【背景】家禽卵泡发育具有独特的等级卵泡发育特性,受自分泌、旁分泌、生长因子和多种功能基因的共同调控。颗粒细胞是卵泡内数量最多的功能细胞,颗粒细胞的存活直接决定了卵泡的生长发育和成熟。铁死亡是一种新发现细胞程序性死亡方式...【背景】家禽卵泡发育具有独特的等级卵泡发育特性,受自分泌、旁分泌、生长因子和多种功能基因的共同调控。颗粒细胞是卵泡内数量最多的功能细胞,颗粒细胞的存活直接决定了卵泡的生长发育和成熟。铁死亡是一种新发现细胞程序性死亡方式,与卵泡发育密切相关。泛素特异性肽酶18(ubiquitin specific peptidase 18,USP18)在铁死亡过程中发挥重要的调控作用,但其在蛋鸭卵巢颗粒细胞中的功能尚不清楚。【目的】通过探究USP18在蛋鸭卵泡颗粒细胞铁死亡过程中的分子调控机制,为家禽产蛋性状的遗传改良提供理论基础和分子靶点。【方法】选取300日龄高、低产蛋鸭各4只,采集等级前卵泡组织,实时荧光定量PCR和Western blotting检测高、低产蛋鸭等级前卵泡组织中USP18表达水平;分离等级前颗粒细胞,设计USP18干扰片段并转染卵巢颗粒细胞,采用CCK-8试剂盒、Calcein-AM/PI细胞双染法检测USP18对颗粒细胞活力的影响,活性氧检测试剂盒和酶联免疫吸附法分析USP18对颗粒细胞内氧化应激水平的影响;利用线粒体膜电位试剂盒、脂质过氧化荧光探针、铁离子FerroOrange探针、普鲁士蓝试剂盒检测USP18对颗粒细胞铁死亡的影响;通过免疫荧光、免疫共沉淀和Western blotting分析USP18与谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白间的互作关系及GPX4蛋白表达水平。【结果】高产蛋鸭等级前卵泡组织中USP18表达量显著高于低产蛋鸭(P<0.01);敲降USP18显著降低颗粒细胞活力(P<0.01),抑制颗粒细胞存活(P<0.05),促进ROS积累(P<0.01),提高丙二醛(malondialdehyde,MDA)含量(P<0.01),抑制超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活性(P<0.01);敲降USP18显著提高颗粒细胞内脂质过氧化水平(P<0.05),降低线粒体膜电位(P<0.05),增强线粒体膜通透性(P<0.01),提高细胞内铁离子水平和铁沉积(P<0.01),促进铁死亡抑制因子A长链酰基辅酶A合成酶4(acyl-coA synthetase long chain family member 4,ACSL4)和GPX4表达并抑制铁死亡驱动因子转铁蛋白受体1(transferrin receptor protein 1,TFR1)表达(P<0.01);免疫共沉淀和免疫荧光证实USP18可与GPX4互作;Western blotting和免疫共沉淀结果显示敲降USP18提高GPX4泛素化水平并促进GPX4蛋白降解。【结论】USP18在高产蛋鸭等级前卵泡组织中高表达;敲降USP18显著提高了蛋鸭卵巢颗粒细胞内氧化应激水平并诱发铁死亡;USP18可与GPX4蛋白发生互作,敲降USP18提高GPX4泛素化水平并促进GPX4蛋白降解。综上所述,USP18通过抑制GPX4泛素化降解调控蛋鸭卵巢颗粒细胞铁死亡。展开更多
目的探究新鱼腥草素钠(sodium new houttuyfonate,SNH)通过泛素特异性肽酶22(ubiquitin-specific peptidase22,USP22)介导的沉默调节蛋白1(silent mating type information regulation 2 homolog 1,SIRT1)去泛素化调控自噬抑制肝细胞癌...目的探究新鱼腥草素钠(sodium new houttuyfonate,SNH)通过泛素特异性肽酶22(ubiquitin-specific peptidase22,USP22)介导的沉默调节蛋白1(silent mating type information regulation 2 homolog 1,SIRT1)去泛素化调控自噬抑制肝细胞癌进展的作用机制。方法人肝癌HepG2细胞给予SNH或索拉非尼处理24 h后,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Transwell实验检测细胞侵袭能力。质粒转染诱导USP22过表达,免疫共沉淀(co-immunoprecipitation,Co-IP)验证USP22和SIRT1之间的相互作用,Western blotting检测USP22、SIRT1蛋白表达和SIRT1泛素化水平。使用SIRT1激活剂处理后,通过m RFP-GFP-LC3双荧光标记、三磷酸腺苷(adenosine triphosphate,ATP)水平测定、乳酸生成测定以及USP22、SIRT1、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)-Ⅱ/Ⅰ、p62的蛋白表达分析评估自噬活性。在体内实验中,40只BALB/c-nu裸鼠异位移植HepG2细胞,给予SNH或索拉非尼干预12 d,每3天监测肿瘤体积和质量。对肿瘤组织进行苏木素-伊红(hematoxylin-eosin,HE)染色、TUNEL染色,采用免疫组化法检测核增殖抗原Ki67、USP22和SIRT1的蛋白表达。结果SNH呈剂量相关性地抑制HepG2细胞活力和侵袭(P<0.01、0.001),诱导细胞凋亡(P<0.001),其中高剂量SNH的作用与索拉非尼相当。Co-IP证实了USP22-SIRT1蛋白相互作用,高剂量SNH显著降低USP22-SIRT1蛋白相互作用(P<0.01)。高剂量SNH显著下调USP22和SIRT1蛋白表达(P<0.01、0.001),增加SIRT1的泛素化(P<0.001),以上作用被USP22过表达逆转(P<0.05、0.001)。SNH抑制HepG2细胞自噬,表现为GFP/mRFP荧光信号增强(P<0.01),ATP水平降低(P<0.001),乳酸水平升高(P<0.001),p62表达增加以及USP22、SIRT1、LC3-Ⅱ/Ⅰ蛋白表达水平降低(P<0.05、0.001)。SIRT1激活部分抵消了SNH的自噬抑制作用(P<0.05、0.01、0.001)。在体内,高剂量SNH显著降低肿瘤体积、质量和恶性程度(P<0.001),诱导肿瘤细胞凋亡以及Ki67、USP22和SIRT1表达降低(P<0.001)。结论SNH调控USP22表达介导SIRT1去泛素化来抑制自噬,从而抑制肝细胞癌的进展,为肝细胞癌的治疗提供了潜在策略。展开更多
Cilia are microtubule-based organelles projecting from the cell surface with important sensory and motility functions.Ciliary defects are associated with diverse diseases collectively known as ciliopathies.However,the...Cilia are microtubule-based organelles projecting from the cell surface with important sensory and motility functions.Ciliary defects are associated with diverse diseases collectively known as ciliopathies.However,the molecular mechanisms that govern ciliogenesis remain not fully understood.Here,we demonstrate that ubiquitin-specific protease 21(USP21)is indispensable for cilium formation through its deubiquitinating activity.Usp21 knockout mice exhibit ciliary defects in multiple organs,such as the kidney,liver,and trachea.Our data also reveal a constant localization of USP21 at the centrosome and basal body during ciliogenesis.Mechanistically,USP21 interacts with dihydropyrimidinase-like 2(DPYSL2)at the centrosome and removes lysine 48-linked ubiquitination from DPYSL2.Loss of USP21 leads to the proteasomal degradation of DPYSL2 and causes a significant reduction in its centrosome abundance,ultimately resulting in ciliary defects.These findings thus identify a critical role for the USP21–DPYSL2 axis in ciliogenesis and have important implications for health and disease.展开更多
Background Follicular atresia,a complex degenerative process regulated by multiple molecular mechanisms,significantly affects female reproductive performance in animals.While granulosa cell(GC)apoptosis has been well ...Background Follicular atresia,a complex degenerative process regulated by multiple molecular mechanisms,significantly affects female reproductive performance in animals.While granulosa cell(GC)apoptosis has been well established as a primary mechanism underlying follicular atresia,the potential involvement of ferroptosis,which is an irondependent form of regulated cell death,remains largely unexplored in chickens.Results Using a tamoxifen(TMX)-induced avian model of follicular atresia,we demonstrated that ferroptosis plays a critical role in follicular degeneration.Inhibition of ferroptosis through pharmacological agents significantly restored follicular function,underscoring its potential as a therapeutic target.Notably,we observed a significant upregulation of ubiquitin-specific peptidase 9,X-linked(USP9X)in GCs during atresia.Through comprehensive in vitro and in vivo investigations,we confirmed that USP9X facilitates follicular atresia by promoting ferroptosis in GCs.Mechanistically,USP9X induces ferroptosis by stabilizing Beclin1 through deubiquitination,thereby activating autophagy-dependent ferroptosis.This pathway was effectively suppressed by autophagy inhibitors,emphasizing the essential role of autophagy in USP9X-mediated ferroptosis.Conclusions Our findings provide the evidence that the USP9X-Beclin1 axis regulates autophagy-dependent ferroptosis during avian follicular atresia.These insights reveal novel molecular targets and potential genetic markers for improving reproductive efficiency in chicken breeding programs.展开更多
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
Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain ...Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain undefined.This study aims to identify ubiquitin-specific proteases(USPs)that deubiquitinate MafB and enhance its stability.Methods We constructed a MafB-conjugated luciferase and overexpressed 40 individual USPs,measuring changes in luciferase activity.The identified USP was overexpressed in human CD14^(+) peripheral blood mononuclear cells(PBMCs)to evaluate its effect.Osteoclast differentiation was assessed through osteoclast marker Integrin alpha-V(CD51)staining and Western blot analysis.Co-immunoprecipitation(co-IP)was performed to assess the interplay.The influence on MafB ubiquitination and degradation was evaluated via immunoprecipitation and Western blot.Finally,MafB was knocked down in the USP-overexpressing PBMCs to analyze its effect on osteoclast differentiation.Results Overexpression of ubiquitin-specific protease 29(USP29)significantly increased MafB expression by approximately 75%(p<0.0001).Elevated USP29 levels strongly inhibited osteoclastic differentiation in CD14^(+) PBMCs(p<0.0001).USP29 was found to interact with MafB,markedly reducing its ubiquitination and subsequent degradation in PBMCs(p<0.001).Knocking down MafB in USP29-overexpressing PBMCs alleviated the inhibitory effect of USP29 on osteoclastogenesis.Conclusion USP29 acts as a potent stabilizer of MafB,inhibiting osteoclastogenesis in human CD14^(+) PBMCs,at least in part,by enhancing MafB stability.These findings expand our understanding of USP29’s role and the post-translational regulation of MafB.Furthermore,USP29 serves as a vital factor that controls osteoclast differentiation,and its regulatory function is at least partially mediated by deubiquitinating and stabilizing MafB.展开更多
本文旨在对3个影响猪达100 kg体重日龄(Age at 100 kg,AGE)和达100 kg体重平均日增重(Average Daily Gain at 100 kg,ADG)的候选基因进行生物信息学分析。运用生物信息学数据库及其软件,分别分析了肉碱O-乙酰转移酶(Carnitine O-acetylt...本文旨在对3个影响猪达100 kg体重日龄(Age at 100 kg,AGE)和达100 kg体重平均日增重(Average Daily Gain at 100 kg,ADG)的候选基因进行生物信息学分析。运用生物信息学数据库及其软件,分别分析了肉碱O-乙酰转移酶(Carnitine O-acetyltransferase,CRAT)、G蛋白偶联受体107(G Protein-Coupled Receptor 107,GPR107)和泛素特异性肽酶20(Ubiquitin Specific Peptidase 20,USP20)3个基因与其编码的产物。结果发现:CRAT基因位于猪的1号染色体,具有15个外显子、14个内含子,共编码626个氨基酸;USP20基因位于猪的1号染色体,具有26个外显子,25个内含子,共编码915个氨基酸;GPR107基因位于猪的1号染色体,具有19个外显子、18个内含子,共编码597个氨基酸;3个蛋白的二级结构主要组成均为无规则卷曲,且均表现出疏水特征,含有磷酸化位点,仅GPR107蛋白检测到信号肽和跨膜结构;猪CRAT、USP20、GPR107在进化过程中与其他哺乳动物之间均具有高度保守性以及序列同源性;CRAT与羟基类固醇17-β脱氢酶4(Hydroxysteroid 17-Beta Dehydrogenase 4,HSD17B4)、囊泡乙酰胆碱转运蛋白(Solute Carrier Family 18 Member A3,SLC18A3)、脂肪酶(Patatin Like Phospholipase Domain Containing 2,PNPLA2)等蛋白互作;USP20与STAM结合蛋白样1(STAM Binding Protein Like 1,STAMBPL1)、β2-肾上腺素能受体(Adrenoceptor Beta 2,ADRB2)、泛素特异性肽酶14(Ubiquitin Specific Peptidase 14,USP14)等蛋白互作;GPR107与LIN52(Lin-52 Homolog,LIN52)、P-选择素(Selectin P,SELP)、G蛋白偶联受体137C(G Protein-Coupled Receptor 137C,GPR137C)等蛋白互作;GO富集分析发现CRAT、USP20、GPR107主要参与脂肪酸的分解代谢、蛋白质稳定性和降解、配子生成过程;CRAT基因在肌肉组织中高表达,USP20基因在下丘脑中高表达,GPR107基因在血管和下丘脑中高表达。本文结果为进一步研究猪生长性状相关的功能基因提供了数据与理论参考。展开更多
文摘【背景】家禽卵泡发育具有独特的等级卵泡发育特性,受自分泌、旁分泌、生长因子和多种功能基因的共同调控。颗粒细胞是卵泡内数量最多的功能细胞,颗粒细胞的存活直接决定了卵泡的生长发育和成熟。铁死亡是一种新发现细胞程序性死亡方式,与卵泡发育密切相关。泛素特异性肽酶18(ubiquitin specific peptidase 18,USP18)在铁死亡过程中发挥重要的调控作用,但其在蛋鸭卵巢颗粒细胞中的功能尚不清楚。【目的】通过探究USP18在蛋鸭卵泡颗粒细胞铁死亡过程中的分子调控机制,为家禽产蛋性状的遗传改良提供理论基础和分子靶点。【方法】选取300日龄高、低产蛋鸭各4只,采集等级前卵泡组织,实时荧光定量PCR和Western blotting检测高、低产蛋鸭等级前卵泡组织中USP18表达水平;分离等级前颗粒细胞,设计USP18干扰片段并转染卵巢颗粒细胞,采用CCK-8试剂盒、Calcein-AM/PI细胞双染法检测USP18对颗粒细胞活力的影响,活性氧检测试剂盒和酶联免疫吸附法分析USP18对颗粒细胞内氧化应激水平的影响;利用线粒体膜电位试剂盒、脂质过氧化荧光探针、铁离子FerroOrange探针、普鲁士蓝试剂盒检测USP18对颗粒细胞铁死亡的影响;通过免疫荧光、免疫共沉淀和Western blotting分析USP18与谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白间的互作关系及GPX4蛋白表达水平。【结果】高产蛋鸭等级前卵泡组织中USP18表达量显著高于低产蛋鸭(P<0.01);敲降USP18显著降低颗粒细胞活力(P<0.01),抑制颗粒细胞存活(P<0.05),促进ROS积累(P<0.01),提高丙二醛(malondialdehyde,MDA)含量(P<0.01),抑制超氧化物歧化酶(superoxide dismutase,SOD)和过氧化氢酶(catalase,CAT)的活性(P<0.01);敲降USP18显著提高颗粒细胞内脂质过氧化水平(P<0.05),降低线粒体膜电位(P<0.05),增强线粒体膜通透性(P<0.01),提高细胞内铁离子水平和铁沉积(P<0.01),促进铁死亡抑制因子A长链酰基辅酶A合成酶4(acyl-coA synthetase long chain family member 4,ACSL4)和GPX4表达并抑制铁死亡驱动因子转铁蛋白受体1(transferrin receptor protein 1,TFR1)表达(P<0.01);免疫共沉淀和免疫荧光证实USP18可与GPX4互作;Western blotting和免疫共沉淀结果显示敲降USP18提高GPX4泛素化水平并促进GPX4蛋白降解。【结论】USP18在高产蛋鸭等级前卵泡组织中高表达;敲降USP18显著提高了蛋鸭卵巢颗粒细胞内氧化应激水平并诱发铁死亡;USP18可与GPX4蛋白发生互作,敲降USP18提高GPX4泛素化水平并促进GPX4蛋白降解。综上所述,USP18通过抑制GPX4泛素化降解调控蛋鸭卵巢颗粒细胞铁死亡。
文摘目的探究新鱼腥草素钠(sodium new houttuyfonate,SNH)通过泛素特异性肽酶22(ubiquitin-specific peptidase22,USP22)介导的沉默调节蛋白1(silent mating type information regulation 2 homolog 1,SIRT1)去泛素化调控自噬抑制肝细胞癌进展的作用机制。方法人肝癌HepG2细胞给予SNH或索拉非尼处理24 h后,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,Transwell实验检测细胞侵袭能力。质粒转染诱导USP22过表达,免疫共沉淀(co-immunoprecipitation,Co-IP)验证USP22和SIRT1之间的相互作用,Western blotting检测USP22、SIRT1蛋白表达和SIRT1泛素化水平。使用SIRT1激活剂处理后,通过m RFP-GFP-LC3双荧光标记、三磷酸腺苷(adenosine triphosphate,ATP)水平测定、乳酸生成测定以及USP22、SIRT1、微管相关蛋白轻链3(microtubule-associated protein light chain 3,LC3)-Ⅱ/Ⅰ、p62的蛋白表达分析评估自噬活性。在体内实验中,40只BALB/c-nu裸鼠异位移植HepG2细胞,给予SNH或索拉非尼干预12 d,每3天监测肿瘤体积和质量。对肿瘤组织进行苏木素-伊红(hematoxylin-eosin,HE)染色、TUNEL染色,采用免疫组化法检测核增殖抗原Ki67、USP22和SIRT1的蛋白表达。结果SNH呈剂量相关性地抑制HepG2细胞活力和侵袭(P<0.01、0.001),诱导细胞凋亡(P<0.001),其中高剂量SNH的作用与索拉非尼相当。Co-IP证实了USP22-SIRT1蛋白相互作用,高剂量SNH显著降低USP22-SIRT1蛋白相互作用(P<0.01)。高剂量SNH显著下调USP22和SIRT1蛋白表达(P<0.01、0.001),增加SIRT1的泛素化(P<0.001),以上作用被USP22过表达逆转(P<0.05、0.001)。SNH抑制HepG2细胞自噬,表现为GFP/mRFP荧光信号增强(P<0.01),ATP水平降低(P<0.001),乳酸水平升高(P<0.001),p62表达增加以及USP22、SIRT1、LC3-Ⅱ/Ⅰ蛋白表达水平降低(P<0.05、0.001)。SIRT1激活部分抵消了SNH的自噬抑制作用(P<0.05、0.01、0.001)。在体内,高剂量SNH显著降低肿瘤体积、质量和恶性程度(P<0.001),诱导肿瘤细胞凋亡以及Ki67、USP22和SIRT1表达降低(P<0.001)。结论SNH调控USP22表达介导SIRT1去泛素化来抑制自噬,从而抑制肝细胞癌的进展,为肝细胞癌的治疗提供了潜在策略。
基金supported by the National Natural Science Foundation of China(32300694,32270807,32170829,and 31900538)the Shandong Natural Science Foundation(2022HWYQ-075)the Taishan Scholar Foundation of Shandong Province(tsqn202211109).
文摘Cilia are microtubule-based organelles projecting from the cell surface with important sensory and motility functions.Ciliary defects are associated with diverse diseases collectively known as ciliopathies.However,the molecular mechanisms that govern ciliogenesis remain not fully understood.Here,we demonstrate that ubiquitin-specific protease 21(USP21)is indispensable for cilium formation through its deubiquitinating activity.Usp21 knockout mice exhibit ciliary defects in multiple organs,such as the kidney,liver,and trachea.Our data also reveal a constant localization of USP21 at the centrosome and basal body during ciliogenesis.Mechanistically,USP21 interacts with dihydropyrimidinase-like 2(DPYSL2)at the centrosome and removes lysine 48-linked ubiquitination from DPYSL2.Loss of USP21 leads to the proteasomal degradation of DPYSL2 and causes a significant reduction in its centrosome abundance,ultimately resulting in ciliary defects.These findings thus identify a critical role for the USP21–DPYSL2 axis in ciliogenesis and have important implications for health and disease.
基金funded by The National Key Research and Development Program of China,grant number 2022YFF1000202Sichuan Science and Technology Program,grant number 2023NSFSC1940,2021YFYZ0007 and 2024YFNH0025+1 种基金National Natural Science Foundation of China Grants,grant number 32402745China Agriculture Research System of MOF and MARA,grant number CARS-40。
文摘Background Follicular atresia,a complex degenerative process regulated by multiple molecular mechanisms,significantly affects female reproductive performance in animals.While granulosa cell(GC)apoptosis has been well established as a primary mechanism underlying follicular atresia,the potential involvement of ferroptosis,which is an irondependent form of regulated cell death,remains largely unexplored in chickens.Results Using a tamoxifen(TMX)-induced avian model of follicular atresia,we demonstrated that ferroptosis plays a critical role in follicular degeneration.Inhibition of ferroptosis through pharmacological agents significantly restored follicular function,underscoring its potential as a therapeutic target.Notably,we observed a significant upregulation of ubiquitin-specific peptidase 9,X-linked(USP9X)in GCs during atresia.Through comprehensive in vitro and in vivo investigations,we confirmed that USP9X facilitates follicular atresia by promoting ferroptosis in GCs.Mechanistically,USP9X induces ferroptosis by stabilizing Beclin1 through deubiquitination,thereby activating autophagy-dependent ferroptosis.This pathway was effectively suppressed by autophagy inhibitors,emphasizing the essential role of autophagy in USP9X-mediated ferroptosis.Conclusions Our findings provide the evidence that the USP9X-Beclin1 axis regulates autophagy-dependent ferroptosis during avian follicular atresia.These insights reveal novel molecular targets and potential genetic markers for improving reproductive efficiency in chicken breeding programs.
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Objectives Dysregulated osteoclast function contributes to skeletal diseases.However,the specific ubiquitination regulators of the osteoclastogenesis repressor MafB,particularly at the post-translational level,remain undefined.This study aims to identify ubiquitin-specific proteases(USPs)that deubiquitinate MafB and enhance its stability.Methods We constructed a MafB-conjugated luciferase and overexpressed 40 individual USPs,measuring changes in luciferase activity.The identified USP was overexpressed in human CD14^(+) peripheral blood mononuclear cells(PBMCs)to evaluate its effect.Osteoclast differentiation was assessed through osteoclast marker Integrin alpha-V(CD51)staining and Western blot analysis.Co-immunoprecipitation(co-IP)was performed to assess the interplay.The influence on MafB ubiquitination and degradation was evaluated via immunoprecipitation and Western blot.Finally,MafB was knocked down in the USP-overexpressing PBMCs to analyze its effect on osteoclast differentiation.Results Overexpression of ubiquitin-specific protease 29(USP29)significantly increased MafB expression by approximately 75%(p<0.0001).Elevated USP29 levels strongly inhibited osteoclastic differentiation in CD14^(+) PBMCs(p<0.0001).USP29 was found to interact with MafB,markedly reducing its ubiquitination and subsequent degradation in PBMCs(p<0.001).Knocking down MafB in USP29-overexpressing PBMCs alleviated the inhibitory effect of USP29 on osteoclastogenesis.Conclusion USP29 acts as a potent stabilizer of MafB,inhibiting osteoclastogenesis in human CD14^(+) PBMCs,at least in part,by enhancing MafB stability.These findings expand our understanding of USP29’s role and the post-translational regulation of MafB.Furthermore,USP29 serves as a vital factor that controls osteoclast differentiation,and its regulatory function is at least partially mediated by deubiquitinating and stabilizing MafB.
文摘本文旨在对3个影响猪达100 kg体重日龄(Age at 100 kg,AGE)和达100 kg体重平均日增重(Average Daily Gain at 100 kg,ADG)的候选基因进行生物信息学分析。运用生物信息学数据库及其软件,分别分析了肉碱O-乙酰转移酶(Carnitine O-acetyltransferase,CRAT)、G蛋白偶联受体107(G Protein-Coupled Receptor 107,GPR107)和泛素特异性肽酶20(Ubiquitin Specific Peptidase 20,USP20)3个基因与其编码的产物。结果发现:CRAT基因位于猪的1号染色体,具有15个外显子、14个内含子,共编码626个氨基酸;USP20基因位于猪的1号染色体,具有26个外显子,25个内含子,共编码915个氨基酸;GPR107基因位于猪的1号染色体,具有19个外显子、18个内含子,共编码597个氨基酸;3个蛋白的二级结构主要组成均为无规则卷曲,且均表现出疏水特征,含有磷酸化位点,仅GPR107蛋白检测到信号肽和跨膜结构;猪CRAT、USP20、GPR107在进化过程中与其他哺乳动物之间均具有高度保守性以及序列同源性;CRAT与羟基类固醇17-β脱氢酶4(Hydroxysteroid 17-Beta Dehydrogenase 4,HSD17B4)、囊泡乙酰胆碱转运蛋白(Solute Carrier Family 18 Member A3,SLC18A3)、脂肪酶(Patatin Like Phospholipase Domain Containing 2,PNPLA2)等蛋白互作;USP20与STAM结合蛋白样1(STAM Binding Protein Like 1,STAMBPL1)、β2-肾上腺素能受体(Adrenoceptor Beta 2,ADRB2)、泛素特异性肽酶14(Ubiquitin Specific Peptidase 14,USP14)等蛋白互作;GPR107与LIN52(Lin-52 Homolog,LIN52)、P-选择素(Selectin P,SELP)、G蛋白偶联受体137C(G Protein-Coupled Receptor 137C,GPR137C)等蛋白互作;GO富集分析发现CRAT、USP20、GPR107主要参与脂肪酸的分解代谢、蛋白质稳定性和降解、配子生成过程;CRAT基因在肌肉组织中高表达,USP20基因在下丘脑中高表达,GPR107基因在血管和下丘脑中高表达。本文结果为进一步研究猪生长性状相关的功能基因提供了数据与理论参考。
