Mechanism of the Rpn13-induced activation of Uch37.
Mechanism of the Rpn13-induced activation of Uch37.
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Rpn13 诱导 Uch37 激活的机制
DOI：
10.1007/s13238-014-0046-z
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发表时间：
2014
期刊：
影响因子：
21.1
通讯作者：
Liu, Zhi-Jie
中科院分区：
文献类型：
--
作者：
Jiao, Lianying;Ouyang, Songying;Shaw, Neil;Song, Gaojie;Feng, Yingang;Niu, Fengfeng;Qiu, Weicheng;Zhu, Hongtao;Hung, Li-Wei;Zuo, Xiaobing;Shtykova, V. Eleonora;Zhu, Ping;Dong, Yu-Hui;Xu, Ruxiang;Liu, Zhi-Jie
Uch 37是一种去泛素化酶，由Rpn 13激活，参与蛋白质的蛋白酶体降解。全长Uch 37显示出低的异肽酶活性，并且被认为是自抑制的。结构比较显示，在Uch 37的同源二聚体中，每个催化结构域都阻断了另一个的泛素（Ub）结合位点。这种阻断可能阻止Ub进入Uch 37的活性位点，并可能形成自抑制的基础。为了清楚地了解自抑制模式并阐明Rpn 13对Uch 37的激活机制，我们使用诱变、生化、NMR和小角X射线散射（SAXS）技术的组合研究了Uch 37-Rpn 13复合物。我们的研究结果还证明，Uch 37在溶液中寡聚化，对去泛素化酶的荧光底物泛素-7-氨基-4-甲基香豆素（Ub-AMC）具有非常低的活性。Uch 37 ΔHb，Hc，KEKE，C-末端延伸区（残基256-329）的截短去除将寡聚Uch 37转化为单体形式，其表现出与仅含截短的Uch 37催化结构域相当的异肽酶活性。我们还证明了Rpn 13 C（Rpn 13残基270-407）可以通过隔离Uch 37并形成Uch 37-Rpn 13复合物来破坏Uch 37的寡聚化。Uch 37在这样的复合物中被激活，表现出比单独的Uch 37高12倍的活性。时间分辨SAXS（TR-SAXS）和FRET实验支持所提出的自抑制模式和Rpn 13激活Uch 37的机制。Rpn 13通过形成1：1的化学计量复合物激活Uch 37，其中Uch 37的活性位点可接近Ub。
Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other’s ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37ΔHb,Hc,KEKE, a truncation removal of the C-terminal extension region (residues 256–329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270–407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
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影响因子：
3.7
作者：
Larzábal M;Mercado EC;Vilte DA;Salazar-González H;Cataldi A;Navarro-Garcia F
通讯作者：
Navarro-Garcia F
DOI：
10.1111/j.1742-4658.2011.08393.x
发表时间：
2011-12
期刊：
The FEBS journal
影响因子：
--
作者：
Maiti TK;Permaul M;Boudreaux DA;Mahanic C;Mauney S;Das C
通讯作者：
Das C
影响因子：
6.1
作者：
Konarev, PV;Volkov, VV;Svergun, DI
通讯作者：
Svergun, DI
影响因子：
6.1
作者：
Kozin, MB;Svergun, DI
通讯作者：
Svergun, DI
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL