[1]苏文成,等.去泛素酶复合体Ubp3/Bre5的制备及与Cdc48作用[J].深圳大学学报理工版,2015,32(1):58-67.[doi:10.3724/SP.J.1249.2015.01058]
 Su Wencheng,Lyu Cao,Shi Lili,et al.Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48[J].Journal of Shenzhen University Science and Engineering,2015,32(1):58-67.[doi:10.3724/SP.J.1249.2015.01058]
点击复制

去泛素酶复合体Ubp3/Bre5的制备及与Cdc48作用()
分享到:

《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第32卷
期数:
2015年第1期
页码:
58-67
栏目:
生物工程
出版日期:
2015-01-20

文章信息/Info

Title:
Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48
文章编号:
201501008
作者:
苏文成1 2吕操12时丽丽3景晓飞12盖园明2张洁2谭焕波2王鹏举2 夏立新4邹培建2秦刚2
1)天津工业技术大学生物技术学院, 天津 300457
2)中国科学院天津工业生物技术研究所,国家工业酶重点实验室,天津300308
3)天津药物研究院天津分子设计与药物发现重点实验室, 天津300193
4)深圳大学医学部,呼吸疾病国家重点实验室深圳大学变态反应分室, 深圳 518060
Author(s):
Su Wencheng12 Lyu Cao12 Shi Lili3 Jing Xiaofei12 Gai Yuanming2 Zhang Jie2 Tan Huanbo2 Wang Pengju2 Xia Lixin4 Zou Peijian2 and Qin Gang2
1) College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, P.R.China
2) National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, P.R.China
3) Tianjin Key Laboratory of Molecular Design and Drug Discovery, Tianjin Institute of Pharmaceutical Research, Tianjin 300193, P.R.China
4) Health Science Center, Shenzhen University, State Key Laboratory of Respiratory Disease for Allergy at Shenzhen University, Shenzhen 518060, P.R.China
关键词:
结合蛋白质Ubp3去泛素化酶结合辅因子Bre5ATP酶Cdc48去泛素化复合体与谷光苷肽巯基转移酶沉淀试验直接相互作用
Keywords:
protein binding deubiquitinase Ubp3 cofactor Bre5 ATPase Cdc48 deubiquitinating complex GST-pulldown direct interaction
分类号:
Q 513
DOI:
10.3724/SP.J.1249.2015.01058
文献标志码:
A
摘要:
泛素化是一种存在于真核细胞中与生理功能密切相关的蛋白修饰,泛素化与去泛素化处于动态调节过程中. Ubp3是与人USP10同源的酵母去泛素化酶,结合辅引子Bre5在细胞内发挥广泛作用.为研究该复合体的工作机制,制备重组蛋白复合体,在大肠杆菌中成功表达并纯化重组Ubp3与Bre5单体及Ubp3/Bre5复合体,首次成功大规模制备重组Ubp3/Bre5复合体.通过一系列pulldown实验,检验Ubp3/Bre5与AAA家族中泛素选择性ATP酶Cdc48的相互作用模式,结果发现,Ubp3及Bre5无法单独与Cdc48结合,但Ubp3/Bre5复合体可以有效与Cdc48相互作用.提出了Ubp3/Bre5-Cdc48相互作用的新模式,制备了高质量重组Ubp3/Bre5复合体.该研究为通过生化及结构生物学进行分子机制探索奠定了基础.
Abstract:
Ubiquitination modification is a dynamic process essential for eukaryotic cell physiology. Ubp3, the Saccharomyces cerevisiae homologue of human deubiquitinase USP10, together with its cofactor Bre5, plays an active role in numerous cellular processes. Although Bre5 is essential for Ubp3 function in vivo, unfortunately, due to difficulty in preparing critical quantities of intact functional Ubp3 and Ubp3/Bre5 reconstitute, systemic characterization on this complex is lacking. Hence, how exactly Bre5 regulates Ubp3 activity still remains elusive. To fill this gap, we report the successful expression and purification of recombinant Ubp3 and Bre5 in Escherichia coli in monomeric and complex form. To our knowledge, this is the first report the successful preparation of full-length Ubp3/Bre5 protein complex in large scale, which allows us to obtain further understanding of molecular bases. The stoichiometric interaction between purified Ubp3 and Bre5 confirmed proper folding of these proteins. To assess the proposed direct interactions between Ubp3 and Bre5 with the ubiquitin selective ATPase associated with a variety of cellular activities (AAA ATPase) Cdc48, series of pull-down assays are performed; results reveal that, neither Ubp3 nor Bre5 alone is able to bind Cdc48. However, the Ubp3/Bre5 complex could bind Cdc48 efficiently, which provids novel insight on Ubp3/Bre5-Cdc48 interaction mode. In summary, our results lay the foundation for future mechanistic evaluation by both biochemical and structural means.

参考文献/References:

[1] Wilkinson K D. Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome[J]. Seminars in Cell & Developmental Biology, 2000, 11(3):141-148.
[2] Nijman S M,Luna-Vargas M P,Velds A,et al.A genomic and functional inventory of deubiquitinating enzymes[J]. Cell, 2005, 123(5):773-786.
[3] Reyes-Turcu F E, Ventii K H, Wilkinson K D. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes[J]. Annual Review of Biochemistry, 2009,78:363-397.
[4] Amerik A Y, Li S J, Hochstrasser M. Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae[J]. The Journal of Biological Chemistry, 2000, 381(9/10): 981 -992.
[5] Poulsen J W, Madsen C T, Young C, et al. Comprehensive profiling of proteome changes upon sequential deletion of deubiquitylating enzymes[J]. Journal of Proteomics, 2012, 75(13):3886-3897.
[6] Bilsland E, Hult M, Bell S D, et al. The Bre5/Ubp3 ubiquitin protease complex from budding yeast contributes to the cellular response to DNA damage[J]. DNA Repair (Amst), 2007, 6(10):1471-1484.
[7] Mao P, Smerdon M J. Yeast deubiquitinase Ubp3 interacts with the 26 S proteasome to facilitate Rad4 degradation[J]. Journal of Biological Chemistry, 2010, 285(48): 37542-37550.
[8] Chew B S, Siew W L, Xiao B, et al. Transcriptional activation requires protection of the TATA-binding protein Tbp1 by the ubiquitin-specific protease Ubp3[J]. Biochemical Journal, 2010, 431(3):391-399.
[9] Kvint K, Uhler J P, Taschner M J, et al. Reversal of RNA polymerase II ubiquitylation by the ubiquitin protease Ubp3[J]. Molecular Cell, 2008, 30(4):498-506.
[10] Li Y, Wang Y. Ras protein/cAMP-dependent protein kinase signaling is negatively regulated by a deubiquitinating enzyme, Ubp3, in yeast[J]. Journal of Biological Chemistry, 2013, 288(16):11358-11365.
[11] Wang Y, Zhu M, Ayalew M, et al. Down-regulation of Pkc1-mediated signaling by the deubiquitinating enzyme Ubp3[J]. Journal of Biological Chemistry, 2008, 283(4):1954-1961.
[12] Cohen M, Stutz F, Belgareh N, et al. Ubp3 requires a cofactor, Bre5, to specifically de-ubiquitinate the COPII protein, Sec23[J]. Nature Cell Biology, 2003, 5(7):661-667.
[13] Kraft C, Deplazes A, Sohrmann M, et al. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease[J]. Nature Cell Biology, 2008, 10(5):602-610.
[14] Li K, Ossareh-Nazari B, Liu X, et al. Molecular basis for bre5 cofactor recognition by the ubp3 deubiquitylating enzyme[J]. Journal of Molecular Biology, 2007,372(1):194-204.
[15] Li K, Zhao K, Ossareh-Nazari B, et al. Structural basis for interaction between the Ubp3 deubiquitinating enzyme and its Bre5 cofactor[J]. Journal of Biological Chemistry, 2005, 280(32):29176-29185.
[16] Ossareh-Nazari B, Bonizec M, Cohen M, et al. Cdc48 and Ufd3, new partners of the ubiquitin protease Ubp3, are required for ribophagy[J]. EMBO reports, 2010, 11(7): 548-554.
[17] Rumpf S, Jentsch S. Functional division of substrate processing cofactors of the ubiquitin-selective Cdc48 chaperone[J]. Molecular Cell, 2006, 21(2):261-269.
[18] Hanzelmann P, Buchberger A, Schindelin H. Hierarchical binding of cofactors to the AAA ATPase p97[J]. Structure, 2011, 19(6):833-843.
[19] Stolz A, Hilt W, Buchberger A, et al. Cdc48: a power machine in protein degradation[J]. Trends in Biochemical Sciences, 2011, 36(10):515-523.

备注/Memo

备注/Memo:
Received:2014-01-17;Revised:2014-12-24;Accepted:2014-12-26
Foundation: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)
Corresponding author:Associate professor Qin Gang, E-mail: qing@genequantum.com
Citation:Su Wencheng, Lyu Cao, Shi Lili, et al.Preparation of full-length deubiquitinating complex Ubp3/Bre5 and characterization of interaction with Cdc48[J]. Journal of Shenzhen University Science and Engineering, 2015, 32(1): 58-67.
基金项目:天津市科技支撑计划资助项目(11ZCZDSY08100); 中国科学院百人计划资助项目(KSCW2-YW-BR-4) ;国家自然科学基金资助项目(81273275)
作者简介:苏文成(1987—),女(汉族),内蒙古自治区呼和浩特市人,天津工业技术大学硕士,E-mail: woshisuwenc@yahoo.com
引文:苏文成,吕操,时丽丽,等. 去泛素酶复合体Ubp3/Bre5的制备及与Cdc48作用[J]. 深圳大学学报理工版,2015,32(1):58-67.(英文版)
更新日期/Last Update: 2015-01-05