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首页> 外文期刊>Journal of Molecular Biology >A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems
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A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems

机译：一种新型的体外CypD介导的p53聚集测定法提出了一种由伴侣系统进行线粒体通透性转变的模型

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摘要

Tissue necrosis as a consequence of ischemia-reperfusion injury and oxidative damage is a leading cause of permanent disability and death worldwide. The complete mechanism by which cells undergo necrosis upon oxidative stress is not understood. In response to an oxidative insult, wild-type p53 has been implicated as a central regulatory component of the mitochondrial permeability transition (mPT), triggering necrosis. This process is associated with cellular stabilization and translocation of p53 into the mitochondrial matrix. Here, we probe the mechanism by which p53 activates the key mPT regulator cyclophilin D (CypD). We explore the involvement of Trap1, an Hsp90-related mitochondrial matrix protein and a member of the mitochondrial unfolded protein response, and its ability to suppress mPT in a p53-dependent manner. Our study finds that catalytically active CypD causes strong aggregation of wild-type p53 protein (both full-length and isolated DNA-binding domain) into amyloid-type fibrils in vitro. The responsible CypD residues for this activity were mapped by NMR to the active site amino acids R55, F60, F113, and W121. The data also present a new proline isomerization assay for CypD by monitoring the aggregation of p53 as an indicator of CypD activity. Moreover, we find that the inhibition of Trap1 by the mitochondria-specific HSP90 ATPase antagonist Gamitrinib strongly sensitizes primary mouse embryonic fibroblasts to mPT and permeability transition pore opening in a p53- and CypD-dependent manner. We propose a mechanism by which the influx of unfolded p53 into the mitochondrial matrix in response to oxidative stress indirectly activates the normally inhibited CypD by displacing it from Trap1 complexes. This activates CypD's isomerase activity. Liberated CypD then isomerizes multiple proteins including p53 (causing p53 aggregation) and the structural components of the mPTP pore, inducing pore opening. This working model can now be tested in the future. (C) 2016 Elsevier Ltd. All rights reserved.
机译：缺血-再灌注损伤和氧化损伤导致的组织坏死是全世界永久性残疾和死亡的主要原因。细胞氧化应激后坏死的完整机制尚不清楚。作为对氧化损伤的响应，野生型p53被认为是线粒体通透性转变（mPT）的中央调节成分，引发坏死。此过程与细胞稳定和p53进入线粒体基质的转运有关。在这里，我们探讨了p53激活关键mPT调节剂亲环蛋白D（CypD）的机制。我们探索Trap1，Hsp90相关的线粒体基质蛋白和线粒体未折叠的蛋白质反应的成员的参与，以及它以p53依赖的方式抑制mPT的能力。我们的研究发现，具有催化活性的CypD会导致野生型p53蛋白（全长和分离的DNA结合域）在体外强烈聚集为淀粉样蛋白原纤维。通过NMR将负责该活性的CypD残基定位到活性位点氨基酸R55，F60，F113和W121。数据还通过监测p53的聚集作为CypD活性的指标，提供了CypD的新脯氨酸异构化检测方法。此外，我们发现线粒体特异的HSP90 ATPase拮抗剂Gamitrinib对Trap1的抑制作用以p53和CypD依赖的方式强烈刺激原代小鼠胚胎成纤维细胞对mPT和通透性转变孔的开放。我们提出了一种机制，通过该机制，未折叠的p53涌入线粒体基质以响应氧化应激，通过将其从Trap1复合物中置换而间接激活正常抑制的CypD。这会激活CypD的异构酶活性。然后，被释放的CypD异构化包括p53（导致p53聚集）和mPTP孔的结构成分在内的多种蛋白质，从而诱导孔的开放。现在可以在将来测试此工作模型。 （C）2016 Elsevier Ltd.保留所有权利。

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