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
The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deub...The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29^(–/–)). Usp29^(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29^(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.展开更多
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.
基金supported by the National Key Research and Developmental Program of China(2018YFC1004700 and 2016YFC1000600)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19000000)+1 种基金the National Natural Science Foundation of China(31401953,31630050,31890780,31871514 and 81571495)Major Program of Development Foundation of Hefei Centre for Physical Science and Technology(2018ZYFX005)
文摘The balanced actions between ubiquitination and deubiquitination precisely control the levels of various proteins vital for spermatogenesis. Ubiquitin-specific processing proteases(USPs) are the largest family of deubiquitinatingenzymes(DUBs),containing more than 50 members. So far, the functions of only a few USPs in male fertility have been studied, the roles of the majority are yet unknown. The present study aimed to explore the function of Usp29(ubiquitin-specific protease 29) in male fertility. We found that Usp29 showed predominant expression in mouse testis, and its m RNA expression started to increase at 14 days postpartum(dpp), with a peak at 28 and 35 dpp. Using CRISPR/Cas9 technology, we generated Usp29 knockout mice(Usp29^(–/–)). Usp29^(–/–)mice exhibited no overt developmental anomalies. Further examination revealed that Usp29^(–/–)mice had normal fertility and showed no detectable difference in the testis/body weight ratio, testicular and epididymal histology as well as epididymal sperm count from the wild-type littermates. Moreover, Usp29 is not a pseudogene in mice. Taken together, our study first reported that though Usp29 is predominantly expressed in the testis, it is not essential for male fertility in mice.
