Human cyclophilin D is a mitochondrial peptidyl-prolyl isomerase that plays a role in regulating the opening of the mitochondrial permeability transition pore. It is considered a viable and promising molecular target for the treatment of diseases for which disease development is associated with pore opening, e.g., Alzheimer's disease or ischemia/reperfusion injury. Currently available and widely used methods based on Kofron's assay for determining cyclophilin D activity suffer from serious drawbacks and limitations. In this study, a completely novel approach for an assay of cyclophilin D activity using RNase T1 refolding is introduced. The method is simple and is more in line with the presumed physiological role of cyclophilin D in protein folding than Kofron's assay, which relies on a peptide substrate. The method is applicable for identifying novel inhibitors of cyclophilin D as potential drugs for the treatment of the diseases mentioned above. Moreover, the description of CypD activity in the RNase T1 refolding assay reveals new possibilities for investigating the role of cyclophilin D in protein folding in cells and may lead to a better understanding of its pathological and physiological roles.
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