Abstract 16765: Cyclophilin-D: A Regulator of Mitochondrial Gene Expression
Background: Cyclophilin-D (Cyp-D) is a peptidyl-prolyl isomerase resident in the mitochondrial matrix; and like other cyclophilins it can catalyze conformational changes of proteins. Cyp-D is best known for its role in lowering the threshold for opening of the mitochondrial permeability transition pore. However, a serendipitous observation by us, whereby Cyp-D silencing halted cell proliferation, and reports by others that Cyp-D could interact with nuclear transcription factors that have translocated to mitochondria, prompted us to hypothesize that Cyp-D could interact with native mitochondrial transcription factors (mTFs; TFB1M, TFB2M, and TFAM) and therefore be a key regulator of mitochondrial gene expression. Given that all 13 mitochondrial-encoded proteins are components of respiratory chain complexes, our hypothesis would support a central role of Cyp-D in cellular respiration.
Methods and Results: We first analyzed the interaction of Cyp-D with mTFs by co-immunoprecipitation (co-IP) and reverse co-IP after transfection of constructs expressing FLAG-tagged Cyp-D and V5-tagged mTFs in HEK 293T cells, showing specific interactions of Cyp-D with TFB1M and with TFB2M but not with TFAM. We then quantitated by real time qPCR mitochondrial-encoded respiratory chain transcripts in HEK 293T cells at day 8 post-infection with lentiviruses encoding shRNA against Cyp-D, showing striking reductions in transcripts ND1, COX1, and ATP6 (initiated from the heavy strand promoter 2) but not in transcript ND6 (initiated from the light strand promoter). Finally, we measured cellular oxygen consumption, also on post-infection day 8, and found it reduced without improvement after uncoupling of electron transport from ATP synthesis with FCCP, consistent with the aforementioned reductions in mitochondrial transcripts of electron transport complexes. At the same time, mitochondrial mass, expression of nuclear genes, and cell viability were all preserved.
Conclusion: In HEK 293 T cells, Cyp-D is required for mitochondrial gene expression modulating transcription possibly through the heavy strand promoter 2 suggesting a novel Cyp-D function as regulator of oxidative phosphorylation and thus of the fundamental process of cellular respiration.
Author Disclosures: J. Radhakrishnan: None. R.J. Gazmuri: None.
- © 2014 by American Heart Association, Inc.