泛素化是一种动态可逆的蛋白质翻译后修饰,泛素分子在泛素激活酶、泛素结合酶和泛素连接酶的级联酶促反应催化下共价连接到底物蛋白上。去泛素化酶将泛素分子从底物上移除,动态可逆地调控泛素化修饰,在成熟泛素的生成、泛素链的移除与...泛素化是一种动态可逆的蛋白质翻译后修饰,泛素分子在泛素激活酶、泛素结合酶和泛素连接酶的级联酶促反应催化下共价连接到底物蛋白上。去泛素化酶将泛素分子从底物上移除,动态可逆地调控泛素化修饰,在成熟泛素的生成、泛素链的移除与修剪、游离泛素链的回收等过程中发挥着关键的调控作用。本文的研究对象是酵母中泛素特异性蛋白酶(ubiquitin specific protease,USP)家族成员Ubp14,负责回收细胞内游离的泛素链。本研究定量比较了酵母细胞中Ubp14缺失对全蛋白质组的影响,进而找出其潜在的调控通路和分子功能。首先,通过同源重组技术构建了ubp14△菌株,发现其生长速度低于野生型酵母。利用稳定同位素氨基酸代谢标记技术结合深度覆盖的蛋白质组学分析技术,系统比较了ubp14△菌株相对于野生型菌株的差异蛋白,共计鉴定3685个蛋白,通过统计学分析筛选得到109个差异蛋白。基因本体论分析发现,Ubp14缺失引起的差异蛋白主要参与了包括氨基酸代谢、氧化还原和热应激等生物学过程。本研究为深入探究去泛素化酶Ubp14的生物学功能,进而深刻理解游离泛素的稳态平衡与生物学过程调控提供了高可信的蛋白质组学数据信息。展开更多
Previous studies have shown that expression of the interferon-sensitive gene （ISG）15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon （IFN）-α therapy. We hypo...Previous studies have shown that expression of the interferon-sensitive gene （ISG）15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon （IFN）-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by HI （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.展开更多
Brassica napus(B.napus)is a globally significant oilseed crop,making a substantial contribution to both human oil and livestock feed production.Enhancing seed weight is crucial for improving rapeseed yield;however,onl...Brassica napus(B.napus)is a globally significant oilseed crop,making a substantial contribution to both human oil and livestock feed production.Enhancing seed weight is crucial for improving rapeseed yield;however,only a limited number of seed weight-related genes have been functionally validated in B.napus thus far.UBIQUITINSPECIFIC PROTEASE 15(UBP15)belongs to the ubiquitin protease pathway and plays a maternal role in prolonging seed development in Arabidopsis.The potential utilization of UBP15 for enhancing seed yield in B.napus has remained unexplored until now.In this study,we identified the orthologs of UBP15 in B.napus and investigated its functionality using the CRISPR-Cas9 system.We generated mutant plants with multiple editing types targeting Bnaubp15s and successfully isolated T-DNA-free homozygous mutant lines that exhibited edits across four homologs of BnaUBP15 in T2 generation plants.Our preliminary data demonstrated that mutation of BnaUBP15s significantly reduced seed size,seed weight,and plant height while noticeably increasing the number of primary branches.These findings not only provide crucial evidence for further elucidating the molecular mechanism underlying the regulation of seed weight and size by BnaUBP15s but also offer promising novel germplasm for enhancing plant architecture.展开更多
As a key transcription factor in the brassinosteroid (BR) signaling pathway, the activity and expression ofBES1 (BRI1-EMS-SUPPRESSOR 1) are stringently regulated. BES1 degradation is mediated by ubiquitinrelated 26S p...As a key transcription factor in the brassinosteroid (BR) signaling pathway, the activity and expression ofBES1 (BRI1-EMS-SUPPRESSOR 1) are stringently regulated. BES1 degradation is mediated by ubiquitinrelated 26S proteasomal and autophagy pathways, which attenuate and terminate BR signaling;however,the opposing deubiquitinases (DUBs) are still unknown. Here, we showed that the ubp12-2w/13-3 doublemutant phenocopies the BR-deficient dwarf mutant, suggesting that the two DUBs UBP12/UBP13 antagonize ubiquitin-mediated degradation to stabilize BES1. These two DUBs can trim tetraubiquitin with K46 and K63 linkages in vitro. UBP12/BES1 and UBP13/BES1 complexes are localized in bothcytosol and nuclei. UBP12/13 can deubiquitinate polyubiquitinated BES1 in vitro and in planta, andUBP12 interacts with and deubiquitinates both inactive, phosphorylated BES1 and active, dephosphorylated BES1 in vivo. UBP12 overexpression in BES1OE plants significantly enhances cell elongation in hypocotyls and petioles and increases the ratio of leaf length to width compared with BES1OE or UBP12OE plants.Hypocotyl elongation and etiolation result from elevated BES1 levels because BES1 degradation is retardedby UBP12 in darkness or in light with BR. Protein degradation inhibitor experiments show that the majorityof BES1 can be degraded by either the proteasomal or the autophagy pathway, but a minor BES1 fractionremains pathway specific. In conclusion, UBP12/UBP13 deubiquitinate BES1 to stabilize the latter as a positive regulator for BR responses.展开更多
Salicylic acid(SA),a defense hormone produced after pathogen challenge,is critical for plant immunity.Arabidopsis NONEXPRESSER OF PR GENES 1(NPR1)and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transd...Salicylic acid(SA),a defense hormone produced after pathogen challenge,is critical for plant immunity.Arabidopsis NONEXPRESSER OF PR GENES 1(NPR1)and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transduction.NPR1 functions as a transcriptional co-activator to promote defense gene expression,whereas NPR3 and NPR4 have been shown to function as negative regulators in the SA signaling pathway.Although the mechanism about NPR1 regulation has been well studied,how NPR3/NPR4 proteins are regulated in immune responses remains largely unknown.Here,we show that the stability of NPR3/NPR4 is enhanced by SA.In the absence of pathogen challenge,NPR3/NPR4 are unstable and degraded by the 26S proteasome,whereas the increase in cellular SA levels upon pathogen infection suppresses NPR3/NPR4 degradation.We found that UBP12 and UBP13,two homologous deubiquitinases from a ubiquitin-specific protease subfamily,negatively regulate plant immunity by promoting NPR3/NPR4 stability.Our genetic results further showed that UBP12/UBP13-mediated immunity suppression is partially dependent on NPR3/NPR4 functions.By interacting with NPR3 in the nucleus in an SA-dependent manner,UBP12 and UBP13 remove ubiquitin from polyubiquitinated NPR3 to protect it from being degraded.The stabilization of NPR3/NPR4 promoted by UBP12/UBP13 is essential for negative regulation of basal and SA-induced immunity.展开更多
植物器官的大小调控是一个重要的生物学过程,直接影响农作物产量。目前对于植物器官大小调控的研究集中在转录调控、激素信号通路及蛋白质合成与修饰等多个水平和途径,但具体的分子机制并不是很清晰。李云海实验室前期研究发现了一个器...植物器官的大小调控是一个重要的生物学过程,直接影响农作物产量。目前对于植物器官大小调控的研究集中在转录调控、激素信号通路及蛋白质合成与修饰等多个水平和途径,但具体的分子机制并不是很清晰。李云海实验室前期研究发现了一个器官大小的关键调控基因UBIQUITIN SPECIFIC PROTEASE 15(UBP15)/SUPPRESSOR2 OF DA1(SOD2)。sod2-1突变体由于UBP15基因缺失导致器官变小。UBP15作为DA1的底物,其蛋白稳定性受DA1的调节,但其下游通路并不清楚。为了进一步了解UBP15调控器官大小的分子机制,通过EMS诱变筛选分离得到了sod2-1的抑制突变体sus40-1D。sus40-1D sod2-1突变体与sod2-1植株相比,子叶、花瓣和种子面积显著增大。遗传分析表明sus40-1D为显性突变。基因组重测序及连锁分析鉴定出2个与sus40-1D紧密连锁的候选基因:At1g05820和At1g08470。At1g05820的突变发生在内含子区域,而At1g08470的突变发生在外显子区。本研究发现了一个调控器官大小的突变体sus40-1D,对该突变体的进一步研究将有助于解析植物器官大小的调控机制。展开更多
Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-...Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified.In this study,we found that the previously identified PWWP-EPCR-ARID-TRB(PEAT)complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2(HAM1/2)to form a larger version of PEAT complex in Arabidopsis thaliana.UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo.HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase com-plex,and are responsible for histone H4K5 acetylation.Within the PEAT complex,the PWWP components(PWWP1,PWWP2,and PWWP3)directly interact with UBP5 and are necessary for UBP5-mediated H2A deu-biquitination,while the EPCR components(EPCR1 and EPCR2)directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5acetylation.Collectively,our study not onlyidentifies dual roles of thePEAT com-plex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.展开更多
文摘泛素化是一种动态可逆的蛋白质翻译后修饰,泛素分子在泛素激活酶、泛素结合酶和泛素连接酶的级联酶促反应催化下共价连接到底物蛋白上。去泛素化酶将泛素分子从底物上移除,动态可逆地调控泛素化修饰,在成熟泛素的生成、泛素链的移除与修剪、游离泛素链的回收等过程中发挥着关键的调控作用。本文的研究对象是酵母中泛素特异性蛋白酶(ubiquitin specific protease,USP)家族成员Ubp14,负责回收细胞内游离的泛素链。本研究定量比较了酵母细胞中Ubp14缺失对全蛋白质组的影响,进而找出其潜在的调控通路和分子功能。首先,通过同源重组技术构建了ubp14△菌株,发现其生长速度低于野生型酵母。利用稳定同位素氨基酸代谢标记技术结合深度覆盖的蛋白质组学分析技术,系统比较了ubp14△菌株相对于野生型菌株的差异蛋白,共计鉴定3685个蛋白,通过统计学分析筛选得到109个差异蛋白。基因本体论分析发现,Ubp14缺失引起的差异蛋白主要参与了包括氨基酸代谢、氧化还原和热应激等生物学过程。本研究为深入探究去泛素化酶Ubp14的生物学功能,进而深刻理解游离泛素的稳态平衡与生物学过程调控提供了高可信的蛋白质组学数据信息。
基金The Program of Tianjin Municipal Science&Technology Project(11ZCZDSY08100)The Program of“One Hundred Talented People”of the Chinese Academy of Sciences(KSCW2-YW-BR-4)National Natural Science Foundation of China(81273275)~~
基金National Science Foundation of China (30271170National Hish Technology Research and Douelopment program of China (2006AA02Z128)
文摘Previous studies have shown that expression of the interferon-sensitive gene （ISG）15 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon （IFN）-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by HI （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by H1 （psiUBP43） could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.
基金This work was supported by the National Key Research and Development Program of China(2022YFD1200400)National Natural Science Foundation of China(32072099,31971977).
文摘Brassica napus(B.napus)is a globally significant oilseed crop,making a substantial contribution to both human oil and livestock feed production.Enhancing seed weight is crucial for improving rapeseed yield;however,only a limited number of seed weight-related genes have been functionally validated in B.napus thus far.UBIQUITINSPECIFIC PROTEASE 15(UBP15)belongs to the ubiquitin protease pathway and plays a maternal role in prolonging seed development in Arabidopsis.The potential utilization of UBP15 for enhancing seed yield in B.napus has remained unexplored until now.In this study,we identified the orthologs of UBP15 in B.napus and investigated its functionality using the CRISPR-Cas9 system.We generated mutant plants with multiple editing types targeting Bnaubp15s and successfully isolated T-DNA-free homozygous mutant lines that exhibited edits across four homologs of BnaUBP15 in T2 generation plants.Our preliminary data demonstrated that mutation of BnaUBP15s significantly reduced seed size,seed weight,and plant height while noticeably increasing the number of primary branches.These findings not only provide crucial evidence for further elucidating the molecular mechanism underlying the regulation of seed weight and size by BnaUBP15s but also offer promising novel germplasm for enhancing plant architecture.
基金This work was supported in part by an RSSS grant(no.NRF-RSSS-002)to N.-H.C.from the National Research Foundation,Singapore.
文摘As a key transcription factor in the brassinosteroid (BR) signaling pathway, the activity and expression ofBES1 (BRI1-EMS-SUPPRESSOR 1) are stringently regulated. BES1 degradation is mediated by ubiquitinrelated 26S proteasomal and autophagy pathways, which attenuate and terminate BR signaling;however,the opposing deubiquitinases (DUBs) are still unknown. Here, we showed that the ubp12-2w/13-3 doublemutant phenocopies the BR-deficient dwarf mutant, suggesting that the two DUBs UBP12/UBP13 antagonize ubiquitin-mediated degradation to stabilize BES1. These two DUBs can trim tetraubiquitin with K46 and K63 linkages in vitro. UBP12/BES1 and UBP13/BES1 complexes are localized in bothcytosol and nuclei. UBP12/13 can deubiquitinate polyubiquitinated BES1 in vitro and in planta, andUBP12 interacts with and deubiquitinates both inactive, phosphorylated BES1 and active, dephosphorylated BES1 in vivo. UBP12 overexpression in BES1OE plants significantly enhances cell elongation in hypocotyls and petioles and increases the ratio of leaf length to width compared with BES1OE or UBP12OE plants.Hypocotyl elongation and etiolation result from elevated BES1 levels because BES1 degradation is retardedby UBP12 in darkness or in light with BR. Protein degradation inhibitor experiments show that the majorityof BES1 can be degraded by either the proteasomal or the autophagy pathway, but a minor BES1 fractionremains pathway specific. In conclusion, UBP12/UBP13 deubiquitinate BES1 to stabilize the latter as a positive regulator for BR responses.
基金National Research Foundation(NRF),Prime Minister's Office,Singapore under its Campus for Research Excel-lence and Technological Enterprise(CREATE)programThe Disruptive and Sustainable Technologies for Agricultural Precision(DiSTAP)is an interdisciplinary research group(IRG)of the Singapore-MIT Alliance for Research and Technology Centre(SMART)+1 种基金National Research Foundation(NRF)Prime Minister's Office,Singapore,under its Campus for Research Excellence and Technological Enterprise(CREATE)program.
文摘Salicylic acid(SA),a defense hormone produced after pathogen challenge,is critical for plant immunity.Arabidopsis NONEXPRESSER OF PR GENES 1(NPR1)and its paralogs NPR3 and NPR4 can bind SA and mediate SA signal transduction.NPR1 functions as a transcriptional co-activator to promote defense gene expression,whereas NPR3 and NPR4 have been shown to function as negative regulators in the SA signaling pathway.Although the mechanism about NPR1 regulation has been well studied,how NPR3/NPR4 proteins are regulated in immune responses remains largely unknown.Here,we show that the stability of NPR3/NPR4 is enhanced by SA.In the absence of pathogen challenge,NPR3/NPR4 are unstable and degraded by the 26S proteasome,whereas the increase in cellular SA levels upon pathogen infection suppresses NPR3/NPR4 degradation.We found that UBP12 and UBP13,two homologous deubiquitinases from a ubiquitin-specific protease subfamily,negatively regulate plant immunity by promoting NPR3/NPR4 stability.Our genetic results further showed that UBP12/UBP13-mediated immunity suppression is partially dependent on NPR3/NPR4 functions.By interacting with NPR3 in the nucleus in an SA-dependent manner,UBP12 and UBP13 remove ubiquitin from polyubiquitinated NPR3 to protect it from being degraded.The stabilization of NPR3/NPR4 promoted by UBP12/UBP13 is essential for negative regulation of basal and SA-induced immunity.
文摘植物器官的大小调控是一个重要的生物学过程,直接影响农作物产量。目前对于植物器官大小调控的研究集中在转录调控、激素信号通路及蛋白质合成与修饰等多个水平和途径,但具体的分子机制并不是很清晰。李云海实验室前期研究发现了一个器官大小的关键调控基因UBIQUITIN SPECIFIC PROTEASE 15(UBP15)/SUPPRESSOR2 OF DA1(SOD2)。sod2-1突变体由于UBP15基因缺失导致器官变小。UBP15作为DA1的底物,其蛋白稳定性受DA1的调节,但其下游通路并不清楚。为了进一步了解UBP15调控器官大小的分子机制,通过EMS诱变筛选分离得到了sod2-1的抑制突变体sus40-1D。sus40-1D sod2-1突变体与sod2-1植株相比,子叶、花瓣和种子面积显著增大。遗传分析表明sus40-1D为显性突变。基因组重测序及连锁分析鉴定出2个与sus40-1D紧密连锁的候选基因:At1g05820和At1g08470。At1g05820的突变发生在内含子区域,而At1g08470的突变发生在外显子区。本研究发现了一个调控器官大小的突变体sus40-1D,对该突变体的进一步研究将有助于解析植物器官大小的调控机制。
基金supported by the National Natural Science Foundation of China(grant number:32025003).
文摘Histone H2A monoubiquitination is associated with transcriptional repression and needs to be removed by deubiquitinases to facilitate gene transcription in eukaryotes.However,the deubiquitinase responsible for genome-wide H2A deubiquitination in plants has yet to be identified.In this study,we found that the previously identified PWWP-EPCR-ARID-TRB(PEAT)complex components interact with both the ubiquitin-specific protease UBP5 and the redundant histone acetyltransferases HAM1 and HAM2(HAM1/2)to form a larger version of PEAT complex in Arabidopsis thaliana.UBP5 functions as an H2A deubiquitinase in a nucleosome substrate-dependent manner in vitro and mediates H2A deubiquitination at the whole-genome level in vivo.HAM1/2 are shared subunits of the PEAT complex and the conserved NuA4 histone acetyltransferase com-plex,and are responsible for histone H4K5 acetylation.Within the PEAT complex,the PWWP components(PWWP1,PWWP2,and PWWP3)directly interact with UBP5 and are necessary for UBP5-mediated H2A deu-biquitination,while the EPCR components(EPCR1 and EPCR2)directly interact with HAM1/2 and are required for HAM1/2-mediated H4K5acetylation.Collectively,our study not onlyidentifies dual roles of thePEAT com-plex in H2A deubiquitination and H4K5 acetylation but also illustrates how these processes collaborate at the whole-genome level to regulate the transcription and development in plants.
