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
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。
