title: Targeting of splice variants of human cytochrome P450 2C8 (CYP2C8) to mitochondria and their role in arachidonic acid metabolism and respiratory dysfunction.
abstract: In this study, we found that the full-length CYP2C8 (WT CYP2C8) and 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 possessed 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, whereas 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 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. Conversely, mitochondrial variant 3 catalyzed the metabolism of arachidonic acid into 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid when reconstituted with adrenodoxin and adrenodoxin reductase. HepG2 cells stably expressing variant 3 generated higher levels of reactive oxygen species and showed a higher level of mitochondrial respiratory dysfunction. This study suggests that mitochondrially targeted variant 3 CYP2C8 may contribute to oxidative stress in various tissues.
authors.affiliation: From the Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and.
authors.forename: Prachi
authors.initials: P
authors.lastname: Bajpai
authors.affiliation: From the Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and.
authors.forename: Satish
authors.initials: S
authors.lastname: Srinivasan
authors.affiliation: From the Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and.
authors.forename: Jyotirmoy
authors.initials: J
authors.lastname: Ghosh
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: Leslie D
authors.initials: LD
authors.lastname: Nagy
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: Shouzou
authors.initials: S
authors.lastname: Wei
authors.affiliation: Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
authors.forename: F Peter
authors.initials: FP
authors.lastname: Guengerich
authors.affiliation: From the Department of Animal Biology and Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 and Narayan@vet.upenn.edu.
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D019556:COS Cells; D019069:Cell Respiration; D002522:Chlorocebus aethiops; D003198:Computer Simulation; D065727:Cytochrome P-450 CYP2C8 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008862:Microsomes, Liver / Q000201:enzymology; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D011485:Protein Binding; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000596:Amino Acids; D007527:Isoenzymes; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D016718:Arachidonic Acid; D006418:Heme; D001189:Aryl Hydrocarbon Hydroxylases; C450259:CYP2C8 protein, human; D065727:Cytochrome P-450 CYP2C8
keywords: Alternative Splicing; Arachidonic Acid (AA) (ARA); Cytochrome P450; Mitochondrial Respiratory Chain Complex; Mitochondrial Transport; Oxidative Stress; Splice Variant