Exploration of the mechanistic role of Cyclophilin D in the mitochondria

Introduction Cyclophilin D (CypD) is an 18 kda protein encoded by the nuclear ppif gene and transported to the mitochondria. Being the only member of the cyclophilin family localized in the mitochondria, its known function is the regulation of the mitochondrial permeability transition pore (MPTP) which usually precedes mitochondrial dysfunction during calcium overload and oxidative stress. However, the mechanism of MPTP induction by CypD is still unknown, therefore, this study is aimed at understanding the generic role of CypD in the mitochondria. Method CypD of murine liver mitochondria was quantitatively determined from recombinant CypD standard curve using densitometric analysis programme, image J. Difference in CypD level between liver and heart tissue mitochondria was quantified. Modulation of the binding stability of recombinant CypD by some mitochondrial metabolites ( in vitro ) was carried out by dynamic scanning fluorimetry (DSF). Mitochondrial swelling and NAD(P)H fluorescence were monitored spectrophotometrically at 540 nm and 355/450 (Ex/Em) respectively. Metabolic differences between CypD WT and KO murine tissues were carried out using HNMR spectroscopy. Results CypD was observed to constitute about 0.1% of the total mitochondrial protein. It was also observed to be more than 2‐fold higher in the liver than heart tissue mitochondria. MPTP activation experiments revealed 2‐fold higher calcium induced swelling in liver than heart tissue mitochondria. Mitochondrial NAD(P)H fluorescence was observed to be highest at 100 uM calcium in the presence of CsA while a massive loss of NAD(P)H was observed when CypD was uninhibited at this concentration. Succinyl CoA and alphaketoglutarate, TCA cycle intermediates enhanced calcium induced mitochondrial swelling however, DSF reveals that Succinyl CoA negatively regulates CypD binding interaction with CsA while alphaketoglutarate had no effect. Global metabolite differences were observed in CypD genotypes and increased level of succinate and fumarate in were observed in ppif KO when compared with WT heart tissues. Conclusion These findings give further insight into the role of CypD in the mitochondria as it revealed differences in MPTP between tissues and impact on cellular reductive equivalents which are associated with the differential amount of CypD in the mitochondria of these tissues. It also reveals the influence of CypD on global metabolism especially in the liver which is the majour site of metabolism and that some of these may play direct or indirect role in MPTP activation. Support or Funding Information OSA acknowledge Osun State University, Nigeria for financial support.YIA is grateful to University of Liverpool for a Postgraduate Bursary This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .