The proteasome-interacting Ecm29 protein disassembles the 26S proteasome in response to oxidative stress
The proteasome-interacting Ecm29 protein disassembles the 26S proteasome in response to oxidative stress
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DOI：
10.1074/jbc.m117.803619
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发表时间：
2017-09-29
影响因子：
4.8
通讯作者：
Huang, Lan
中科院分区：
文献类型：
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作者：
Wang, Xiaorong;Chemmama, Ilan E.;Huang, Lan
氧化应激与多种人类神经系统和其他疾病有关。蛋白酶体是多亚基蛋白酶，对于去除氧化损伤的蛋白质至关重要。为了了解应激相关的人类病理学，重要的是要揭示氧化应激后蛋白酶体调节的分子事件。为此，我们研究了H2 O2应激诱导的人26 S蛋白酶体的分子变化，并确定了应激诱导的26 S蛋白酶体解体是保守的从酵母到人类。此外，我们开发并采用了一种新的蛋白质组学方法，XAP（在体内交联辅助亲和纯化），加上稳定同位素标记与细胞培养（SILAC）为基础的定量MS中的氨基酸，捕获和定量几个弱结合蛋白酶体相互作用的蛋白质，并检查它们在应激介导的蛋白酶体重塑的作用。我们的研究结果表明，衔接蛋白Ecm 29是主要的蛋白酶体相互作用蛋白，负责在人类细胞中的26 S蛋白酶体的应力触发重塑。重要的是，使用基于二琥珀酰亚胺基亚砜的交联MS平台，我们绘制了Ecm 29自身内和与蛋白酶体亚基的相互作用，并确定了Ecm 29-蛋白酶体复合物的结构与综合结构建模。这些结果使我们能够提出一种结构模型，其中Ecm 29侵入26 S蛋白酶体中20 S核心颗粒和19 S调节颗粒之间的相互作用，破坏蛋白酶体结构以响应氧化应激。
Oxidative stress has been implicated in multiple human neurological and other disorders. Proteasomes are multi-subunit proteases critical for the removal of oxidatively damaged proteins. To understand stress-associated human pathologies, it is important to uncover the molecular events underlying the regulation of proteasomes upon oxidative stress. To this end, we investigated H2O2 stress-induced molecular changes of the human 26S proteasome and determined that stress-induced 26S proteasome disassembly is conserved from yeast to human. Moreover, we developed and employed a new proteomic approach, XAP (in vivo cross-linking-assisted affinity purification), coupled with stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative MS, to capture and quantify several weakly bound proteasome-interacting proteins and examine their roles in stress-mediated proteasomal remodeling. Our results indicate that the adapter protein Ecm29 is the main proteasome-interacting protein responsible for stress-triggered remodeling of the 26S proteasome in human cells. Importantly, using a disuccinimidyl sulfoxide-based cross-linking MS platform, we mapped the interactions of Ecm29 within itself and with proteasome subunits and determined the architecture of the Ecm29-proteasome complex with integrative structure modeling. These results enabled us to propose a structural model in which Ecm29 intrudes on the interaction between the 20S core particle and the 19S regulatory particle in the 26S proteasome, disrupting the proteasome structure in response to oxidative stress.