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1.
Med Oncol ; 36(11): 95, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31637536

RESUMO

Ovarian cancer is one of the most lethal gynecological cancers; owning to its late detection and chemoresistance, understanding the pathogenesis of this malignant tumor is much critical. Previous studies have reported that ubiquitin-specific peptidase 39 (USP39) is generally overexpressed in a variety of cancers, including hepatocellular carcinoma, gastric cancer and so forth. Furthermore, USP39 is proved to be associated with the proliferation of malignant tumors. However, the function and mechanism of USP39 in ovarian cancer have not been elucidated. In the present study, we observed that USP39 was frequently overexpressed in human ovarian cancer and was highly correlated with TNM stage. Suppression of USP39 markedly inhibited the growth and migration of ovarian cancer cell lines HO-8910 and SKOV3 and induced cell cycle G2/M arrest. Moreover, knockdown of USP39 inhibited ovarian tumor growth in a xenograft model. In addition, our findings indicated that cell cycle arrest induced by USP39 knockdown might be involved in p53/p21 signaling pathway. Furthermore, we found that the depletion of USP39 inhibited the migration of ovarian cancer cells via blocking epithelial-mesenchymal transition. Taken together, these results suggest that USP39 may play vital roles in the genesis and progression and may serve as a potential biomarker for diagnosis and therapeutic target of ovarian cancer.


Assuntos
Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Transição Epitelial-Mesenquimal , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Xenoenxertos , Humanos , Imuno-Histoquímica , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Camundongos Nus , Estadiamento de Neoplasias , Neoplasias Ovarianas/enzimologia , Transdução de Sinais , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/genética
2.
Gene ; 721: 144100, 2019 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-31493508

RESUMO

BACKGROUND: Breast cancer (BRCA) is the most prevalent cancer that threatens female health. A growing body of evidence has demonstrated the non-negligible effects of messenger RNAs (mRNAs) on biological processes involved in cancers; however, there is no definite conclusion regarding the role of mRNAs in predicting the prognosis of BRCA patients. MATERIALS AND METHODS: We systematically screened the mRNA expression landscape and clinical data of samples from the Cancer Genome Atlas (TCGA). Univariate Cox analysis and robust likelihood-based survival analysis were conducted to identify key mRNAs associated with BRCA. Furthermore, risk scores based on multivariate Cox analysis divided the training set into high-risk and low-risk groups. ROC analysis determined the optimal cut-off point for patient classification of risk levels. The prognostic model was additionally validated in the testing set and complete dataset. Finally, we plotted the survival curves for the mRNAs used in our model. RESULTS: We obtained the original expression data of 13,617 mRNAs from a total of 1088 samples. After comprehensive survival analysis, the four-mRNA (ACSL1, OTUD3, PKD1L2, and WISP1) prognosis risk assessment model was constructed. Furthermore, the area under cure (AUC) was 0.834, indicating that the model was meaningful and reasonable. In each dataset, analysis based on the four-mRNA signature risk score indicated that the survival status of the group with high risk score was worse than that of the group with low risk scores. Patients with strong mRNA expression of OTUD3, PKD1L2, and WISP1 tended to have good prognosis, whereas patients with high ACSL1 expression tended to have poor prognosis. CONCLUSION: In summary, we constructed a four-mRNA prognosis risk assessment model for BRCA. The newly developed model offers more possibilities for assessing prognosis and guiding the selection of better treatment strategies for BRCA.


Assuntos
Neoplasias da Mama , Bases de Dados de Ácidos Nucleicos , Modelos Biológicos , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Proteínas de Sinalização Intercelular CCN/biossíntese , Proteínas de Sinalização Intercelular CCN/genética , Coenzima A Ligases/biossíntese , Coenzima A Ligases/genética , Intervalo Livre de Doença , Feminino , Humanos , Valor Preditivo dos Testes , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA Neoplásico/genética , Receptores Acoplados a Proteínas-G/biossíntese , Receptores Acoplados a Proteínas-G/genética , Taxa de Sobrevida , Proteases Específicas de Ubiquitina/biossíntese , Proteases Específicas de Ubiquitina/genética
3.
Oncogene ; 38(37): 6414-6428, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31332287

RESUMO

Increasing evidence demonstrates that ubiquitin specific protease 39 (USP39) plays an oncogenic role in various human tumors. Here, using expression analysis of the publicly available Oncomine database, clinical glioma patient samples, and glioma cells, we found that USP39 was overexpressed in human gliomas. Knockdown of USP39 in glioma cells demonstrated that the protein promoted cell growth, invasion and migration in vitro and in a tumor model in nude mice. To identify mediators of USP39 growth-promoting properties, we used luciferase reporter constructs under transcriptional control of various promoters specific to seven canonical cancer-associated pathways. Luciferase activity from a synthetic TEAD-dependent YAP/TAZ-responsive reporter, as a direct readout of the Hippo signaling pathway, was decreased by 92% in cells with USP39 knockdown, whereas the luciferase activities from the other six cancer pathways, including MAPK/ERK, MAPK/JNK, NFκB, Notch, TGFß, and Wnt, remained unchanged. TAZ protein expression however was decreased independent of canonical Hippo signaling. Immunohistochemistry revealed a positive correlation between USP39 and TAZ proteins in orthotopic xenografts derived from modified glioma cells expressing USP39 shRNAs and primary human glioma samples (p < 0.05). Finally, loss of USP39 decreased TAZ pre-mRNA splicing efficiency in glioma cells in vitro, which led to reduced levels of TAZ protein. In summary, USP39 has oncogenic properties that increase TAZ protein levels by inducing maturation of its mRNA. USP39 therefore provides a novel therapeutic target for the treatment of human glioma.


Assuntos
Neoplasias Encefálicas/patologia , Glioma/patologia , Processamento de RNA/genética , Transativadores/genética , Proteases Específicas de Ubiquitina/fisiologia , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Glioma/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Proteases Específicas de Ubiquitina/genética
4.
Nat Commun ; 10(1): 2914, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266968

RESUMO

The deubiquitylase OTUD3 plays a suppressive role in breast tumorigenesis through stabilizing PTEN protein, but its role in lung cancer remains unclear. Here, we demonstrate that in vivo deletion of OTUD3 indeed promotes breast cancer development in mice, but by contrast, it slows down KrasG12D-driven lung adenocarcinoma (ADC) initiation and progression and markedly increases survival in mice. Moreover, OTUD3 is highly expressed in human lung cancer tissues and its higher expression correlates with poorer survival of patients. Further mechanistic studies reveal that OTUD3 interacts with, deubiquitylates and stabilizes the glucose-regulated protein GRP78. Knockdown of OTUD3 results in a decrease in the level of GRP78 protein, suppression of cell growth and migration, and tumorigenesis in lung cancer. Collectively, our results reveal a previously unappreciated pro-oncogenic role of OTUD3 in lung cancer and indicate that deubiquitylases could elicit tumor-suppressing or tumor-promoting activities in a cell- and tissue-dependent context.


Assuntos
Proteínas de Choque Térmico/metabolismo , Neoplasias Pulmonares/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Proteínas de Choque Térmico/genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteases Específicas de Ubiquitina/genética
5.
Mol Cell ; 75(3): 483-497.e9, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253574

RESUMO

In mammals, ∼100 deubiquitinases act on ∼20,000 intracellular ubiquitination sites. Deubiquitinases are commonly regarded as constitutively active, with limited regulatory and targeting capacity. The BRCA1-A and BRISC complexes serve in DNA double-strand break repair and immune signaling and contain the lysine-63 linkage-specific BRCC36 subunit that is functionalized by scaffold subunits ABRAXAS and ABRO1, respectively. The molecular basis underlying BRCA1-A and BRISC function is currently unknown. Here we show that in the BRCA1-A complex structure, ABRAXAS integrates the DNA repair protein RAP80 and provides a high-affinity binding site that sequesters the tumor suppressor BRCA1 away from the break site. In the BRISC structure, ABRO1 binds SHMT2α, a metabolic enzyme enabling cancer growth in hypoxic environments, which we find prevents BRCC36 from binding and cleaving ubiquitin chains. Our work explains modularity in the BRCC36 DUB family, with different adaptor subunits conferring diversified targeting and regulatory functions.


Assuntos
Proteína BRCA1/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Enzimas Desubiquitinantes/genética , Chaperonas de Histonas/genética , Neoplasias/genética , Sítios de Ligação/genética , Proteínas de Transporte/genética , Núcleo Celular/genética , Núcleo Celular/imunologia , Citoplasma/genética , Citoplasma/imunologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA/imunologia , Enzimas Desubiquitinantes/imunologia , Células HeLa , Humanos , Imunidade Celular/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Neoplasias/imunologia , Proteínas Associadas à Matriz Nuclear/genética , Ligação Proteica/genética , Ubiquitina/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
6.
Nat Commun ; 10(1): 2849, 2019 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253762

RESUMO

Fanconi anemia (FA) is a multigenic disease of bone marrow failure and cancer susceptibility stemming from a failure to remove DNA crosslinks and other chromosomal lesions. Within the FA DNA damage response pathway, DNA-dependent monoubiquitinaton of FANCD2 licenses downstream events, while timely FANCD2 deubiquitination serves to extinguish the response. Here, we show with reconstituted biochemical systems, which we developed, that efficient FANCD2 deubiquitination by the USP1-UAF1 complex is dependent on DNA and DNA binding by UAF1. Surprisingly, we find that the DNA binding activity of the UAF1-associated protein RAD51AP1 can substitute for that of UAF1 in FANCD2 deubiquitination in our biochemical system. We also reveal the importance of DNA binding by UAF1 and RAD51AP1 in FANCD2 deubiquitination in the cellular setting. Our results provide insights into a key step in the FA pathway and help define the multifaceted role of the USP1-UAF1-RAD51AP1 complex in DNA damage tolerance and genome repair.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Anemia de Fanconi/genética , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Mutação , Proteínas Nucleares/genética , Ligação Proteica , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
7.
Mol Med Rep ; 20(2): 1429-1435, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173246

RESUMO

Pathological scarring is a result of the hypertrophy of scar tissue during tissue repair following trauma. The aim of the present study was to assess the effect of ubiquitin­specific protease 4 (USP4) silencing on pathological scarring, and to evaluate the mechanistic basis for the effect. An MTT assay was used to assess cell viability. Immunoprecipitation (IP) was used to determine ubiquitination levels of the TGF­ß receptor (TßR)I and Smad7. Tumor formation was assessed by injecting keloid fibroblasts. Hematoxylin and eosin staining was used to detect pathological changes in tumor tissue. Reverse transcription quantitative polymerase chain reaction and western blot analysis assays were used to evaluate the expression levels of TßRI and Smad7. Compared with the untreated control animals, cell viability and the expression of TßRI and Smad7 increased significantly in animals treated with TGF­ß. Short hairpin RNA for USP4 (shUSP4) decreased the cell viability of negative control cells, TGF­ß­induced cellular proliferation, and the expression of TßRI and Smad7. IP experiments indicated that the ubiquitination level of TßRI was decreased following USP4 silencing. There was no remarkable difference in the structure of scar tissue among the various animal groups at 14 days following treatment, while the necrotic area of the scar tissue in the shUSP4 and vialinin A (USP inhibitor)­treated animals increased significantly at the 28th and 42nd day compared with the control animals. At days 14, 28 and 42, the expression levels of TßRI and Smad7 in the shUSP4 and vialinin A­treated animals were significantly decreased compared with the control animals (P<0.05). In summary, interference with or inhibition of USP4 prevented the activity of the TGF­ß/Smad pathway signaling and inhibited the formation of pathological scars.


Assuntos
Cicatriz/genética , Queloide/genética , Receptor do Fator de Crescimento Transformador beta Tipo I/genética , Proteína Smad7/genética , Fator de Crescimento Transformador beta/genética , Proteases Específicas de Ubiquitina/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cicatriz/metabolismo , Cicatriz/patologia , Cicatriz/prevenção & controle , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibroblastos/transplante , Regulação da Expressão Gênica , Humanos , Queloide/metabolismo , Queloide/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais , Proteína Smad7/metabolismo , Compostos de Terfenil/farmacologia , Fator de Crescimento Transformador beta/metabolismo , Transplante Heterólogo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo
8.
J Exp Clin Cancer Res ; 38(1): 277, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31234902

RESUMO

BACKGROUND: The deubiquitinating enzyme ubiquitin-specific protease 3 (USP3) plays a crucial role in numerous biological processes. The aberrant expression of USP3 may have an important role in tumor development. However, the mechanism by which USP3 promotes gastric cancer (GC) metastasis remains largely unknown. METHODS: Effects of USP3 on the progression of GC in vivo and in vitro and the potential underlying mechanisms have been investigated utilizing proteomics, RT-PCR, western blotting, immunohistochemistry, immunofluorescence, cell invasion and migration assays and xenograft tumor models. RESULTS: USP3 expression was upregulated in GC compared with matched normal tissues and was predictive of poor survival. USP3 also promoted migration and epithelial-to-mesenchymal transition (EMT) in GC cells. Moreover, TGF-ß1 induced USP3 expression, and USP3 knockdown inhibited TGF-ß1-induced EMT. Furthermore, we utilized Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to identify differentially expressed proteins in USP3-overexpressing cells compared with control cells. Importantly, we found that SUZ12 is indispensable for USP3-mediated oncogenic activity in GC. We observed that USP3 interacted with and stabilized SUZ12 via deubiquitination. SUZ12 knockdown inhibited USP3-induced migration and invasion, as well as EMT in GC cells. Examination of clinical samples confirmed that USP3 expression was positively correlated with SUZ12 protein expression and that the levels of USP3 or SUZ12 protein were negatively correlated with the levels of E-cadherin protein. CONCLUSIONS: These findings identify USP3 as a critical regulator. The USP3-SUZ12 axis might promote tumor progression and could be a potential therapeutic candidate for human GC.


Assuntos
Transição Epitelial-Mesenquimal , Complexo Repressor Polycomb 2/metabolismo , Neoplasias Gástricas/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Caderinas/metabolismo , Carcinoma/patologia , Carcinoma/secundário , Linhagem Celular Tumoral , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Complexo Repressor Polycomb 2/biossíntese , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fator de Crescimento Transformador beta1/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
9.
G3 (Bethesda) ; 9(7): 2287-2302, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31088904

RESUMO

Ectopic expression of master regulatory transcription factors can reprogram the identity of specific cell types. The effectiveness of such induced cellular reprogramming is generally greatly reduced if the cellular substrates are fully differentiated cells. For example, in the nematode C. elegans, the ectopic expression of a neuronal identity-inducing transcription factor, CHE-1, can effectively induce CHE-1 target genes in immature cells but not in fully mature non-neuronal cells. To understand the molecular basis of this progressive restriction of cellular plasticity, we screened for C. elegans mutants in which ectopically expressed CHE-1 is able to induce neuronal effector genes in epidermal cells. We identified a ubiquitin hydrolase, usp-48, that restricts cellular plasticity with a notable cellular specificity. Even though we find usp-48 to be very broadly expressed in all tissue types, usp-48 null mutants specifically make epidermal cells susceptible to CHE-1-mediated activation of neuronal target genes. We screened for additional genes that allow epidermal cells to be at least partially reprogrammed by ectopic che-1 expression and identified many additional proteins that restrict cellular plasticity of epidermal cells, including a chromatin-related factor (H3K79 methyltransferase, DOT-1.1), a transcription factor (nuclear hormone receptor NHR-48), two MAPK-type protein kinases (SEK-1 and PMK-1), a nuclear localized O-GlcNAc transferase (OGT-1) and a member of large family of nuclear proteins related to the Rb-associated LIN-8 chromatin factor. These findings provide novel insights into the control of cellular plasticity.


Assuntos
Plasticidade Celular/genética , Cromatina/genética , Proteínas Quinases/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Caenorhabditis elegans/genética , Diferenciação Celular , Reprogramação Celular/genética , Cromatina/metabolismo , Biologia Computacional/métodos , Epiderme/metabolismo , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Família Multigênica , Mutagênese , Mutação , Transporte Proteico , Interferência de RNA , Transgenes , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Sequenciamento Completo do Genoma
10.
Gene ; 707: 44-52, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30898716

RESUMO

Long non-coding RNAs (lncRNAs) have been wildly verified to modulate multiple tumorigenesis, especially nasopharyngeal carcinoma (NPC). In present study, we aims to investigate the role and mechanism of LINC00520 in the NPC carcinogenesis. Results indicated that LINC00520 was significantly increasing in NPC tissues and cells in comparison to their corresponding controls. Moreover, the aberrant overexpression of LINC00520 indicated the poor prognosis of NPC patients. Silence of LINC00520 was able to repress NPC cell growth in vitro while overexpression of LINC00520 inversed this process. Moreover, in vivo tumor xenografts were establishing using CNE-1/SUNE-1 cells to investigate the function of LINC00520 in NPC tumorigenesis. Rescue assay was conducting to further confirm that LINC00520 contributed to NPC progression by regulating miR-26b-3p/ubiquitin-specific protease 39 (USP39) signal pathway. Taken together, our study discovered the oncogenic role of LINC00520 in clinical specimens and cellular experiments, showing the potential LINC00520/miR-26b-3p/USP39 pathway. This results and findings provide a novel insight for NPC tumorigenesis.


Assuntos
MicroRNAs/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , RNA Longo não Codificante/genética , Proteases Específicas de Ubiquitina/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Transplante de Neoplasias , Prognóstico , Proteases Específicas de Ubiquitina/metabolismo , Regulação para Cima
11.
Nat Commun ; 10(1): 1224, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30874560

RESUMO

Poly-(ADP-ribose) polymerase inhibitors (PARPi) selectively kill breast and ovarian cancers with defects in homologous recombination (HR) caused by BRCA1/2 mutations. There is also clinical evidence for the utility of PARPi in breast and ovarian cancers without BRCA mutations, but the underlying mechanism is not clear. Here, we report that the deubiquitylating enzyme USP15 affects cancer cell response to PARPi by regulating HR. Mechanistically, USP15 is recruited to DNA double-strand breaks (DSBs) by MDC1, which requires the FHA domain of MDC1 and phosphorylated Ser678 of USP15. Subsequently, USP15 deubiquitinates BARD1 BRCT domain, and promotes BARD1-HP1γ interaction, resulting in BRCA1/BARD1 retention at DSBs. USP15 knockout mice exhibit genomic instability in vivo. Furthermore, cancer-associated USP15 mutations, with decreased USP15-BARD1 interaction, increases PARP inhibitor sensitivity in cancer cells. Thus, our results identify a novel regulator of HR, which is a potential biomarker for therapeutic treatment using PARP inhibitors in cancers.


Assuntos
Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Estimativa de Kaplan-Meier , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/genética , Neoplasias Experimentais/mortalidade , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Transativadores/genética , Transativadores/metabolismo , Resultado do Tratamento , Proteases Específicas de Ubiquitina/genética , Irradiação Corporal Total
12.
Biol Pharm Bull ; 42(4): 573-579, 2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30686807

RESUMO

Pancreatic cancer is known to be a fatal disease, which is difficult to be diagnosed in its early stages. Ubiquitin-Specific Protease 34 (USP34) are closely related to human cancers in the development and progression. However, there are rarely studies about the role of USP34 in pancreatic cancer. Thus, we aimed to investigate the effect of USP34 in human pancreatic cancer. Short-hairpin RNA targeting USP34 (USP34-shRNA) and USP34 overexpression lentivirus were used in the current study. The level of USP34 in human pancreatic cancer (PANC-1) cells were then analyzed by quantitative (q)RT-PCR. In addition, Western blotting was used to examine phosphorylated (p)-AKT, p-protein kinase C (PKC) and p-extracellular signal-regulated kinase (ERK) protein levels. CCK-8 assay, flow cytometry, and migration assay were used to detect cell proliferation, apoptosis and migration, respectively in vitro. According to the result of qRT-PCR and Western blotting, USP34-shRNA1 significantly downregulated USP34 gene level in PANC-1 cell. Subsequently, Western blotting assay indicated that USP34 silencing significantly down-regulated the expression of p-AKT and p-PKC in cells. On the other hand, USP34 overexpressing remarkably up-regulated the expression of p-AKT and p-PKC in cells. In addition, USP34 overexpression promoted PANC-1 cell proliferation and migration via up-regulating the proteins of p-AKT and p-PKC. Moreover, USP34 overexpression reversed AKT inhibitor and PKC inhibitor induced PACN-1 cell apoptosis. Our results indicated USP34 regulated h PANC-1 cell survival via AKT and PKC pathways, and which played a pro-survival role in human pancreatic cancer. Therefore, we suggested USP34 could be a potential therapeutic target for pancreatic cancer.


Assuntos
Regulação Enzimológica da Expressão Gênica , Neoplasias Pancreáticas/metabolismo , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteases Específicas de Ubiquitina/genética , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Sobrevivência Celular/genética , Técnicas de Silenciamento de Genes , Humanos , Lentivirus/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Plasmídeos , RNA Interferente Pequeno/genética , Transdução de Sinais
13.
Clin Sci (Lond) ; 133(2): 335-349, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30622220

RESUMO

Ubiquitin-specific peptidase 4 (USP4) protein is a type of deubiquitination enzyme that is correlated with many important biological processes. However, the function of USP4 in hepatic ischaemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to explore the role of USP4 in hepatic I/R injury. USP4 gene knockout mice and primary hepatocytes were used to construct hepatic I/R models. The effect of USP4 on hepatic I/R injury was examined via pathological and molecular analyses. Our results indicated that USP4 was significantly up-regulated in liver of mice subjected to hepatic I/R injury. USP4 knockout mice exhibited exacerbated hepatic I/R injury, as evidenced by enhanced liver inflammation via the nuclear factor κB (NF-κB) signalling pathway and increased hepatocyte apoptosis. Additionally, USP4 overexpression inhibited hepatocyte inflammation and apoptosis on hepatic I/R stimulation. Mechanistically, our study demonstrates that USP4 deficiency exerts its detrimental effects on hepatic I/R injury by inducing activation of the transforming growth factor ß-activated kinase 1 (TAK1)/JNK signalling pathways. TAK1 was required for USP4 function in hepatic I/R injury as TAK1 inhibition abolished USP4 function in vitro In conclusion, our study demonstrates that USP4 deficiency plays a detrimental role in hepatic I/R injury by promoting activation of the TAK1/JNK signalling pathways. Modulation of this axis may be a novel strategy to alleviate the pathological process of hepatic I/R injury.


Assuntos
Fígado/irrigação sanguínea , Fígado/enzimologia , MAP Quinase Quinase Quinases/metabolismo , Traumatismo por Reperfusão/enzimologia , Proteases Específicas de Ubiquitina/deficiência , Animais , Apoptose , Modelos Animais de Doenças , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/patologia , MAP Quinase Quinase Quinases/genética , Masculino , Camundongos Knockout , NF-kappa B/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Proteases Específicas de Ubiquitina/genética
14.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626683

RESUMO

Hepatitis C virus (HCV) utilizes cellular factors for efficient propagation. Ubiquitin is covalently conjugated to the substrate to alter its stability or to modulate signal transduction. In this study, we examined the importance of ubiquitination for HCV propagation. We found that inhibition of deubiquitinating enzymes (DUBs) or overexpression of nonspecific DUBs impaired HCV replication, suggesting that ubiquitination regulates HCV replication. To identify specific DUBs involved in HCV propagation, we set up RNA interference (RNAi) screening against DUBs and successfully identified ubiquitin-specific protease 15 (USP15) as a novel host factor for HCV propagation. Our studies showed that USP15 is involved in translation of HCV RNA and production of infectious HCV particles. In addition, deficiency of USP15 in human hepatic cell lines (Huh7 and Hep3B/miR-122 cells) but not in a nonhepatic cell line (293T cells) impaired HCV propagation, suggesting that USP15 participates in HCV propagation through the regulation of hepatocyte-specific functions. Moreover, we showed that loss of USP15 had no effect on innate immune responses in vitro and in vivo We also found that USP15-deficient Huh7 cells showed reductions in the amounts of lipid droplets (LDs), and the addition of palmitic acids restored the production of infectious HCV particles. Taken together, these data suggest that USP15 participates in HCV propagation by regulating the translation of HCV RNA and the formation of LDs.IMPORTANCE Although ubiquitination has been shown to play important roles in the HCV life cycle, the roles of deubiquitinating enzymes (DUBs), which cleave ubiquitin chains from their substrates, in HCV propagation have not been investigated. Here, we identified USP15 as a DUB regulating HCV propagation. USP15 showed no interaction with viral proteins and no participation in innate immune responses. Deficiency of USP15 in Huh7 cells resulted in suppression of the translation of HCV RNA and reduction in the amounts of lipid droplets, and the addition of fatty acids partially restored the production of infectious HCV particles. These data suggest that USP15 participates in HCV propagation in hepatic cells through the regulation of viral RNA translation and lipid metabolism.


Assuntos
Hepacivirus/genética , Hepatite C/metabolismo , Hepatite C/virologia , Gotículas Lipídicas/metabolismo , RNA Viral/genética , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Regulação da Expressão Gênica/fisiologia , Células HEK293 , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Metabolismo dos Lipídeos/fisiologia , Fígado/metabolismo , Fígado/virologia , Interferência de RNA/fisiologia , Transdução de Sinais/genética , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Células Vero , Replicação Viral/genética
15.
Hum Mol Genet ; 28(4): 548-560, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30329047

RESUMO

Ubiquitin specific proteases (USPs) are de-ubiquitinases (DUBs) that control protein ubiquitination cycle. The role of DUBs is poorly understood in neurodegenerative diseases. We found that USP13 is overexpressed in post-mortem Parkinson's disease (PD) brains. We investigated whether changes in USP13 levels can affect two molecules, parkin and alpha-synuclein, that are implicated in PD pathogenesis. Parkin is an E3 ubiquitin ligase that is regulated by ubiquitination and targets certain proteins for degradation, and alpha-synuclein may be ubiquitinated and recycled in the normal brain. We found that USP13 independently regulates parkin and alpha-synuclein ubiquitination in models of alpha-synucleinopathies. USP13 shRNA knockdown increases alpha-synuclein ubiquitination and clearance, in a parkin-independent manner. Furthermore, USP13 overexpression counteracts the effects of a tyrosine kinase inhibitor, Nilotinib, while USP13 knockdown facilitates Nilotinib effects on alpha-synculein clearance, suggesting that alpha-synuclein ubiquitnation is important for its clearance. These studies provide novel evidence of USP13 effects on parkin and alpha-synuclein metabolism and suggest that USP13 is a potential therapeutic target in the alpha-synucleinopathies.


Assuntos
Endopeptidases/genética , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , alfa-Sinucleína/genética , Autopsia , Encéfalo/metabolismo , Encéfalo/patologia , Endopeptidases/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Doença de Parkinson/patologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética
16.
Cancer Res ; 79(1): 72-85, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30425057

RESUMO

Aberrant activation of ß-catenin signaling is a critical driver for tumorigenesis, but the mechanism underlying this activation is not completely understood. In this study, we demonstrate a critical role of ß-catenin signaling in stabilization of enhancer of zeste homolog 2 (EZH2) and control of EZH2-mediated gene repression in oncogenesis. ß-Catenin/TCF4 activated the transcription of the deubiquitinase USP1, which then interacted with and deubiquitinated EZH2 directly. USP1-mediated stabilization of EZH2 promoted its recruitment to the promoters of CDKN1B, RUNX3, and HOXA5, resulting in enhanced enrichment of histone H3K27me3 and repression of target gene expression. In human glioma specimens, expression levels of nuclear ß-catenin, USP1, and EZH2 correlated with one another. Depletion of ß-catenin/USP1/EZH2 repressed glioma cell proliferation in vitro and tumor formation in vivo. Our findings indicate that a ß-catenin-USP1-EZH2 axis orchestrates the interplay between dysregulated ß-catenin signaling and EZH2-mediated gene epigenetic silencing during glioma tumorigenesis. SIGNIFICANCE: These findings identify the ß-catenin-USP1-EZH2 signaling axis as a critical mechanism for glioma tumorigenesis that may serve as a new therapeutic target in glioblastoma.


Assuntos
Carcinogênese/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/química , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Proteases Específicas de Ubiquitina/metabolismo , beta Catenina/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/metabolismo , Proliferação de Células , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética , Glioma/genética , Glioma/metabolismo , Humanos , Camundongos , Camundongos Nus , Prognóstico , Estabilidade Proteica , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
17.
Parasitol Res ; 118(1): 47-55, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30415394

RESUMO

Ubiquitination is an important post-translational modification process that regulates many cellular processes. Proteins can be modified at single or multiple lysine residues by a single ubiquitin protein or by ubiquitin oligomers. It is important to note that the type of ubiquitin chains determines the functional outcome of the modification. Ubiquitin or ubiquitin chains can be removed by deubiquitinases (DUBs). In our previous study, the Eimeria tenella ovarian tumour (Et-OTU) DUB was shown to regulate the telomerase activity of E. tenella and affect E. tenella proliferation. The amino acid sequences of Et-OTU (GenBank: XP_013229759.1) and Eimeria acervulina (E. acervulina) ovarian tumour (Ea-OTUD3) DUB (XP_013250378.1) are 74% identical. Although Et-OTU may regulate E. tenella telomerase activity, whether Ea-OTUD3 affects E. acervulina growth and reproduction remains unclear. We show here that Ea-OTUD3 belongs to the OTU domain class of cysteine protease deubiquitinating enzymes. Ea-OTUD3 is highly linkage-specific, cleaving K48 (Lys48)-, K63-, and K6-linked diubiquitin but not K29-, K33-, and K11-linked diubiquitin. The precise linkage preference of Ea-OTUD3 among these three nonlinear diubiquitin chains is K6 > K48 > K63. Recombinant Ea-OTUD3, but not its catalytic-site mutant Ea-OTUD3 (C247A), exhibits activity against diubiquitin. Ea-OTUD3 removes ubiquitin from the K48-, but to a lesser extent from the K63-linked ubiquitinated E. acervulina proteins of the modified target protein, thereby exhibiting the characteristics of deubiquitinase. This study reveals that the Ea-OTUD3 is a novel functional deubiquitinating enzyme. Furthermore, the Ea-OTUD3 protein may regulate the stability of some K48-linked ubiquitinated E. acervulina proteins.


Assuntos
Coccidiose/parasitologia , Enzimas Desubiquitinantes/metabolismo , Eimeria/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Sequência de Aminoácidos , Biologia Computacional , Enzimas Desubiquitinantes/genética , Eimeria/genética , Humanos , Lisina/metabolismo , Mutagênese Sítio-Dirigida , Filogenia , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
18.
Cancer Res ; 79(1): 33-46, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30341066

RESUMO

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFß during EMT and was required for TGFß-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFß-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Fatores de Transcrição da Família Snail/química , Fator de Crescimento Transformador beta/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Estabilidade Proteica , RNA Interferente Pequeno/genética , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/genética , Células Tumorais Cultivadas , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Biochem Biophys Res Commun ; 509(2): 348-353, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30585151

RESUMO

The E3 ubiquitin (Ub) ligase gp78 plays an important role in endoplasmic reticulum (ER)-associated degradation (ERAD) and regulation of lipid biogenesis. Although a variety of substrates of gp78 have been described, the regulation of the degradation of gp78 itself remains poorly understood. To address this problem, we used co-immunoprecipitation-coupled liquid chromatography-tandem mass spectrometry (Co-IP/LC-MS/MS) to identify novel proteins interacting with gp78. One of the proteins identified in this study is the deubiquitylating (DUB) enzyme USP34 (Ub-specific protease 34). We demonstrate that knockdown of USP34 facilitates proteasomal degradation of gp78 and consequently impairs the function of gp78 in regulating lipid droplet formation. This study unveils a previously unknown function of USP34 in regulating the metabolic stability of gp78 and adds to our understanding of the relevance of partnering of DUBs and E3s in regulation of protein ubiquitylation.


Assuntos
Degradação Associada com o Retículo Endoplasmático , Hepatócitos/metabolismo , Receptores do Fator Autócrino de Motilidade/genética , Proteases Específicas de Ubiquitina/genética , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Células HEK293 , Hepatócitos/citologia , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores do Fator Autócrino de Motilidade/metabolismo , Transdução de Sinais , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação
20.
Mol Cell ; 72(6): 925-941.e4, 2018 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-30576655

RESUMO

BRCA1-deficient tumor cells have defects in homologous-recombination repair and replication fork stability, resulting in PARP inhibitor sensitivity. Here, we demonstrate that a deubiquitinase, USP1, is upregulated in tumors with mutations in BRCA1. Knockdown or inhibition of USP1 resulted in replication fork destabilization and decreased viability of BRCA1-deficient cells, revealing a synthetic lethal relationship. USP1 binds to and is stimulated by fork DNA. A truncated form of USP1, lacking its DNA-binding region, was not stimulated by DNA and failed to localize and protect replication forks. Persistence of monoubiquitinated PCNA at the replication fork was the mechanism of cell death in the absence of USP1. Taken together, USP1 exhibits DNA-mediated activation at the replication fork, protects the fork, and promotes survival in BRCA1-deficient cells. Inhibition of USP1 may be a useful treatment for a subset of PARP-inhibitor-resistant BRCA1-deficient tumors with acquired replication fork stabilization.


Assuntos
Proteína BRCA1/deficiência , Neoplasias da Mama/enzimologia , Replicação do DNA , DNA de Neoplasias/biossíntese , Proteases Específicas de Ubiquitina/metabolismo , Neoplasias do Colo do Útero/enzimologia , Animais , Proteína BRCA1/genética , Sítios de Ligação , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células , Sobrevivência Celular , DNA de Neoplasias/genética , Resistência a Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos Nus , Mutação , Desnaturação de Ácido Nucleico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/genética , Ubiquitinação , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
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