Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.
Structure-function analysis of histone H2B and PCNA ubiquitination dynamics using deubiquitinase-deficient strains.
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使用去泛素酶缺陷菌株对组蛋白H2B和PCNA泛素化动力学的结构 - 功能分析。
DOI：
10.1038/s41598-023-43969-z
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发表时间：
2023-10-04
影响因子：
4.6
通讯作者：
Chandrasekharan, Mahesh B.
中科院分区：
文献类型：
--
作者：
Radmall, Kaitlin S.;Shukla, Prakash K.;Leng, Andrew M.;Chandrasekharan, Mahesh B.
泛素在蛋白质上的翻译后共价结合或泛素化在几乎所有的细胞过程中都很重要。体内单个蛋白质的稳态泛素化通过两种相反的酶活性来维持：E1，E2和E3酶的泛素缀合和去泛素化酶的去除。在这里，我们删除了一个或多个基因编码的去泛素化酶在酵母和评估的要求泛素结合到一个目标蛋白。我们的原理验证研究表明，相关的去泛素化酶的缺乏提供了一种简便和通用的方法，可用于研究体内靶蛋白的泛素缀合和去泛素化的细微差别。我们验证了我们的方法，使用突变体缺乏去泛素化酶Ubp 8和/或Ubp 10，去除泛素从组蛋白H2 B或PCNA。我们的研究表明，接头蛋白Lge 1的C-末端卷曲结构域和Rad 6 E2的C-末端酸性尾有助于组蛋白H2 BK 123的单泛素化，而Rad 6的螺旋-4的远端酸性残基，而不是酸性尾，是PCNA的单泛素化所必需的。此外，H2 B C-末端螺旋中丙氨酸-120的带电取代通过抑制Rad 6-Bre 1介导的泛素缀合和促进Ubp 8/Ubp 10介导的去泛素化而不利地影响组蛋白H2 BK 123单泛素化。总之，酵母去泛素化酶UBP 8和/或UBP 10的缺乏允许揭示体内泛素添加和从其生理底物如组蛋白H2 B或PCNA去除的调节和要求。
Post-translational covalent conjugation of ubiquitin onto proteins or ubiquitination is important in nearly all cellular processes. Steady-state ubiquitination of individual proteins in vivo is maintained by two countering enzymatic activities: conjugation of ubiquitin by E1, E2 and E3 enzymes and removal by deubiquitinases. Here, we deleted one or more genes encoding deubiquitinases in yeast and evaluated the requirements for ubiquitin conjugation onto a target protein. Our proof-of-principle studies demonstrate that absence of relevant deubiquitinase(s) provides a facile and versatile method that can be used to study the nuances of ubiquitin conjugation and deubiquitination of target proteins in vivo. We verified our method using mutants lacking the deubiquitinases Ubp8 and/or Ubp10 that remove ubiquitin from histone H2B or PCNA. Our studies reveal that the C-terminal coiled-domain of the adapter protein Lge1 and the C-terminal acidic tail of Rad6 E2 contribute to monoubiquitination of histone H2BK123, whereas the distal acidic residues of helix-4 of Rad6, but not the acidic tail, is required for monoubiquitination of PCNA. Further, charged substitution at alanine-120 in the H2B C-terminal helix adversely affected histone H2BK123 monoubiquitination by inhibiting Rad6-Bre1-mediated ubiquitin conjugation and by promoting Ubp8/Ubp10-mediated deubiquitination. In summary, absence of yeast deubiquitinases UBP8 and/or UBP10 allows uncovering the regulation of and requirements for ubiquitin addition and removal from their physiological substrates such as histone H2B or PCNA in vivo.
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影响因子：
4.8
作者：
Atkin G;Paulson H
通讯作者：
Paulson H
影响因子：
64.8
作者：
Jumper J;Evans R;Pritzel A;Green T;Figurnov M;Ronneberger O;Tunyasuvunakool K;Bates R;Žídek A;Potapenko A;Bridgland A;Meyer C;Kohl SAA;Ballard AJ;Cowie A;Romera-Paredes B;Nikolov S;Jain R;Adler J;Back T;Petersen S;Reiman D;Clancy E;Zielinski M;Steinegger M;Pacholska M;Berghammer T;Bodenstein S;Silver D;Vinyals O;Senior AW;Kavukcuoglu K;Kohli P;Hassabis D
通讯作者：
Hassabis D
影响因子：
4.8
作者：
Chandrasekharan, Mahesh B.;Huang, Fu;Sun, Zu-Wen
通讯作者：
Sun, Zu-Wen
影响因子：
64.8
作者：
Gallego, Laura D.;Schneider, Maren;Koehler, Alwin
通讯作者：
Koehler, Alwin
影响因子：
4.8
作者：
Daniel, JA;Torok, MS;Grant, PA
通讯作者：
Grant, PA