Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite ou...Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite outgrowth remains unknown.Here,we found that spastin interacted with ubiquitin and was significantly degraded by K48-mediated poly-ubiquitination.Cullin3 facilitated spastin degradation and ubiquitination.RING-box protein 1,but not RING-box protein 2,acted synergistically with Cullin3 protein to regulate spastin degradation.Overexpression of Culin3 or BRX1 markedly suppressed spastin expression,and inhibited spastin-mediated microtubule severing and promotion of neurite outgrowth.Moreover,USP14 interacted directly with spastin to mediate its deubiquitination.USP14 overexpression significantly increased spastin expression and suppressed its ubiquitination and degradation.Although co-expression of spastin and USP14 did not enhance microtubule severing,it did increase neurite length in hippocampal neurons.Taken together,these findings elucidate the intricate regulatory mechanisms of spastin turnover,highlighting the roles of the Cullin-3–Ring E3 ubiquitin ligase complex and USP14 in orchestrating its ubiquitination and degradation.The dynamic interplay between these factors governs spastin stability and function,ultimately influencing microtubule dynamics and neuronal morphology.These insights shed light on potential therapeutic targets for neurodegenerative disorders associated with spastin defects.展开更多
MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses. In this study, we found that activation of toll-like receptor 4 （TLR4） rapidly increased the level of microRNA-124 （miR- ...MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses. In this study, we found that activation of toll-like receptor 4 （TLR4） rapidly increased the level of microRNA-124 （miR- 124） in lipopolysaccharide （LPS）-treated macrophages and mice. MiR-124 knockdown significantly increased the production of the pro-inflammatory cytokine TNF-oL at the post-transcriptional level in LPS-triggered macrophages. Furthermore, miR-124 knockdown or overexpression significanly increased or decreased the protein stability of TNF- α. We found miR-124 directly targeted ubiquitin-specific proteases 2 （USP2） and 14 （USP14）, two components of deubiquitinating enzymes. Knockdown of USP2 and USP14 attenuated the miR-124-mediated protein degradation of TNF-α. Together, our data identify miR-124 as an important feedback negative regulator for LPS-induced pro- duction of TNF-α by targeting USP2 and USP14, thus outlining new mechanisms for fine-tuning the TLR-triggered inflammatory response.展开更多
胰腺癌仍然是最致命的癌症之一,而且较少发现有效的治疗方案。虽然在许多肿瘤细胞,包括胰腺癌细胞中观察到泛素特异性蛋白酶14(ubiquitin specific peptidase 14,USP14)的过度表达,但它在胰腺癌中的确切作用仍未得到较好的阐明。我们研...胰腺癌仍然是最致命的癌症之一,而且较少发现有效的治疗方案。虽然在许多肿瘤细胞,包括胰腺癌细胞中观察到泛素特异性蛋白酶14(ubiquitin specific peptidase 14,USP14)的过度表达,但它在胰腺癌中的确切作用仍未得到较好的阐明。我们研究了USP14在胰腺癌中的生物学功能及其分子机制。对癌症基因组图谱数据库(The Cancer Genome Atlas,TCGA)的分析显示USP14在胰腺癌组织中高度表达,进一步探究发现,其表达水平与患者的预后呈负相关。利用shRNAUSP14慢病毒建立了稳定的USP14敲低胰腺癌细胞系,通过CCK8、克隆形成实验、划痕和Transwell试验发现,USP14敲低抑制了胰腺癌细胞的增殖和迁移能力。在胰腺癌细胞SW1990和MIAPaCa2中蛋白质印迹结果表明,下调USP14的表达导致细胞周期蛋白D3(cyclin D3)水平下降,而过表达USP14会增加细胞周期蛋白D3表达水平。此外,免疫共沉淀证明,USP14与细胞周期蛋白D3相互作用,泛素化结果显示,USP14的过表达降低了细胞周期蛋白D3的泛素化水平。并且在SW1990胰腺癌细胞中,CRISPR/Cas9介导的USP14敲除导致细胞周期蛋白D3水平下降。这些发现表明,USP14通过与细胞周期蛋白D3的相互作用促进了胰腺癌细胞的增殖和迁移能力,凸显了USP14作为胰腺癌的潜在治疗靶点。展开更多
基金supported by the National Natural Science Foundation of China,No.32071033(to MT)Basic and Applied Basic Research Foundation of Guangdong Province,Nos.2023A1515010140(to MT),2022A1515140169(to MT),2022A1515111096(to ZC)+3 种基金Science and Technology Project of Guangzhou,Nos.202201010015(to YL),2023A03J0790(to TJ)Basic and Applied Basic Research Foundation of Guangzhou,No.2023A04J1285(to ZC)Medical Research Foundation of Guangdong Province,No.A2023147(to ZC)Health Science and Technology Project of Guangzhou,No.20221A011039(to TJ)。
文摘Post-translational modification of spastin enables precise spatiotemporal control of its microtubule severing activity.However,the detailed mechanism by which spastin turnover is regulated in the context of neurite outgrowth remains unknown.Here,we found that spastin interacted with ubiquitin and was significantly degraded by K48-mediated poly-ubiquitination.Cullin3 facilitated spastin degradation and ubiquitination.RING-box protein 1,but not RING-box protein 2,acted synergistically with Cullin3 protein to regulate spastin degradation.Overexpression of Culin3 or BRX1 markedly suppressed spastin expression,and inhibited spastin-mediated microtubule severing and promotion of neurite outgrowth.Moreover,USP14 interacted directly with spastin to mediate its deubiquitination.USP14 overexpression significantly increased spastin expression and suppressed its ubiquitination and degradation.Although co-expression of spastin and USP14 did not enhance microtubule severing,it did increase neurite length in hippocampal neurons.Taken together,these findings elucidate the intricate regulatory mechanisms of spastin turnover,highlighting the roles of the Cullin-3–Ring E3 ubiquitin ligase complex and USP14 in orchestrating its ubiquitination and degradation.The dynamic interplay between these factors governs spastin stability and function,ultimately influencing microtubule dynamics and neuronal morphology.These insights shed light on potential therapeutic targets for neurodegenerative disorders associated with spastin defects.
文摘MicroRNAs play pivotal roles in the regulation of both innate and adaptive immune responses. In this study, we found that activation of toll-like receptor 4 （TLR4） rapidly increased the level of microRNA-124 （miR- 124） in lipopolysaccharide （LPS）-treated macrophages and mice. MiR-124 knockdown significantly increased the production of the pro-inflammatory cytokine TNF-oL at the post-transcriptional level in LPS-triggered macrophages. Furthermore, miR-124 knockdown or overexpression significanly increased or decreased the protein stability of TNF- α. We found miR-124 directly targeted ubiquitin-specific proteases 2 （USP2） and 14 （USP14）, two components of deubiquitinating enzymes. Knockdown of USP2 and USP14 attenuated the miR-124-mediated protein degradation of TNF-α. Together, our data identify miR-124 as an important feedback negative regulator for LPS-induced pro- duction of TNF-α by targeting USP2 and USP14, thus outlining new mechanisms for fine-tuning the TLR-triggered inflammatory response.
文摘胰腺癌仍然是最致命的癌症之一,而且较少发现有效的治疗方案。虽然在许多肿瘤细胞,包括胰腺癌细胞中观察到泛素特异性蛋白酶14(ubiquitin specific peptidase 14,USP14)的过度表达,但它在胰腺癌中的确切作用仍未得到较好的阐明。我们研究了USP14在胰腺癌中的生物学功能及其分子机制。对癌症基因组图谱数据库(The Cancer Genome Atlas,TCGA)的分析显示USP14在胰腺癌组织中高度表达,进一步探究发现,其表达水平与患者的预后呈负相关。利用shRNAUSP14慢病毒建立了稳定的USP14敲低胰腺癌细胞系,通过CCK8、克隆形成实验、划痕和Transwell试验发现,USP14敲低抑制了胰腺癌细胞的增殖和迁移能力。在胰腺癌细胞SW1990和MIAPaCa2中蛋白质印迹结果表明,下调USP14的表达导致细胞周期蛋白D3(cyclin D3)水平下降,而过表达USP14会增加细胞周期蛋白D3表达水平。此外,免疫共沉淀证明,USP14与细胞周期蛋白D3相互作用,泛素化结果显示,USP14的过表达降低了细胞周期蛋白D3的泛素化水平。并且在SW1990胰腺癌细胞中,CRISPR/Cas9介导的USP14敲除导致细胞周期蛋白D3水平下降。这些发现表明,USP14通过与细胞周期蛋白D3的相互作用促进了胰腺癌细胞的增殖和迁移能力,凸显了USP14作为胰腺癌的潜在治疗靶点。
