Lebedev Ivan, Nemajerova Alice, Foda Zachariah H, Kornaj Maja, Tong Michael, Moll Ute M, Seeliger Markus A
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA.
Department of Pathology, Stony Brook University, Stony Brook, NY 11794, USA.
J Mol Biol. 2016 Oct 9;428(20):4154-4167. doi: 10.1016/j.jmb.2016.08.001. Epub 2016 Aug 8.
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.
缺血再灌注损伤和氧化损伤导致的组织坏死是全球永久性残疾和死亡的主要原因。细胞在氧化应激下发生坏死的完整机制尚不清楚。在氧化损伤反应中，野生型p53被认为是线粒体通透性转换（mPT）的核心调节成分，触发坏死。这个过程与细胞稳定以及p53转位到线粒体基质有关。在这里，我们探究p53激活关键mPT调节因子亲环蛋白D（CypD）的机制。我们研究了Trap1的作用，Trap1是一种与热休克蛋白90相关的线粒体基质蛋白，也是线粒体未折叠蛋白反应的成员，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在体外会导致野生型p53蛋白（全长和分离的DNA结合结构域）强烈聚集成淀粉样纤维。通过核磁共振将这种活性的负责CypD残基定位到活性位点氨基酸R55、F60、F113和W121。这些数据还通过监测p53的聚集作为CypD活性的指标，提出了一种新的CypD脯氨酸异构化检测方法。此外，我们发现线粒体特异性热休克蛋白90 ATP酶拮抗剂Gamitrinib对Trap1的抑制以p53和CypD依赖的方式使原代小鼠胚胎成纤维细胞对mPT和通透性转换孔开放高度敏感。我们提出了一种机制，即响应氧化应激时未折叠的p53流入线粒体基质，通过将其从Trap1复合物中置换出来，间接激活通常被抑制的CypD。这激活了CypD的异构酶活性。释放的CypD然后使多种蛋白质异构化，包括p53（导致p53聚集）和mPTP孔的结构成分，诱导孔开放。这个工作模型现在可以在未来进行测试。
