Structural characterization of the interaction of Ubp6 with the 26S proteasome
Structural characterization of the interaction of Ubp6 with the 26S proteasome
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DOI：
10.1073/pnas.1510449112
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发表时间：
2015-07-14
影响因子：
11.1
通讯作者：
Foerster, Friedrich
中科院分区：
文献类型：
--
作者：
Aufderheide, Antje;Beck, Florian;Foerster, Friedrich
在真核细胞中，26S 蛋白酶体负责调节细胞内蛋白质的降解。几个辅助因子与这个大分子机器短暂相互作用并调节其功能。去泛素化酶泛素 C 末端水解酶 6 [Ubp6;哺乳动物中的泛素特异性蛋白酶 (USP) 14] 是最丰富的蛋白酶体相互作用蛋白，在调节蛋白酶体功能方面具有多种作用。在这里，我们研究了在抑制剂泛素醛存在和不存在的情况下 Ubp6 和 26S 蛋白酶体之间相互作用的结构基础。为此，我们将单粒子电子冷冻显微镜与交联和质谱分析相结合。 Ubp6 通过其 N 端泛素样结构域与调节颗粒非 ATP 酶 (Rpn) 1 结合，而其催化 USP 结构域的位置各不相同。添加泛素醛可以稳定 USP 结构域在连接蛋白酶体亚基 Rpn1 和调节颗粒三 A ATP 酶 (Rpt) 1 的位置上的结合。USP 结构域在紧邻 Ubp6 活性位点的位置与 Rpt1 结合，这可能会影响其激活。催化三联体位于 ATP 酶模块口和去泛素化酶 Rpn11 附近，强烈暗示它们的功能联系。在蛋白酶体方面，Ubp6 的结合有利于 26S 蛋白酶体构象转换为中间能量构象状态，特别是在添加泛素醛后。 Ubp6 对 26S 蛋白酶体构象空间的这种调节解释了 Ubp6 对蛋白酶体降解动力学的影响。
In eukaryotic cells, the 26S proteasome is responsible for the regulated degradation of intracellular proteins. Several cofactors interact transiently with this large macromolecular machine and modulate its function. The deubiquitylating enzyme ubiquitin C-terminal hydrolase 6 [Ubp6; ubiquitin-specific protease (USP) 14 in mammals] is the most abundant proteasome-interacting protein and has multiple roles in regulating proteasome function. Here, we investigate the structural basis of the interaction between Ubp6 and the 26S proteasome in the presence and absence of the inhibitor ubiquitin aldehyde. To this end we have used single-particle electron cryomicroscopy in combination with cross-linking and mass spectrometry. Ubp6 binds to the regulatory particle non-ATPase (Rpn) 1 via its N-terminal ubiquitin-like domain, whereas its catalytic USP domain is positioned variably. Addition of ubiquitin aldehyde stabilizes the binding of the USP domain in a position where it bridges the proteasome subunits Rpn1 and the regulatory particle triple-A ATPase (Rpt) 1. The USP domain binds to Rpt1 in the immediate vicinity of the Ubp6 active site, which may effect its activation. The catalytic triad is positioned in proximity to the mouth of the ATPase module and to the deubiquitylating enzyme Rpn11, strongly implying their functional linkage. On the proteasome side, binding of Ubp6 favors conformational switching of the 26S proteasome into an intermediate-energy conformational state, in particular upon the addition of ubiquitin aldehyde. This modulation of the conformational space of the 26S proteasome by Ubp6 explains the effects of Ubp6 on the kinetics of proteasomal degradation.