Targeting of Splice Variants of Human Cytochrome P450 2C8 (CYP2C8) to Mitochondria and Their Role in Arachidonic Acid Metabolism and Respiratory Dysfunction [Membrane Biology]

August 26th, 2014 by Bajpai, P., Srinivasan, S., Ghosh, J., Nagy, L., Wei, S., Guengerich, F. P., Avadhani, N. G.

In this study we found that the full length CYP2C8 (WT CYP2C8) and also an N-terminal truncated splice variant 3 (~44 kDa mass) are localized in mitochondria in addition to the endoplasmic reticulum. Analysis of human livers showed that the mitochondrial levels of these two forms varied markedly. Molecular modeling based on the X-ray crystal structure coordinates of CYP2D6 and CYP2C8 showed that despite lacking the N-terminal 102 residues, variant 3 had nearly complete substrate binding and heme binding pockets. Stable expression of cDNAs in HepG2 cells showed that the WT protein is mostly targeted to the endoplasmic reticulum and at low levels to mitochondria, while the variant 3 is primarily targeted to mitochondria and at low levels to the endoplasmic reticulum. Enzyme reconstitution experiments showed that both microsomal and mitochondrial WT CYP2C8 efficiently catalyzed the paclitaxel 6-hydroxylation. However, mitochondrial variant 3 was unable to catalyze this reaction possibly because of its inability to stabilize the large 854 Da substrate. The mitochondrial variant 3, on the other hand, catalyzed the metabolism of arachidonic acid into 8-9, 11-12, and 14-15 EETs (epoxyeicosatrienoic acids), and also 20-hydroxyeicosatetraenoic acid (20-HETE) when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondria-targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.
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