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Current Drug Metabolism, 2010, 11, 830-838

Molecular Mechanisms Regulating the Mitochondrial Targeting of Microsomal Cytochrome P450 Enzymes
Taeho Ahn1,* and Chul-Ho Yun2,*
1

Department of Biochemistry, College of Veterinary Medicine, Chonnam National University and 2School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea
Abstract: Cytochrome P450 enzymes (CYPs) are a superfamily of monooxygenases found in almost all living organisms. CYPs are predominantly localized in the endoplasmic reticulum membranes as integral membrane proteins, where they metabolize a variety of endogenous and xenobiotic compounds. CYPs also reside in other subcellular compartments, including the plasma membranes and mitochondria. CYP localization in mitochondria is regulated in one of two ways: (1) direct targeting of inherent CYPs with canonical mitochondrial signals in their protein sequence after synthesis in the cytosol or (2) mitochondrial localization of microsomal CYPs after processing of the NH2-terminal region. Microsomal CYPs targeted to mitochondria demonstrate conventional or altered catalytic activities using electrons provided by the mitochondrial electron transport system. Mechanisms of microsomal CYP targeting to mitochondria, regulation of localization, and the implications of these in drug metabolism are described in the present review.

Keywords: cytochrome P450, drug metabolism, microsome, mitochondria, signal sequence, targeting. INTRODUCTION General Aspects of CYP Enzymes The cytochrome P450 (CYP) enzymes are a superfamily of monooxygenases (EC 1.14.14.1) found in almost all living organisms, from archaea to humans (http://drnelson.uthsc.edu/ cytochrome P450.html). The CYPs are heme-containing proteins with molecular weights ranging from 45,000 to 60,000 Da. They absorb light at 450 nm, hence the name P450, when the reduced hemeprotein is bound to carbon monoxide. In mammals, CYPs exist primarily in the endoplasmic reticulum (ER) membranes of hepatic cells. They comprise the major enzymatic system that catalyzes the oxidation of various endogenous compounds, including fatty acids, hormones, and eicosanoids, as well as many exogenous substances, such as drugs, carcinogens, and other xenobiotic chemicals [1, 2]. The CYP redox system necessarily includes phospholipids and the NADPH-cytochrome P450 reductase (CPR), which is an electron donor to the CYPs. The system encompasses more than 60 different chemical reactions including aromatic and aliphatic hydroxylation, N- and S- oxidation, epoxidation, and dealkylation [3, 4]. CYP enzymes also appear to play key roles in the detoxification of many chemicals, thus forming a defense system against hazardous substances in humans [5]. However, CYP metabolism of some chemicals may result in compounds with increased toxicity, as in procarcinogen transformation into carcinogens [5, 6]. Drug-metabolizing CYPs are also present and functionally active in most extrahepatic tissues and are involved in the metabolism of endogenous compounds as well as in that of drugs and environmental chemicals. Similar to hepatic CYPs, the expression and catalytic activity of extrahepatic CYPs may be regulated by factors present in the tissue in which they are expressed [7]. Each mammalian species has between 40 60 different CYP enzymes. Within the human genome, 57 active P450 genes and 58 pseudogenes are present (see http://drnelson.uthsc.edu/cytochrome P450.html). Among the numerous CYP subtypes, the metabolism of most drugs in humans is performed by the P450 families 1, 2, and 3. Other CYPs are involved in metabolism of endogenous compounds such as steroids and fatty acids. Typical human CYP activities are summarized in a previous review [8]. In mammals, the expression levels of CYPs and their activities differ by age, gender, ethnicity, individual, diet, and disease state [9-11]. Microsomal CYPs Microsomal CYPs are integral membrane proteins that are anchored to the ER membrane through an NH2-terminal region, and the large catalytic domain is exposed to the cytoplasm. Regardless of the subtypes, this membrane topology is applied to all microsomal CYPs for functional coupling with CPR and phase II enzymes in this compartment. The NH2-terminal region comprises a long stretch of hydrophobic amino acids usually proceeded by a negatively charged amino acid Fig. (1). The charged and hydrophobic regions in the NH2-terminus are followed by basic amino acid residues and a well-conserved proline-rich region that is important for the correct folding of the membrane-integrated protein and is essential for catalytic activity [12]. Microsomal CYPs are targeted and inserted into the endoplasmic reticulum (ER) membrane by cotranslation in a signal recognition particle (SRP)-dependent manner. This process is highly conserved and regulated and involves several proteins and protein complexes present in the cytoplasm and the ER membrane. Microsomal CYPs are translated on membrane-free ribosomes in the cytosol; once the hydrophobic NH2-terminus emerges from the ribosome where the SRP has bound, translation is halted and the complex is subsequently targeted to the ER. In the ER, translation then continues after binding with the SRP receptor present in the ER membrane [13]. The protein translocation through the ER membrane is inhibited after the hydrophobic NH2-terminus is inserted in the membrane, leading to translation of the remaining CYP protein on the cytoplasmic side of the ER membrane [14, 15]. Several studies have demonstrated the roles of the NH2-terminus of CYPs during these processes: the NH2-terminal region acts as the signal sequence to which the SRP binds, targeting the translational complex to the ER membrane. It also acts as a signal-anchor sequence responsible for the CYP binding to the ER membrane (ER retention) and, consequently, inhibition of ER-mediated protein targeting to other compartments in the cell. In addition, several studies have reported that this region determines the topological orientation of CYP proteins in the ER membrane, where a large part of the polypeptide is exposed on the cytoplasmic side and the NH2terminal end is anchored to the ER membrane [16-18]. The replacement of the acidic amino acids with basic residues results in
2010 Bentham Science Publishers Ltd.

*Address correspondence to these authors at the Department of Biochemistry, College of Veterinary Medicine, Chonnam National University Tel: 8262-530-2823/Fax: 82-62-530-2809; E-mail: thahn@jnu.ac.kr and School of Biological Sciences and Technology and Hormone Research Center, Chonnam National University, Gwangju 500-757, Republic of Korea; Tel: 82-62-530-2194; Fax: 82-62-530-2199; E-mail: chyun@jnu.ac.kr 1389-2002/10 $55.00+.00

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Fig. (1). Comparison of characteristic NH2-terminal sequences between human CYP2C9 (microsomal CYP) and human CYP11A1 (mitochondrial CYP). Positively (red color) and negatively (blue color) charged amino acids are marked with plus and minus signs, respectively.

protein translocation into the ER lumen as demonstrated for CYP2C2 and CYP2C11 [18, 19]. However, the molecular properties of the NH2-terminal region responsible for the ER retention still remain uncharacterized, as the conventional ER retention and retrieval signals, KDEL and KKXX motifs, are absent from microsomal CYPs. The NH2-terminus of some CYP2C enzymes mediates direct retention in the ER, whereas CYP2E1 resides in the membranes through a retrieval mechanism from post-ER compartments [20]. Some ER-retention receptors may be involved in the CYP retention: BAP31 (B cell-associated protein 31) interacts with CYP2C2, and the downregulation of BAP31 results in a redistribution of CYP2C2 to the nuclear membrane, Golgi apparatus, and plasma membrane, suggesting that BAP31 is essential for the enzymes localization [21]. However, it is unknown whether BAP31 is involved in the retention of other CYPs. It is hypothesized that different CYPs are retained in the ER by different retention mechanisms and these differences are partially imposed by molecular properties of the NH2-terminal region such as hydrophobicity, length, and charge. In summary, the NH2-terminal 20- to 40-amino acid residues of CYPs are crucial for protein synthesis, for correct targeting to and incorporation into the ER membrane, and for appropriate folding to form the catalytically active protein. Mitochondrial CYPs CYPs were shown to be present in mitochondria isolated from steroidogenic organs and later in mitochondria from other organs. Mitochondrial CYPs differ from the microsomal CYPs not only with respect to localization, but also with regard to substrate specificity and structural characteristics [22]. Mitochondrial CYPs, including CYP11A, 24A, and 27A, are imported into the mitochondria through translocation channels that are present in the mitochondrial outer membrane (TOM, translocase of the outer membrane) and inner membrane (TIM, translocase of the inner membrane) by means of a canonical mitochondrial-targeting sequence. The signal sequence is also present at the NH2-terminus of these CYPs and consists of a few positively charged amino acids, arginine or lysine, and a stretch of neutral or hydrophobic amino acids Fig. (1). The mitochondrial-targeting sequence is proteolytically removed after import into the mitochondrial matrix, and the mature protein is associated with the inner membrane, whereas the catalytic domain remains exposed to the matrix space. Mitochondrial CYPs receive electrons for the catalysis of monooxygenation reactions from NADPH via NADPH-

adrenodoxin reductase (AdR) and adrenodoxin (Adx), which are both soluble matrix proteins whereas microsomal P450s receive electrons from NADPH via ER membrane-bound CPR. The apparent difference in the electron transport from NADPH to CYPs has long been considered to be an important distinction between mitochondrial and microsomal CYPs. However, recent studies have suggested that some microsomal CYPs can receive electrons from Adx [23]. In addition, it was reported that truncated forms of a rat microsomal CYP1A1 were imported into mitochondria and received electrons from Adx [24]. Microsomal CYP2E1 targeted to mitochondria by deletion of the NH2-terminal hydrophobic transmembrane domain was also enzymatically active with AdR and Adx as the electron donor [25]. Conversely, a rat mitochondrial CYP27A was targeted to the ER-membrane in yeast cells through modification of the amino-terminal targeting sequence, and efficiently received electrons from CPR for the catalysis of 27hydroxylation of cholesterol [26]. Therefore, both mitochondrial and microsomal CYPs seem to be able to receive electrons from either the CPR or Adx system depending on the enzyme localization although it was suggested that the different electron transfer proteins may differently modulate the CYP enzyme activity [24]. Mitochondrial CYPs play essential roles in the biosynthesis of steroid hormones from cholesterol. The first and the rate-limiting step of steroid hormone biosynthesis in mammalian steroidogenic organs is the side chain cleavage of cholesterol catalyzed by CYP11A (CYPscc), a mitochondrial CYP that produces pregnenolone. The subsequent reactions in the synthesis of various steroid hormones, adrenal cortex hormones, and sex steroid hormones are catalyzed by two other mitochondrial CYPs (11B1 and 11B2) and three microsomal CYPs (17A, 19A, and 21A) together with hydroxysteroid dehydrogenases [27]. CYP11B1 and CYP11B2 catalyze the final steps in the synthesis of glucocorticoids and mineral corticoids in the adrenal cortex. All steps for the bile acid biosyntheses from cholesterol are also catalyzed by microsomal and mitochondrial CYPs. Mitochondrial CYP27A catalyzes one of the final steps of bile acid synthesis; 27-hydroxylation of 5 cholestane-3 ,7 ,12 -triol and 5 -cholestane-3 ,7 -diol in the synthesis of cholic acid and chenodeoxycholic acid, respectively, in the classic pathway of bile acid synthesis in the liver. Mitochondrial CYP27A also catalyzes the 27-hydroxylation of cholesterol in the alternative pathway of bile acid synthesis [28, 29]. Several microsomal CYPs (7A, 7B, 8B, 39A, and 46A) participate in the synthesis of bile acids from cholesterol. 7 -Hydroxylation of cholesterol catalyzed by CYP7A is the rate-limiting step in the biosynthesis of

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bile acids in the liver. The metabolic conversion of Vitamin D, D2, and D3, to the active form is catalyzed by mitochondrial CYP27A and 27B. CYP27A catalyzes the 25-hydroxylation of Vitamin D in the liver in addition to its participation in bile acid synthesis. CYP27B catalyzes the 1 -hydroxylation of 25-hydroxyvitamin D3 in the kidney to form 1 ,25-dihydroxyvitamin D3, the active form of Vitamin D. Another mitochondrial CYP in the kidney, CYP24A, catalyzes 24-hydroxylation of 25-hydroxyvitamin D3 and 1 ,25dihydroxyvitamin D3, which regulates the level of the active form of Vitamin D [30]. General Mechanisms for Mitochondrial Targeting of Microsomal CYPs Mitochondria isolated from rat liver and brain were shown to metabolize xenobiotics, such as aflatoxin B1 and benzo[a]pyrene (BaP) [31, 32]. Subsequent studies identified the presence of several microsomal CYPs, including CYP1A, CYP2B, CYP2D6, CYP2E1, and CYP3A in mitochondria isolated from the liver, brain, and lungs in rats [33]. Similar CYP subtypes and associated monooxygenase activities were also identified in human brain mitochondrial samples obtained at autopsy [34]. Further characterization revealed that, similar to the inherent mitochondrial CYPs, the microsomal CYPs were located inside the mitochondrial matrix and were dependent on Adx and AdR for catalytic activity [35-37]. Quantitatively, the amount of CYPs targeted to the mitochondria was lower compared with the total content of CYPs reside in the ER [16, 38]. However, higher total or inducible CYP levels than those of corresponding microsomes have been reported in rat brain or lung mitochondria [39, 40]. The molecular mechanisms responsible for the mitochondrial targeting of microsomal CYPs (mtCYPs) involve phosphorylation or proteolytic processing of the NH2-terminus, both of which result in a decrease in SRP affinity for the NH2-terminus of the CYPs, diminishing the subsequent targeting and incorporation into the ER [25, 38, 41]. Rather than being targeted to the ER, a cryptic mitochondrial-targeting signal is exposed and interacts with cytosolic chaperones. The protein is then post-translationally imported into mitochondria in a manner similar to that observed for the mitochondrial CYPs [42]. The protease responsible for the NH2terminal processing of CYPs and subsequent mitochondrial import has been identified as a dimeric cytosolic serine protease expressed in both the liver and the kidneys [43]. Other proteins, such as p53 and glucocorticoid receptors, have also been suggested to act as substrates for this serine protease by exposing a non-canonical mitochondrial-targeting signal after processing, leading to mitochondrial import [43]. In addition to the NH2-terminal deletion, the phosphorylation of CYP2E1 (COS cells), CYP2B1 (rat liver), and CYP1A1 (mouse liver) resulted in mitochondrial targeting and import of these proteins during protein synthesis and prevented ER-membrane incorporation [35, 41, 44]. These phosphorylations are mediated by protein kinase A or C (PKA or PKC), which have been reported to be associated with the signaling pathway of mitochondrial targeting [45]. However, a different study suggested that a truncated form of CYP2E1 was targeted and imported into the mitochondria [25]. This truncated CYP2E1 was detected in rat liver mitochondria, although at low levels [25, 36]. The phosphorylation of CYPs also induced the degradation of certain CYPs as well as the mitochondrial targeting of microsomal CYPs. CYP proteins are degraded by two pathways: the lysosomal pathway (for long-lived proteins) and the ubiquitin-dependent 26S proteasomal pathway (for short-lived proteins) [46]. The phosphorylation of CYPs in situ in the ER membrane has been shown to accelerate the degradation of CYP2E1 and CYP3A by the proteasomal system, indicating that this post-translational modification is important for the regulation and targeting of CYPs [46, 47]. cAMP-dependent protein kinase has been implicated in the phosphorylation of CYP2E1 (in cultured

hepatocytes) and CYP3A1 (in rat liver), resulting in a loss of catalytic activity followed by degradation [47, 48]. This was confirmed in a recent study in which cAMP-dependent PKA and PKCmediated phosphorylation of CYP3A4 resulted in ubiquitindependent proteasomal degradation, suggesting a direct link between phosphorylation, ubiquitination, and degradation [49]. In a proteomics study involving mass spectrometry analysis, several CYPs, including CYP1A2, CYP2E1, CYP2D6, and CYP3A4, were demonstrated to be constitutively expressed as a phosphorylated form in human liver tissue [50], suggesting that the phosphorylation-induced targeting and degradation mechanisms are also operative in vivo. As a detailed mechanism for mitochondrial targeting of microsomal CYP, it has been suggested that CYPs with noncanonical targeting signals, including phosphorylation or cleavable chimeric sequence, interact with Hsp70 and Hsp90 and bypass a part of TOMs whereas those with canonical signals (e.g., CYP27) interact only with Hsp70 and depend on a TOMs system for proper translocation [51, 52]. Mitochondrial targeting generally seems to be proportional to the expression levels of microsomal CYPs. However, on grounds that microsomal CYP targeting requires specific translocation machineries in mitochondria, cytosolic endoprotease activity, and chaperones, the targeting may not be dependent on the law of mass action. The targeting is not attributed to the escape from SRPbinding resulting from massive production of microsomal CYPs beyond the processing capacity of both the SRP and ER translocation systems. The mitochondrial targeting mechanisms for microsomal CYPs are summarized in Fig. (2) as an illustration. Circumstances Driving the Targeting and Functional Consequences CYP1A1/2 CYP1A may be targeted to mitochondria through its chimeric NH2-terminal signal that facilitates the protein targeting to both the ER and mitochondria [38, 53]. An approximately 30-amino acid NH2-terminal stretch of CYP1A1 is thought to provide signals for ER membrane insertion and stop transfer. In the case of CYP1A1, after cleavage of the ER retention signal by a cytosolic endoprotease to expose a cryptic mitochondrial targeting signal, a sequence motif (residues 33-44), which is immediately preceded by the transmembrane domain, functions as a mitochondrial targeting signal both in vivo (rat liver) and in vitro, producing mtCYP1A1 (CYPMT2b, +33/1A1), a major mitochondrial form [38]. Another mtCYP1A1 (CYPMT2a, +5/1A1) was identified and represented 15 25% of the total mitochondrial 1A1 antibody reactive protein pool [38]. These mtCYP1A1s (CYPMT2a and CYPMT2b) were localized within the inner membrane compartment and demonstrated distinct substrate specificities and preferences for different electron transport proteins [24]. As revealed by site-directed mutagenesis, the positively charged residues at positions 34 and 39 were also critical for the mitochondrial targeting. Although the delivery mechanisms and involvement of mitochondrial proteins are still unknown for CYP1A1 targeting, it seems that decreased affinity for SRP binding may be an important regulatory step: mutations of Pro-2 to Leu and Tyr-5 to Leu in nascent CYP1A1, which increases the signal recognition particle (SRP) binding, diminished mitochondrial targeting of the protein in COS cells [44]. By contrast, mutations of Leu-7 to Asn and Leu-17 to Asn, which decreased SRP-binding affinity, enhanced the targeting. PKCmediated phosphorylation at Thr-35 vastly decreased the affinity for SRP binding and consequently increased CYP1A1 targeting to mitochondria in vitro [44]. The NH2-terminally processed CYP1A1 in mitochondria displayed high levels of Adx/AdR-mediated N-demethylation of several non-typical substrates, such as erythromycin, diazepam and morphine, which are known to be metabolized by other microsomal CYPs including CYP3A4 and 2C19 [44, 54]. These observations

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Fig. (2). Illustration of the mitochondrial targeting of microsomal CYPs. The NH2-terminal phosphorylation or proteolysis of microsomal CYPs results in posttranslational targeting to the mitochondria by a serial interaction with cytosolic chaperones (e.g., Hsp70) and the mitochondrial import machineries, TOM/TIM.

led to the hypothesis that microsomal CYPs in mitochondria may contribute to the metabolism of certain drugs and may result in CYP-induced toxicity. Using knock-in mouse lines and treatment with benzo[a]pyrene (BaP), the functional differences between mitochondrial and microsomal CYP1A1 were partially elucidated [55]: wild-type and CYP1A1(mc/mc) mice expressing abolished mitochondrial targeting signals (R34D and K39I) were completely protected against the toxic effects of dietary benzo[a]pyrene. However, CYP1A1(-/-) knock-out and CYP1A1(mtp/mtp) mice, which lack the SRPbinding site (L7N and L17N), targeted exclusively to mitochondria and displayed striking toxicity and compensatory up-regulation of CYP1A2 and CYP1B1 mRNA in several tissues. -naphthoflavone (BNF), a xenobiotic inducer for CYP1A1 expression, induced the expression of mitochondrial CYP1A1 in various pig brain regions and in liver cells [56]. Approximately, 25% of total nascent CYP1A1 was processed by the endoprotease and localized to mitochondria in BNF-treated rat liver cells [38]. Chronic treatment with BNF also induced mtCYP1A1 content and reached 50 and 95% of the total cellular pool, respectively, in rat brain and C6 glioma cells [57]. Another investigation suggested that BNF treatment induced the increase in ethoxylresorufin O-deethylase activity about 40-fold in the microsomal fraction and 25-fold in the mitochondrial fraction [40]. Similar to BNF, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also induced the accumulation of CYP1A1 and 1A2 in mitochondria of mouse livers via activation of the aromatic hydrocarbon receptor [58, 59]. Therefore, xenobiotic inducers seem to be closely associated with the escape from ER retention and subsequent translocation of CYP1A into mitochondria as well as accumulation in ER membranes. Regarding the catalytic activities of the enzyme in mitochondria, it was demonstrated that co-expression of native CYP1A1 and Adx cDNAs resulted in 5 7-fold higher erythromycin Ndemethylation in the mitochondrial fraction but minimal changes in the microsomal fraction of transfected cells [54]. In addition, coexpression of CYP1A1 and Adx in COS cells conferred protection against erythromycin-mediated mitochondrial translation inhibition,

suggesting a physiological function for the xenobiotic-inducible CYP1A1 against drug-mediated mitochondrial toxicity [54]. However, the contribution of mitochondrial CYP1A1 to erythromycin metabolism was minor compared with that of microsomal CYP3A in transgenic mice in vivo [60]. Also, the presence of mitochondrial CYP1A1 alone could not protect against BaP-induced toxicity in knock-in mice, most likely reflecting the amount of total CYP1A1 present in mitochondria versus that observed in microsomes [55]. A previous study in mice demonstrated that CYP1A1 induction prevented toxicity and lethality following oral exposure to BaP, through increased BaP metabolism and clearance from the circulation [61]. Mice with microsomal CYP1A1 were protected from BaP-induced toxicity and metabolized BaP in a similar manner to wild-type animals, displaying low blood BaP levels [55]. In contrast, mice expressing the mitochondrial CYP1A1 form alone displayed significant toxicity following oral BaP exposure, as illustrated by a decrease in body weight, immunosuppression and an increase in liver toxicity. These data confirm that mitochondrial CYP1A1 is functionally different from microsomal CYP1A1, as only the latter is able to metabolize BaP and prevent BaP-induced toxicity. These observations might be of importance in drug discovery and development, as the typical screening assays for microsomal drug metabolism and toxicity do not consider that mitochondrial CYP forms exhibit altered substrate specificities. Interestingly, mtCYP1A1 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic antidepressants, anti-convulsants, opiates, and erythromycin [57]. These results provide new insights on the role of mtCYP1A1 in the modulation of pharmacological potencies of different neuroactive drugs in chronically exposed individuals. The overexpression of wild-type and alternatively spliced CYP1A1 has been observed in various types of ovarian cancer, but not in benign tissue [62]. The alternatively spliced form of CYP1A1 was demonstrated to localize to the nuclear membrane and in mitochondria and to be catalytically active in ovarian cancer cells, suggesting that this form might contribute to the initiation and progression of ovarian cancer [62].

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CYP2B and 2D6 CYP2B1 also has a chimeric mitochondrial and ER targeting signal at its NH2-terminus. However, unlike CYP1A, full-length CYP2B1 was identified in yeast and rat liver mitochondria [35, 63]. Phosphorylation at the Ser128 residue by cAMP-dependent PKA was suggested as a translocation signal for the enzyme in an in vitro transport assay [35]. The phosphorylation reduced the affinity of the NH2-terminal region for SRP binding but increased the affinity to mitochondrial translocase proteins including TOM40/44 and Hsp70 [51,52]. Therefore, cAMP modulates CYP2B1 targeting to two distinct organelles in cells. Xenobiotic-induced targeting patterns of the enzyme are dependent on the different tissue; BNF administration caused a 50 to 80% reduction in CYP2B1/2-associated pentoxyresorufin Odeethylase and benzyloxyresorufin O-dealkylase activities in rat lung mitochondria, whereas the inducer increased mitochondrial expression as well as microsomes in rat liver [33]. Phenobarbital (PB) also caused induction of aminopyrine N-demethylase activity in rat brain mitochondria [39]. However, it is not known whether the changes in subcellular activity of CYP2B represent the overall expression and/or the targeted amounts of the enzyme. Furthermore, the functional consequences of mitochondrial targeting of CYP2B1/2 are also not clear. However, as microsomal CYPs as well as CYP2B in mitochondria can receive electrons from the Adx and AdR redox systems and show catalytic activities [64], it is clear that mtCYP2B is involved in drug metabolism (and possibly physiological changes) in mitochondria. Human CYP2D6 is responsible for the metabolism of approximately 20 25% of currently-used drugs. The targeting mechanism for CYP2D6 to mitochondria is similar to that of CYP1A1 in the human liver. CYP2D6 contains an NH2-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both NH2-terminal deletions and point mutations suggested that the mitochondrial targeting signal is localized between residues 23 33 and that the positively-charged residues at positions 24, 25, 26, 28, and 32 are required for mitochondrial targeting [65]. The mtCYP2D6 from human liver oxidized 7-methoxy-4-aminomethylcoumarin as a substrate and both the mitochondria and the microsomal enzyme exhibited bufuralol 1'-hydroxylation activity [65]. There was also significant variability in the level of the mitochondrial enzyme and its metabolic activity between individuals [65]. Human brain microsomal and mitochondrial fractions metabolized arachidonoyl ethanolamide (anandamide) into hydroxylated and epoxygenated products such as epoxyeicosatrienoic acid ethanolamides (EET-EAs) in microsomes and mitochondria, respectively [66]. An inhibitory antibody against CYP2D6 decreased the mitochondrial formation of EET-EAs, implying that anandamide may be a physiological substrate for brain mtCYP2D6. As anandamide is an important signaling mediator of the endocannabinoid system, a pharmacological target of cannabinoid receptor ligands or drugs altering endocannabinoid synthesis and inactivation [67], these results may be applied to understand the CYP-mediated pathways of anandamide metabolism in the brain. Together with the identification of genetic variants of human CYP2D6 with the impaired mitochondrial targeting [68], these investigations suggest the hypothesis that neuropsychiatric phenotype differences could be related to genetic polymorphisms and concomitant mitochondrial targeting defects of CYPs. In contrast to molecular mechanisms regulating CYP2D6 localization, however, xenobiotic inducers or physiological conditions that stimulate the enzyme translocation are still unclear. CYP2E1 CYP2E1 is readily induced by acute and chronic alcohol ingestion, and is known to actively metabolize alcohol and acetaldehyde. The enzyme activity is also affected by pathophysiological condi-

tions such as diabetes, starvation, and obesity [69]. Although CYP2E1 is involved in the oxidative metabolism of a small range of substrates, there are many important functions mediated by CYP2E1 including drug interactions and production of reactive oxygen species. Similar to CYP2B1, full-length CYP2E1 was also identified in both microsomes and mitochondria in COS cells and Saccharomyces cerevisiae [63] and was also purified from mitochondria of pyrazole-induced rat livers [37]. However, it appeared that phosphorylation was required for the mitochondrial targeting, because phosphorylated CYP2E1 was found in rat liver mitochondria at a higher level compared with the microsomal counterpart [37]. Bimodal targeting through the phosphorylation of the NH2-terminal region was then suggested by the observation that mitochondrial targeting of the protein was severely impaired in cAMP-dependent PKA-deficient cells [41]. Similarly, the phosphorylation site mutant CYP2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance of PKA-mediated protein phosphorylation in mitochondrial targeting [63]. In the phosphorylationdependent targeting of full-length CYP2E1, an amino acid stretch including the B-helix from Gly 82 to Asp 95 was responsible for mitochondrial association of the enzyme and the same region in the B-helix was required for membrane interactions of CYP2E1 by electrostatic associations in yeast [70]. The mtCYP2E1 are localized in the matrix compartment peripherally associated with the inner membrane [63]. However, some reports have suggested that NH2-terminal truncation can be used for the mitochondrial targeting of CYP2E1 in vitro, and this deleted form is also present in rat liver [36]. The NH2-terminal region of CYP2E1 is a transmembrane domain inserted in the ER. Therefore, it was suggested that the truncation produced a cryptic signal for mitochondrial targeting and a soluble enzyme with a molecular weight of approximately 40 kDa localized in the matrix. Regardless of the targeting mechanisms and the final forms of mtCYP2E1, they all displayed the same catalytic activity as the microsomal counterpart either by an artificial electron supply or by mitochondrial electron transfer including Adx and AdR [36, 37]. At present, it is not thought that one of these is a prevailing mechanism for the enzyme translocation. The circumstances that drive the enzyme targeting to mitochondria and the functional consequences are not completely known. Robin et al. reported that ethanol, a known inducer of microsomal CYP2E1, also increased the amount of the mitochondrial enzyme in rat hepatocytes and in the liver of lean mice [71]. This increased targeting was associated with decreased glutathione levels in mitochondria and consequently oxidative stress production. Similarly, chronic alcohol consumption induced rat hepatic mtCYP2E1 [72]. In successive studies, HepG2 cell lines overexpressing mtCYP2E1 (NH2-terminally truncated form with molecular weight of 40 kDa) induced elevated oxidative stress in the mitochondria, damaged the membrane potential and eventually caused a loss of cell viability [72]. Recently, it was proposed that altered structural features of the truncated CYP2E1 in the mitochondrial environment, which are different from the cytosol-exposed microsomal organization, may be responsible for the induction of oxidative stress [73]. Mitochondria are subcellular organelles that are vulnerable to reactive oxygen species inducing cell death. Therefore, mtCYP2E1 may be involved in alcohol liver disease. CYP3A CYP3A enzymes are responsible for the oxidative metabolism of approximately 50% of the current drugs on the market [74]. CYP3A enzymes are embedded in the endoplasmic reticulum where they can catalyze a wide variety of biochemical reactions including hydroxylation, N-demethylation, O-dealkylation, Soxidation, deamination or epoxidation of substrates. Unlike mitochondrial targeting for CYP1A, 2B, and 2E1, CYP3A enzymes are likely expressed and targeted constitutively to

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brain mitochondria in mouse and rat [75, 76]. The brain regions containing mtCYP3A encompass neurons of hippocampus and hypothalamic areas that are sensitive to steroid hormones and cells contributing to the blood-brain barrier. CYP3A expression is also responsive to chemical inducers. However, xenobiotic treatmentdependent induction patterns are complicated. CYP3A1/2associated erythromycin N-demethylase (ERND) activity and antibody reactive proteins were reduced by 50 to 70% in both rat lung mitochondria and microsomes upon BNF administration, whereas the activity was increased in both fractions after dexamethasone treatment [40]. In contrary to lung tissue, BNF and PB treatment caused a 2-fold increase in the mitochondrial ERND activity in rat liver [33]. These results are similar to the tissue-dependent variations of CYP2B1/2 expression and localization upon xenobiotic treatment. Therefore, we suggest that microsomal CYPs present in different tissues and the corresponding cells react to xenobiotic inducers in different ways, and this consequently results in variable expression and localization patterns. The mitochondrial targeting mechanism(s) for microsomal CYP3As are still unclear. The NH2-terminal hydrophobic segment (positions 7 26) of rat liver CYP3A1 has been shown to be essential for enzyme insertion and retention in ER membranes [77]. The NH2-terminal truncated CYP3A4 was detected in cytoplasmic fractions of human liver cells and showed catalytic activity when reconstituted with CPR and cytochrome b5 [78]. However, no NH2teminal region with phosphorylation has been found for CYP3A enzymes. Instead, cAMP-dependent phosphorylation of CYP3As accelerates enzyme degradation [46, 49]. Therefore, CYP3As may be targeted to mitochondria by different mechanisms possibly including the unprocessed microsomal form. The molecular mechanisms, species, organs, and xenobiotic inducers for the mitochondrial targeting of microsomal CYP enzymes are summarized in Table 1.

CONCLUSION Microsomal CYPs are mostly present at the ER membrane, where these enzymes metabolize substrates that include both endogenous and exogenous compounds. A significant amount of these microsomal CYPs can be targeted to mitochondria as well as other subcellular compartments where they are catalytically active. At present, the reason for the presence of catalytically active microsomal CYPs outside the ER remains unknown. Xenobiotic inducers including BNF, TCDD, and PB may induce the mitochondrial targeting of several CYP enzymes. The physiological significance of microsomal CYPs localized in mitochondria and the circumstances stimulating the targeting are also still unknown. However, given the facts that microsomal CYPs in mitochondria can receive electrons from the Adx and AdR redox system and exhibit catalytic activities, it is clear that the enzymes play a role in drug metabolism in mitochondria. Regarding the physiological functions of mitochondrial targeting CYPs, it is interesting that the substrate specificity of targeted CYP1A1 was altered compared with the ER counterpart, and the enzyme could protect cells against erythromycin-mediated toxicity. It is also suggestive that mtCYP2E1 overexpression could produce elevated oxidative stress in the mitochondria, damage the membrane potential and eventually cause cell death. These results can be applied in drug metabolism and toxicity studies although the possible changes in substrate binding and catalytic activity remains an interesting research field for mitochondrial targeting CYPs. Furthermore, these investigations should consider CYPs possible drug targets in drug discovery and development. The presence of microsomal CYPs in other cellular compartments and under certain pathological conditions, such as cancer, could provide the basis for novel applications for new and existing drugs that utilize CYPs as targets. In addition, the presence of CYPs with altered substrate specificity in mitochondria should be considered in drug discovery and development because of the formation of organelle-specific metabolites.

Table 1.

Mitochondrial Targeting of Microsomal CYP Enzymes

CYP Species, Organ Rat, liver In vitro assay CYP1A1 COS cells Mouse, liver Pig, brain/liver CYP1A1/2 CYP2B1 CYP2B1/2 Rat, brain Human, liver CYP2D6 In vitro assay Human, brain H2.35 cells & Rat, liver Rat, liver CYP2E1 Yeast In vitro assay & COS cells Mouse, liver & rat hepatocytes Rat, liver CYP3A1/2 Rat, lung CYP3A4 Human, liver Unknown NH2-terminal deletion Dexamethasone Unknown [40] [78] Unknown Unknown NH2-terminal deletion & Phosphorylation by PKA Unknown NH2-terminal deletion Phosphorylation Phosphorylation by PKA Phosphorylation by PKA Unknown Unknown Ethanol BNF, PB Unknown Unknown Pyrazole Unknown PB Unknown [39] [65, 68] [65] [66] [36] [37] [63] [41] [71] [33] Mouse, liver In vitro assay Rat, liver Targeting Mechanism NH2-terminal deletion NH2-terminal deletion NH2-terminal deletion & Phosphorylation by PKC NH2-terminal deletion Unknown Unknown Phosphorylation by PKA Unknown BNF Unknown BNF BNF TCDD Targeting Inducer Unknown References [38] [38, 44] [38, 44] [44] [56] [58, 59] [35] [33]

836 Current Drug Metabolism, 2010, Vol. 11, No. 10

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ACKNOWLEDGEMENTS We thank Dr. Leaf Huang (University of North Carolina at Chapel Hill, NC) for his helpful comments and suggestions. This work was supported by the National Research Foundation of Korea Grants funded by the Korean Government (NRF-2010-013-C00021 AND NRF-2010-0028244). DISCLOSURE The authors state no conflict or duality of interest in regards to this work. REFERENCES
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Received: November 05, 2010

Revised: December 16, 2010 Accepted: December 21, 2010