Background: Tumor angiogenesis is related to solid tumor occurrence. Ubiquitin-specific peptidase 13 (USP13) is a deubiquitinating enzyme with a pivotal effect on tumor proliferation, metastasis, and tumorigenesis. No...Background: Tumor angiogenesis is related to solid tumor occurrence. Ubiquitin-specific peptidase 13 (USP13) is a deubiquitinating enzyme with a pivotal effect on tumor proliferation, metastasis, and tumorigenesis. Nonetheless, its effect on colorectal cancer (CRC) angiogenesis remains poorly understood. Methods: Human umbilical vein endothelial cells (HUVECs) and CRC cells were cultivated, followed by USP13 knockdown/overexpression using shRNA lentiviral vectors or plasmids. Conditioned media (CM) from treated CRC cells were collected to assess HUVEC migration, invasion, and tube formation. Phosphatase and tensin homolog (PTEN) overexpression and recombinant vascular endothelial growth factor A (VEGFA) rescue experiments were performed. Molecular mechanisms were analyzed via Western blot (PTEN, p-AKT, VEGFA), co-immunoprecipitation (PTEN ubiquitination), and in vivo nude mice study to detect the role of USP13 in tumor angiogenesis. Results: USP13 expression in CRC cells is downregulated and negatively related to platelet endothelial cell adhesion molecule-1 (CD31) expression. Furthermore, the conditioned medium from CRC cells with USP13 knockdown significantly promoted HUVEC migration, invasion, and tube formation, while USP13 overexpression exerted the opposite effect. Additionally, USP13 overexpression significantly increased PTEN expression while decreasing protein kinase B (AKT) phosphorylation levels. Concurrently, USP13 overexpression significantly reduced PTEN ubiquitination, whereas USP13 knockdown remarkably increased this modification. Overexpression of PTEN in sh-USP13 CRC cells decreased the expression levels of VEGFA and p-AKT. USP13 also inhibited tumor angiogenesis through downregulating VEGFA, and recombinant VEGFA blocked the inhibition of the conditioned medium from USP13-overexpressing CRC cells against HUVEC angiogenesis in vivo. Conclusions: USP13 downregulated VEGFA and inhibited tumor angiogenesis via the PTEN-AKT pathway.展开更多
为了探究去泛素化酶USP13在前列腺癌中发挥的功能及其相关分子机制,采用PCR、酶切、酶连和转化大肠杆菌等方法,成功构建USP13基因真核表达重组质粒.同时,在前列腺癌细胞PC-3和DU145中转染USP13质粒,进行CCK-8实验、克隆形成、划痕以及Tr...为了探究去泛素化酶USP13在前列腺癌中发挥的功能及其相关分子机制,采用PCR、酶切、酶连和转化大肠杆菌等方法,成功构建USP13基因真核表达重组质粒.同时,在前列腺癌细胞PC-3和DU145中转染USP13质粒,进行CCK-8实验、克隆形成、划痕以及Transwell迁移实验;利用逆转录PCR、蛋白质免疫印迹法和泛素化Co-IP等实验,分析USP13过表达对信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)的影响.实验结果发现,USP13可以抑制前列腺癌细胞的增殖、克隆和迁移能力;过表达USP13不影响STAT1 mRNA水平,但上调STAT1蛋白水平,而敲低USP13则下调STAT1蛋白水平;同时,过表达USP13降低了STAT1泛素化水平,敲低USP13则提高STAT1泛素化水平.展开更多
Doxorubicin(Dox)is an anthracycline drug widely applied in various malignancies.However,the fatal cardiotoxicity induced by Dox limits its clinical application.Post-transcriptional protein modification via ubiquitinat...Doxorubicin(Dox)is an anthracycline drug widely applied in various malignancies.However,the fatal cardiotoxicity induced by Dox limits its clinical application.Post-transcriptional protein modification via ubiquitination/deubiquitination in cardiomyocytes mediates the pathophysiological process in Dox-induced cardiotoxicity(DIC).In this study,we aimed to clarify the regulatory role and mechanism of a deubiquitinating enzyme,ubiquitin-specific peptidase 13(USP13),in DIC.RNA-seq analysis and experimental examinations identified that cardiomyocyte-derived USP13 positively correlated with DIC.Mice with cardiac-specific deletion of USP13 were subjected to Dox modeling.Adeno-associated virus serotype 9(AAV9)carrying cTNT promoter was constructed to overexpress USP13 in mouse heart tissues.Cardiomyocyte-specific knockout of USP13 exacerbated DIC,while its overexpression mitigated DIC in mice.Mechanistically,USP13 deubiquitinates the stimulator of interferon genes(STING)and promotes the autolysosome-related degradation of STING,subsequently alleviating cardiomyocyte inflammation and death.Our study suggests that USP13 serves a cardioprotective role in DIC and indicates USP13 as a potential therapeutic target for DIC treatment.展开更多
Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis(NASH)have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated.Inactive rhomboid p...Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis(NASH)have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated.Inactive rhomboid protein 2(IRHOM2),a promising target for treatment of inflammation-related diseases,contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis(NASH)progression.However,the molecular mechanism underlying Irhom2 regulation is still not completely understood.In this work,we identify the ubiquitin-specific protease 13(USP13)as a critical and novel endogenous blocker of IRHOM2,and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes.Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis,followed by glycometabolic disorder,lipid deposition,increased inflammation,and markedly promotes NASH development.Conversely,transgenic mice with Usp13 overexpression,lentivirus(LV)-or adeno-associated virus(AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent.Mechanistically,in response to metabolic stresses,USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N(UBC13),a ubiquitin E2 conjugating enzyme,and thus prevents its activation of downstream cascade pathway.USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.展开更多
基金supported by the Clinical Frontier Technology Program of the First Affiliated Hospital of Jinan University,China(No.JNU1AF-CFTP-2022-a01223)Guangzhou Science and Technology Plan City-School Joint Funding Project(2024A03J0825)+2 种基金Science and Technology Projects in Guangzhou(202201020566)Medical Joint Fund of Jinan University(YXJC2022005)the Fundamental Research Funds for the Central Universities(21623319).
文摘Background: Tumor angiogenesis is related to solid tumor occurrence. Ubiquitin-specific peptidase 13 (USP13) is a deubiquitinating enzyme with a pivotal effect on tumor proliferation, metastasis, and tumorigenesis. Nonetheless, its effect on colorectal cancer (CRC) angiogenesis remains poorly understood. Methods: Human umbilical vein endothelial cells (HUVECs) and CRC cells were cultivated, followed by USP13 knockdown/overexpression using shRNA lentiviral vectors or plasmids. Conditioned media (CM) from treated CRC cells were collected to assess HUVEC migration, invasion, and tube formation. Phosphatase and tensin homolog (PTEN) overexpression and recombinant vascular endothelial growth factor A (VEGFA) rescue experiments were performed. Molecular mechanisms were analyzed via Western blot (PTEN, p-AKT, VEGFA), co-immunoprecipitation (PTEN ubiquitination), and in vivo nude mice study to detect the role of USP13 in tumor angiogenesis. Results: USP13 expression in CRC cells is downregulated and negatively related to platelet endothelial cell adhesion molecule-1 (CD31) expression. Furthermore, the conditioned medium from CRC cells with USP13 knockdown significantly promoted HUVEC migration, invasion, and tube formation, while USP13 overexpression exerted the opposite effect. Additionally, USP13 overexpression significantly increased PTEN expression while decreasing protein kinase B (AKT) phosphorylation levels. Concurrently, USP13 overexpression significantly reduced PTEN ubiquitination, whereas USP13 knockdown remarkably increased this modification. Overexpression of PTEN in sh-USP13 CRC cells decreased the expression levels of VEGFA and p-AKT. USP13 also inhibited tumor angiogenesis through downregulating VEGFA, and recombinant VEGFA blocked the inhibition of the conditioned medium from USP13-overexpressing CRC cells against HUVEC angiogenesis in vivo. Conclusions: USP13 downregulated VEGFA and inhibited tumor angiogenesis via the PTEN-AKT pathway.
文摘为了探究去泛素化酶USP13在前列腺癌中发挥的功能及其相关分子机制,采用PCR、酶切、酶连和转化大肠杆菌等方法,成功构建USP13基因真核表达重组质粒.同时,在前列腺癌细胞PC-3和DU145中转染USP13质粒,进行CCK-8实验、克隆形成、划痕以及Transwell迁移实验;利用逆转录PCR、蛋白质免疫印迹法和泛素化Co-IP等实验,分析USP13过表达对信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)的影响.实验结果发现,USP13可以抑制前列腺癌细胞的增殖、克隆和迁移能力;过表达USP13不影响STAT1 mRNA水平,但上调STAT1蛋白水平,而敲低USP13则下调STAT1蛋白水平;同时,过表达USP13降低了STAT1泛素化水平,敲低USP13则提高STAT1泛素化水平.
基金supported by National Natural Science Foundation of China(82271347 to Gaojun Wu and U24A20814 to Guang Liang).
文摘Doxorubicin(Dox)is an anthracycline drug widely applied in various malignancies.However,the fatal cardiotoxicity induced by Dox limits its clinical application.Post-transcriptional protein modification via ubiquitination/deubiquitination in cardiomyocytes mediates the pathophysiological process in Dox-induced cardiotoxicity(DIC).In this study,we aimed to clarify the regulatory role and mechanism of a deubiquitinating enzyme,ubiquitin-specific peptidase 13(USP13),in DIC.RNA-seq analysis and experimental examinations identified that cardiomyocyte-derived USP13 positively correlated with DIC.Mice with cardiac-specific deletion of USP13 were subjected to Dox modeling.Adeno-associated virus serotype 9(AAV9)carrying cTNT promoter was constructed to overexpress USP13 in mouse heart tissues.Cardiomyocyte-specific knockout of USP13 exacerbated DIC,while its overexpression mitigated DIC in mice.Mechanistically,USP13 deubiquitinates the stimulator of interferon genes(STING)and promotes the autolysosome-related degradation of STING,subsequently alleviating cardiomyocyte inflammation and death.Our study suggests that USP13 serves a cardioprotective role in DIC and indicates USP13 as a potential therapeutic target for DIC treatment.
基金supported by National Natural Science Foundation of China(NSFC Grant Nos.81703527 and 82200652)Chongqing Research Program of Basic Research and Frontier Technology(Grant Nos.cstc2017jcyjAX0356,cstc2018jcyjA3686,cstc2018jcyjAX0784,cstc2018jcyjA1472,cstc2018jcyjAX0811,cstc2018jcyjA3533,and KJZD-M201801601,China)+4 种基金School-level Research Program of Chongqing University of Education(Grant Nos.KY201710B and 17GZKP01,China)Advanced Programs of Post-doctor of Chongqing(Grant No.2017LY39,China)Science and Technology Research Program of Chongqing Education Commission of China(Grant Nos.KJQN201901608,KJQN201901615,KJ1601402,and KJZDK202001603)Children's Research Institute of National Center for Schooling Development Programme and Chongqing University of Education(Grant No.CSDP19FSO1108,China)Chongqing Professional Talents Plan for Innovation and Entrepreneurship Demonstration Team(CQCY201903258,China).
文摘Nowadays potential preclinical drugs for the treatment of nonalcoholic steatohepatitis(NASH)have failed to achieve expected therapeutic efficacy because the pathogenic mechanisms are underestimated.Inactive rhomboid protein 2(IRHOM2),a promising target for treatment of inflammation-related diseases,contributes to deregulated hepatocyte metabolism-associated nonalcoholic steatohepatitis(NASH)progression.However,the molecular mechanism underlying Irhom2 regulation is still not completely understood.In this work,we identify the ubiquitin-specific protease 13(USP13)as a critical and novel endogenous blocker of IRHOM2,and we also indicate that USP13 is an IRHOM2-interacting protein that catalyzes deubiquitination of Irhom2 in hepatocytes.Hepatocyte-specific loss of the Usp13 disrupts liver metabolic homeostasis,followed by glycometabolic disorder,lipid deposition,increased inflammation,and markedly promotes NASH development.Conversely,transgenic mice with Usp13 overexpression,lentivirus(LV)-or adeno-associated virus(AAV)-driven Usp13 gene therapeutics mitigates NASH in 3 models of rodent.Mechanistically,in response to metabolic stresses,USP13 directly interacts with IRHOM2 and removes its K63-linked ubiquitination induced by ubiquitin-conjugating enzyme E2N(UBC13),a ubiquitin E2 conjugating enzyme,and thus prevents its activation of downstream cascade pathway.USP13 is a potential treatment target for NASH therapy by targeting the Irhom2 signaling pathway.
