RESUMEN
Alternative lengthening of telomeres (ALT) is mediated by break-induced replication (BIR), but how BIR is regulated at telomeres is poorly understood. Here, we show that telomeric BIR is a self-perpetuating process. By tethering PML-IV to telomeres, we induced telomere clustering in ALT-associated PML bodies (APBs) and a POLD3-dependent ATR response at telomeres, showing that BIR generates replication stress. Ablation of BLM helicase activity in APBs abolishes telomere synthesis but causes multiple chromosome bridges between telomeres, revealing a function of BLM in processing inter-telomere BIR intermediates. Interestingly, the accumulation of BLM in APBs requires its own helicase activity and POLD3, suggesting that BIR triggers a feedforward loop to further recruit BLM. Enhancing BIR induces PIAS4-mediated TRF2 SUMOylation, and PIAS4 loss deprives APBs of repair proteins and compromises ALT telomere synthesis. Thus, a BLM-driven and PIAS4-mediated feedforward loop operates in APBs to perpetuate BIR, providing a critical mechanism to extend ALT telomeres.
Asunto(s)
Proteínas del Grupo de Complementación de la Anemia de Fanconi/genética , Retroalimentación Fisiológica , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas Inhibidoras de STAT Activados/genética , ARN Helicasas/genética , Homeostasis del Telómero , Telómero/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Línea Celular , Línea Celular Tumoral , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/antagonistas & inhibidores , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Cuerpos de Inclusión Intranucleares/genética , Cuerpos de Inclusión Intranucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/antagonistas & inhibidores , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Transducción de Señal , Sumoilación , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genéticaRESUMEN
DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.
Asunto(s)
Enzimas Reparadoras del ADN/genética , Reparación del ADN , ADN/genética , Proteína Huntingtina/genética , Enfermedad de Huntington/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Inhibidoras de STAT Activados/genética , Procesamiento Proteico-Postraduccional , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Animales , Diferenciación Celular , ADN/metabolismo , Daño del ADN , Enzimas Reparadoras del ADN/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Proteína Huntingtina/metabolismo , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Cultivo Primario de Células , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Transcripción GenéticaRESUMEN
Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.
Asunto(s)
Daño del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Secuencias de Aminoácidos , Dominio Catalítico , Línea Celular Tumoral , Reparación del ADN , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Humanos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Péptidos/análisis , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Oxidative stress serves important roles in cardiac injury during the process of ischemia/reperfusion (I/R). Ybox protein 1 (YB1), a member of the highly conserved Ybox protein family, is closely associated with inflammation and stress responses by regulating gene transcription, RNA splicing and mRNA translation. However, the roles of YB1 in oxidative stressinduced myocardialI/R (MI/R) injury are unknown. The aim of the present study was to examine the effects of YB1 on H2O2induced cardiomyocyte injury and its underlying mechanism. The results demonstrated that YB1 expression was upregulated during H2O2induced myocardial injury. YB1 knockdown through transfection of small interfering RNA significantly aggravated cardiac cell apoptosis. Furthermore, YB1 knockdown significantly reversed the H2O2mediated increase in phosphorylated signal transducer and activator of transcription (STAT)3, but did not affect the phosphorylation of P38, extracellular signalregulated kinases 1/2, cJun Nterminal kinases, P65, Janus kinase 1 and 2 or STAT1. Moreover, protein coimmunoprecipitation and RNAbinding protein immunoprecipitation assays revealed that YB1 interacted with protein inhibitor of activated STAT 3 (PIAS3) mRNA but not its translated protein. YB1 overexpression may have promoted PIAS3 mRNA decay, decreasing PIAS3 protein levels, and therefore increased the levels of phosphorylated STAT3. Finally, YB1 knockdown, mediated by a lentivirus carrying YB1 targeted short hairpin RNA, significantly decreased left ventricle percentage fractional shortening and ejection fraction values, while increasing the infarct sizes in a rat model of MI/R injury. These results demonstrated for the first time (to the best of our knowledge) that YB1 may protect cardiac myocytes against H2O2 or MI/Rinduced injury by binding to PIAS3 mRNA and resulting in the phosphorylation of STAT3.
Asunto(s)
Peróxido de Hidrógeno/toxicidad , Chaperonas Moleculares/genética , Miocitos Cardíacos/efectos de los fármacos , Proteínas Inhibidoras de STAT Activados/genética , Factor de Transcripción STAT3/genética , Proteína 1 de Unión a la Caja Y/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica , Masculino , Chaperonas Moleculares/antagonistas & inhibidores , Chaperonas Moleculares/metabolismo , Daño por Reperfusión Miocárdica/inducido químicamente , Daño por Reperfusión Miocárdica/tratamiento farmacológico , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas , Sustancias Protectoras/farmacología , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismoRESUMEN
Amyloid-ß (Aß) produces neurotoxicity in the brain and causes neuronal death, but the endogenous defense mechanism that is activated on Aß insult is less well known. Here we found that acute Aß increases the expression of PIAS1 and Mcl-1 via activation of MAPK/ERK, and Aß induction of PIAS1 enhances HDAC1 SUMOylation in rat hippocampus. Knockdown of PIAS1 decreases endogenous HDAC1 SUMOylation and blocks Aß induction of Mcl-1. Sumoylated HDAC1 reduces it association with CREB, increases CREB binding to the Mcl-1 promoter and mediates Aß induction of Mcl-1 expression. Transduction of SUMO-modified lenti-HDAC1 vector to the hippocampus of APP/PS1 mice rescues spatial learning and memory deficit and long-term potentiation impairment in APP/PS1 mice. It also reduces the amount of amyloid plaque and the number of apoptotic cells in CA1 area of APP/PS1 mice. Meanwhile, HDAC1 SUMOylation decreases HDAC1 binding to the neprilysin promoter. These results together reveal an important role of HDAC1 SUMOylation as a naturally occurring defense mechanism protecting against Aß toxicity and provide an alternative therapeutic strategy against AD.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Epigénesis Genética , Histona Desacetilasa 1/metabolismo , Neuroprotección/efectos de los fármacos , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Apoptosis/efectos de los fármacos , Butadienos/farmacología , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Células HEK293 , Hipocampo/metabolismo , Histona Desacetilasa 1/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Nitrilos/farmacología , Unión Proteica , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , SumoilaciónRESUMEN
Interstitial fibrosis represents a key pathological process in non-alcoholic steatohepatitis (NASH). In the liver, fibrogenesis is primarily mediated by activated hepatic stellate cells (HSCs) transitioning from a quiescent state in response to a host of stimuli. The molecular mechanism underlying HSC activation is not completely understood. Here we report that there was a simultaneous up-regulation of PIAS4 expression and down-regulation of SIRT1 expression accompanying increased hepatic fibrogenesis in an MCD-diet induced mouse model of NASH. In cultured primary mouse HSCs, stimulation with high glucose activated PIAS4 while at the same time repressed SIRT1. Over-expression of PIAS4 directly repressed SIRT1 promoter activity. In contrast, depletion of PIAS4 restored SIRT1 expression in HSCs treated with high glucose. Estrogen, a known NASH-protective hormone, antagonized HSC activation by targeting PIAS4. Lentivirus-mediated delivery of short hairpin RNA (shRNA) targeting PIAS4 in mice ameliorated MCD diet induced liver fibrosis by normalizing SIRT1 expression in vivo. PIAS4 promoted HSC activation in a SIRT1-dependent manner in vitro. Mechanistically, PIAS4 mediated SIRT1 repression led to SMAD3 hyperacetylation and enhanced SMAD3 binding to fibrogenic gene promoters. Taken together, our data suggest SIRT1 trans-repression by PIAS4 plays an important role in HSC activation and liver fibrosis.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/patología , Proteínas Inhibidoras de STAT Activados/metabolismo , Sirtuina 1/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Estrógenos/farmacología , Glucosa/farmacología , Células Estrelladas Hepáticas/citología , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/etiología , Cirrosis Hepática/metabolismo , Masculino , Ratones , Ratones Obesos , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Sirtuina 1/genética , Proteína smad3/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Protein arginine methyltransferase 1 (PRMT1) catalyzes methylation of histones and other cellular proteins, and thus regulates gene transcription and protein activity. In antigen-induced pulmonary inflammation (AIPI) PRMT1 was up-regulated in the epithelium, while in chronic AIPI, increased PRMT1 shifted to fibroblasts. In this study we investigated the cell type specific regulatory mechanism of PRMT1. Epithelial cells and fibroblasts were stimulated with IL-4 or IL-1ß. Gene and protein expression were determined by RT-qPCR, immunohistochemistry staining and Western blotting. Signaling pathway inhibitors, siRNAs and shRNA were used to determine the regulatory mechanism of PRMT1. The results showed that IL-4 up-regulated PRMT1 through STAT6 signaling in epithelial cells, while IL-1ß regulated PRMT1 through NF-κB in fibroblasts. The NF-kB inhibitor protein RKIP was highly expressed in epithelial cells and blocked IL-1ß induced PRMT1 up-regulation; while the STAT6 inhibitor protein PIAS1 was expressed in fibroblasts and suppressed IL-4 induced PRMT1 expression. Furthermore, IL-4 stimulated epithelial cells to release IL-1ß which up-regulated PRMT1 expression in fibroblasts. In conclusion, the inhibitor proteins RKIP and PIAS1 regulated the cell type and signaling specific expression of PRMT1. Thus PRMT1 expression in structural lung cells in asthma can be considered as potential target for new therapeutic intervention.
Asunto(s)
Proteínas de Unión a Fosfatidiletanolamina/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Células A549 , Sitios de Unión , Western Blotting , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Inmunoprecipitación , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-4/farmacología , Pulmón/metabolismo , Pulmón/patología , Microscopía Fluorescente , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/antagonistas & inhibidores , Proteínas de Unión a Fosfatidiletanolamina/genética , Fosforilación/efectos de los fármacos , Neumonía/metabolismo , Neumonía/patología , Regiones Promotoras Genéticas , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/genética , Interferencia de ARN , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Factor de Transcripción STAT6/antagonistas & inhibidores , Factor de Transcripción STAT6/genética , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/antagonistas & inhibidores , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Regulación hacia Arriba/efectos de los fármacos , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Obesity is associated with chronic low-level inflammation, especially in fat tissues, which contributes to insulin resistance and type 2 diabetes mellitus (T2DM). Protein inhibitor of activated STAT 1 (PIAS1) modulates a variety of cellular processes such as cell proliferation and DNA damage responses. Particularly, PIAS1 functions in the innate immune system and is a key regulator of the inflammation cascade. However, whether PIAS1 is involved in the regulation of insulin sensitivity remains unknown. Here, we demonstrated that PIAS1 expression in white adipose tissue (WAT) was downregulated by c-Jun N-terminal kinase in prediabetic mice models. Overexpression of PIAS1 in inguinal WAT of prediabetic mice significantly improved systemic insulin sensitivity, whereas knockdown of PIAS1 in wild-type mice led to insulin resistance. Mechanistically, PIAS1 inhibited the activation of stress-induced kinases and the expression of nuclear factor-κB target genes in adipocytes, mainly including proinflammatory and chemotactic factors. In doing so, PIAS1 inhibited macrophage infiltration in adipose tissue, thus suppressing amplification of the inflammation cascade, which in turn improved insulin sensitivity. These results were further verified in a fat transplantation model. Our findings shed light on the critical role of PIAS1 in controlling insulin sensitivity and suggest a therapeutic potential of PIAS1 in T2DM.
Asunto(s)
Tejido Adiposo Blanco/metabolismo , Diabetes Mellitus Tipo 2/etiología , Resistencia a la Insulina , Macrófagos/metabolismo , Obesidad/metabolismo , Estado Prediabético/etiología , Proteínas Inhibidoras de STAT Activados/metabolismo , Tejido Adiposo Blanco/inmunología , Animales , Quimiotaxis , Diabetes Mellitus Tipo 2/prevención & control , Dieta Alta en Grasa/efectos adversos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas , Activación de Macrófagos , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Obesidad/inmunología , Obesidad/fisiopatología , Obesidad/terapia , Estado Prediabético/prevención & control , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Células RAW 264.7 , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450-480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.
Asunto(s)
Astrocitos/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de la radiación , Muerte Celular/genética , Muerte Celular/fisiología , Muerte Celular/efectos de la radiación , Núcleo Celular/metabolismo , Células Cultivadas , Daño del ADN , Rayos gamma , Técnicas de Silenciamiento del Gen , Células HEK293 , Haploinsuficiencia , Proteínas de Choque Térmico/deficiencia , Proteínas de Choque Térmico/genética , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Mapas de Interacción de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estrés Fisiológico , Sumoilación , Técnicas del Sistema de Dos HíbridosRESUMEN
Proto-oncogene Akt plays essential roles in cell proliferation and tumorigenesis. Full activation of Akt is regulated by phosphorylation, ubiquitination, and acetylation. Here we report that SUMOylation of Akt is a novel mechanism for its activation. Systematically analyzing the role of lysine residues in Akt activation revealed that K276, which is located in a SUMOylation consensus motif, is essential for Akt activation. Ectopic or endogenous Akt1 could be modified by SUMOylation. RNA interference-mediated silencing of UBC9 reduced Akt SUMOylation, which was promoted by SUMO E3 ligase PIAS1 and reversed by the SUMO-specific protease SENP1. Although multiple sites on Akt could be SUMOylated, K276 was identified as a major SUMO acceptor site. K276R or E278A mutation reduced SUMOylation of Akt but had little effect on its ubiquitination. Strikingly, these mutations also completely abolished Akt kinase activity. In support of these results, we found that expression of PIAS1 and SUMO1 increased Akt activity, whereas expression of SENP1 reduced Akt1 activity. Interestingly, the cancer-derived mutant E17K in Akt1 that occurs in various cancers was more efficiently SUMOylated than wild-type Akt. Moreover, SUMOylation loss dramatically reduced Akt1 E17K-mediated cell proliferation, cell migration, and tumorigenesis. Collectively, our findings establish that Akt SUMOylation provides a novel regulatory mechanism for activating Akt function.
Asunto(s)
Adenocarcinoma/patología , Proliferación Celular , Transformación Celular Neoplásica/patología , Neoplasias Pulmonares/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Apoptosis , Western Blotting , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Fosforilación , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-akt/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sumoilación , Enzimas Ubiquitina-Conjugadoras/antagonistas & inhibidores , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación , Cicatrización de HeridasRESUMEN
Tumorigenesis results from dysregulation of oncogenes and tumor suppressors that influence cellular proliferation, differentiation, apoptosis, and/or senescence. Many gene products involved in these processes are substrates of the E3 ubiquitin ligase Mule/Huwe1/Arf-BP1 (Mule), but whether Mule acts as an oncogene or tumor suppressor in vivo remains controversial. We generated K14Cre;Mule(flox/flox(y)) (Mule kKO) mice and subjected them to DMBA/PMA-induced skin carcinogenesis, which depends on oncogenic Ras signaling. Mule deficiency resulted in increased penetrance, number, and severity of skin tumors, which could be reversed by concomitant genetic knockout of c-Myc but not by knockout of p53 or p19Arf. Notably, in the absence of Mule, c-Myc/Miz1 transcriptional complexes accumulated, and levels of p21CDKN1A (p21) and p15INK4B (p15) were down-regulated. In vitro, Mule-deficient primary keratinocytes exhibited increased proliferation that could be reversed by Miz1 knockdown. Transfer of Mule-deficient transformed cells to nude mice resulted in enhanced tumor growth that again could be abrogated by Miz1 knockdown. Our data demonstrate in vivo that Mule suppresses Ras-mediated tumorigenesis by preventing an accumulation of c-Myc/Miz1 complexes that mediates p21 and p15 down-regulation.
Asunto(s)
Transformación Celular Neoplásica , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Proteínas Nucleares/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/metabolismo , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/metabolismo , 9,10-Dimetil-1,2-benzantraceno/farmacología , Animales , Transformación Celular Neoplásica/genética , Células Cultivadas , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Genes ras , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/patología , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Oncogénica p21(ras)/antagonistas & inhibidores , Proteína Oncogénica p21(ras)/genética , Proteínas Inhibidoras de STAT Activados/deficiencia , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Proto-Oncogénicas c-myc/deficiencia , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Acetato de Tetradecanoilforbol/farmacología , Proteína p53 Supresora de Tumor , Proteínas Supresoras de Tumor , Ubiquitina-Proteína Ligasas/deficiencia , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Data presented here extends our previous observations on α-globin transcriptional regulation by the CP2 and PIAS1 proteins. Using RNAi knockdown, we have now shown that CP2b, CP2c and PIAS1 are each necessary for synergistic activation of endogenous α-globin gene expression in differentiating MEL cells. In this system, truncated PIAS1 mutants lacking the ring finger domain recruited CP2c to the nucleus, as did wild-type PIAS1, demonstrating that this is a sumoylation-independent process. In vitro, recombinant CP2c, CP2b and PIAS1 bound DNA as a stable CBP (CP2c/CP2b/PIAS1) complex. Following PIAS1 knockdown in MEL cells, however, the association of endogenous CP2c and CP2b with the α-globin promoter simultaneously decreased. By mapping the CP2b- and CP2c-binding domains on PIAS1, and the PIAS1-binding domains on CP2b and CP2c, we found that two regions of PIAS1 that interact with CP2c/CP2b are required for its co-activator function. We propose that CP2c, CP2b, and PIAS1 form a hexametric complex with two units each of CP2c, CP2b, and PIAS1, in which PIAS1 serves as a clamp between two CP2 proteins, while CP2c binds directly to the target DNA and CP2b mediates strong transactivation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Eritroides/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Globinas alfa/genética , Animales , Línea Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Humanos , Ratones , Regiones Promotoras Genéticas , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/química , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/químicaRESUMEN
Sma- and MAD-related protein 3 (Smad3) plays crucial roles in the transforming growth factor-beta (TGF-beta)-mediated signaling pathway, which produce a variety of cellular responses, including cell proliferation and differentiation. In our previous study, we demonstrated that protein inhibitor of activated STATy (PIASy) suppresses TGF-beta signaling by interacting with and sumoylating Smad3. In the present study, we examined the molecular mechanisms of Smad3 sumoylation during PIASy-mediated suppression of TGF-beta signaling. We found that small-interfering RNA-mediated reduction of endogenous PIASy expression enhanced TGF-beta-induced gene expression. Importantly, coexpression of Smad3 with PIASy and SUMO1 affected the DNA-binding activity of Smad3. Furthermore, coexpression of Smad3 with PIASy and SUMO1 stimulated the nuclear export of Smad3. Finally, fluorescence resonance energy transfer analyses revealed that Smad3 interacted with SUMO1 in the cytoplasm. These results suggest that PIASy regulates TGF-beta/Smad3-mediated signaling by stimulating sumoylation and nuclear export of Smad3.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína SUMO-1/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Línea Celular Tumoral , Transferencia Resonante de Energía de Fluorescencia , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Transducción de Señal , Proteína smad3/análisis , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Shear stress-induced extracellular signal-regulated kinase (ERK)5 activation and the consequent regulation of Kruppel-like factor 2 and endothelial nitric oxide synthase expression represents one of the antiinflammatory and vascular tone regulatory mechanisms maintaining normal endothelial function. Endothelial dysfunction is a major initiator of atherosclerosis, a vascular pathology often associated with diabetes. Small ubiquitin-like modifier (SUMO) covalently attaches to certain residues of specific target transcription factors and could inhibit its activity. We investigated whether H(2)O(2) and AGE (advanced glycation end products), 2 well-known mediators of diabetes, negatively regulated ERK5 transcriptional activity and laminar flow-induced endothelial nitric oxide synthase expression through ERK5 SUMOylation. H(2)O(2) and AGE induced endogenous ERK5 SUMOylation. In addition, ERK5 SUMOylation was increased in the aortas from diabetic mice. ERK5 transcriptional activity, but not kinase activity, was inhibited by expression of Ubc9 (SUMO E2 conjugase) or PIAS1 (E3 ligase), suggesting the involvement of ERK5 SUMOylation on its transcriptional activity. Point-mutation analyses showed that ERK5 is covalently modified by SUMO at 2 conserved sites, Lys6 and Lys22, and that the SUMOylation defective mutant of ERK5, dominant negative form of Ubc9 (DN-Ubc9), and small interfering RNA PIAS1 reversed H(2)O(2) and AGE-mediated reduction of shear stress-mediated ERK5/myocyte enhancer factor 2 transcriptional activity, as well as promoter activity of Kruppel-like factor 2. Finally, PIAS1 knockdown reversed the inhibitory effect of H(2)O(2) in shear stress-induced Kruppel-like factor 2 and endothelial nitric oxide synthase expression. These data clearly defined SUMOylation-dependent ERK5 transcriptional repression independent of kinase activity and suggested this process as among the molecular mechanisms of diabetes-mediated endothelial dysfunction.
Asunto(s)
Células Endoteliales/metabolismo , Inflamación/metabolismo , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteína SUMO-1/metabolismo , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Cultivadas , Diabetes Mellitus/inducido químicamente , Diabetes Mellitus/metabolismo , Modelos Animales de Enfermedad , Inducción Enzimática/efectos de los fármacos , Productos Finales de Glicación Avanzada/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Masculino , Ratones , Ratones Endogámicos , Proteína Quinasa 7 Activada por Mitógenos/genética , Oxidantes/farmacología , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/metabolismo , ARN Interferente Pequeño/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Estreptozocina , Estrés Mecánico , Transcripción Genética/efectos de los fármacosRESUMEN
The Myc-associated zinc-finger protein, Miz1, is a negative regulator of cell proliferation and induces expression of the cell-cycle inhibitors p15(Ink4b) and p21(Cip1). Here we identify the ribosomal protein L23 as a negative regulator of Miz1-dependent transactivation. L23 exerts this function by retaining nucleophosmin, an essential co-activator of Miz1 required for Miz1-induced cell-cycle arrest, in the nucleolus. Mutant forms of nucleophosmin found in acute myeloid leukaemia fail to co-activate Miz1 and re-localize it to the cytosol. As L23 is encoded by a direct target gene of Myc, this regulatory circuit may provide a feedback mechanism that links translation of Myc target genes and cell growth to Miz1-dependent cell-cycle arrest.
Asunto(s)
Proteínas Nucleares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Ribosómicas/metabolismo , Alelos , Animales , Proliferación Celular , Retroalimentación Fisiológica , Células HeLa , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Nucleofosmina , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/metabolismo , RatasRESUMEN
Th2 cytokine expression is dependent on the transcription factor GATA-3. However, the molecular interactions of GATA-3 leading to Th2 cytokine gene activation have not been well characterized. Here, we reported a number of GATA-3 associated proteins in Th2 cells, and one of such proteins Pias1 functioned as a positive transcriptional coregulator for GATA-3. When overexpressed in Th2 cells, Pias1 enhanced the expression of IL-13, and to lesser degrees, IL-4 and -5. Conversely, Pias1 siRNA down-regulated the Th2 cytokine expression. In Leishmania major infection, manipulating Pias1 expression in parasite-reactive CD4 T cells altered severity of disease caused by Th2 responses. Mechanistically, Pias1 markedly potentiated GATA-3-mediated activation of the IL-13 promoter by facilitating the recruitment of GATA-3 to the promoter. In contrast, IL-5 promoter was modestly enhanced by Pias1 and no effect was observed on IL-4 promoter. Thus, both promoter activation and additional mechanisms are responsible for regulation by Pias1.
Asunto(s)
Factor de Transcripción GATA3/metabolismo , Inmunidad/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Células Th2/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Citocinas/antagonistas & inhibidores , Citocinas/genética , Humanos , Interleucina-13/genética , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Interleucina-5/metabolismo , Leishmania major , Leishmaniasis Cutánea/genética , Leishmaniasis Cutánea/inmunología , Ratones , Regiones Promotoras Genéticas , Proteínas Inhibidoras de STAT Activados/antagonistas & inhibidores , Proteínas Inhibidoras de STAT Activados/genética , ARN Interferente Pequeño/genética , Retroviridae/genética , Transcripción Genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: The precision of the metaphase-anaphase transition ensures stable genetic inheritance. The spindle checkpoint blocks anaphase onset until the last chromosome biorients at metaphase plate, then the bonds between sister chromatids are removed and disjoined chromatids segregate to the spindle poles. But, how sister separation is triggered is not fully understood. PRINCIPAL FINDINGS: We identify PIASgamma as a human E3 sumo ligase required for timely and efficient sister chromatid separation. In cells lacking PIASgamma, normal metaphase plates form, but the spindle checkpoint is activated, leading to a prolonged metaphase block. Sister chromatids remain cohered even if cohesin is removed by depletion of hSgo1, because DNA catenations persist at centromeres. PIASgamma-depleted cells cannot properly localize Topoisomerase II at centromeres or in the cores of mitotic chromosomes, providing a functional link between PIASgamma and Topoisomerase II. CONCLUSIONS: PIASgamma directs Topoisomerase II to specific chromosome regions that require efficient removal of DNA catenations prior to anaphase. The lack of this activity activates the spindle checkpoint, protecting cells from non-disjunction. Because DNA catenations persist without PIASgamma in the absence of cohesin, removal of catenations and cohesin rings must be regulated in parallel.