BPS2026 – Mechanism of cyclophilin modulation of the mitochondrial permeability transition pore

The mitochondrial permeability transition pore (MPTP), which plays an important role in programmed cell death, is a large, non-selective channel located on the mitochondrial inner membrane. During cardiac ischemia-reperfusion, excessive calcium entry into mitochondria induces MPTP opening, leading to the collapse of the mitochondrial membrane potential and triggering a cascade of pathways leading to cell death. Cyclophilin D (CypD), a peptidyl-prolyl isomerase located in the mitochondrial matrix, is a sensitizer of the MPTP. Physiological studies have shown that deleting the CypD gene or inhibiting CypD with cyclosporin A (CsA) significantly reduced tissue damage following ischemia-reperfusion injury in mice. Currently, the molecular mechanism by which CypD enhances PTP opening remains unclear. This motivated us to conduct biochemical characterization of CypD and investigate how it regulates PTP. Interestingly, I found that CypD associates with not only mitochondrial membrane but also Xenopus oocyte membranes, suggesting that it is a peripheral membrane protein. Proteinase K digestion further demonstrated that CypD is localized to the mitochondrial matrix. I then generated CypD knockout HEK 293 cell lines and systematically expressed CypD homologs from a wide range of organisms. Using a mitochondrial calcium-buffering assay, I identified several homologs that cannot sensitize PTP opening. Follow-up experiments using chimera strategies enabled identification of key area that allows CypD to regulate the PTP. This finding provides new insights into the molecular mechanism by which the physiologically crucial PTP-opening process is regulated.