A Novel In Vitro CypD-Mediated p53 Aggregation Assay Suggests a Model for Mitochondrial Permeability Transition by Chaperone Systems

Abstract

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, wildtype 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 member of the mitochondrial unfolded protein response (mtUPR), and its ability to suppress mPT in a p53-dependent manner. Our study finds that catalytically active CypD causes strong aggregation of wildtype 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 inhibition of Trap1 by the mitochondria-specific HSP90 ATPase antagonist gamitrinib strongly sensitizes primary mouse embryonic fibroblasts (MEFs) to mPT and permeability transition pore (mPTP) opening in a p53- and CypD-dependent manner. We propose a mechanism by which 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.

The path from mitochondrial ROS to aging runs through the mitochondrial permeability transition pore

“…In young animals, CypD is largely inhibited as a result of a number of post‐translational modifications (Elrod & Molkentin, ). CypD also interacts with inhibitory proteins such as HSP90 (TRAP1 in humans), which was reported to aggregate CypD (Lam et al ., ) and prevent its interaction with activating proteins (e.g., P53) and the mPTP (Lebedev et al ., ). In aged cells, the UPRmt is suppressed, inhibiting HSP90 synthesis and its translocation to the matrix, while P53 is translocated to the matrix to further enhance CypD activation (Vaseva et al ., ; Priami et al ., ).…”

Mitochondria and aging: A role for the mitochondrial transition pore?

“…Recently, Lebedev et al. (2016) suggested a model in which p53 activates CypD by displacing it from Trap1, an Hsp90‐related mitochondrial matrix protein that complexes CypD, and maintains it inactive. This process may occur during aging and favors mPTP opening (Figure 2).…”

Transgenic Expression of the Mitochondrial Chaperone TNFR-associated Protein 1 (TRAP1) Accelerates Prostate Cancer Development

“…These molecules preserve the folding of key regulators of oxidative phosphorylation, membrane permeability transition, and redox balance (7). Accordingly, structural requirements of TRAP1 regulation of mitochondrial cell death have been elucidated recently (8). In this context, pharmacologic or genetic interference with Hsp90/ TRAP1-directed protein folding causes collapse of multiple mitochondrial functions (7,9) with suppression of tumor cell proliferation (10,11), induction of apoptosis (9,12,13), and inhibition of cell invasion (14,15).…”