RESUMEN
Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1ß (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3.
Asunto(s)
Citocinas , Glucocorticoides , Humanos , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Citocinas/metabolismo , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , FN-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Budesonida/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Células Epiteliales/metabolismo , ARN Mensajero/metabolismo , Dexametasona/farmacología , Dexametasona/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Increasing evidence indicates that long noncoding RNAs (lncRNAs) play crucial roles in the resistance to endoplasmic reticulum (ER) stress in many cancers. However, ER stress-regulated lncRNAs are still unknown in glioma. In the present study, we investigated the altered lncRNAs upon ER stress in glioma and found that small nucleolar RNA host gene 1 (SNHG1) was markedly increased in response to ER stress. Increased SNHG1 suppressed ER stress-induced apoptosis and promoted tumorigenesis in vitro and in vivo. Further mechanistic studies indicated that SNHG1 elevated BIRC3 mRNA stability and enhanced BIRC3 expression. We also found that KLF4 transcriptionally upregulated SNHG1 expression and contributed to the ER stress-induced SNHG1 increase. Collectively, the present findings indicated that SNHG1 is a KLF4-regulated lncRNA that suppresses ER stress-induced apoptosis and facilitates gliomagenesis by elevating BIRC3 expression.
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Glioma , MicroARNs , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/metabolismo , Supervivencia Celular , Carcinogénesis/genética , Transformación Celular Neoplásica/genética , Glioma/genética , MicroARNs/genética , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Apoptosis/genética , Línea Celular Tumoral , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismoRESUMEN
Colon cancer is a cancer with high morbidity and mortality worldwide. Receptor interacting serine/threonine kinase 2 (RIPK2) has been identified as a proto-oncogene, but its role in colon cancer is largely unknown. Herein, we found that RIPK2 interference could inhibit the proliferation and invasion of colon cancer cells, and promote apoptosis. Baculoviral IAP repeat containing 3 (BIRC3) is an E3 ubiquitin ligase, which was found highly expressed in colon cancer cells. Co-immunoprecipitation (Co-IP) experiments showed that RIPK2 could directly bind with BIRC3. Then, we demonstrated that RIPK2 overexpression promoted the expression of BIRC3, BIRC3 interference could eliminate RIPK2-dependent cell proliferation and invasion, and BIRC3 overexpression rescued the suppressive effect of RIPK2 interference on cell proliferation and invasion. We further identified IKBKG, an inhibitor of nuclear factor kappa B, as a ubiquitination substrate targeted by BIRC3. IKBKG interference could eliminate the inhibitory effect of BIRC3 interference on cell invasion. RIPK2 could promote BIRC3-mediated ubiquitination of IKBKG, inhibit the expression of IKBKG protein, and promote the expression of NF-κB subunits p50 and p65 proteins. In addition, DLD-1 cells transfected with sh-RIPK2 or/and sh-BIRC3 were injected into mice to establish a tumor xenograft model, and we found that administration of sh-RIPK2 or sh-BIRC3 impeded the growth of xenograft tumors in vivo, and co-administration displayed a better inhibitory effect. In general, RIPK2 promotes the progression of colon cancer by promoting BIRC3-mediated ubiquitination of IKBKG and activating the NF-κB signaling pathway.
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Neoplasias del Colon , FN-kappa B , Humanos , Animales , Ratones , FN-kappa B/metabolismo , Ubiquitinación , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Neoplasias del Colon/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/genética , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismoRESUMEN
BACKGROUND/AIM: Head and neck squamous cell carcinoma (HNSCC) has poor prognosis, with survival rates that have not significantly improved over the past several decades. Therefore, prediction of HNSCC prognosis is of clinical importance. Baculoviral IAP Repeat containing 2 (BIRC2) and Baculoviral IAP Repeat containing 3 (BIRC3) are involved in oncogenic activity by modulating cell proliferation, apoptosis and invasion in HNSCC. This study aimed to develop and validate a predictive gene signature for BIRC2 and BIRC3. MATERIALS AND METHODS: The genomic copy number and gene expression for BIRC2 and BIRC3 were systematically explored in patients with HNSCC to investigate the clinical relevance of BIRC2 and BIRC3 activation. A prognostic signature was developed based on correlations associated with BIRC2 and BIRC3 mRNA expression and copy number alterations. Hierarchical clustering was used to classify the clusters (Clusters 1 and 2). Moreover, independent validation of the BIRC2-BIRC3 gene signature was performed using the Leipzig, MDACC, FHCRC, and KHU datasets. To explore the biological functions of the BIRC2-BIRC3 gene signature, string analysis and pathway annotation were also performed. RESULTS: BIRC2-BIRC3 gene signature-derived cluster 2 patients exhibited significantly poor survival. This signature also predicted survival in three independent cohorts. Interestingly, the BIRC2-BIRC3 gene signature additionally permitted the identification of survival in advanced tumor stages with excellent accuracy in all three cohorts. Multivariate Cox regression analysis identified that the BIRC2-BIRC3 signature was an independent predictor associated with the survival of patients with HNSCC. Moreover, Inhibition of BIRC2 modulated the NF-B signaling pathway via upregulation of CBR1 expression. CONCLUSION: The BIRC2-BIRC3 gene signature was found to be associated with the prognosis of HNSCC. Thus, BIRC2 and BIRC3 could be potential targets for improving HNSCC prognosis.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Carcinogénesis , Neoplasias de Cabeza y Cuello , Proteínas Inhibidoras de la Apoptosis , Ubiquitina-Proteína Ligasas , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Pronóstico , Transducción de Señal/genética , Carcinoma de Células Escamosas de Cabeza y Cuello/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Objective: To explore the correlation of CD49d expression patterns with molecular genetics and hotspot gene mutants in patients with chronic lymphocytic leukemia. Methods: The expression of CD49d was detected by flow cytometry and grouped into homogeneous, bimodal, negative and positive expression. Panel fluorescence in situ hybridization (FISH) was used for molecular genetics analysis and next-generation sequencing (NGS) was conducted for gene mutation detection. Results: There were 43 patients (23.89% ) with positive CD49d expression, 137 patients (76.11% ) with negative CD49d expression, 96 patients (53.33% ) with homogeneous CD49d expression and 84 patients (46.67% ) with bimodal CD49d expression. Compared with patients in the CD49d negative group, patients in the CD49d positive group had higher Rai stage (P=0.048) and higher proportion of spleen enlargement (P=0.030) . Compared with patients with homogeneous expression of CD49d, patients with bimodal expression of CD49d had a higher proportion of spleen enlargement (P=0.009) . The expression rate of 11q22- in bimodal CD49d(-) group was significantly higher than that in homogeneous CD49d(-) group (24.29% vs 10.45% , P=0.043) . The incidence of +12 in homogeneous CD49d group was higher than that in bimodal CD49d group (16.67% vs 5.95% , P=0.035) . The incidence of +12 in homogeneous CD49d(+) group was higher than that in bimodal CD49d(-) group (17.24% vs 4.29% , P=0.045) . The incidence of +12 in homogeneous CD49d(-) group was higher than that in bimodal CD49d(-) group (16.42% vs 4.29% , P=0.024) . BIRC3 mutation rate in CD49d positive group was higher than that in CD49d negative group (11.63% vs 2.92% , P=0.037) . Conclusion: There were significant correlations between CD49d and 11q22-, +12 and BIRC3 gene mutation. Patients with bimodal CD49d were more correlated with poor prognosis indexes.
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Integrina alfa4 , Leucemia Linfocítica Crónica de Células B , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Biomarcadores de Tumor/metabolismo , Humanos , Hibridación Fluorescente in Situ , Integrina alfa4/genética , Integrina alfa4/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/metabolismo , Biología Molecular , PronósticoRESUMEN
BACKGROUND: Immune activation, neuroinflammation, and cell death are the hallmarks of multiple sclerosis (MS), which is an autoimmune demyelinating disease of the central nervous system (CNS). It is well-documented that the cellular inhibitor of apoptosis 2 (cIAP2) is induced by inflammatory stimuli and regulates adaptive and innate immune responses, cell death, and the production of inflammatory mediators. However, the impact of cIAP2 on neuroinflammation associated with MS and disease severity remains unknown. METHODS: We used experimental autoimmune encephalomyelitis (EAE), a widely used mouse model of MS, to assess the effect of cIAP2 deletion on disease outcomes. We performed a detailed analysis on the histological, cellular, and molecular levels. We generated and examined bone-marrow chimeras to identify the cIAP2-deficient cells that are critical to the disease outcomes. RESULTS: cIAP2-/- mice exhibited increased EAE severity, increased CD4+ T cell infiltration, enhanced proinflammatory cytokine/chemokine expression, and augmented demyelination. This phenotype was driven by cIAP2-deficient non-hematopoietic cells. cIAP2 protected oligodendrocytes from cell death during EAE by limiting proliferation and activation of brain microglia. This protective role was likely exerted by cIAP2-mediated inhibition of the non-canonical NLRP3/caspase-8-dependent myeloid cell activation during EAE. CONCLUSIONS: Our findings suggest that cIAP2 is needed to modulate neuroinflammation, cell death, and survival during EAE. Significantly, our data demonstrate the critical role of cIAP2 in limiting the activation of microglia during EAE, which could be explored for developing MS therapeutics in the future.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Sistema Nervioso Central/patología , Encefalomielitis Autoinmune Experimental/patología , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Esclerosis Múltiple/patología , Enfermedades NeuroinflamatoriasRESUMEN
BACKGROUND: Baculoviral IAP repeat-containing 3 (BIRC3) which encodes a member of the IAP family of proteins upregulated in the asthma expression profile dataset. However, there was few research on studying the clinical implication of BIRC3 in asthma. OBJECTIVE: To validate BIRC3 expression and its clinical implications in induced sputum of asthma. METHODS: Based on the GSE76262 (118 asthma cases and 21 healthy controls) dataset, differentially expressed genes were screened using R software. Subsequently, BIRC3 mRNA and protein were clinically verified in induced sputum samples through quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Besides, the correlations between BIRC3 expression and asthmatic eosinophilic/allergic inflammation indicators (FeNO, IgE, and EOS%), pulmonary function (FEV1, FEV1% pred, FVC% pred, and FEV1/FVC), and inflammatory cytokines (IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP) were analyzed. Finally, BIRC3 mRNA was detected in human primary bronchial epithelial cells stimulated by cytokines (IL-4 or IL-13). RESULTS: BIRC3 was screened as a candidate gene in the GSE76262, which was highly expressed in asthma. Highly expressed BIRC3 was positively correlated with eosinophilic and allergic indicators, including FeNO, blood eosinophil, and serum IgE. Moreover, BIRC3 protein was positively associated with inflammation cytokines, like IL-4, IL-5, IL-13, IL-25, IL-10, IL-33, and TSLP, while negatively correlated with FEV1, FEV1%pred, FVC% pred, and FEV1/FVC. Furthermore, the expression of BIRC3 could be induced in primary bronchial epithelial cells treated by cytokines IL-4 or IL-13. CONCLUSIONS: BIRC3 significantly increased in induced sputum of asthma and positively correlated with airway eosinophilic and peripheral blood allergic inflammation, type 2 cytokines, and airway obstruction. Increased BIRC3 might be involved in the pathogenesis of asthma by affecting the eosinophilic and allergic inflammation.
Asunto(s)
Asma , Esputo , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Citocinas/metabolismo , Eosinófilos/metabolismo , Humanos , Pruebas de Función Respiratoria , Esputo/metabolismoRESUMEN
Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.
Asunto(s)
Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Anemia de Fanconi/tratamiento farmacológico , Anemia de Fanconi/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Alquinos/farmacología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/antagonistas & inhibidores , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Anemia de Fanconi/complicaciones , Anemia de Fanconi/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/complicaciones , Neoplasias de Cabeza y Cuello/inmunología , Humanos , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Oligopéptidos/farmacología , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , Proteínas Señalizadoras YAP/genética , Proteínas Señalizadoras YAP/metabolismoRESUMEN
Cellular inhibitor of apoptosis protein-1 (cIAP-1) is member of inhibitor of apoptosis proteins (IAPs) which can affect apoptosis through interactions with caspases. cIAP-1 is a multi-domain protein and able to regulate apoptosis through interactions with proteins such as caspases and possesses E3 ligase activity. Human cIAP-1 contains three baculovirus IAP repeat (BIR) domains which are critical for protein-protein interactions. Here, we report NMR resonance assignments of the first BIR domain of human cIAP. Its secondary structures in solution were determined based on the assigned resonances. The dynamics of this domain was obtained, and our hydrogen-deuterium exchange experiment reveals that the first helix in BIR1 is exposed to the solvent. The availability of assignments of backbone and side chain resonances will be useful for probing protein-protein interactions.
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Baculoviridae , Proteínas Inhibidoras de la Apoptosis , Apoptosis/fisiología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Baculoviridae/metabolismo , Caspasas/metabolismo , Línea Celular , Humanos , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión ProteicaRESUMEN
Although inhibitor of apoptosis proteinlike protein2 (ILP2) is considered to be a novel enhancer of breast cancer proliferation, its underlying mechanism of action remains unknown. Therefore, the present study aimed to investigate the expression profile of ILP2related proteins in MCF7 cells to reveal their effect on promoting breast cancer cell proliferation. The isobaric tags for relative and absolute quantification (iTRAQ) method was used to analyse the expression profile of ILP2related proteins in MCF7 breast cancer cells transfected with small interfering (si)RNA against ILP2 (siRNA5 group) and the negative control (NC) siRNA. The analysis of the iTRAQ data was carried out using western blotting and reverse transcriptionquantitative PCR. A total of 4,065 proteins were identified in MCF7 cells, including 241 differentially expressed proteins (DEPs; fold change ≥1.20 or ≤0.83; P<0.05). Among them, 156 proteins were upregulated and 85 were downregulated in the siRNA5 group compared with in the NC group. The aforementioned DEPs were mainly enriched in 'ECMreceptor interaction'. In addition, the top 10 biological processes related to these proteins were associated with signal transduction, cell proliferation and immune system processes. Furthermore, ILP2 silencing upregulated N(4)(ßNacetylglucosaminyl)Lasparaginase, metallothionein1E and tryptophan 2,3dioxygenase, whereas ILP2 overexpression exerted the opposite effect. The results of the present study suggested that ILP2 could promote breast cancer growth via regulating cell proliferation, signal transduction, immune system processes and other cellular physiological activities.
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Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Neoplasias de la Mama/metabolismo , Proliferación Celular/fisiología , Proteómica/métodos , Transducción de Señal/fisiología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proliferación Celular/genética , Cromatografía Liquida , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Células MCF-7 , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genética , Espectrometría de Masas en TándemRESUMEN
A hallmark of infection by the pathogen Helicobacter pylori, which colonizes the human gastric epithelium, is the simultaneous activation of the classical and alternative nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways, underlying inflammation and cell survival. Here, we report that the classical NF-κB target gene product A20 contributes to the negative regulation of alternative NF-κB signaling in gastric epithelial cells infected by H. pylori. Mechanistically, the de novo synthesized A20 protein interacts with tumor necrosis factor receptor-associated factor-interacting protein with forkhead-associated domain (TIFA) and thereby interferes with the association of TIFA with the NIK regulatory complex. We also show that alternative NF-κB activity contributes to the up-regulation of anti-apoptotic genes, such as baculoviral IAP repeat containing 2 (BIRC2), BIRC3 and B-cell lymphoma 2-related protein A1 (BCL2A1) in gastric epithelial cells. Furthermore, the observed over-expression of RelB in human gastric biopsies with type B gastritis and RelB-dependent suppression of apoptotic cell death emphasize an important role of the alternative NF-κB pathway in H. pylori infection.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Mucosa Gástrica/metabolismo , Infecciones por Helicobacter/metabolismo , FN-kappa B/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Línea Celular Tumoral , Mucosa Gástrica/microbiología , Gastritis/genética , Gastritis/metabolismo , Gastritis/microbiología , Expresión Génica , Técnicas de Inactivación de Genes , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Helicobacter pylori/fisiología , Interacciones Huésped-Patógeno , Humanos , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , FN-kappa B/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/genética , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIB/genética , Factor de Transcripción ReIB/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genéticaRESUMEN
AIMS: Liver fibrosis is a growing public health concern without effective medical treatment. Recent reports have indicated that inhibitors of apoptosis proteins (IAPs) were potential targets for idiopathic pulmonary fibrosis therapy. However, their roles have not been well identified in liver fibrosis. METHODS: The expression of IAPs were examined in human liver tissue and experimental mouse models. Liver fibrosis in CCl4-induced mouse models were investigated by Sirius red staining, RT-PCR, Western blotting after hepatocytes-specific cIAP2 knockout or IAPs inhibitor APG-1387 treatment. The underlying molecular mechanism of APG-1387 action was explored by apoptosis analysis, matrix metalloprotein 9 (MMP9) inhibition, neutrophils depletion, and CC Motif Chemokine Ligand 5 (CCL5) gene knockout in vitro and in vivo. FINDINGS: Our study showed that increased expression of cIAP2 was associated with liver fibrosis severity in liver tissues. Deletion of cIAP2 from hepatocytes or degrading cIAPs by APG-1387 ameliorated liver fibrosis induced by CCl4. APG-1387 treatment exhibited increased expression of MMP9 and resulted in higher ratio of MMP9 to tissue inhibitor of metalloproteinase-1. MMP9 was mainly derived from CCL5 chemotactic neutrophils. Further, MMP9 inhibition by CTT peptide, neutrophil depletion by Ly6G antibody or CCL5 deficiency blocked the anti-fibrotic effects of APG-1387 in vivo. SIGNIFICANCE: These results suggested that cIAPs, especially cIAP2, might play a novel role in the pathogenesis of liver fibrosis, and targeting cIAPs represented a promising therapeutic strategy for liver fibrosis by increasing MMP9 expression induced by CCL5 chemotactic neutrophils.
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Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Intoxicación por Tetracloruro de Carbono/metabolismo , Regulación Enzimológica de la Expresión Génica , Cirrosis Hepática/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Neutrófilos/metabolismo , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Intoxicación por Tetracloruro de Carbono/genética , Intoxicación por Tetracloruro de Carbono/patología , Eliminación de Gen , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Noqueados , Neutrófilos/patologíaRESUMEN
Macrophages serve as viral reservoirs due to their resistance to apoptosis and HIV-cytopathic effects. We have previously shown that inhibitor of apoptosis proteins (IAPs) confer resistance to HIV-Vpr-induced apoptosis in normal macrophages. Herein, we show that second mitochondrial activator of caspases (SMAC) mimetics (SM) induce apoptosis of monocyte-derived macrophages (MDMs) infected in vitro with a R5-tropic laboratory strain expressing heat stable antigen, chronically infected U1 cells, and ex-vivo derived MDMs from HIV-infected individuals. To understand the mechanism governing SM-induced cell death, we show that SM-induced cell death of primary HIV-infected macrophages was independent of the acquisition of M1 phenotype following HIV infection of macrophages. Instead, SM-induced cell death was found to be mediated by IAPs as downregulation of IAPs by siRNAs induced cell death of HIV-infected macrophages. Moreover, HIV infection caused receptor interacting protein kinase-1 (RIPK1) degradation which in concert with IAP1/2 downregulation following SM treatment may result in apoptosis of macrophages. Altogether, our results show that SM selectively induce apoptosis in primary human macrophages infected in vitro with HIV possibly through RIPK1. Moreover, modulation of the IAP pathways may be a potential strategy for selective killing of HIV-infected macrophages in vivo.
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Fármacos Anti-VIH/farmacología , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/efectos de los fármacos , Infecciones por VIH/tratamiento farmacológico , VIH-1/patogenicidad , Macrófagos/efectos de los fármacos , Imitación Molecular , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Reguladoras de la Apoptosis/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Efecto Citopatogénico Viral , Infecciones por VIH/enzimología , Infecciones por VIH/patología , Infecciones por VIH/virología , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Macrófagos/enzimología , Macrófagos/patología , Macrófagos/virología , Fenotipo , Células U937 , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.
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Apoptosis/genética , Necroptosis/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína de Dominio de Muerte Asociada a Receptor de TNF/genética , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis/efectos de los fármacos , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Sistemas CRISPR-Cas , Cicloheximida/farmacología , Eliminación de Gen , Regulación de la Expresión Génica , Células HeLa , Humanos , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , FN-kappa B/genética , FN-kappa B/metabolismo , Necroptosis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Transducción de Señal , Proteína de Dominio de Muerte Asociada a Receptor de TNF/deficiencia , Factor 2 Asociado a Receptor de TNF/genética , Factor 2 Asociado a Receptor de TNF/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , UbiquitinaciónRESUMEN
This study probed the largely unexplored regulation and role of fibronectin in Angiotensin II-stimulated cardiac fibroblasts. Using gene knockdown and overexpression approaches, Western blotting, and promoter pull-down assay, we show that collagen type I-activated Discoidin Domain Receptor 2 (DDR2) mediates Angiotensin II-dependent transcriptional upregulation of fibronectin by Yes-activated Protein in cardiac fibroblasts. Furthermore, siRNA-mediated fibronectin knockdown attenuated Angiotensin II-stimulated expression of collagen type I and anti-apoptotic cIAP2, and enhanced cardiac fibroblast susceptibility to apoptosis. Importantly, an obligate role for fibronectin was observed in Angiotensin II-stimulated expression of AT1R, the Angiotensin II receptor, which would link extracellular matrix (ECM) signaling and Angiotensin II signaling in cardiac fibroblasts. The role of fibronectin in Angiotensin II-stimulated cIAP2, collagen type I, and AT1R expression was mediated by Integrin-ß1-integrin-linked kinase signaling. In vivo, we observed modestly reduced basal levels of AT1R in DDR2-null mouse myocardium, which were associated with the previously reported reduction in myocardial Integrin-ß1 levels. The role of fibronectin, downstream of DDR2, could be a critical determinant of cardiac fibroblast-mediated wound healing following myocardial injury. In summary, our findings suggest a complex mechanism of regulation of cardiac fibroblast function involving two major ECM proteins, collagen type I and fibronectin, and their receptors, DDR2 and Integrin-ß1.
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Receptor con Dominio Discoidina 2/deficiencia , Receptor con Dominio Discoidina 2/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Integrina beta1/metabolismo , Miocardio/metabolismo , Receptor de Angiotensina Tipo 1/metabolismo , Angiotensina II/farmacología , Animales , Apoptosis/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Colágeno Tipo I/antagonistas & inhibidores , Colágeno Tipo I/metabolismo , Receptor con Dominio Discoidina 2/genética , Fibroblastos/efectos de los fármacos , Fibronectinas/genética , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Corazón/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Masculino , Ratones , Ratones Noqueados , Cultivo Primario de Células , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Angiotensina Tipo 1/genética , Transducción de Señal , Proteínas Señalizadoras YAPRESUMEN
Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IMn) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IMn-MS experiments also show evidence of interconversion between compact and extended structures. IMn-MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.
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Proteína 3 que Contiene Repeticiones IAP de Baculovirus/química , Citocromos c/química , Espectrometría de Masas/métodos , Desplegamiento Proteico , Animales , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Citocromos c/metabolismo , Gases/química , Caballos , Humanos , Espectrometría de Movilidad Iónica/métodos , IonesRESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant disease worldwide. Although the diagnosis and treatment of HCC have greatly improved in the recent years, there is still a lack of accurate methods to predict the prognosis of patients. Evidence has shown that Hippo signaling in tissues adjacent to HCC plays a significant role in HCC development. In the present study, we aimed to construct a model based on the expression of Hippo-related genes (HRGs) in tissues adjacent to HCC to predict the prognosis of HCC patients. METHODS: Gene expression data of paired normal tissues adjacent to HCC (PNTAH) and clinical information were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. The HRG signature was constructed using four canonical Hippo-related pathways. Univariate Cox regression analysis was used to screen survival-related HRGs. LASSO and multivariate Cox regression analyses were used to construct the prognostic model. The true and false positive rates of the model were confirmed using receiver operating characteristic (ROC) analysis. RESULTS: The prognostic model was constructed based on the expression levels of five HRGs (NF2, MYC, BIRC3, CSNK1E, and MINK1) in PNTAH. The mortality rate of HCC patients increased as the risk score determined by the model increased. Furthermore, the risk score was found to be an independent risk factor for the survival of patients. ROC analysis showed that the prognostic model had a better predictive value than the other conventional clinical parameters. Moreover, the reliability of the prognostic model was confirmed in TCGA-LIHC cohort. A nomogram was generated to predict patient survival. An exploration of the predictive value of the model in HCC tissues indicated that the model is PNTAH-specific. CONCLUSIONS: We developed and validated a prognostic model based on the expression levels of five HRGs in PNTAH, and this model should be helpful in predicting the prognosis of patients with HCC.
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Carcinoma Hepatocelular/genética , Expresión Génica , Neoplasias Hepáticas/genética , Hígado , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Caseína Cinasa 1 épsilon/genética , Caseína Cinasa 1 épsilon/metabolismo , Bases de Datos Genéticas , Femenino , Perfilación de la Expresión Génica , Vía de Señalización Hippo , Humanos , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/metabolismo , Neurofibromina 2/genética , Neurofibromina 2/metabolismo , Nomogramas , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Curva ROC , Factores de Riesgo , Transcriptoma , Adulto JovenRESUMEN
Preeclampsia is a gestational hypertensive disease; however, preeclampsia remains poorly understood. Bioinformatics analysis was applied to find novel genes involved in the pathogenesis of preeclampsia and identified CLDN1 as one of the most differentially expressed genes when comparing patients with preeclampsia and healthy controls. The results of the qRT-PCR, Western blotting and immunohistochemistry experiments demonstrated that CLDN1 was significantly downregulated in the chorionic villi in samples from patients with preeclampsia. Furthermore, knockdown of CLDN1 in HTR-8/SVneo cells resulted in the inhibition of proliferation and induction of apoptosis, and overexpression of CLDN1 reversed these effects. In addition, RNA-seq assays demonstrated that the gene BIRC3 is potentially downstream of CLDN1 and is involved in the regulation of apoptosis. Knockdown of CLDN1 confirmed that the expression level of BIRC3 was obviously decreased and was associated with a significant increase in cleaved PARP. Interestingly, the apoptotic effect in CLDN1 knockdown cells was rescued after BIRC3 overexpression. Overall, these results indicate that a decrease in CLDN1 inhibits BIRC3 expression and increases cleaved PARP levels thus participating in the pathogenesis of preeclampsia.
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Apoptosis , Proliferación Celular , Claudina-1/metabolismo , Regulación del Desarrollo de la Expresión Génica , Preeclampsia/patología , Trofoblastos/patología , Adulto , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Estudios de Casos y Controles , Movimiento Celular , Claudina-1/genética , Femenino , Humanos , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Preeclampsia/genética , Preeclampsia/metabolismo , Embarazo , Trofoblastos/metabolismoRESUMEN
Pigmented villonodular synovitis (PVNS) is a rare hyperplasia disease of the synovium with a predilection for the knee in either a localized (LPVNS) or a diffuse form (DPVNS). But the exact cause is not clear. The aim of this study was to explore the relationship between the expression of cellular inhibitor of apoptosis 2 (cIAP2) and proliferation, apoptosis, invasive growth and postoperative recurrence in PVNS. Clinical significance of cIAP2 expression in synovium from 63 patients' knee joints with PVNS (40 DPVNS; 23 LPVNS) were investigated with 20 normal subjects acting as controls. The cIAP2 gene was screened by Human Cancer Pathway Finder PCR Array and real-time polymerase chain reaction (RT-PCR). We also used immunohistochemistry to detect cIAP2 and proliferating cell nuclear antigen (PCNA) protein expression and analyzed their relationship with PVNS type, invasive growth, and postoperative recurrence. The expression of cIAP2, PCNA, caspase-8, caspase-9 and caspase-3 protein was tested in Western blot. Screening results of Human Cancer Pathway Finder PCR array and RT-PCR showed significantly more cIAP2 mRNA in DPVNS synovium than in normal or LPVNS synovium (P < 0.05). Immunohistochemistry and western blot showed that the cIAP2 protein expression level in DPVNS was significantly higher than in LPVNS tissue (P < 0.01). As cIAP2 expression increased, the expression of PCNA increased (P < 0.05) and expression of cleaved caspase-3, -8, -9 decreased (P < 0.01). cIAP2 and PCNA overexpression were found to be related to ligament and bone erosion in PVNS and to disease recurrence (P < 0.05). This study suggested that cIAP2 overexpression plays an important role in the anti-apoptotic, proliferative and invasive growth of PVNS, which may account for the recurrence and poor prognosis of DPVNS.
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Proteína 3 que Contiene Repeticiones IAP de Baculovirus/metabolismo , Sinovitis Pigmentada Vellonodular/metabolismo , Sinovitis Pigmentada Vellonodular/patología , Adolescente , Adulto , Anciano , Proteína 3 que Contiene Repeticiones IAP de Baculovirus/genética , Western Blotting , Femenino , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Sinovitis Pigmentada Vellonodular/genética , Adulto JovenRESUMEN
Ubiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.