|本期目录/Table of Contents|

[1]石萌,向嵩.核小体泛素连接酶Bre1的eRING结构域与泛素转移酶Rad6的相互作用研究[J].天津医科大学学报,2023,29(01):31-35.
　SHI Meng,XIANG Song.Biochemical charterization of the interaction between the eRING domain in the nucleosome ubiquitin ligase Bre1 and the ubiquitin conjugating enzyme Rad6[J].Journal of Tianjin Medical University,2023,29(01):31-35.
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核小体泛素连接酶Bre1的eRING结构域与泛素转移酶Rad6的相互作用研究(PDF)
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《天津医科大学学报》[ISSN:1006-8147/CN:12-1259/R]

卷:
29卷
期数:
2023年01期
页码:
31-35
栏目:
基础医学
出版日期:
2023-01-20

文章信息/Info

Title:
Biochemical charterization of the interaction between the eRING domain in the nucleosome ubiquitin ligase Bre1 and the ubiquitin conjugating enzyme Rad6
文章编号:
1006-8147(2023)01-0031-05
作者:
石萌; 向嵩
（天津医科大学基础医学院生物化学与分子生物学系，天津 300070）
Author(s):
SHI Meng; XIANG Song
（Department of Biochemistry and Molecular Biology，School of Basic Medical Sciences，Tianjin Medical University，Tianjin 300070，China）
关键词:
Bre1; 泛素; 谷胱甘肽转移酶; 蛋白质-蛋白质相互作用
Keywords:
Bre1; Ubiquitin; glutathione S-transferase; protein-protein interaction
分类号:
Q51
DOI:
-
文献标志码:
A
摘要:
目的：探究乳酸克鲁维酵母核小体泛素连接酶Bre1的eRING结构域与泛素转移酶Rad6的相互作用，解析Bre1的RBD结构域对该相互作用的影响。方法：在大肠杆菌中表达乳酸克鲁维酵母的GST-Rad6、Rad6、Bre1 eRING等蛋白并将它们纯化至纯度>95%，通过GST-Rad6下拉实验，结合蛋白免疫印迹实验研究GST-Rad6和Bre1的eRING结构域的相互作用及Bre1的RBD结构域对其相互作用的影响；通过SPR实验研究Rad6与Bre1的eRING结构域之间的相互作用。结果：乳酸克鲁维酵母的GST-Rad6与Bre1的eRING结构域存在相互作用，该相互作用可以被Bre1 RBD所抑制；GST蛋白与乳酸克鲁维酵母Bre1的eRING存在相互作用，不可作为该蛋白的下拉标签使用。结论：Rad6与Bre1的eRING结构域存在相互作用，该相互作用可以被RBD所抑制，GST标签不适于乳酸克鲁维酵母的eRING相互作用研究。
Abstract:
Objective： To explore the interaction between the eRING domain of the nucleosome ribosome ubiquitin ligase Bre1 and the ubiquitin transferase Rad6 of S. cerevisiae，elucidate the influence of RBD domain of Bre1 to this interaction. Methods：GST-tagged K. lactis Rad6，Rad6，Bre1 eRING domain and related proteins were expressed in E. coli and purified to a purity of>95%. GST Rad6 pull-down experiments and Western blotting were performed to probe the interaction between the K. lactis Bre1 eRING domain and GST-Rad6 and the effect of the Bre1 RBD domain on their interaction. SPR experiments were performed to probe the interaction between the K. lactis Bre1 eRING domain and Rad6. Results：GST-tagged K. lactis Rad6 interacted strongly with the K. lactis Bre1 eRING domain，the interaction was inhibited by the K. lactis Bre1 RBD. The GST protein alone interacts with the K. lactis Bre1 eRING domain or its N-terminal CCNR domain，so the GST protein was not suitable for the pull-down tag in this experiment. Conclusion：Rad6 interacts with Bre1-eRING domain，and the interaction can be inhibited by RBD. GST tag is not suitable for the pull-down assay in K. lactis.

参考文献/References:

[1] ZHOU K，GAULLIER G，LUGER K. Nucleosome structure and dynamics are coming of age[J]. Nat Struct Mol Biol，2019，26（1）：3-13.
[2] CUTLER J A，PERNER F，ARMSTRONG S A. Histone PTM crosstalk stimulates dot1 methyltransferase activity[J]. Trends Biochem Sci，2021，46（7）：522-524.
[3] KUNADIS E，LAKIOTAKI E，KORKOLOPOULOU P，et al. Targeting post-translational histone modifying enzymes in glioblastoma [J].Pharmacol Ther，2021，220：107721.
[4] BONNET J，DEVYS D，TORA L. Histone H2B ubiquitination：signaling not scrapping [J]. Drug Discov Today，2014，12：e19-e27.
[5] PAG?魪 V，CHEN J J，DURAND-DUBIEF M，et al. Histone H2B ubiquitylation regulates histone gene expression by suppressing antisense transcription in fission yeast[J]. Genetics，2019，213（1）：161-172.
[6] SANS?譫 M，PARUA P K，PINTO D，et al. Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription[J]. Nucleic Acids Res，2020，48（13）：7154-7168.
[7] HSU P L，SHI H，LEONEN C，et al. Structural basis of H2B ubiquitination-dependent H3K4 methylation by compass[J]. Mol Cell，2019，76（5）：712-723，e4.
[8] WORDEN E J，HOFFMANN N A，HICKS C W，et al. Mechanism of cross-talk between H2B ubiquitination and H3 methylation by dot1L [J].Cell，2019，176（6）：1490-1501，e12.
[9] MOYAL L，LERENTHAL Y，GANA-WEISZ M，et al. Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks[J].Mol Cell，2011，41（5）：529-542.
[10] JEUSSET L M，MCMANUS K J. Characterizing and exploiting the many roles of aberrant H2B monoubiquitination in cancer pathogenesis[J]. Semin Cancer Biol，2021，86（3）：782-798.
[11] COLE A J，DICKSON K A，LIDDLE C，et al. Ubiquitin chromatin remodelling after DNA damage is associated with the expression of key cancer genes and pathways[J]. Cell Mol Life Sci，2021，78（3）：1011-1027.
[12] WOOD A，KROGAN N J，DOVER J，et al. Bre1，an E3 ubiquitin ligase required for recruitment and substrate selection of Rad6 at a promoter [J]. Mol Cell，2003，11（1）：267-274.
[13] HWANG W W，VENKATASUBRAHMANYAM S，IANCULESCU A G，et al. A conserved RING finger protein required for histone H2B monoubiquitination and cell size control[J]. Mol Cell，2003，11（1）：261-266.
[14] ROBZYK K，RECHT J，OSLEY M A. Rad6-dependent ubiquitination of histone H2B in yeast [J]. Science，2000，287（5452）：501-504.
[15] HORN V，UCKELMANN M，ZHANG H，et al. Structural basis of specific H2A K13/K15 ubiquitination by RNF168[J]. Nat Commun， 2019，10（1）：1751.
[16] TURCO E，GALLEGOL D，SCHNEIDER M，et al. Monoubiquitination of histone H2B is intrinsic to the Bre1 RING domain-Rad6 interaction and augmented by a second Rad6-binding site on Bre1[J]. J Biol Chem，2015，290（9）：5298-5310.
[17] KIM J，ROEDER R G. Direct Bre1-Paf1 complex interactions and RING finger-independent Bre1-Rad6 interactions mediate histone H2B ubiquitylation in yeast[J]. J Biol Chem，2009，284（31）：20582-20592.
[18] FREEMONT P S，HANSON I M，TROWSDALE J. A novel cysteine-rich sequence motif [J]. Cell，1991，64（3）：483-484.
[19] BARLOW P N，LUISI B，MILNER A，et al. Structure of the C3HC4 domain by 1H-nuclear magnetic resonance spectroscopy. A new structural class of zinc-finger[J]. J Mol Biol，1994，237（2）：201-211.
[20] BORDEN K L，BODDY M N，LALLY J，et al. The solution structure of the RING finger domain from the acute promyelocytic leukaemia proto-oncoprotein PML [J].EMBO J，1995，14（7）：1532-1541.
[21] MATTIROLI F，PENENGO L. Histone ubiquitination：an integrative signaling platform in genome stability[J]. Trends Genet，2021，37（6）：566-581.
[22] LI S，LIANG Y H，MARIANO J，et al. Insights into ubiquitination from the unique clamp-like binding of the RING E3 AO7 to the E2 UbcH5B [J]. J Biol Chem，2015，290（51）：30225-30239.
[23] HIBBERT R G，HUANG A，BOELENS R，et al. E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6[J]. Proc Nat Acad Sci，2011，108（14）：5590-5595.
[24] LOPATA A，KNISS A，L?魻HR F，et al. Ubiquitination in the ERAD process [J]. Int J Mol Sci，2020，21（15）：E5369.
[25] DAS R，LIANG Y H，MARIANO J，et al. Allosteric regulation of E2：E3 interactions promote a processive ubiquitination machine[J].EMBO J，2013，32（18）：2504-2516.
[26] PLECHANOVOV?魣 A，JAFFRAY E G，TATHAM M H，et al. Structure of aRING E3 ligase and ubiquitin-loaded E2 primed for catalysis [J]. Nature，2012，489（7414）：115-120.
[27] KUMAR P，WOLBERGER C. Structure of the yeast Bre1 RING domain [J]. Proteins，2015，83（6）：1185-1190.
[28] DENG Z H，AI H S，LU C P，et al. The Bre1/Rad6 machinery：writing the central histone ubiquitin mark on H2B and beyond [J]. Chromosome Res，2020，28（3/4）：247-258.

相似文献/References:

[1]赵佳奇,石萌,向嵩.Bre1与Rad6形成的复合物的表达、纯化和结晶[J].天津医科大学学报,2023,29(01):83.
　ZHAO Jia-qi,SHI Meng,XIANG Song.Expression，purification and crystallization of Bre1-Rad6 complex[J].Journal of Tianjin Medical University,2023,29(01):83.

备注/Memo

备注/Memo:
基金项目: 国家自然科学基金面上项目（32071205）
作者简介: 石萌（1994-），女，硕士在读，研究方向：生物学；通信作者：向嵩，E-mail：xiangsong@tmu.edu.cn。
更新日期/Last Update: 2023-02-01