RESUMEN
Matrix metalloproteinase (MMP) 2 and 9 are the family members of proteases normally up-regulated in tumor to enhance the invasion and metastatic of tumor cells, and are associated with poor outcome of head and neck squamous cell carcinomas (HNSCCs). In the present work, MMPs-degradable gelatin nanoparticles (GNPs) are simultaneously loaded with photosensitizer indocyanine green (ICG) along with signal transducer activator of transcription 3 (STAT3) inhibitor NSC74859 (NSC, N) for efficient photothermal therapy (PTT) and immunotherapy of HNSCCs. In the tumor tissue, Gel-N-ICG nanoparticle was degraded and encapsulated ICG and NSC were effectively released. Under near-infrared (NIR) irradiation, the released ICG nanoparticles enabled effective photothermal destruction of tumors, and the STAT3 inhibitor NSC elicited potent antitumor immunity for enhanced cancer therapy. Based on two HNSCC mouse models, we demonstrated that Gel-N-ICG significantly delayed tumor growth without any appreciable body weight loss. Taken together, the strategy reported here may contribute that the stimuli-responsive proteases triggered nanoplatform could reduce tumor size more effectively in complex tumor microenvironment (TME) through combination of PTT and immunotherapy.
Asunto(s)
Gelatinasas/metabolismo , Nanopartículas , Fármacos Fotosensibilizantes , Proteínas Inhibidoras de STAT Activados , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Inmunoterapia , Verde de Indocianina/química , Verde de Indocianina/farmacocinética , Ratones , Nanopartículas/química , Nanopartículas/metabolismo , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacocinética , Fármacos Fotosensibilizantes/farmacología , Terapia Fototérmica , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/farmacocinética , Proteínas Inhibidoras de STAT Activados/farmacología , Factor de Transcripción STAT3/antagonistas & inhibidoresRESUMEN
The human PIAS proteins are small ubiquitin-like modifier (SUMO) E3 ligases that participate in important cellular functions. Several of these functions depend on a conserved SUMO-interacting motif (SIM) located in the central region of all PIAS proteins (SIM1). Recently, it was determined that Siz2, a yeast homolog of PIAS proteins, possesses a second SIM at its C terminus (SIM2). Sequence alignment indicates that a SIM2 is also present in PIAS1-3, but not PIAS4. Using biochemical and structural studies, we demonstrate PIAS-SIM2 binds to SUMO1, but that phosphorylation of the PIAS-SIM2 or acetylation of SUMO1 alter this interaction in a manner distinct from what is observed for the PIAS-SIM1. We also show that the PIAS-SIM2 plays a key role in formation of a UBC9-PIAS1-SUMO1 complex. These results provide insights into how post-translational modifications selectively regulate the specificity of multiple SIMs found in the PIAS proteins by exploiting the plasticity built into the SUMO-SIM binding interface.
Asunto(s)
Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína SUMO-1/metabolismo , Acetilación , Secuencias de Aminoácidos , Cristalografía por Rayos X , Células HEK293 , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosforilación , Proteínas de Unión a Poli-ADP-Ribosa/química , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/genética , Dominios y Motivos de Interacción de Proteínas , Serina/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
Aims: Neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) signaling have been implicated in learning, memory, and underlying long-lasting synaptic plasticity. In this study, we aimed at detecting whether nNOS is a target protein of SUMOylation in the hippocampus and its contributions to hippocampal long-term potentiation (LTP) of synaptic transmission. Results: We showed that N-methyl-d-aspartate receptor-dependent neuronal activity enhancement induced the attachment of small ubiquitin-like modifier 1 (SUMO1) to nNOS. Protein inhibitor of activated STAT3 (PIAS3) promoted SUMO1 conjugation at K725 and K739 on nNOS, which upregulated NO production and nNOS S1412 phosphorylation (activation). In addition, the N-terminus (amino acids 43-86) of PIAS3 bound nNOS directly. Tat-tagged PIAS3 segment representing amino acids 43-86, a cell-permeable peptide containing PIAS3 residues 43-86, suppressed activity-induced nNOS SUMOylation by disrupting PIAS3-nNOS association. It also decreased LTP-related expression of Arc and brain-derived neurotrophic factor and blocked signaling via extracellular signal-regulated kinase (ERK) 1/2 and Elk-1 in the hippocampus. More importantly, PIAS3-mediated nNOS SUMOylation was required for activity-regulated ERK1/2 activation in nNOS-positive neurons and hippocampal LTP induction. Innovation and Conclusion: These findings indicated that network activity-regulated nNOS SUMOylation underlies excitatory synaptic LTP by facilitating nNOS-NO-ERK1/2 signal cascades.
Asunto(s)
Hipocampo/citología , Chaperonas Moleculares/metabolismo , Óxido Nítrico Sintasa de Tipo I/química , Óxido Nítrico Sintasa de Tipo I/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína SUMO-1/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Hipocampo/metabolismo , Potenciación a Largo Plazo , Sistema de Señalización de MAP Quinasas , Masculino , Modelos Moleculares , Chaperonas Moleculares/química , Óxido Nítrico/metabolismo , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Ratas , Sumoilación , Transmisión SinápticaRESUMEN
Insulin plays a central role in glucose homeostasis and is produced exclusively by pancreatic islet ß-cells. Insulin gene transcription is regulated by a set of ß-cell-enriched transcription factors that bind to cis-regulatory elements within the promoter region, and regulation of the insulin gene promoter is closely linked to ß-cell functionality. PIASy, a member of the PIAS family of SUMO E3 ligases, is thought to affect insulin gene transcription, but its mechanism of action is not fully understood. Here, we demonstrate that PIASy interacts with MafA and represses insulin gene promoter activity. MafA is a ß-cell-restricted basic leucine-zipper transcriptional activator that binds to the C1 element of the insulin gene promoter. In line with previous studies showing the transactivator domain of MafA is SUMOylated, PIASy enhanced the SUMOylation of MafA. However, a SUMOylation-deficient mutant of MafA was still repressed by PIASy, indicating that this modification is dispensable for repression. Using a series of MafA and PIASy mutants, we found that the basic domain of MafA and the amino-terminal region of PIASy containing the SAP domain are necessary for their interaction. In addition, SUMO-interacting motif 1 (SIM1) at the carboxyl-terminal region of PIASy was required to repress the synergistic transactivation of MafA, Pdx1, and Beta2, transcription factors playing central roles in ß-cell differentiation and function. The PINIT and SP-RING domains in the middle region of PIASy were dispensable. These findings suggest that PIASy binds to MafA through the SAP domain and negatively regulates the insulin gene promoter through a novel SIM1-dependent mechanism.
Asunto(s)
Insulina/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Animales , Regulación de la Expresión Génica , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ratones , Proteínas de Unión a Poli-ADP-Ribosa/química , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Dominios y Motivos de Interacción de Proteínas , Sumoilación , Transactivadores/metabolismo , Activación Transcripcional , Dedos de ZincRESUMEN
Protein inhibitor of activated STAT (PIAS) plays a critical role in the feedback modulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway as a negative regulator in mammals and Drosophila, but the function of PIAS in crustaceans is still unclear. In this study, a PIAS termed LvPIAS was cloned and characterized from Litopenaeus vannamei. The full length of LvPIAS was 3065 bp, including a 2361 bp open reading frame (ORF) coding for a protein of 786 aa. LvPIAS expression was most abundant in muscle and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly I: C and white spot syndrome virus (WSSV). LvPIAS could be induced by the transcription factor LvSTAT, but LvPIAS could inhibit the transcriptional activity of LvSTAT to the LvPIAS promoter conversely, which indicated that there was a negative feedback loop between LvSTAT and LvPIAS. Furthermore, RNAi-mediated knockdown of LvPIAS shrimps showed higher survival rate to WSSV infection than those in the control group (dsGFP injection), suggesting that LvPIAS may play a negatively role against WSSV infection.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Penaeidae/genética , Penaeidae/inmunología , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Secuencia de Bases , Perfilación de la Expresión Génica , Lipopolisacáridos/farmacología , Filogenia , Poli I-C/farmacología , Proteínas Inhibidoras de STAT Activados/química , Alineación de Secuencia , Staphylococcus aureus/fisiología , Vibrio parahaemolyticus/fisiología , Virus del Síndrome de la Mancha Blanca 1/fisiologíaRESUMEN
Porcine reproductive and respiratory syndrome virus (PRRSV) activates NF-κB during infection. We examined the ability of all 22 PRRSV genes for NF-κB regulation and determined the nucleocapsid (N) protein as the NF-κB activator. Protein inhibitor of activated STAT1 (signal transducer and activator of transcription 1) (PIAS1) was identified as a cellular protein binding to N. PIAS1 is known to bind to p65 (RelA) in the nucleus and blocks its DNA binding, thus functions as a repressor of NF-κB. Binding of N to PIAS1 released p65 for NF-κB activation. The N-terminal half of PIAS1 was mapped as the N-binding domain, and this region overlapped its p65-binding domain. For N, the region between 37 and 72 aa was identified as the binding domain to PIAS1, and this domain alone was able to activate NF-κB. A nuclear localization signal (NLS) knock-out mutant N did not activate NF-κB, and this is mostly likely due to the lack of its interaction with PIAS1 in the nucleus, demonstrating the positive correlation between the binding of N to PIAS1 and the NF-κB activation. Our study reveals a role of N in the nucleus for NF-κB activation and proinflammatory cytokine production during infection.
Asunto(s)
Proteínas de la Cápside/metabolismo , FN-kappa B/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/metabolismo , Virus del Síndrome Respiratorio y Reproductivo Porcino/patogenicidad , Proteínas Inhibidoras de STAT Activados/metabolismo , Animales , Sitios de Unión , Células Cultivadas , Células HeLa , Humanos , Macrófagos/metabolismo , Macrófagos/virología , FN-kappa B/genética , Señales de Localización Nuclear , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/genética , PorcinosRESUMEN
Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.
Asunto(s)
Modelos Moleculares , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Represoras/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitinas/metabolismo , Proteínas de Xenopus/metabolismo , Secuencias de Aminoácidos , Animales , Eliminación de Gen , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Ligandos , Mutagénesis Sitio-Dirigida , Mutación , Isótopos de Nitrógeno , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Ubiquitinas/química , Ubiquitinas/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
Fusion tyrosine kinases play a crucial role in the development of hematological malignancies. FIP1L1-PDGFRA is a leukemogenic fusion kinase that causes chronic eosinophilic leukemia. As a constitutively active kinase, FIP1L1-PDGFRA stimulates downstream signaling molecules, leading to cellular proliferation and the generation of an anti-apoptotic state. Contribution of the N-terminal FIP1L1 portion is necessary for FIP1L1-PDGFRA to exert its full transforming activity, but the underlying mechanisms have not been fully characterized. We identified PIAS1 as a FIP1L1-PDGFRA association molecule by yeast two-hybrid screening. Our analyses indicate that the FIP1L1 portion of FIP1L1-PDGFRA is required for efficient association with PIAS1. As a consequence of the association, FIP1L1-PDGFRA phosphorylates PIAS1. Moreover, the kinase activity of FIP1L1-PDGFRA stabilizes PIAS1. Therefore, PIAS1 is one of the downstream targets of FIP1L1-PDGFRA. Moreover, we found that PIAS1, as a SUMO E3 ligase, sumoylates and stabilizes FIP1L1-PDGFRA. In addition, suppression of PIAS1 activity by a knockdown experiment resulted in destabilization of FIP1L1-PDGFRA. Therefore, FIP1L1-PDGFRA and PIAS1 form a positive cross-talk through their enzymatic activities. Suppression of sumoylation by ginkgolic acid, a small molecule compound inhibiting a SUMO E1-activating enzyme, also destabilizes FIP1L1-PDGFRA, and while the tyrosine kinase inhibitor imatinib suppresses FIP1L1-PDGFRA-dependent cell growth, ginkgolic acid or siRNA of PIAS1 has a synergistic effect with imatinib. In conclusion, our results suggest that sumoylation by PIAS1 is a potential target in the treatment of FIP1L1-PDGFRA-positive chronic eosinophilic leukemia.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Fusión Oncogénica/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Transcripción STAT1/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Apoptosis , Células HEK293 , Humanos , Síndrome Hipereosinofílico/tratamiento farmacológico , Síndrome Hipereosinofílico/metabolismo , Mesilato de Imatinib/uso terapéutico , Immunoblotting , Inmunoprecipitación , Proteínas de Fusión Oncogénica/química , Proteínas Inhibidoras de STAT Activados/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/química , Factor de Transcripción STAT1/química , Transducción de Señal , Sumoilación , Transfección/métodos , Factores de Escisión y Poliadenilación de ARNm/químicaRESUMEN
Cell fate specification requires precise coordination of transcription factors and their regulators to achieve fidelity and flexibility in lineage allocation. The transcriptional repressor growth factor independence 1 (GFI1) is comprised of conserved Snail/Slug/Gfi1 (SNAG) and zinc finger motifs separated by a linker region poorly conserved with GFI1B, its closest homolog. Moreover, GFI1 and GFI1B coordinate distinct developmental fates in hematopoiesis, suggesting that their functional differences may derive from structures within their linkers. We show a binding interface between the GFI1 linker and the SP-RING domain of PIAS3, an E3-SUMO (small ubiquitin-related modifier) ligase. The PIAS3 binding region in GFI1 contains a conserved type I SUMOylation consensus element, centered on lysine-239 (K239). In silico prediction algorithms identify K239 as the only high-probability site for SUMO modification. We show that GFI1 is modified by SUMO at K239. SUMOylation-resistant derivatives of GFI1 fail to complement Gfi1 depletion phenotypes in zebrafish primitive erythropoiesis and granulocytic differentiation in cultured human cells. LSD1/CoREST recruitment and MYC repression by GFI1 are profoundly impaired for SUMOylation-resistant GFI1 derivatives, while enforced expression of MYC blocks granulocytic differentiation. These findings suggest that SUMOylation within the GFI1 linker favors LSD1/CoREST recruitment and MYC repression to govern hematopoietic differentiation.
Asunto(s)
Hematopoyesis , Histona Demetilasas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Sitios de Unión , Células COS , Diferenciación Celular , Chlorocebus aethiops , Regulación de la Expresión Génica , Células HEK293 , Células HL-60 , Humanos , Lisina/metabolismo , Ratones , Chaperonas Moleculares/química , Células 3T3 NIH , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Proteínas Proto-Oncogénicas/química , Proteínas Represoras/química , SumoilaciónRESUMEN
UNLABELLED: Small ubiquitin-like modifier (SUMO) is used by the intrinsic antiviral immune response to restrict viral pathogens, such as herpes simplex virus 1 (HSV-1). Despite characterization of the host factors that rely on SUMOylation to exert their antiviral effects, the enzymes that mediate these SUMOylation events remain to be defined. We show that unconjugated SUMO levels are largely maintained throughout infection regardless of the presence of ICP0, the HSV-1 SUMO-targeted ubiquitin ligase. Moreover, in the absence of ICP0, high-molecular-weight SUMO-conjugated proteins do not accumulate if HSV-1 DNA does not replicate. These data highlight the continued importance for SUMO signaling throughout infection. We show that the SUMO ligase protein inhibitor of activated STAT 4 (PIAS4) is upregulated during HSV-1 infection and localizes to nuclear domains that contain viral DNA. PIAS4 is recruited to sites associated with HSV-1 genome entry through SUMO interaction motif (SIM)-dependent mechanisms that are destabilized by ICP0. In contrast, PIAS4 accumulates in replication compartments through SIM-independent mechanisms irrespective of ICP0 expression. Depletion of PIAS4 enhances the replication of ICP0-null mutant HSV-1, which is susceptible to restriction by the intrinsic antiviral immune response. The mechanisms of PIAS4-mediated restriction are synergistic with the restriction mechanisms of a characterized intrinsic antiviral factor, promyelocytic leukemia protein, and are antagonized by ICP0. We provide the first evidence that PIAS4 is an intrinsic antiviral factor. This novel role for PIAS4 in intrinsic antiviral immunity contrasts with the known roles of PIAS proteins as suppressors of innate immunity. IMPORTANCE: Posttranslational modifications with small ubiquitin-like modifier (SUMO) proteins regulate multiple aspects of host immunity and viral replication. The protein inhibitor of activated STAT (PIAS) family of SUMO ligases is predominantly associated with the suppression of innate immune signaling. We now identify a unique and contrasting role for PIAS proteins as positive regulators of the intrinsic antiviral immune response to herpes simplex virus 1 (HSV-1) infection. We show that PIAS4 relocalizes to nuclear domains that contain viral DNA throughout infection. Depletion of PIAS4, either alone or in combination with the intrinsic antiviral factor promyelocytic leukemia protein, significantly impairs the intrinsic antiviral immune response to HSV-1 infection. Our data reveal a novel and dynamic role for PIAS4 in the cellular-mediated restriction of herpesviruses and establish a new functional role for the PIAS family of SUMO ligases in the intrinsic antiviral immune response to DNA virus infection.
Asunto(s)
Herpes Simple/genética , Herpes Simple/inmunología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Inmunidad Innata/genética , Proteínas Inhibidoras de STAT Activados/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Línea Celular , Replicación del ADN , ADN Viral , Progresión de la Enfermedad , Expresión Génica , Genoma Viral , Herpes Simple/metabolismo , Herpes Simple/virología , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Mutación , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Recombinantes de Fusión , Proteína SUMO-1/metabolismo , Sumoilación , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Replicación ViralRESUMEN
Post-translational modification of substrate proteins by small ubiquitin-like modifier (SUMO) regulates a vast array of cellular processes. SUMOylation occurs through three sequential enzymatic steps termed E1, E2 and E3. Substrate selection can be determined through interactions between the target protein and the SUMO E2 conjugating enzyme Ubc9 and specificity can be enhanced by substrate interactions with E3 ligase enzymes. We used the putative substrate recognition (PINIT) domain from the SUMO E3 PIAS3 as bait to identify potential SUMO substrates. One protein identified was Argonaute-2 (Ago2), which mediates RNA-induced gene silencing through binding small RNAs and promoting degradation of complimentary target mRNAs. We show that Ago2 can be SUMOylated in mammalian cells by both SUMO1 and SUMO2. SUMOylation occurs primarily at K402, and mutation of the SUMO consensus site surrounding this lysine reduces Ago2-mediated siRNA-induced silencing in a luciferase-based reporter assay. These results identify SUMOylation as a potential regulator of Ago2 activity and open new avenues for research into the mechanisms underlying the regulation of RNA-induced gene silencing.
Asunto(s)
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Interferencia de ARN , Sumoilación , Secuencia de Aminoácidos , Animales , Proteínas Argonautas/deficiencia , Proteínas Argonautas/genética , Sitios de Unión/genética , Células Cultivadas , Secuencia de Consenso , Técnicas de Inactivación de Genes , Humanos , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Especificidad por SustratoRESUMEN
PURPOSE: Deregulation of STAT3 activation is a hallmark of many cancer cells, and the underlying mechanisms are subject to intense investigation. We examined the extent of PIAS3 expression in mesothelioma cells and human tumor samples and determined the functional effects of PIAS3 expression on STAT3 signaling. EXPERIMENTAL DESIGN: We evaluated the expression of PIAS3 in mesothelioma tumors from patients and correlated the expression levels with the course of the disease. We also measured the effects of enhanced PIAS3 activity on STAT3 signaling, cellular growth, and viability in cultured mesothelioma cells. RESULTS: Gene expression databases revealed that mesotheliomas have the lowest levels of PIAS3 transcripts among solid tumors. PIAS3 expression in human mesothelioma tumors is significantly correlated with overall survival intervals (P = 0.058). The high expression of PIAS3 is predictive of a favorable prognosis and decreases the probability of death within one year after diagnosis by 44%. PIAS3 expression is functionally linked to STAT3 activation in mesothelioma cell lines. STAT3 downregulation with siRNA or enhanced expression of PIAS3 both inhibited mesothelioma cell growth and induced apoptosis. Mesothelioma cells are sensitive to curcumin and respond by the induction of PIAS3. Corroborative evidence has been obtained from STAT3 inhibition experiments. Exposure of the cells to a peptide derived from the PIAS3 protein that interferes with STAT3 function resulted in apoptosis induction and the inhibition of cell growth. CONCLUSION: These results suggest that PIAS3 protein expression impacts survival in patients with mesothelioma and that PIAS3 activation could become a therapeutic strategy. Clin Cancer Res; 20(19); 5124-32. ©2014 AACR.
Asunto(s)
Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Mesotelioma/genética , Mesotelioma/metabolismo , Chaperonas Moleculares/genética , Proteínas Inhibidoras de STAT Activados/genética , Factor de Transcripción STAT3/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Curcumina/farmacología , Expresión Génica , Humanos , Neoplasias Pulmonares/mortalidad , Mesotelioma/mortalidad , Mesotelioma Maligno , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/farmacología , Fragmentos de Péptidos/farmacología , Pronóstico , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Inhibidoras de STAT Activados/farmacología , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genéticaRESUMEN
Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.
Asunto(s)
Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Inhibidoras de STAT Activados/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Línea Celular , Humanos , Antígenos Específicos del Melanoma/metabolismo , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Especificidad por Sustrato , Sumoilación , UbiquitinaciónRESUMEN
Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.
Asunto(s)
Regulación de la Expresión Génica , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteína SUMO-1/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Transcripción Genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/genética , Multimerización de Proteína , Proteína SUMO-1/química , Proteína SUMO-1/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/química , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
The protein inhibitor of activated signal transducer and activator of transcription 3 (PIAS3) regulates the transcriptional activity of signal transducer and activator of transcription 3 (STAT3) which regulates transcription of genes involved in cell growth, proliferation and apoptosis. The conserved proline, isoleucine, asparagine, isoleucine, threonine (PINIT) domain of PIAS3 is thought to promote STAT3-PIAS3 interaction. The (His)(7) -PINIT domain (PIAS3(85-272) ) was heterologously expressed and purified to homogeneity by nickel affinity and size exclusion chromatography, and shown to be a folded monomer in solution. Using surface plasmon resonance spectroscopy (SPR) the PINIT domain (PIAS3(85-272) ) alone was shown to specifically bind to STAT3 in a concentration dependent manner. L97A, R99N and R99Q mutations of the PINIT domain were found to abrogate binding to STAT3, suggesting that these residues were part of a potential binding surface. An homology model for the PINIT domain was calculated to analyse the potential locations of L97 and R99 in the structure, and to evaluate the potential role of these residues in interactions with STAT3.
Asunto(s)
Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/metabolismo , Factor de Transcripción STAT3/química , Factor de Transcripción STAT3/metabolismo , Animales , Cromatografía en Gel , Ratones , Unión Proteica , Proteínas Inhibidoras de STAT Activados/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factor de Transcripción STAT3/genética , Resonancia por Plasmón de SuperficieRESUMEN
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
Signaling components, which confer an "addiction" phenotype on cancer cells, represent promising drug targets. The transcription factor signal transducers and activators of transcription 3 (STAT3) is constitutively activated in many different types of tumor cells and its activity is indispensible in a large fraction. We found that the expression of the endogenous inhibitor of STAT3, protein inhibitor of activated STAT3 (PIAS3), positively correlates with STAT3 activation in normal cells. This suggests that PIAS3 controls the extent and the duration of STAT3 activity in normal cells and thus prevents its oncogenic function. In cancer cells, however, the expression of PIAS3 is posttranscriptionally suppressed, possibly enhancing the oncogenic effects of activated STAT3. We delimited the interacting domains of STAT3 and PIAS3 and identified a short fragment of the COOH-terminal acidic region of PIAS3, which binds strongly to the coiled-coil domain of STAT3. This PIAS3 fragment was used to derive the recombinant STAT3-specific inhibitor rPP-C8. The addition of a protein transduction domain allowed the efficient internalization of rPP-C8 into cancer cells. This resulted in the suppression of STAT3 target gene expression, in the inhibition of migration and proliferation, and in the induction of apoptosis at low concentrations [half maximal effective concentration (EC(50)), <3 micromol/L]. rPP-C8 did not affect normal fibroblasts and represents an interesting lead for the development of novel cancer drugs targeting the coiled-coil domain of STAT3.
Asunto(s)
Terapia Genética/métodos , Chaperonas Moleculares/genética , Neoplasias/terapia , Péptidos/genética , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Recombinantes/genética , Factor de Transcripción STAT3/genética , Animales , Muerte Celular/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Ratones , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Péptidos/síntesis química , Unión Proteica/genética , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/metabolismo , Estructura Terciaria de Proteína/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/genética , Transducción Genética/métodos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The function of transcription factors can be critically regulated by SUMOylation. c-Maf, the cellular counterpart of v-maf oncogene, is a potent transactivator of the IL-4 gene in Th2 cells. We found in a yeast two-hybrid screen that c-Maf can interact with Ubc9 and PIAS1, two key enzymes of the SUMOylation pathway. In this study, we report that c-Maf co-localized with these two SUMO (small ubiquitin-like modifier) ligases in the nucleus and that c-Maf can be SUMOylated in vitro and also in primary Th2 cells. We also demonstrated that lysine-33 is the dominant, if not the only, SUMO acceptor site of c-Maf. SUMOylation of c-Maf attenuated its transcriptional activity. Reciprocally, a SUMOylation resistant c-Maf was more potent than WT-c-Maf in driving IL-4 production in c-Maf-deficient Th2 cells. Furthermore, we showed that ablation of the SUMO site did not alter the subcellular localization or the stability of c-Maf protein but instead enhanced its recruitment to the Il4-promoter. We conclude that SUMOylation at lysine-33 is a functionally critical post-translational modification event of c-Maf in Th cells.
Asunto(s)
Interleucina-4/biosíntesis , Proteínas Inhibidoras de STAT Activados/fisiología , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-maf/fisiología , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/fisiología , Células Th2/metabolismo , Enzimas Ubiquitina-Conjugadoras/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas/metabolismo , Humanos , Interleucina-4/genética , Riñón , Lisina/química , Ratones , Datos de Secuencia Molecular , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/aislamiento & purificación , Mapeo de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-maf/química , Proteínas Recombinantes de Fusión/fisiología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/química , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/aislamiento & purificación , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Enzimas Ubiquitina-Conjugadoras/químicaRESUMEN
DNA double-strand breaks (DSBs) are highly cytotoxic lesions that are generated by ionizing radiation and various DNA-damaging chemicals. Following DSB formation, cells activate the DNA-damage response (DDR) protein kinases ATM, ATR and DNA-PK (also known as PRKDC). These then trigger histone H2AX (also known as H2AFX) phosphorylation and the accumulation of proteins such as MDC1, 53BP1 (also known as TP53BP1), BRCA1, CtIP (also known as RBBP8), RNF8 and RNF168/RIDDLIN into ionizing radiation-induced foci (IRIF) that amplify DSB signalling and promote DSB repair. Attachment of small ubiquitin-related modifier (SUMO) to target proteins controls diverse cellular functions. Here, we show that SUMO1, SUMO2 and SUMO3 accumulate at DSB sites in mammalian cells, with SUMO1 and SUMO2/3 accrual requiring the E3 ligase enzymes PIAS4 and PIAS1. We also establish that PIAS1 and PIAS4 are recruited to damage sites via mechanisms requiring their SAP domains, and are needed for the productive association of 53BP1, BRCA1 and RNF168 with such regions. Furthermore, we show that PIAS1 and PIAS4 promote DSB repair and confer ionizing radiation resistance. Finally, we establish that PIAS1 and PIAS4 are required for effective ubiquitin-adduct formation mediated by RNF8, RNF168 and BRCA1 at sites of DNA damage. These findings thus identify PIAS1 and PIAS4 as components of the DDR and reveal how protein recruitment to DSB sites is controlled by coordinated SUMOylation and ubiquitylation.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Animales , Proteína BRCA1/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Biológicos , Fosforilación , Proteínas Inhibidoras de STAT Activados/química , Proteínas Inhibidoras de STAT Activados/genética , Estructura Terciaria de Proteína , Proteína de Replicación A/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Protein inhibitor of activated STAT3 (PIAS3) functions in vivo as a key molecule in suppressing the transcriptional activity of both microphthalmia transcription factor (MITF) and STAT3, two transcription factors that play a major role in the development, phenotypic expression, and survival of mast cells and melanocytes. In the present study we have investigated the role played by PIAS3 in the regulation of cell cycle in mast cells and melanocytes. We have characterized the biological role of a 23-aa domain derived from PIAS3 that induces apoptosis in these cells by inhibiting the transcriptional activity of both MITF and STAT3. This PIAS3 inhibitor peptide could serve as the beginning of an in depth study for the development of peptide inhibitors for MITF and STAT3.