impairment of distal V-DJ joining with extensive degradation of unrepaired coding ends and episomal signal joint reintegration at V(D)J junctions. This results in apoptosis, loss of T-cell receptor alpha locus integrity and lymphopenia. Further impairment of the apoptotic checkpoint causes propagation of lymphocytes that have antigen receptor breaks. These data suggest a more general role for 53BP1 in maintaining genomic stability during long-range joining of DNA breaks.


Asunto(s)
ADN/metabolismo , Reordenamiento Génico de Linfocito T/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Recombinación Genética , Animales , Apoptosis , Proteínas Cromosómicas no Histona , ADN/genética , Roturas del ADN , Proteínas de Unión al ADN , Genes Codificadores de la Cadena alfa de los Receptores de Linfocito T/genética , Inestabilidad Genómica , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Linfopenia/genética , Linfopenia/patología , Ratones , Modelos Genéticos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Homología de Secuencia , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Proteína 1 de Unión al Supresor Tumoral P53
18.
Mol Cell Biol ; 44(3): 103-122, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38506112

RESUMEN

EWSR1 is a member of the FET family of nucleic acid binding proteins that includes FUS and TAF15. Here, we report the systematic analysis of endogenous EWSR1's cellular organization in human cells. We demonstrate that EWSR1, which contains low complexity and nucleic acid binding domains, is present in cells in faster and slower-recovering fractions, indicative of a protein undergoing both rapid exchange and longer-term interactions. The employment of complementary high-resolution imaging approaches shows EWSR1 exists in two visual modalities, a distributed state which is present throughout the nucleoplasm, and a concentrated state consistent with the formation of foci. Both EWSR1 visual modalities localize with nascent RNA. EWSR1 foci concentrate in regions of euchromatin, adjacent to protein markers of transcriptional activation, and significantly colocalize with phosphorylated RNA polymerase II. Our results contribute to bridging the gap between our understanding of the biophysical and biochemical properties of FET proteins, including EWSR1, their functions as transcriptional regulators, and the participation of these proteins in tumorigenesis and neurodegenerative disease.


Asunto(s)
Enfermedades Neurodegenerativas , Ácidos Nucleicos , Proteína EWS de Unión a ARN , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , ARN Polimerasa II/metabolismo , Proteína EWS de Unión a ARN/genética , Proteína EWS de Unión a ARN/metabolismo
19.
Cancer Res Commun ; 4(8): 2101-2111, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-39041239

RESUMEN

Triple-negative breast cancer (TNBC) is clinically aggressive and relatively unresponsive to current therapies. Therefore, the development of new anticancer agents is needed to satisfy clinical needs. Oxyphenisatin acetate (Acetalax), which had been used as a laxative, has recently been reported to have anticancer activity in murine models. In this study, we demonstrate that Acetalax and its diphenolic laxative structural analogue bisacodyl (Dulcolax) exhibit potent antiproliferative activity in TNBC cell lines and cause oncosis, a nonapoptotic cell death characterized by cellular and nuclear swelling and cell membrane blebbing, leading to mitochondrial dysfunction, ATP depletion, and enhanced immune and inflammatory responses. Mechanistically, we provide evidence that transient receptor potential melastatin member 4 (TRPM4) is poisoned by Acetalax and bisacodyl in MDA-MB468, BT549, and HS578T TNBC cells. MDA-MB231 and MDA-MB436 TNBC cells without endogenous TRPM4 expression as well as TRPM4-knockout TNBC cells were found to be Acetalax- and bisacodyl-resistant. Conversely, ectopic expression of TRPM4 sensitized MDA-MB231 and MDA-MB436 cells to Acetalax. TRPM4 was also lost in cells with acquired Acetalax resistance. Moreover, TRPM4 is rapidly degraded by the ubiquitin-proteasome system upon acute exposure to Acetalax and bisacodyl. Together, these results demonstrate that TRPM4 is a previously unknown target of Acetalax and bisacodyl and that TRPM4 expression in cancer cells is a predictor of Acetalax and bisacodyl efficacy and could be used for the clinical development of these drugs as anticancer agents. SIGNIFICANCE: Acetalax and bisacodyl kill cancer cells by causing oncosis following poisoning of the plasma membrane sodium transporter TRPM4 and represent a new therapeutic approach for TNBC.


Asunto(s)
Canales Catiónicos TRPM , Neoplasias de la Mama Triple Negativas , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/genética , Humanos , Línea Celular Tumoral , Femenino , Proliferación Celular/efectos de los fármacos , Animales , Ratones , Antineoplásicos/farmacología
20.
Neuro Oncol ; 26(6): 1067-1082, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38363979

RESUMEN

BACKGROUND: The aim of this study is an improved understanding of drug distribution in brain metastases. Rather than single point snapshots, we analyzed the time course and route of drug/probe elimination (clearance), focusing on the intramural periarterial drainage (IPAD) pathway. METHODS: Mice with JIMT1-BR HER2+ experimental brain metastases were injected with biocytin-TMR and either trastuzumab or human IgG. Drugs/probes circulated for 5 min to 48 h, followed by perfusion. Brain sections were stained for human IgG, vascular basement membrane proteins laminin or collagen IV, and periarterial α-SMA. A machine learning algorithm was developed to identify metastases, metastatic microenvironment, and uninvolved brain in confocally scanned brain sections. Drug/probe intensity over time and total imaged drug exposure (iAUC) were calculated for 27,249 lesions and co-immunofluorescence with IPAD-vascular matrix analyzed in 11,668 metastases. RESULTS: In metastases, peak trastuzumab levels were 5-fold higher than human IgG but 4-fold less than biocytin-TMR. The elimination phase constituted 85-93% of total iAUC for all drugs/probes tested. For trastuzumab, total iAUC during uptake was similar to the small molecule drug probe biocytin-TMR, but slower trastuzumab elimination resulted in a 1.7-fold higher total iAUC. During elimination trastuzumab and IgG were preferentially enriched in the α-SMA+ periarterial vascular matrix, consistent with the IPAD clearance route; biocytin-TMR showed heterogeneous elimination pathways. CONCLUSIONS: Drug/probe elimination is an important component of drug development for brain metastases. We identified a prolonged elimination pathway for systemically administered antibodies through the periarterial vascular matrix that may contribute to the sustained presence and efficacy of large antibody therapeutics.


Asunto(s)
Neoplasias Encefálicas , Neoplasias de la Mama , Inmunoglobulina G , Receptor ErbB-2 , Trastuzumab , Trastuzumab/farmacocinética , Animales , Ratones , Humanos , Femenino , Neoplasias Encefálicas/secundario , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Inmunoglobulina G/metabolismo , Receptor ErbB-2/metabolismo , Antineoplásicos Inmunológicos/farmacocinética , Ensayos Antitumor por Modelo de Xenoinjerto
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