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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.
Oncogene ; 38(32): 6051-6064, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31292489

RESUMO

Impaired Wnt signaling pathway plays a crucial role in the development of colorectal cancer through activation of the ß-catenin/TCF7L2 complex. Although genes upregulated by Wnt/ß-catenin signaling have been intensively studied, the roles of downregulated genes are poorly understood. Previously, we reported that interferon-induced proteins with tetratricopeptide repeats 2 (IFIT2) was downregulated by the Wnt/ß-catenin signaling, and that the suppressed expression of IFIT2 conferred antiapoptotic property to colorectal cancer (CRC) cells. However, the mechanisms underlying how Wnt/ß-catenin signaling regulates IFIT2 remain to be elucidated. In this study, we have uncovered that the expression of IFIT2 is induced by IRF1, which is negatively regulated by the Wnt/ß-catenin signaling. In addition, we found that downregulation of IRF1 is mediated by its degradation through the ubiquitination-proteasome pathway, and that decreased activity of a deubiquitinase complex containing USP1 and UAF1 is involved in the degradation of IRF1 by Wnt/ß-catenin signaling. These data should provide better understanding of the Wnt signaling pathway and human carcinogenesis.


Assuntos
Apoptose/fisiologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Fator Regulador 1 de Interferon/metabolismo , Proteólise , Apoptose/genética , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação/genética , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo
3.
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
4.
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
5.
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
6.
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
7.
Int J Oncol ; 55(1): 277-288, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31180526

RESUMO

Ubiquitin­specific protease 39 (USP39), as one of the deubiquitinating enzymes (DUBs), exhibits aberrant an expression and has oncogenic functions in several types of cancer. However, the function and underlying molecular mechanisms of action of USP39 in ovarian cancer remain largely undetermined. The present study thus aimed to investigate whether USP39 is a promising tumor­associated gene and whether it could be a viable target for overcoming chemotherapeutic resistance in ovarian cancer. The present study identified that USP39 was highly expressed in ovarian cancer samples with carboplatin resistance. A series of functional assays revealed that the knockdown of USP39 in ES2 and SKOV3 cells significantly decreased cell proliferation, induced cell cycle arrest at the G2/M phase and impaired the cell colony formation ability. USP39 deficiency enhanced the carboplatin­induced apoptosis of the SKOV3 cells via the activation of poly­ADP ribose polymerase and caspase­3. USP39 knockdown led to the inhibition of cell migration and invasion. The opposite effects were observed when USP39 was overexpressed in the ES2 and SKOV3 cells. In vivo animal models revealed that the subcutaneous transplantation and intraperitoneal injection of USP39­overexpressing ES2 cells increased tumor burden with or without treatment with carboplatin. However, the knockdown of USP39 suppressed SKOV3 cell growth in vivo. Mechanistic analyses also demonstrated that USP39 induced the phosphorylation of extracellular signal­regulated kinase and AKT and increased the expression of epidermal growth factor receptor and cyclin B1. Collectively, the findings of this study suggest that USP39 may paly a vital role in regulating ovarian cancer malignant phenotypes and carboplatin resistance. Therefore, USP39 may prove to be a promising therapeutic target for patients with ovarian cancer.


Assuntos
Carboplatina/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Células HEK293 , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Ovarianas/patologia , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Distribuição Aleatória , Proteases Específicas de Ubiquitina/biossíntese
8.
Cell Physiol Biochem ; 53(1): 157-171, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31251006

RESUMO

BACKGROUND/AIMS: Dysregulation of deubiquitinating enzymes (DUBs), which regulate the stability of key proteins, has been implicated in many human diseases, including cancers. Thus, DUBs can be considered as potential therapeutic targets for many diseases. Among them, USP4 has been proposed as a promising target for colon cancer drugs since USP4 controls the stability of ß-catenin, a key factor in the Wnt signaling involved in the tumorigenesis of colorectal cancer. However, developing potential DUB inhibitors has been hindered because many DUBs harbor similar active site structures and show broad substrate specificities. METHODS: By performing in vitro deubiquitinating activity assays using a chemical library, we identified several potential DUB inhibitors. Among them, only neutral red (NR) showed selective inhibitory activity on USP4 in a cell-based assay system. In colon cancer cells, NR affected the protein stability of ß-catenin, as shown by immunoblotting, and it affected the target gene expression of ß-catenin, as shown by quantitative real-time PCR. NR's potential as an anticancer drug was further estimated by colony formation and cell migration assays and by using a mouse xenograft model. RESULTS: We identified NR as an uncompetitive inhibitor of USP4 and validated its effects in colorectal cancer. NR-treated cells showed decreased ß-catenin stability and reduced expression of ß-catenin target genes. Additionally, treating colon cancer cells with NR significantly reduced colony formation and cell migration, and injecting NR into a mouse xenograft model reduced the tumor volume. CONCLUSION: The current results suggest that NR could be developed as an anticancer drug targeting USP4, and they support the possibility of developing specific DUB inhibitors as therapeutic agents.


Assuntos
Vermelho Neutro/farmacologia , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ciclina D1/genética , Ciclina D1/metabolismo , Progressão da Doença , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Vermelho Neutro/química , Vermelho Neutro/uso terapêutico , Transplante Heterólogo , Proteases Específicas de Ubiquitina/metabolismo
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.
J Biomed Sci ; 26(1): 42, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133011

RESUMO

Protein ubiquitination is an important mechanism for regulating the activity and levels of proteins under physiological conditions. Loss of regulation by protein ubiquitination leads to various diseases, such as cancer. Two types of enzymes, namely, E1/E2/E3 ligases and deubiquitinases, are responsible for controlling protein ubiquitination. The ubiquitin-specific peptidases (USPs) are the main members of the deubiquitinase family. Many studies have addressed the roles of USPs in various diseases. An increasing number of studies have indicated that USPs are critical for cancer progression, and some USPs have been used as targets to develop inhibitors for cancer prevention. Herein we collect and organize most of the recent studies on the roles of USPs in cancer progression and discuss the development of USP inhibitors for cancer therapy in the future.


Assuntos
Progressão da Doença , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Proteases Específicas de Ubiquitina/antagonistas & inibidores , Proteases Específicas de Ubiquitina/metabolismo , Humanos , Ubiquitinação
11.
Cells ; 8(3)2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901874

RESUMO

Aquaporin 2 (AQP2) mediates the osmotic water permeability of the kidney collecting duct in response to arginine vasopressin (VP) and is essential for body water homeostasis. VP effects on AQP2 occur via long-term alterations in AQP2 abundance and short-term changes in AQP2 localization. Several of the effects of VP on AQP2 are dependent on AQP2 phosphorylation and ubiquitylation; post-translational modifications (PTM) that modulate AQP2 subcellular distribution and function. Although several protein kinases, phosphatases, and ubiquitin E3 ligases have been implicated in AQP2 PTM, how AQP2 is deubiquitylated or the role of deubiquitylases (DUBS) in AQP2 function is unknown. Here, we report a novel role of the ubiquitin-specific protease USP4 in modulating AQP2 function. USP4 co-localized with AQP2 in the mouse kidney, and in mpkCCD14 cells USP4 and AQP2 abundance are increased by VP. AQP2 and USP4 co-immunoprecipitated from mpkCCD14 cells and mouse kidney, and in vitro, USP4 can deubiquitylate AQP2. In mpkCCD14 cells, shRNA mediated knockdown of USP4 decreased AQP2 protein abundance, whereas no changes in AQP2 mRNA levels or VP-induced cAMP production were detected. VP-induced AQP2 membrane accumulation in knockdown cells was significantly reduced, which was associated with higher levels of ubiquitylated AQP2. AQP2 protein half-life was also significantly reduced in USP4 knockdown cells. Taken together, the data suggest that USP4 is a key regulator of AQP2 deubiquitylation and that loss of USP4 leads to increased AQP2 ubiquitylation, decreased AQP2 levels, and decreased cell surface AQP2 accumulation upon VP treatment. These studies have implications for understanding body water homeostasis.


Assuntos
Aquaporina 2/metabolismo , Membrana Celular/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Meia-Vida , Rim/citologia , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica , Ubiquitinação/efeitos dos fármacos , Vasopressinas/farmacologia
12.
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
13.
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
14.
mBio ; 10(2)2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890612

RESUMO

Human papillomaviruses (HPVs) are important pathogens with a significant medical burden. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism. In this report, we provide evidence that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 deubiquitinating enzyme complex, a member of the Fanconi anemia DNA damage response pathway, is required for the completion of the bidirectional theta replication of the HPV11 genome and the subsequent initiation of the unidirectional replication. We show that unidirectional replication proceeds via theta structures and is supported by the cellular Bloom helicase, which interacts directly with E1 and whose engagement in HPV11 replication requires UAF1-USP1 activity. We propose that the unidirectional replication of the HPV11 genome initiates from replication fork restart events. These findings suggest a new role for the Fanconi anemia pathway in HPV replication.IMPORTANCE Human papillomaviruses (HPVs) are important pathogens that replicate their double-stranded circular DNA genome in the nucleus of infected cells. HPV genomes replicate in infected cells via bidirectional theta replication and a poorly understood unidirectional mechanism, and the onset of viral replication requires the engagement of cellular DNA damage response pathways. In this study, we showed that the previously described interaction between the viral E1 helicase and the cellular UAF1-USP1 complex is necessary for the completion of bidirectional replication and the subsequent initiation of the unidirectional replication mechanism. Our results suggest HPVs may use the cellular Fanconi anemia DNA damage pathway to achieve the separation of daughter molecules generated by bidirectional theta replication. Additionally, our results indicate that the unidirectional replication of the HPV genome is initiated from restarted bidirectional theta replication forks.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Interações Hospedeiro-Patógeno , Papillomavirus Humano 11/fisiologia , Proteínas Nucleares/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Linhagem Celular , Humanos , Ligação Proteica
15.
Methods Enzymol ; 618: 281-319, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850056

RESUMO

Ubiquitin-specific proteases (USPs) are an important class of deubiquitinating enzymes (DUBs) that carry out critical roles in cellular physiology and are regulated at multiple levels. Quantitative characterization of USP activity is crucial for mechanistic understanding of USP function and regulation. This requires kinetic analysis using in vitro activity assays on minimal and natural substrates with purified proteins. In this chapter we give advice for efficient design of USP constructs and their optimal expression, followed by a series of purification strategies. We then present protocols for studying USP activity quantitatively on minimal and more natural substrates, and we discuss how to include possible regulatory elements such as internal USP domains or external interacting proteins. Lastly, we examine different binding assays for studying USP interactions and discuss how these can be included in full kinetic analyses.


Assuntos
Proteases Específicas de Ubiquitina/metabolismo , Animais , Ensaios Enzimáticos/métodos , Humanos , Cinética , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas , Especificidade por Substrato , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/isolamento & purificação
16.
Blood ; 133(14): 1560-1571, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30755420

RESUMO

Hematopoietic stem cell (HSC) homeostasis is controlled by cytokine receptor-mediated Janus kinase 2 (JAK2) signaling. We previously found that JAK2 is promptly ubiquitinated upon cytokine stimulation. Whether a competing JAK2 deubiquitination activity exists is unknown. LNK is an essential adaptor protein that constrains HSC expansion through dampening thrombopoietin (TPO)-induced JAK2 signaling. We show here that a LNK-associated lysine-63 (K63)-deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling. We pinpoint a direct interaction between the LNK SH2 domain and a phosphorylated tyrosine residue in KIAA0157 (Abraxas2), a unique and defining BRISC component. Kiaa0157 deficiency in mice led to an expansion of phenotypic and functional HSCs. Endogenous JAK2 and phospho-JAK2 were rapidly K63-ubiquitinated upon TPO stimulation, and this action was augmented in cells depleted of the BRISC core components KIAA0157, MERIT40, or BRCC36. This increase in JAK2 ubiquitination after BRISC knockdown was associated with increased TPO-mediated JAK2 activation and protein levels, and increased MPL receptor presence at the cell surface. In addition, BRISC depletion promoted membrane proximal association between the MPL receptor and pJAK2/JAK2, thus enhancing activated JAK2/MPL at the cell membrane. These findings define a novel pathway by which K63-ubiquitination promotes JAK2 stability and activation in a proteasome-independent manner. Moreover, mutations in BRCC36 are found in clonal hematopoiesis in humans. This research may shed light on the mechanistic understanding of a potential role of BRCC36 in human HSCs.


Assuntos
Proliferação de Células , Enzimas Desubiquitinantes/fisiologia , Células-Tronco Hematopoéticas/citologia , Janus Quinase 2/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Enzimas Desubiquitinantes/genética , Humanos , Camundongos , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores de Trombopoetina/metabolismo , Transdução de Sinais , Trombopoetina/farmacologia , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitinação , Domínios de Homologia de src
17.
Cancer Lett ; 449: 114-124, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30771428

RESUMO

The serine/threonine kinase, CHK2 (checkpoint kinase 2), is a key mediator in DNA damage response and a tumor suppressor, which is implicated in promoting cell cycle arrest, apoptosis and DNA repair. Accumulating evidence suggests that these functions are primarily exerted through phosphorylation downstream factors such as p53 and BRCA1. Recent studies have shown that ubiquitination is an important mode of regulation of CHK2. However, it remains largely unclear whether deubiquitinases participate in regulation of CHK2. Here, we report that a deubiquitinase, USP39, is a new regulator of CHK2. Mechanistically, USP39 deubiquitinates and stabilizes CHK2, which in turn enhances CHK2 stability. Short hairpin RNA (shRNA) mediated knockdown of USP39 led to deregulate CHK2, which resulted in compromising the DNA damage-induced G2/M checkpoint, decreasing apoptosis, and conferring cancer cells resistance to chemotherapy drugs and radiation treatment. Collectively, we identify USP39 as a novel regulator of CHK2 in the DNA damage response.


Assuntos
Quinase do Ponto de Checagem 2/química , Quinase do Ponto de Checagem 2/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/metabolismo , Tolerância a Radiação , Proteases Específicas de Ubiquitina/metabolismo , Células A549 , Ciclo Celular , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Estabilidade Proteica , Ubiquitinação , Regulação para Cima
18.
World J Gastroenterol ; 25(7): 824-836, 2019 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-30809082

RESUMO

BACKGROUND: Intestinal ischemia reperfusion (I/R) injury is a serious but common pathophysiological process of many diseases, resulting in a high mortality rate in clinical practice. Ubiquitin-specific protease 22 (USP22) acts as regulator of cell cycle progression, proliferation, and tumor invasion. Depleted USP22 expression has been reported to contribute to arrested cell cycle and disrupted generation of differentiated cell types in crypts and villi. However, the role of USP22 in intestinal damage recovery has not been investigated. Therefore, elucidation of the underlying mechanism of USP22 in intestinal I/R injury may help to improve the tissue repair and patient prognosis in clinical practice. AIM: To investigate the role of USP22 in intestinal cell proliferation and regeneration after intestinal I/R injury. METHODS: An animal model of intestinal I/R injury was generated in male Sprague-Dawley rats by occlusion of the superior mesenteric artery followed by reperfusion. Chiu's scoring system was used to grade the damage to the intestinal mucosa. An in vitro model was developed by incubating rat intestinal epithelial IEC-6 cells in hypoxia/reoxygenation conditions in order to simulate I/R in vivo. siRNA and overexpression plasmid were used to regulate the expression of USP22. USP22, Cyclin D1, and proliferating cell nuclear antigen (PCNA) expression levels were measured by Western blot analysis and immunohistochemistry staining. Cell survival (viability) and cell cycle were evaluated using the Cell Counting Kit-8 and flow cytometry, respectively. RESULTS: USP22 expression was positively correlated with the expression levels of PCNA and Cyclin D1 both in vivo and in vitro, which confirmed that USP22 was involved in cell proliferation and intestinal regeneration after intestinal I/R injury. Decreased levels of Cyclin D1 and cell cycle arrest were observed in the USP22 knockdown group (P < 0.05), while opposite results were observed in the USP22 overexpression group (P < 0.05). In addition, increased expression of USP22 was related to improved intestinal pathology or IEC-6 cell viability after I/R or hypoxia/reoxygenation. These results suggested that USP22 may exert a protective effect on intestinal I/R injury by regulating cell proliferation and facilitating tissue regeneration. CONCLUSION: USP22 is correlated with promoting intestinal cell proliferation and accelerating intestinal tissue regeneration after intestinal I/R injury and may serve as a potential target for therapeutic development for tissue repair during intestinal I/R injury.


Assuntos
Proliferação de Células , Enzimas Desubiquitinantes/metabolismo , Mucosa Intestinal/patologia , Regeneração , Traumatismo por Reperfusão/patologia , Proteases Específicas de Ubiquitina/metabolismo , Animais , Linhagem Celular , Enzimas Desubiquitinantes/genética , Modelos Animais de Doenças , Humanos , Mucosa Intestinal/citologia , Masculino , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/etiologia
19.
Rheumatology (Oxford) ; 58(4): 708-718, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30608617

RESUMO

OBJECTIVE: Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-ß signalling in primary fibroblast cell lines. METHODS: High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-ß stimulation. RESULTS: Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-ß signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-ß induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-ß-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action. CONCLUSION: Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-ß, and is a potential therapeutic target in SSc.


Assuntos
Fibroblastos/metabolismo , Escleroderma Sistêmico/enzimologia , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Humanos , Escleroderma Sistêmico/tratamento farmacológico
20.
Mol Cells ; 42(1): 17-27, 2019 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-30622230

RESUMO

Ubiquitin-specific protease 44 (USP44) has been implicated in tumor progression and metastasis across various tumors. However, the function of USP44 in prostate cancers and regulatory mechanism of histone-modifying enzymes by USP44 in tumors is not well-understood. Here, we found that enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 methyltransferase, is regulated by USP44. We showed that EZH2 is a novel target of USP44 and that the protein stability of EZH2 is upregulated by USP44-mediated deubiquitination. In USP44 knockdown prostate cancer cells, the EZH2 protein level and its gene silencing activity were decreased. Furthermore, USP44 knockdown inhibited the tumorigenic characteristics and cancer stem cell-like behaviors of prostate cancer cells. Inhibition of tumorigenesis caused by USP44 knockdown was recovered by ectopic introduction of EZH2. Additionally, USP44 regulates the protein stability of oncogenic EZH2 mutants. Taken together, our results suggest that USP44 promotes the tumorigenesis of prostate cancer cells partly by stabilizing EZH2 and that USP44 is a viable therapeutic target for treating EZH2-dependent cancers.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteases Específicas de Ubiquitina/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Proteínas Mutantes/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias da Próstata/genética , Ligação Proteica , Estabilidade Proteica , Ubiquitinação
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