HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, includin...HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).展开更多
泛素化连接酶是泛素蛋白酶体系统中的关键酶之一,主要负责靶蛋白特异性识别以及泛素化系统活性的调控。HUWE1是一类具有HETC(homologous to E6AP C terminus)功能域的泛素化连接酶。研究发现,HUWE1参与细胞凋亡、基因组DNA损伤、肿瘤抑...泛素化连接酶是泛素蛋白酶体系统中的关键酶之一,主要负责靶蛋白特异性识别以及泛素化系统活性的调控。HUWE1是一类具有HETC(homologous to E6AP C terminus)功能域的泛素化连接酶。研究发现,HUWE1参与细胞凋亡、基因组DNA损伤、肿瘤抑制因子调控和神经细胞分化增殖等重要生理过程。现主要针对泛素化连接酶HUWE1在肿瘤发生、男性生殖系统损伤、胚胎发育异常与神经发育疾病中的研究进展以及调节机制进行综述。展开更多
Background:Elucidation of the post-transcriptional modification has led to novel strategies to treat intractable tumors,especially glioblastoma(GBM).The ubiquitin-proteasome system(UPS)mediates a reversible,stringent ...Background:Elucidation of the post-transcriptional modification has led to novel strategies to treat intractable tumors,especially glioblastoma(GBM).The ubiquitin-proteasome system(UPS)mediates a reversible,stringent and stepwise post-translational modification which is closely associated with malignant processes of GBM.To this end,developing novel therapeutic approaches to target the UPS may contribute to the treatment of this disease.This study aimed to screen the vital and aberrantly regulated component of the UPS in GBM.Based on the molecular identification,functional characterization,and mechanism investigation,we sought to elaborate a novel therapeutic strategy to target this vital factor to combat GBM.Methods:We combined glioma datasets and human patient samples to screen and identify aberrantly regulated E3 ubiquitin ligase.Multidimensional database analysis and molecular and functional experiments in vivo and in vitro were used to evaluate the roles of HECT,UBA and WWE domain-containing E3 ubiquitin ligase 1(HUWE1)in GBM.dCas9 synergistic activation mediator system and recombinant adeno-associated virus(rAAV)were used to endogenously overexpress full-length HUWE1 in vitro and in glioma orthotopic xenografts.Results:Low expression of HUWE1 was closely associated with worse prognosis of GBM patients.The ubiquitination and subsequent degradation of N-Myc mediated by HUWE1,leading to the inactivation of downstream Delta-like 1(DLL1)-NOTCH1 signaling pathways,inhibited the proliferation,invasion,and migration of GBM cells in vitro and in vivo.A rAAV dual-vector system for packaging and delivery of dCas9-VP64 was used to augment endogenous HUWE1 expression in vivo and showed an antitumor activity in glioma orthotopic xenografts.Conclusions:The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress GBM progression.Antitumor activity of rAAV dual-vector delivering dCas9-HUWE1 system uncovers a promising therapeutic strategy for GBM.展开更多
泛素-蛋白酶体系统在蛋白质降解时发挥着重要的作用。泛素化过程需要E1泛素激活酶、E2泛素结合酶、E3泛素连接酶协同完成。本研究组前期研究证明E3泛素连接酶HUWE1(HECT,UBA and WWE domain containing 1)可降解表皮生长因子受体(epider...泛素-蛋白酶体系统在蛋白质降解时发挥着重要的作用。泛素化过程需要E1泛素激活酶、E2泛素结合酶、E3泛素连接酶协同完成。本研究组前期研究证明E3泛素连接酶HUWE1(HECT,UBA and WWE domain containing 1)可降解表皮生长因子受体(epidermal growth factor receptor,EGFR),抑制肾小管间质纤维化。为了进一步明确HUWE1抑制肾小管间质纤维化的机制,本研究鉴定了参与HUWE1降解EGFR过程的E2泛素结合酶。通过real-time PCR观察可能与HUWE1发生相互作用的候选E2泛素结合酶在肾脏损伤模型中的表达变化。用小鼠单侧输尿管结扎(unilateral ureteral obstruction,UUO)模型的肾组织和转化生长因子-β(transforming growth factor-β,TGF-β)刺激的人肾脏近端小管上皮细胞(HK-2细胞)来检测候选E2泛素结合酶的表达量变化。结果显示,与对照组相比,E2泛素结合酶UBE2Q2在UUO术后小鼠肾脏组织中mRNA与蛋白水平均显著下调,在TGF-β刺激的HK-2细胞中UBE2Q2 mRNA和蛋白表达水平也显著下调,与HUWE1表达变化趋势一致,表明在肾脏损伤时E2泛素结合酶UBE2Q2表达水平与HUWE1具有协同性。免疫共沉淀(co-immunoprecipitation,Co-IP)和细胞免疫荧光染色结果验证了HUWE1与UBE2Q2的相互作用。在HK-2细胞中敲低UBE2Q2后,HUWE1、EGFR与泛素的结合均显著降低。上述结果提示,UBE2Q2可能是与HUWE1相互作用的E2泛素结合酶,参与HUWE1对肾小管间质纤维化的抑制作用。展开更多
More than 80%of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons,and a lack of regeneration of these cells leads to permanent hearing los...More than 80%of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons,and a lack of regeneration of these cells leads to permanent hearing loss.Therefore,the regeneration of lost hair cells is an important goal for the treatment of deafness.Atoh1 is a basic helix-loop-helix(bHLH)transcription factor that is critical in both the development and regeneration of cochlear hair cells.Atoh1 is transcriptionally regulated by several signaling pathways,including Notch and Wnt signalings.At the post-translational level,it is regulated through the ubiquitin-proteasome pathway.In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development,but also leads to the regeneration of cochlear hair cells after damage.Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells.This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea.展开更多
基金supported by the National Natural Science Foundation of China,No.81771642(to WMX)the New Bud Research Foundation of West China Second University Hospital of China(to GQH)
文摘HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).
文摘泛素化连接酶是泛素蛋白酶体系统中的关键酶之一,主要负责靶蛋白特异性识别以及泛素化系统活性的调控。HUWE1是一类具有HETC(homologous to E6AP C terminus)功能域的泛素化连接酶。研究发现,HUWE1参与细胞凋亡、基因组DNA损伤、肿瘤抑制因子调控和神经细胞分化增殖等重要生理过程。现主要针对泛素化连接酶HUWE1在肿瘤发生、男性生殖系统损伤、胚胎发育异常与神经发育疾病中的研究进展以及调节机制进行综述。
基金from National Key R&D Program of China(2016YFA0101200 to XWB)the National Natural Science Foundation of China(81602196 to TL)+1 种基金the Special Grant for Chongqing Postdoctoral Researcher Research Project(xmT2017001 to TL)the Postdoctoral Support Program for Innovative Talent(BX201600022 to TL)'Open Project of Key Laboratory of Tumor Immunopathology of Ministry of Education(2020jsz603 to YY).
文摘Background:Elucidation of the post-transcriptional modification has led to novel strategies to treat intractable tumors,especially glioblastoma(GBM).The ubiquitin-proteasome system(UPS)mediates a reversible,stringent and stepwise post-translational modification which is closely associated with malignant processes of GBM.To this end,developing novel therapeutic approaches to target the UPS may contribute to the treatment of this disease.This study aimed to screen the vital and aberrantly regulated component of the UPS in GBM.Based on the molecular identification,functional characterization,and mechanism investigation,we sought to elaborate a novel therapeutic strategy to target this vital factor to combat GBM.Methods:We combined glioma datasets and human patient samples to screen and identify aberrantly regulated E3 ubiquitin ligase.Multidimensional database analysis and molecular and functional experiments in vivo and in vitro were used to evaluate the roles of HECT,UBA and WWE domain-containing E3 ubiquitin ligase 1(HUWE1)in GBM.dCas9 synergistic activation mediator system and recombinant adeno-associated virus(rAAV)were used to endogenously overexpress full-length HUWE1 in vitro and in glioma orthotopic xenografts.Results:Low expression of HUWE1 was closely associated with worse prognosis of GBM patients.The ubiquitination and subsequent degradation of N-Myc mediated by HUWE1,leading to the inactivation of downstream Delta-like 1(DLL1)-NOTCH1 signaling pathways,inhibited the proliferation,invasion,and migration of GBM cells in vitro and in vivo.A rAAV dual-vector system for packaging and delivery of dCas9-VP64 was used to augment endogenous HUWE1 expression in vivo and showed an antitumor activity in glioma orthotopic xenografts.Conclusions:The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress GBM progression.Antitumor activity of rAAV dual-vector delivering dCas9-HUWE1 system uncovers a promising therapeutic strategy for GBM.
文摘More than 80%of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons,and a lack of regeneration of these cells leads to permanent hearing loss.Therefore,the regeneration of lost hair cells is an important goal for the treatment of deafness.Atoh1 is a basic helix-loop-helix(bHLH)transcription factor that is critical in both the development and regeneration of cochlear hair cells.Atoh1 is transcriptionally regulated by several signaling pathways,including Notch and Wnt signalings.At the post-translational level,it is regulated through the ubiquitin-proteasome pathway.In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development,but also leads to the regeneration of cochlear hair cells after damage.Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells.This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea.
