RESUMO
The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.
Assuntos
Peptídeos/química , Técnicas de Síntese em Fase Sólida/métodos , Compostos de Sulfidrila/química , Ubiquitina/química , Ubiquitinação , Cisteína/química , Escherichia coli/genética , Ésteres/química , Expressão Gênica , Hidroxilaminas/química , Imidas/química , Inteínas , Espectroscopia de Ressonância Magnética , Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ubiquitina/biossíntese , Ubiquitina/metabolismo , Proteínas Ubiquitinadas/biossíntese , Proteínas Ubiquitinadas/química , Proteínas Ubiquitinadas/isolamento & purificação , Ubiquitinas/biossíntese , Ubiquitinas/química , Zinco/metabolismoRESUMO
INTRODUCTION: The tight regulation of the cytokine network during macrophage activation is of prime importance to enable a fast and potent innate immune response against exogenous pathogens. The inflammation mediating ubiquitin-like protein HLA-F adjacent transcript number 10 (FAT10) was shown to be transcriptionally regulated by and also regulate the nuclear factor-κB (NFκB) signaling pathway. However, very little is known about the regulation of FAT10 gene expression during macrophage activation. RESULTS: RNA sequencing of interferon (IFN)γ-stimulated mouse peritoneal macrophages analyzed by ingenuity pathway analysis revealed significant involvement of tumor necrosis factor receptor 1 (TNFR1) signaling in addition to IFNγ signaling. Subsequently, IFNγ robustly upregulated FAT10 expression compared to a milder induction seen with TNFα or lipopolysaccharide (LPS) stimulation. While low dose IFNγ with TNFα synergistically elevated FAT10 expression, preincubation of macrophages with IFNγ strongly augmented TNFα-induced FAT10 expression. Moreover, a short preincubation with IFNγ, which did not elevate FAT10, was sufficient to potentiate the induction of FAT10 by TNFα. A double augmentation mechanism of TNFα signaling was demonstrated, where IFNγ rapidly induced the expression of TNFα and TNFR1, which further augmented the induction of TNFα and TNFR1 expression by TNFα. Importantly, the induction of FAT10 by IFNγ in macrophages from TNFα-deficient or TNFR1-deficient mice was completely inhibited compared to macrophages from wild type (WT) mice. Finally, we show that TNFα-induced FAT10 expression is dependent on NFκB signaling. CONCLUSION: IFNγ potentiates the TNFα/TNFR1 signaling pathway to induce FAT10 expression in mouse macrophages, mediated through NFκB network.
Assuntos
Regulação da Expressão Gênica/imunologia , Interferon gama/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Ubiquitinas/biossíntese , Animais , Imunidade Inata/imunologia , Interferon gama/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/imunologia , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/imunologia , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Breast cancer is a common cancer and could result in a substantial mortality. The study aimed to screen gene signatures associated with the development and metastasis of breast cancer and explore their regulation mechanisms. Three datasets of GSE10797, GSE8977 and GSE3744 were downloaded from GEO (Gene Expression Omnibus) database, containing 55 breast cancer samples and 27 normal samples. After data preprocessing using limma software and RMA (robust multi-array average) algorithm, DEGs (differentially expressed genes) between breast tumor and normal tissues in three individual experiments were identified using MADAM package. Function and pathway enrichment analyses were performed for the DEGs. Transcription factors and TAGs (tumor associated genes) among the DEGs were recognized and the PPI (protein-protein-interaction) network for the DEGs was constructed using Cytoscape software. The mRNA expression was analyzed via real-time quantitative PCR and protein expression was measured by western blotting. Totally, 100 DEGs were identified, including 33 up-regulated genes and 67 down-regulated genes. Among them, up-regulated DEGs such as CD80 was enriched in toll-like receptor (TLR) interaction pathway and the TAG, ISG15 was related to RIG-I-like receptor signaling pathway, while CXCL10 was involved in both of the two pathways. Whereas, the down-regulated DEG, CXCL12 was significantly associated with axon guidance pathway. Additionally, these DEGs were also pivotal nodes in the PPI network with high degrees. Besides, CXCL10 and CD80 were both interacted with IFNG. The mRNA expression of ISG15 was obviously enhanced in human breast cancer cells MCF-7, while no significant difference of CXCL10 mRNA level was found between MCF10A and MCF-7 cells. Moreover, the proteins expression levels of CD80 and ISG15 were significantly increased in MCF-7, MDA-MB-468 and MDA-MB-231 breast cancer cells than in normal MCF10A cells. CD80 might be responsible for the breast cancer's progression and metastasis via regulating innate immune system. In addition, ISG15 is identified as a crucial gene signature associated with breast cancer development and metastasis via RIG-I-like receptor signaling pathway.
Assuntos
Antígeno B7-1/biossíntese , Biomarcadores Tumorais/análise , Neoplasias da Mama/patologia , Citocinas/biossíntese , Invasividade Neoplásica/genética , Ubiquitinas/biossíntese , Neoplasias da Mama/genética , Conjuntos de Dados como Assunto , Progressão da Doença , Feminino , Humanos , Invasividade Neoplásica/patologia , TranscriptomaRESUMO
BAG3 is a member of the cochaperone BAG family and often highly expressed in various cancers. Recently, evidences show that BAG3 promotes stemness of human cancer cells. IFN-stimulated genes 15 (ISG15) is an ubiquitin-like molecule, which is covalently conjugated with substrates to form ISGylated proteins. Global screening BAG3 interacting partners demonstrated that ISG15 might be a potential binding partner. The current study revealed that BAG3 did not interact with ISG15, but positively regulated ISG15 expression in pancreatic ductal adenocarcinoma cancer (PDAC). It was further found that BAG3 deletion stabilized ISG15 mRNAs, while suppressed its translation via increasing Serine phosphorylation of Ago2 at position 387 (S387). Both BAG3 deletion and ISG15 knockdown suppressed stem cell-like phenotypes of PDAC cells, including clonogenicity, invasiveness and spheroid formation. In addition, ectopic ISG15 expression rescued the suppressive role of BAG3 deletion in cancer stem cell (CSC)-like phenotypes of PDAC cells, and this effect of ISG15 was independent of its ISGylation function. The current study implies that BAG3 and ISG15 may provide a therapeutic advantage for PDAC.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Reguladoras de Apoptose , Carcinoma Ductal Pancreático , Citocinas , Deleção de Genes , Proteínas de Neoplasias , Neoplasias Pancreáticas , Biossíntese de Proteínas , Ubiquitinas , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Citocinas/biossíntese , Citocinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células-Tronco Neoplásicas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Ubiquitinas/biossíntese , Ubiquitinas/genéticaRESUMO
Epithelial to mesenchymal transition (EMT) serves important roles in tumor invasion, metastasis, formation of cancer initiating cells (CICs) and drug resistance. HLAF adjacent transcript 10 (FAT10) has been proposed as an oncogene in bladder cancer. However, the functional contribution of FAT10 to EMT and the formation of CICs remains unclear in bladder cancer. The present study reports that FAT10 protein expression is upregulated in bladder cancer cell lines, and the overexpression of FAT10 promotes EMT and the formation of CICs in bladder cancer UMUC3 cells. In addition, increased expression of FAT10 in tumor tissue was associated with shorter overall survival and progression free survival in Chinese patients with bladder cancer. Overexpression of FAT10 promotes cisplatinresistant bladder cancer formation. These results indicated FAT10 may be a novel target for the treatment of bladder cancer.
Assuntos
Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Proteínas Oncogênicas/biossíntese , Ubiquitinas/biossíntese , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Intervalo Livre de Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida , Neoplasias da Bexiga Urinária/tratamento farmacológicoRESUMO
Proteins belonging to the linear ubiquitin assembly complex (LUBAC) are believed to be important in tumorigenesis. LUBAC has been demonstrated to be composed of RBCK1, RNF31 and SHARPIN. The aim of this study was to explore all members of the LUBAC complex as novel biomarkers in breast cancer. We have already reported that RNF31 mRNA levels are higher in breast cancer samples compared to adjacent non-tumor tissue. In this study we extend these findings by demonstrating that the mRNA levels of RBCK1 and SHARPIN are also higher in tumors compared to adjacent non-tumor tissue in the same cross sectional study of samples (p < 0.001). In addition, up-regulated mRNA expression of all three members of the LUBAC complex displayed high predictive value in distinguishing tumor tissues from adjacent non-tumor tissue as determined by ROC curve analysis. Furthermore, we investigated whether there is an association between the mRNA and protein expression levels of RBCK1, RNF31 and SHARPIN and clinicopathological parameters including estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor (HER2) status and found that RNF31 protein is significantly higher in ERalpha-negative tumors than ERalpha-positive tumors (p = 0.034). Collectively, our findings indicate that up-regulated mRNA expression of RNF31, RBCK1 and SHARPIN could potentially be diagnostic biomarkers of breast cancer and RNF31 might be a drug target for ERalpha-negative breast cancers.
Assuntos
Neoplasias da Mama/metabolismo , Regulação da Expressão Gênica , Proteínas de Neoplasias/biossíntese , Fatores de Transcrição/biossíntese , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitinas/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/patologia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
Purpose: ISG15, a di-ubiquitin-like protein, is critical for controlling certain viral and bacterial infections. We sought to determine if ISG15 plays a role in corneal innate immunity against Candida albicans (C. albicans) using a C57BL/6 (B6) mouse model of human fungal keratitis. Methods: Scarified corneas of adult B6 mice were pretreated with TLR5 ligand flagellin and then inoculated with C. albicans. The expression of ISG15 and other genes involved in ISG15 conjugation (ISGylation) was determined by real-time PCR. ISG15 expression and distribution in infected corneas were assessed by immunohistochemistry. ISGylation was examined by Western blotting. siRNA knockdown and recombinant ISG15 were used to elucidate the effects of ISG15 on controlling fungal keratitis by clinical scoring, fungal number plate counting, ELISA cytokine determination, and polymorphonuclear leukocytes (PMN) infiltration measurement. Results: Heat-killed C. albicans induced expression of ISG15, and hBD2 was markedly enhanced by flagellin-pretreatment in cultured human primary corneal epithelial cells (CECs). In vivo, C. albicans infection induced the expression of ISG15, ISGylation-associated genes (UBE1L, UBCH8, and HERC5), and ISGylation in mouse CECs, all of which were enhanced by flagellin-pretreatment. siRNA knockdown of ISG15 increased keratitis severity, dampened flagellin-induced protection, and greatly suppressed the expressions of ISGylation enzymes, IFN-γ, but not CXCL2 in B6 mouse CECs. Recombinant ISG15, on the other hand, enhanced corneal innate immunity against C. albicans and suppressed infection-induced IL-1ß, but not IL-Ra expression. ISG15 alone induced the expression of IL-1Ra, CXCL10, and CRAMP in mouse CECs. ISG15 was upregulated and secreted in cultured human CECs in response to challenge in a type 1 IFN-dependent manner. Conclusions: Our data, for the first time, demonstrate that ISG15 acts as an immunomodulator in the cornea and plays a critical role in controlling fungal keratitis.
Assuntos
Candida albicans/isolamento & purificação , Candidíase/genética , Córnea/metabolismo , Citocinas/genética , Infecções Oculares Fúngicas/genética , Regulação da Expressão Gênica , Ceratite/genética , Animais , Western Blotting , Candidíase/metabolismo , Candidíase/microbiologia , Proteínas de Transporte , Células Cultivadas , Córnea/microbiologia , Córnea/patologia , Citocinas/biossíntese , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Infecções Oculares Fúngicas/metabolismo , Infecções Oculares Fúngicas/microbiologia , Feminino , Humanos , Imuno-Histoquímica , Ceratite/metabolismo , Ceratite/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Ubiquitinas/biossíntese , Ubiquitinas/genéticaRESUMO
In this study, we constructed for the first time a full-length cDNA clone of pig-original bovine viral diarrhea virus 2 (BVDV-2) strain SH-28, modified the cDNA clone (pASH28) for mutant pASHΔNpro and derived virus strain vASHΔNpro by deleting the genomic region encoding the Npro polypeptide, and examined significance of protein Npro for antiviral responses in vitro. Data showed that Npro-deletion mutant virus vASHΔNpro led to significant overexpression of oligo adenylate synthetase (OAS), myxovirus-resistant protein 1 (Mx1), and ubiquitin-like protein 15 (ISG15). Data also revealed that overexpression of Npro, but not NS2 and NS3 proteins, resulted in significant down-regulation of OAS, Mx1, and ISG15 production (p ≤ 0.05) in bovine cells as well as porcine cells transfected with Npro recombinant eukaryotic expression plasmids. Npro (but not NS2 and NS3) was also found to inhibit poly(IC) from inducing production of type I interferon (IFN-I). These results indicated that protein Npro may play multiple roles in regulating antiviral response in host cells interfered by pig BVDV-2 strain, and provided useful information to understand better the mechanism of BVDV-2 persistent infection in pigs.
Assuntos
Vírus da Diarreia Viral Bovina Tipo 2/genética , Proteínas de Resistência a Myxovirus/genética , Infecções por Pestivirus/genética , Proteínas não Estruturais Virais/genética , Animais , Bovinos , Linhagem Celular , Vírus da Diarreia Viral Bovina Tipo 2/patogenicidade , Regulação Viral da Expressão Gênica , Proteínas de Resistência a Myxovirus/biossíntese , Infecções por Pestivirus/virologia , Suínos/virologia , Ubiquitinas/biossíntese , Ubiquitinas/genética , Proteínas não Estruturais Virais/biossínteseRESUMO
Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Neurônios , Ubiquitinas/biossíntese , Proteínas Adaptadoras de Transdução de Sinal , Animais , Autofagia/genética , Proteínas Relacionadas à Autofagia , Encéfalo/patologia , Proteínas de Ciclo Celular/genética , Morte Celular , Humanos , Deficiências da Aprendizagem/genética , Deficiências da Aprendizagem/psicologia , Mutação/genética , Neurônios/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Proteína Sequestossoma-1/biossíntese , Proteína Sequestossoma-1/genética , Aprendizagem Espacial , Ubiquitinas/genéticaRESUMO
Metastasis is the main challenge of osteosarcoma treatment. Herein, we first reveal the oncogenic role of FAT10 in metastasis of osteosarcoma. FAT10 was upregulated in osteosarcoma, especially in metastatic osteosarcoma. High level of FAT10 was associated with poorer prognosis of osteosarcoma patients. Moreover, Transwell and Matrigel assays revealed that silencing FAT10 significantly inhibited the invasive and migratory abilities of osteosarcoma cells. Metastasis assay in vivo showed that silencing FAT10 decreased the number of mice with distant metastasis. We also found that FAT10 may act its oncogenic functions through regulating HOXB9. Collectively, the results suggested that FAT10 may be a novel therapeutic target for osteosarcoma patients.
Assuntos
Proteínas de Homeodomínio/genética , Osteossarcoma/genética , Ubiquitinas/biossíntese , Adulto , Animais , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Proteínas de Homeodomínio/biossíntese , Humanos , Masculino , Camundongos , Metástase Neoplásica , Osteossarcoma/patologia , Ubiquitinas/antagonistas & inibidoresRESUMO
STAT3 plays a pivotal role in the hematopoietic system, which constitutively activated by BCR-ABL via JAK and Erk/MAP-kinase pathways. Phospho-STAT3 was overexpressed in imatinib-resistant CML patients as relative to imatinib responsive ones. By activation of the STAT3 pathway, BCR-ABL can promote cell cycling, and inhibit differentiation and apoptosis. Ribosomal protein S27a (RPS27a) performs extra-ribosomal functions besides imparting a role in ribosome biogenesis and post-translational modifications of proteins. RPS27a can promote proliferation, regulate cell cycle progression and inhibit apoptosis of leukemia cells. However, the relationship between STAT3 and RPS27a has not been reported. In this study, we detected a significantly increased expression of STAT3 and RPS27a in bone marrow samples from CML-AP/BP patients compared with those from CML-CP. In addition, we also demonstrated that it was a positive correlation between the level of STAT3 and that of RPS27a. Imatinib-resistant K562/G01 cells expressed significantly higher levels of STAT3 and RPS27a compared with those of K562 cells. RPS27a could be transactivated by p-STAT3 through the specific p-STAT3-binding site located nt -633 to -625 and -486 to -478 of the RPS27a gene promoter in a dose-dependent manner. The transactivated RPS27a could decrease the percentage of apoptotic CML cells induced by imatinib. And the effect of STAT3 overexpression could be counteracted by the p-STAT3 inhibitor WP1066 or RPS27a knockdown. These results suggest that drugs targeting STAT3/p-STAT3/RPS27a combining with TKI might represent a novel therapy strategy in patients with TKI-resistant CML.
Assuntos
Mesilato de Imatinib/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Proteínas Ribossômicas/biossíntese , Ubiquitinas/biossíntese , Adulto , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Masculino , Pessoa de Meia-Idade , Fator de Transcrição STAT3/biossíntese , Fator de Transcrição STAT3/genética , Ativação TranscricionalRESUMO
Ubiquitin and polyubiquitin chains target proteins for a wide variety of cellular processes. Ubiquitin-mediated targeting is regulated by the lysine through which the ubiquitins are linked as well as the broader ubiquitin landscape on the protein. The mechanisms of this regulation are not fully understood. For example, the canonical proteasome targeting signal is a lysine 48-linked polyubiquitin chain, and the canonical endocytosis signal is a lysine 63-linked polyubiquitin chain. However, lysine 63-linked polyubiquitin chains can also target substrates for degradation. Biochemical studies of ubiquitinated proteins have been limited by the difficulty of building proteins with well-defined polyubiquitin chains. Here we describe an efficient and versatile method for synthesizing ubiquitin chains of defined linkage and length. The synthesized ubiquitin chains are then attached to any protein containing a ubiquitin moiety. These proteins can be used to study ubiquitin targeting in in vitro assays in the tightly controlled manner required for biochemical studies.
Assuntos
Poliubiquitina/biossíntese , Complexo de Endopeptidases do Proteassoma/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Ubiquitinas/biossíntese , Endocitose/fisiologia , Humanos , Lisina/biossíntese , Lisina/química , Poliubiquitina/química , Complexo de Endopeptidases do Proteassoma/química , Proteínas de Saccharomyces cerevisiae/química , Fatores de Tempo , Ubiquitinas/químicaRESUMO
The interferon-stimulated gene 15 (ISG15) plays an important role in the pathogenesis of hepatitis C virus (HCV) infection. ISG15-regulated proteins have previously been identified that putatively affect this proviral interaction. The present observational study aimed to elucidate the relation between ISG15 and these host factors during HCV infection. Transcriptomic and proteomic analyses were performed using liver samples of HCV-infected (n = 54) and uninfected (n = 10) or HBV-infected controls (n = 23). Primary human hepatocytes (PHH) were treated with Toll-like receptor ligands, interferons and kinase inhibitors. Expression of ISG15 and proteasome subunit alpha type-6 (PSMA6) was suppressed in subgenomic HCV replicon cell lines using specific siRNAs. Comparison of hepatic expression patterns revealed significantly increased signals for ISG15, IFIT1, HNRNPK and PSMA6 on the protein level as well as ISG15, IFIT1 and PSMA6 on the mRNA level in HCV-infected patients. In contrast to interferon-stimulated genes, PSMA6 expression occurred independent of HCV load and genotype. In PHH, the expression of ISG15 and PSMA6 was distinctly induced by poly(I:C), depending on IRF3 activation or PI3K/AKT signalling, respectively. Suppression of PSMA6 in HCV replicon cells led to significant induction of ISG15 expression, thus combined knock-down of both genes abrogated the antiviral effect induced by the separate suppression of ISG15. These data indicate that hepatic expression of PSMA6, which is upregulated during viral hepatitis, likely depends on TLR3 activation. PSMA6 affects the expression of immunoregulatory ISG15, a proviral factor in the pathogenesis of HCV infection. Therefore, the proteasome might be involved in the enigmatic interaction between ISG15 and HCV.
Assuntos
Citocinas/biossíntese , Expressão Gênica , Hepatite C/patologia , Complexo de Endopeptidases do Proteassoma/biossíntese , Ubiquitinas/biossíntese , Adulto , Biópsia , Células Cultivadas , Feminino , Perfilação da Expressão Gênica , Hepatócitos/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Proteoma/análiseRESUMO
ISG15, the protein encoded by interferon (IFN)-stimulated gene 15, was the first identified ubiquitin-like protein, which could be strongly upregulated by type I interferons as a primary response to diverse microbial and cellular stress stimuli. Although the biological activities of ISG15 have yet to be fully elucidated, it is frequently overexpressed in various cancers. As the role of ISG15 in esophageal squamous cell cancer (ESCC) has not been well reported, the current study aimed to elucidate the role of ISG15 in predicting outcomes of ESCC patients. Samples were collected from 153 ESCC patients, including 54 pairs of tumor tissues and non-tumor tissues. Compared with the paired non-tumor tissues, higher expression of ISG15 mRNA were detected in ESCC tissues. The cut-off value 1.28 determined by ROC curve analysis divided the ESCC patients into high and low ISG15 mRNA expression group. High-ISG15 mRNA expression appeared with more frequency in ever-drinkers (P = 0.018). Kaplan-Meier analysis indicated that Low-ISG15 mRNA expression group had a longer cancer-specific survival (CSS) compared with High-ISG15 mRNA expression group. Multivariate analysis revealed that ISG15 mRNA (P = 0.024; hazard ratio, 2.759, 95% CI, 1.841-4.134) as well as Pathological staging (P < 0.001; hazard ratio, 1.634, 95% CI, 1.065-2.505) were independent prognostic factors. Subgroup analysis revealed that the discernibility of ISG15 mRNA level on ESCC outcomes was only pronounced in ever-drinkers (P = 0.026) not in never-drinkers (P = 0.138). ISG15 might serve as a novel prognostic biomarker in drinkers with ESCC.
Assuntos
Consumo de Bebidas Alcoólicas/efeitos adversos , Carcinoma de Células Escamosas/metabolismo , Citocinas/biossíntese , Neoplasias Esofágicas/metabolismo , Ubiquitinas/biossíntese , Adulto , Idoso , Área Sob a Curva , Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro , Curva ROC , Reação em Cadeia da Polimerase em Tempo RealRESUMO
UNLABELLED: Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE: Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.
Assuntos
Células Dendríticas/virologia , Flaviviridae/fisiologia , Queratinócitos/virologia , Internalização do Vírus , Replicação Viral , Aedes/virologia , Animais , Autofagia/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Citocinas/biossíntese , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Células Dendríticas/imunologia , Fibroblastos/virologia , Flaviviridae/imunologia , Infecções por Flaviviridae/imunologia , Infecções por Flaviviridae/virologia , Células HEK293 , Receptor Celular 1 do Vírus da Hepatite A , Humanos , Insetos Vetores/virologia , Helicase IFIH1 Induzida por Interferon , Interferon beta/biossíntese , Interferon beta/imunologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Resistência a Myxovirus/biossíntese , Fagossomos/imunologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos , Receptores Virais/genética , Receptores Virais/metabolismo , Pele/imunologia , Pele/virologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/imunologia , Receptor 3 Toll-Like/metabolismo , Receptor 7 Toll-Like/imunologia , Ubiquitinas/biossíntese , Células Vero , Receptor Tirosina Quinase AxlRESUMO
Recent studies indicate that post-translational protein neddylation is required for the maintenance of cell viability in several lymphoma cell lines, while inhibition of the neddylation pathway with an NEDD8-activating enzyme (NAE) inhibitor MLN4924 induces apoptosis in lymphoma cells. However, the mechanism by which neddylation inhibition induces apoptosis in lymphoma cells has not been fully elucidated. Moreover, it is unknown whether neddylation inhibition triggers non-apoptotic cell-killing responses, such as cell senescence, in lymphoma cells. Here, we report that MLN4924 specifically inhibited protein neddylation, inactivated cullin-RING E3 ligase (CRL), the best-known neddylation substrate, and induced the accumulation of tumor-suppressive CRL substrates in lymphoma cells. Moreover, MLN4924 potently suppressed the growth of lymphoma cells by inducing G2 cell-cycle arrest, followed by apoptosis or senescence in a cell line-dependent manner. MLN4924-induced apoptosis was mediated by intrinsic apoptotic signaling with substantial up-regulation of pro-apoptotic Bik and Noxa as well as down-regulation of anti-apoptotic XIAP, c-IAP1 and c-IAP2, while senescence induction upon neddylation inhibition seemed dependent on the expression of tumor suppressor p21/p27. Together, these findings expand our understanding on how lymphoma cells respond to neddylation inhibition and support the development of neddylation inhibitors (e.g. MLN4924) for the treatment of lymphoma.
Assuntos
Proteínas Culina/biossíntese , Ciclopentanos/administração & dosagem , Linfoma/tratamento farmacológico , Pirimidinas/administração & dosagem , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitinas/biossíntese , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteínas Culina/antagonistas & inibidores , Proteínas Culina/genética , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Linfoma/genética , Linfoma/patologia , Proteína NEDD8 , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitinas/antagonistas & inibidoresRESUMO
Cancer stem cells (CSC) are thought to play a major role in the development and metastatic progression of pancreatic ductal adenocarcinoma (PDAC), one of the deadliest solid tumors. Likewise, the tumor microenvironment contributes critical support in this setting, including from tumor stromal cells and tumor-associated macrophages (TAM) that contribute structural and paracrine-mediated supports, respectively. Here, we show that TAMs secrete the IFN-stimulated factor ISG15, which enhances CSC phenotypes in PDAC in vitro and in vivo. ISG15 was preferentially and highly expressed by TAM present in primary PDAC tumors resected from patients. ISG15 was secreted by macrophages in response to secretion of IFNß by CSC, thereby reinforcing CSC self-renewal, invasive capacity, and tumorigenic potential. Overall, our work demonstrates that ISG15 is a previously unrecognized support factor for CSC in the PDAC microenvironment with a key role in pathogenesis and progression.
Assuntos
Carcinogênese/genética , Carcinoma Ductal Pancreático/genética , Citocinas/genética , Células-Tronco Neoplásicas/patologia , Ubiquitinas/genética , Biomarcadores Tumorais , Carcinoma Ductal Pancreático/patologia , Citocinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Macrófagos/metabolismo , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral/genética , Ubiquitinas/biossínteseRESUMO
Type 1 diabetes, one of two major forms of diabetes, results from the complete destruction of pancreatic beta cells. Viral infection has been suggested to be a trigger of beta cell destruction, the pathogenesis of type 1 diabetes. The aim of this study was to clarify the role of the protein encoded by intherferon stimulated gene (ISG) 15, an antiviral effector, in the development of this clinical entity. We used the mouse beta cell line MIN6 to investigate the role of ISG15 and paid special attention to apoptosis. Although not detected in native MIN6 cells, free ISG15 and ISG15 conjugated proteins were both present in dose-dependently increased amounts following stimulation with interferon alpha. As assessed both by caspase 3/7 activity and an annexin V assay, the percentage of apoptotic MIN6 cells (after exposure to the inflammatory cytokines of interleukin-1beta plus interferon gamma or tumor necrosis factor alpha) was decreased by pretreatment with adenovirus-expressing ISG15 and increased by expressing a short hairpin RNA directed against ISG15. In conclusion, ISG15 has an anti-apoptotic effect on MIN6 cells. Thus, promoting ISG15 expression in the pancreatic beta cells could be a potential therapeutic approach for patients with type 1 diabetes.
Assuntos
Apoptose/efeitos dos fármacos , Citocinas/fisiologia , Células Secretoras de Insulina/patologia , Animais , Linhagem Celular , Citocinas/biossíntese , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Interferon-alfa/farmacologia , Interferon gama/farmacologia , Interleucina-1beta/farmacologia , Camundongos , Ubiquitinas/biossíntese , Ubiquitinas/fisiologiaRESUMO
UNLABELLED: The antimicrobial peptide NZ17074, which is derived from arenicin-3 isolated from Arenicola marina, displayed high activity against a broad range of pathogenic bacteria and fungi. However, NZ17074 has not been produced using fermentation technology. The aim of this work was to study the expression of difficult-to-express NZ17074 in Pichia pastoris by fusing with SUMO3. The DNA fragments of NZ17074 and SUMO3 were fused into SUMO3-NZ17074 using overlap PCR and cloned into the pPICZαA vector to construct the pPICZ-SUMO3-NZ17074 expression vector. The rSUMO3-NZ17074 fusion protein, purified by Ni(2) (+) -chelating affinity chromatography, was cleaved by 50% formic acid at 50°C for 28 h to release recombinant NZ17074 (rNZ17074). After purification with second affinity column, 4·1 mg rNZ17074 peptide with the purity over 90% was obtained from per litre fermentation culture. The rNZ17074 peptide exhibited the significant inhibition activity against Gram-negative bacteria: its minimal inhibitory concentrations (MICs) against Escherichia coli, Salmonella enteritidis and Pseudomonas aeruginosa were 2-4, 2 and 8-16 µg ml(-1) , respectively, which indicated that SUMO3 is a good fusion partner for the expression of the toxic peptide. SIGNIFICANCE AND IMPACT OF THE STUDY: Recombinant active NZ17074 was produced with Pichia pastoris by using high-density fermentation technology for the first time. Our findings demonstrated the usefulness of SUMO-fusion technology as an effective expression strategy for synthesizing peptides in yeast. This SUMO3 expression system with a lower cost would likely be widely used for the production of other cytotoxic proteins including antimicrobial peptides.
Assuntos
Antibacterianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Ubiquitinas/biossíntese , Sequência de Aminoácidos , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Sequência de Bases , Candida albicans/efeitos dos fármacos , Clonagem Molecular , Escherichia coli/efeitos dos fármacos , Fermentação , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Pichia , Proteólise , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Recombinantes de Fusão/isolamento & purificação , Salmonella enteritidis/efeitos dos fármacos , Ubiquitinas/isolamento & purificaçãoRESUMO
We showed in our previous study that cancer upregulated gene (CUG) 2, a novel oncogene, confers resistance to infection of oncolytic vesicular stomatitis virus (VSV) by activating Stat1-mediated signal transduction. Since many studies have reported that autophagy is involved in virus replication, we investigated whether autophagy also plays a role in the antiviral activity in A549 cells overexpressing CUG2 (A549-CUG2). We suppressed Atg5 or Beclin 1 expression using siRNA and examined its effect on the susceptibility of cells to infection by oncolytic VSV. We found that A549-CUG2 cells treated with Atg5 or Beclin 1 siRNA became susceptible to VSV infection, whereas A549-CUG2 cells treated with control siRNA were resistant. This result suggests that autophagy is involved in the antiviral response of A549-CUG2 cells. Further investigation revealed that autophagy impairment enhanced the generation of reactive oxygen species (ROS), which resulted in inactivation of S6 kinase. Under these conditions, the levels of ISG15 transcript and protein decreased, which conferred on A549-CUG2 cell susceptibility to VSV infection. Finally, we found that overloading of H2O2 sensitized control A549-CUG2 cells to VSV-induced apoptosis. Taken together, these results indicate that autophagy impairment induces excessive ROS formation, which decreases S6 kinase activity and ISG15 expression, ultimately rendering the A549-CUG2 cells susceptible to VSV infection. We propose that autophagy impairment is a potential strategy for successful VSV virotherapy of CUG2-overexpressing tumors.