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Drug Metabolism Reviews, 37:379^04, 2005 Copyright © Taylor & Francis Inc. ISSN: 0360-2532 print / 1097-9883 online DOI: 10.1081/DMR-200046136 I Taylor & Francis Taylor 6. Francis Croup REGULATION OF CYTOCHROME P450 BY POSTTRANSLATIONAL MODIFICATION Mike Aguiar Applied R&D, MDS Pharma Services, St. Laurent (Montreal), Quebec, Canada Department of Medicine, McGill University, Montreal, Quebec, Canada Robert Masse Applied R&D, MDS Pharma Services, St. Laurent (Montreal), Quebec, Canada Bernard F. Gibbs Applied R&D, MDS Pharma Services, St. Laurent (Montreal), Quebec, Canada Department of Medicine, McGill University, Montreal, Quebec, Canada Cytochrome P450s are a family of enzymes represented in all kingdoms with expression in many species. Over 3,000 enzymes have been identified in nature. Humans express 57 putatively functional enzymes with a variety of critical physiological roles. They are involved in the metabolic oxidation, peroxidation, and reduction of many endogenous and exogenous compounds including xenobiotics, steroids, bile acids, fatty acids, eicosanoids, environmental pollutants, and carcinogens [Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, Af. J., Gunsalus, I. C, Gotoh, O. (1993) The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell Biol. 12(1):I—51.] The development of numerous diseases and disorders including cancer and cardiovascular and endocrine dysfunction has been linked to P450s. Several levels of regulation, including transcription, translation, and posttranslational modification, participate in maintaining the proper function of P450s. Modifications including phosphorylation, glycosylation, nitration, and ubiquitination have been described for P450s. Their physiological significance includes modulation of enzyme activity, targeting to specific cellular compartments, and tagging for proteasomal degradation. Knowledge of P450 posttranslational regulation is derived from studies with relatively few enzymes. In many cases, there is only enough evidence to suggest the occurrence and a possible role for the modification. Thus, many P450 enzymes have not been fully characterized. With the introduction of current proteomics tools, we are primed to answer many important questions regarding regulation of P450 in response to a posttranslational modification. This review considers regulation ofP450 in a context that describes the potential role and physiological significance of each modification. Key Words: Cytochrome P450; Posttranslational modification; Phosphorylation; Ubiquitination; Nitration; Glycosylation. Address correspondence to Bernard F. Gibbs, Applied R&D, MDS Pharma Services, 2350 Cohen Street, St. Laurent (Montreal), Quebec, H4R 2N6, Canada and Department of Medicine, McGill University, Montreal, Quebec, Canada; E-mail: [email protected] 379 380 M. AGUIAR ET AL. INTRODUCTION Background Information on P450 Since the introduction of modern molecular biology techniques allowing for the sequencing of entire genomes, researchers have identified and continue to discover many P450 enzymes in a wide variety of organisms. In order to systematically identify and categorize this growing family of enzymes, a leading group of researchers in the field established the current system of nomenclature (Nelson et al., 1996). P450s are named with the prefix CYP or P450 followed by an Arabic number defining the family, an uppercase letter defining the subfamily, and another Arabic number defining an individual enzyme (e.g., P450 3A4). In cases where only a single member exists in a given family, it may be identified simply as P450 followed only by the family number (e.g., P450 51). The reader may refer to the cytochrome P450 home page (http:// dmelson.utmem.edu/CytochromeP450.html) for the latest nomenclature updates and links to related sites. All mammalian tissues examined express some P450 enzyme system (Porter and Coon, 1991). In addition, mammals express multiple enzymes simultaneously in a variety of tissues, including liver, kidney, lung, and adrenal (Bhagwat et al., 1999a; Guengerich, 2001; Lohr et al., 1998; Parker and Schimmer, 1997). It is also appreciated that various enzymes are found not only in different cell and tissue types, but also in different subcellular compartments, such as the outer nuclear membrane, endoplasmic reticulum (ER), mitochondria, golgi, peroxisome, and plasma membrane. Certain enzymes are found in several different subcellular compartments simultaneously (Guengerich, 2001). All cytochrome P450s, with the exception of bacterial enzymes, are membrane bound. Microsomal enzymes are tethered to the membrane through a hydrophobic transmembrane helix at the N-terminus of the protein, which also serves as a targeting sequence for the signal recognition particle dependent cotranslational incorporation of a nascent P450 into the ER membrane (Bar-Nun et al., 1980; Sakaguchi et al., 1984). Insertion continues until a halt-transfer signal is reached that effectively anchors the P450 to the membrane with the bulk of the protein exposed on the cytosolic side (Monier et al., 1988; Sakaguchi et al., 1987; Szczesna-Skorupa and Kemper, 1989; Szczesna-Skorupa et al., 1988). Because detergents are required for solubilizing membrane-bound P450s, all native mammalian P450s have evaded crystallization and x-ray crystallographic study. To avoid this problem, researchers have modified several P450s resulting in soluble mutants that can be crystallized and that have structures that were solved. The first mammalian P450 structure reported was that of rabbit P450 2C5 (Williams et al., 2000), which was also crystallized in complexes with two different substrates (Wester et al., 2003a,b). Interestingly, one of the two substrates binds in two different orientations (Wester et al., 2003a). These studies of P450 2C5 offer insightful information on the fiexibility of the enzyme and shed light on the ability of P450s to accommodate a variety of substrates with different shapes and sizes. The structure of human P450 2C8 was recently reported, revealing an active site volume twice that of P450 2C5, consistent with the size of its preferred substrates (Schoch et al., 2004). Human P450 2C9 has also been crystallized, with and without bound warfarin, a known anticoagulant substrate (Williams et al., 2003). The structure reveals a new binding pocket that may accommodate several substrate molecules simultaneously and possibly REGULATION OF CYTOCHROME P450 381 account for some complex drug-drug interactions. Another group has also reported a structure for P450 2C9 complexed with flurbiprofen (Wester et al,, 2004), The structure was obtained without modifications to the catalytic domain, revealing some significant conformational differences. The more recent structure helps explain some experimental observations in terms of the substrate selectivity of the enzyme, which were not easily explained with the earlier structure, A crystal structure for rabbit P450 2B4, one of the first P450s to be purified and studied in detail, has been reported in a wide open conformation (Scott et al,, 2003) and in a closed form complexed with the specific inhibitor 4-(4-chlorophenyl)-imidazole (Scott et al,, 2004), These structures may help to understand how mammalian P450s "open/close," allowing substrate to access the buried active site (Poulos, 2003), Two groups have recently reported crystal structures for human P450 3A4: one unliganded structure from each group and two structures hound to different substrates (Williams et al, 2004; Yano et al,, 2004a), The collection of structures should provide a better understanding of the substrate selectivity and unusual kinetics of this important enzyme. Finally, a structure for P450 2A6 has been reported in a meeting abstract (Yano et al,, 2004b), Humans express 57 putatively functional genes and 58 pseudogenes (Nelson et al,, 2004), These may be divided roughly into two groups based on their substrate specificity, P450s involved in the metabolism of most drugs and carcinogens are derived from families 1-3, This group demonstrates wide substrate specificity, with certain enzymes acting on a large number of structurally varied substrates such as in the case of human P450 3A4, The second group demonstrates high substrate specificity, catalyzing the biosynthesis and metabolism of endogenous substrates including cholesterol, steroids, vitamins, and eicosanoids. Several steroidogenic P450 enzymes are outlined in Fig, 1, Lanosterol I P450 51 Cholesterol ^ ^ Pregnenolone ^ ^ * 17-OH Pregnenolone ^ ^ * Dehydroepiandrosterone P450 11AI P45017A1 P450 17A1 J P450 7A1 I 3P HSD 7-OH Cholesterol Progesterone ^ ^ ^ P450 17A1 Bile Acids 11-Deoxycorticosterone |P45O11BI Corticosterone J 3P HSD 17-OH Progesterone i 3P HSD ^ ^ * Androstenedione ^ ^ * Estrone P450 17A1 P45019A1 11-Deoxycortisol Testosterone ^^^^ Estradiol P450 19AI JP45O11B1 Cortisol Aldosterone |P45O11B2 18-OH Corticosterone Figure 1. Cholesterol, bile acid, and steroid hormone biosynthesis: phosphorylated P450 enzymes are indicated in boldface. 382 M. AGUIAR ET AL. Classes of P450 P450s fall into three classes hased on the reduction system transferring electrons. Class I P450s are associated with the inner mitochondrial memhrane and some hacterial systems. Reducing equivalents from NADPH or NADH are transferred two electrons at a time to redoxin reductase, which carries a flavin adenine dinucleotide (FAD) prosthetic group. The isoalloxazine ring of FAD may exist in several oxidation states, which allows for the subsequent transfer of electrons individually to a mobile Fe2S2-containing protein called redoxin. Reduced redoxin is thought to shuttle electrons to the P450, returning to the reductase in the oxidized form for additional cycles. Class II P450s are those that reside in the ER and receive reducing equivalents from NADPH via P450 reductase or cytochrome b^ (a heme protein associated with the ER), P450 reductase is a membrane-bound protein containing a FAD and a flavin mononucleotide (FMN) prosthetic group that also has an isoalloxazine ring with properties similar to FAD, An electron pair from NADPH is received by FAD, which relays the electrons to FMN, finally transferring electrons in single file to the P450, In certain reactions, the first electron is transferred from P450 reductase, while the second electron is transferred from cytochrome b^. Cytochrome b^ may be reduced either by P450 reductase, or cytochrome b^ reductase. Interestingly, apo-^s (cytochrome ^5 devoid of heme), which cannot transfer electrons, is required for optimal activity in a number of P450 enzymes and only with certain substrates (Hlavica and Lewis, 2001; Yamazaki et al,, 2001), Class III P450s are isomerases instead of monooxygenases. They do not require the participation of any redox partners or donors of reducing equivalents. Water is not produced, and therefore, molecular oxygen is not required for catalysis. Substrates of class III P450s are simply rearranged into products. Examples of P450s from this class include P450 8A1 [prostacyclin [PGI2] synthase] and P450 5 [thromboxane [TXA2] synthase]. POSTTRANSLATIOIMAL MODIFICATION A posttranslational modification may be defined as "any difference between a functional protein and the linear polypeptide sequence encoded between the initiation and the termination codons of its structural gene" (Han and Martinage, 1992), Examples of noncovalent modifications include incorporation of cofactors such as heme, protein folding, and the association of subunits to form an oligomeric protein, Allosteric phenomena manifested as deviations from Michaelis-Menten kinetics have been demonstrated for numerous P450 enzymes. Various components known to interact with P450, including substrates, inhibitors, membrane lipids, and redox partners (such as the previously mentioned cytochrome ^5), have been shown to act as homotropic and heterotropic effectors (Hlavica and Lewis, 2001), P450 2E1 is stabilized by ethanol, leading to increased cellular levels of the P450 (Roberts et al,, 1995), Covalent modifications, including cleavage of a signal peptide, formation of disulfide bonds, and an array of modifications to amino acid residues, including phosphorylation, nitration, glycosylation, methylation, sulfation, acetylation, and prenylation, provide another means of posttranslational modification. The remainder of the present article is dedicated to the identification and characterization of covalent P450 posttranslational modifications with an emphasis placed on describing the physiological role of each modification. REGULATION OF CYTOCHROME P450 383 PHOSPHORYLATIOIM An article by Cohen (2002), opens with the following statement, "Protein phosphorylation regulates most aspects of cell life, whereas abnormal phosphorylation is a cause or consequence of disease." Cascades that activate the production of cyclic adenosine monophosphate (cAMP), leading to activation of protein kinase A (PKA) in eukaryotic cells illustrate a remarkable example. Once activated, PKA can phosphorylate many target proteins, resulting in a wide variety of cellular responses, including regulation of gene transcription, modulation of enzyme activity, targeting of protein for degradation, and targeting to various intracellular locations. MODULATION OF P450 ACTIVITY BY PHOSPHORYLATION A number of reports over the last two decades describe the phosphorylation of over twenty P450 enzymes in microsomes, intact hepatocytes, cell culture, and in vivo (Table 1). Whereas some studies demonstrate stimulation of phosphorylation by addition of hormones and intracellular second messengers, other reports correlate phosphorylation with modulation of enzyme activity (Koch and Waxman, 1989; Oesch-Bartlomowicz et al., 1998, 2001; Pyerin and Taniguchi, 1989). Much of the work has focused on members of family 1-3 P450s. Unfortunately, studies regarding regulation by posttranslational modification of the major drug metabolizing P450s, with the exception of P450 3A4 and P450 2E1, which will be discussed below (i.e., P450 1A2, P450 2B6, P450 2C8, P450 2C19, P450 2C9, P450 2D6), have not appeared in the literature. However, evidence for the modification and regulation of P450 enzymes involved specifically in cholesterol and steroid homeostasis has been reported. PHOSPHORYLATION OF STEROID HORMONE SYNTHESIZING P450 ENZYMES Steroid hormones play an essential role in stress response, water and electrolyte balance, sexual differentiation and reproduction, bone and tissue homeostasis, cognitive function, and numerous other key physiological processes (Evans, 1988; Parker and Schimmer, 1993). Androgens and estrogens are known to participate in the development of breast and prostate cancer. Given their potent effects, steroid hormone levels must be very carefully regulated. The adrenal cortex and the gonads are the key sites of glucocorticoid, mineralocorticoid, and sex steroid biosynthesis. Synthesis of steroids requires the concerted action of a number of cytochrome P450 steroid hydroxylases (Fig. 1). Transcdptional regulation of the genes encoding these steroid hydroxylases occurs in response to trophic hormones and transcription factors such as adrenocorticotropin (ACTH) and steroidogenic factor 1 (SF-1), with selective expression in steroidogenic cells (Parker and Schimmer, 1993, 1996, 1997; Stocco, 2000). In addition to transcdptional regulation, they are regulated by posttranslational modification including phosphorylation. The underlying theme involves signal transduction pathways resulting in the activation of cyclic nucleotide and other second messenger molecules ultimately activating kinase/phosphatases that act on P450s as well as ferredoxin/adrenodoxin. 384 M. AGUIAR ET AL. Table 1 Phosphorylated P450 enzymes P450 Species P450 1A2 Rat Rabbit Rat P450 2B1 P450 2B2 Rat P450 2B4 P450 2C6 Rabbit Rat P450 P450 P450 P450 Rat Rat Rat Mouse 2C7 2C11 2C12 2E1 Liver microsomes Liver microsomes Liver microsomes Hepatocytes Liver (in vivo) Liver microsomes Hepatocytes Quail Liver {in vivo) Liver microsomes Liver microsomes Liver {in vivo) Liver microsomes Liver microsomes Liver microsomes COS 7 V79 Liver microsomes Hepatocytes Liver {in vivo) Hepatocytes, Liver microsomes E-coli expressed Liver microsomes Liver microsomes E-coli expressed E-coli expressed Corpus luteum mitochondria Adrenal cortex mitochondria! inner membrane Adrenal cortex mitochondrial inner membrane NCI-H295, COS-1, Kin 8 expressed, Adrenal mierosomes Testis microsomes NCI-H295R, NCI-H295R expressed Placenta MCF-7 cells Brain Rat Chicken Liver microsomes Kidney mitochondria Rat P450 3A1 Rat P450 3A4 P450 3A6 P450 7A1 Human Rabbit Rat P450 llAl Human Bovine P450 UBl Bovine P450 17A1 Human P450 19A1 Rat Human Human* P450 51 P450 27A1** Source Reference Pyerin et al., 1987 Pyerin et al., 1987 Jansson et al., 1990 Oesch-Bartlomowicz et al., Oesch-Bartlomowicz et al., Pyerin et al., 1987 Oesch-Bartlomowicz and Oesch, 1990 Koch and Waxman, 1989 Epstein et al., 1989 Epstein et al., 1989 Koch and Waxman, 1989 Epstein et al., 1989 Pyerin et al., 1987 Epstein et al., 1989 Freeman and Wolf, 1994 Oesch-Bartlomowicz et al., Menez et al., 1993 Oesch-Bartlomowicz et al., Koch and Waxman, 1989 Eliasson et al., 1994 Wang et al., 2001 Pyerin et al, 1987 Tang and Chiang, 1986 Nguyen et al., 1996 Nguyen et al., 1996 Caron et al., 1975 Vilgrain et al., 1984 2001 2001 1998 1998 Dcfaye et al., 1982 Zhang et al., 1995 Lohr and Kuhn-Velten, 1997 Biason-Lauber, 2000 Bellino and Holben, 1989 Yue et al., 2003 Balthazart et al., 2001a; Balthazart et al., 2001b Sonoda et al., 1995 Ghazarian et al., 1985 *Putatively phosphorylated. **Chicken 25-OH Vitamin D 1-hydroxylase sequence not yet reported. Cholesterol Biosynthesis The uptake and synthesis of cholesterol is carefully regulated by cholesterol feedback mechanisms on a number of different enzymes, including acetoacetyl-CoA reductase, HMG-CoA synthase, HMG-CoA reductase, prenyl transferase, squalene REGULATION OF CYTOCHROME P450 385 synthetase, squalene epoxidase, and low-density lipoprotein (LDL) receptor (Sonoda et al,, 1995), When dietary cholesterol is low, various organisms including humans synthesize cholesterol entirely from Acetyl-CoA, One of the biosynthetic steps leading up to cholesterol involves the removal of the ]4a-methyl group from lanosterol and 24, 25-dihydroxylanosterol (DHL), Sterol 14a-demethylase (P450 51) is the microsomal cytochrome P450 that catalyzes this reaction. The human analog of this enzyme is expressed in testis, ovary, adrenal, prostate, liver, kidney, and lung. Experiments performed with preparations of purified rat liver P450 51 have demonstrated that enzyme activity increases when dephosphorylated. When purified enzyme is pretreated with type III bacterial alkaline phosphatase, followed by reconstitution with the remaining system components (NADPH-P450 reductase, NADPH, and 24,25-dihydroxylanosterol), the result is an increase in enzyme activity relative to a control (preparation that was not treated with phosphatase). Thus, it has been proposed that phosphorylation-dephosphorylation of P450 51 may be involved in regulation of the enzyme activity (Sonoda et al,, 1995), and consequently, may represent an important regulatory mechanism of cholesterol biosynthesis and potentially a molecular target for cholesterol-lowering drugs. Cholesterol Metabolism and the Synthesis of Bile Acids Cholesterol is the precursor of all steroids, hence its requirement for maintaining proper endocrine function. However, excessive blood cholesterol levels may lead to medical disorders such as gallstones. The conversion of cholesterol to hydrophilic bile acids in the liver provides an important pathway for its elimination. The rate-limiting step in the synthesis of bile acids from cholesterol is catalyzed by cholesterol 7a-hydroxylase (P450 7A]), a "rheostat" in cholesterol homeostasis. Researchers have established the regulation of P450 7A1 activity by hormones, cytosolic factors, bile acids, and diurnal rhythm (Myant and Mitropoulos, 1977), In addition, literature has appeared mostly arguing for, but with a few reports against, the modulation of P450 7A1 activity by posttranslational phosphorylation (Berglund et al,, 1986; Diven et al, 1988; Einarsson et al,, 1986; Holsztynska and Waxman, 1987; Nguyen et al,, 1996), The main shortcoming of all but the most recent report (Nguyen et al,, 1996) was that the findings were all based on indirect evidence, relating perturbations to the enzyme preparation with kinases, phosphatases, phosphatase inhibitors, etc, to changes in enzyme activity without directly observing corresponding changes to the phosphorylation state of P450 7AI, The study employing Escherichia coll {E. coll) expressed rat and human P450 7A1 has demonstrated modulation of enzyme activity in vitro by phosphorylation and dephosphorylation with a direct corresponding change in phosphorylation state of P450 7A1 as demonstrated hy incorporation of •'^P phosphate into purified enzyme applied to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Nguyen et al,, 1996), Taken together, these results demonstrate that phosphorylation and dephosphorylation are reversible modes of regulation of P450 7A1 activity in vitro. The physiological significance of these phenomena remains to be established, as no reports have appeared demonstrating the importance of this mode of regulation in whole cells or in vivo. 386 M. AGUIAR ET AL. Biosynthesis of Pregnenolone, a Checkpoint in Steroid Hormone Biosynthesis The first step in the conversion of cholesterol to steroids is the synthesis of pregnenolone. The reaction involves three separate hydroxylation steps, with three equivalents of O2 and NADPH. The enzyme that catalyzes this reaction (P450 l l A l ) is expressed in all tissues that synthesize steroids from cholesterol. P450 11 Al resides in the inner mitochondrial membrane. Transcriptional regulation of P450 l l A l and other steroidogenic P450s is well documented (Parker and Schimmer, 1993, 1996, 1997; Stocco, 2000). Activity of P450 11 Al is also regulated by phosphorylation. Initial evidence for stimulation of P450 l l A l activity by phosphorylation was reported several years ago (Caron et al., 1975). A crude preparation of P450 l l A l was isolated from bovine corpus luteum mitochondria. Limiting P450 was reconstituted with excess redoxin and redoxin reductase, and the reconstituted system was subjected to treatment with PKA, cAMP, and ATP. The resulting system demonstrated increased P450 l l A l activity. Decisive evidence for the posttranslational phosphorylation of P450 l l A l came nearly a decade later (Vilgrain et al., 1984). A demonstration with purified bovine adrenocortical P450 l l A l showed that the P450 is efficiently phosphorylated by Ca^"^-activated phospholipid-sensitive protein kinase C (PKC). Four moles of phosphate were incorporated per mole of P450 11 Al, with serine and threonine as the target amino acids phosphorylated in a ratio of 1:1 as revealed by amino acid analysis. Interestingly, PKC activity is also found to be associated with bovine adrenocortical inner mitochondrial membrane (Vilgrain et al., 1984). Thus, it appears that phosphorylation of P450 l l A l in a reconstituted system results in increased enzyme activity. Additional experiments would be required to demonstrate the physiological relevance of the phenomenon. Adrenarche, Androgen Biosynthesis, and Cancer P450 17A1 is a Class II P450 expressed in all primary steroidogenic tissues. Activity of this enzyme is required for the biosynthesis of precursors of glucocorticoids and androgens (Fig. 1). Much interest has been focused on developing inhibitors of P450 17A1, because activity of this one enzyme can direct the course of an androgen-dependent malignancy, such as prostate cancer. Understanding all factors, including posttranslational modification, that regulate P450 17A1 activity is therefore of great importance. Two consecutive reactions in steroid biosynthesis are catalyzed by this enzyme; the 17a-hydroxylation of pregnenolone and progesterone and the subsequent cleavage of the 17-20 steroid bond (lyase activity) of 17-OH-pregnenolone and 17-OH-progesterone (Nakajin and Hall, 1981). Both of these reactions are catalyzed at a single bifunctional active site (Nakajin et al., 1981). The two catalytic steps may be coupled (17hydroxylation followed immediately by 17-20 lyase activity), or catalysis may stop after the initial hydroxylation step, resulting in the formation of androgens and precursors of glucocorticoids, respectively. The adrenals of children between one and eight years of age secrete cortisol (a C21 steroid) but very little sex steroid precursors (C19 steroids). This is due to the adrenal 17ahydroxylation activity of P450 17A1 with a lack of lyase activity. Between seven and nine years of age, the adrenals begin to produce and secrete increasing levels of C19 387 REGULATION OF CYTOCHROME P450 Steroids (Fig, 2) without a corresponding increase in levels of cortisol or adrenocorticotropin secretion (Cutler et al,, 1978; Korth-Schutz et al,, 1976; Parker and Odell, 1980), The secretion of increasing levels of sex steroids continues until the age of 25-35, after which time they begin to drop gradually until they return to childhood levels at 70-80 years of age (Orentreich et al,, 1984), This programmed development of adrenal P450 17A1 17,20-lyase activity is known as adrenarche. In an effort to explain this phenomenon, experiments were performed to determine if the change in lyase activity could be associated with an increase in posttranslational modification of P450 17A1, Human P450 17A1 is phosphorylated on serine and threonine residues by PKA, Phosphorylation increases 17,20-lyase activity, while dephosphorylation has the opposite effect (Zhang et al,, 1995), Phosphorylation of rat testicular P450 17A1 increases the ligand-binding efficiency of the enzyme for its natural ligand (progesterone) as well as the rate of P450 17A1 proteolytic degradation (Lohr and Kuhn-Velten, 1997), These findings have led researchers to propose that pituitary hormones (corticotropin, lutropin, or human chorionic gonadotropin) known to activate the G protein-coupled receptor (GPCR)/adenylate cyclase/PKA signal transduction pathway, may account for adrenarche and the proteolytic regulation of P450 17A1 levels (Lohr and Kuhn-Velten, 1997), Recently, leptin has been identified as a hormone capable of stimulating the lyase activity of P450 17A1 in intact human adrenocortical carcinoma cells in a manner consistent with the development of adrenarche. As may he observed in Fig, 3, physiological levels of leptin, acting through its receptor and the downstream signal 400- GIRLS: Closed symbols BOYS: Open symbols ^ ° a LU' • a 300- O DC LLI IV> O CC o a. a 0 t/) ai • e 250* • 200' 160 140 fr / CC Q I UJ Q 100 80 60 40 20 O ' A 0 n * • / J a 0 , o 0 LU O • / 0 o ^ ' D • t, 0 ^ 12 i // 3 CA MONTHS • . o" • • •8* O • " 3 4 5 0 <^ 05on ^o 1 2 6 7 8 ; 9 10 11 12 13 14 15 16 17 18 19 20 BONE AGE YEARS Figure 2. Serum DHEAS concentration in normal children. Points represent single entries; lines represent serial entries. The period between vertical lines represents average age of puberty onset in girls. The shaded period represents the average age of puberty onset in boys, [Adapted from Korth-Schutz et al,, 1976 with permission,] 388 M. AGUIAR ET AL. 17a-hydroxylase activity 200 200-, 180 ISO160 ISO140 140120 120 10O 100 8080606040402020000 5 10 15 20 25 30 35 40 45 50 55 60 65 -.-OBR-(AP) —•-OBR+ (control) -^OBR-f (inhib) 2 4 __^_^_^__^__^__^ 10 12 14 16 18 20 Incubation time (min) Alkaline phosphatase (AP) B 17,20-lyase activity — — -•-»- £ I OBR+ (AP) OBR- (AP) OBR-^ (control) OBR-f (inhib) Incubation time (min) Alkaline phosphatase (AP) Figure 3. Effect of leptin and dephosphorylation on P450 17A1 enzyme activity. Short-term treatment with 30 pM leptin of human adrenal NCI-H295R cells (leptin receptor+ B ; leptin receptor- A)- A 17a-hydroxylase and B 17,20-lyase activity was analyzed in intact cells. Bottom right: Phosphate removal with alkaline phosphatase (AP) selectively abolishes DHEA production. [Adapted from Biason-Lauber et al., 2000 with permission.] transduction pathway, stimulate the phosphorylation of P450 17A1 leading to an acute and long-term stimulation of P450 17A1 lyase activity in the human adrenal cell (BiasonLauber et al., 2000). Biosynthesis of Glucocortlcoid and Mineralocorticoid P450 l l B l , a Class I P450 expressed only in the adrenal cortex, catalyzes two additional steps in steroid hormone biosynthesis. These steps are the oxidation of 11deoxycorticosterone to corticosterone (a mineralocorticoid precursor) and oxidation of 11-deoxycortisol to cortisol (a glucocorticoid). Studies with bovine P450 l l B l purified from mitochondrial adrenal cortex have demonstrated that P450 l l B l is phosphorylated by skeletal muscle PKA. Enzyme activity of P450 l l B l does not change as a result of phosphorylation if the system is reconstituted with excess adrenodoxin. However, kinetic studies demonstrate that P450 l l B l phosphorylation strikingly increased the affinity between P450 l l B l and adrenodoxin. As limiting adrenodoxin is the normal state in adrenocortieal mitochondria, phosphorylation may be a physiologically relevant factor in stimulating P450 llBl activity (Defaye et al., 1982; Estabrook et al., 1972). Additional studies would be required to clearly demonstrate this hypothesis. Estrogen Biosynthesis, Neuromodulation, and Cancer P450 19A1 is the enzyme required for the biosynthesis of estrogen from androgen. Because the reaction catalyzed by P450 19A1 results in the A-ring aromatization of REGULATION OF CYTOCHROME P450 389 androgens, P450 19A1 is also commonly referred to as aromatase. P450 19A1 is expressed in tissues associated with primary steroid synthesis, as well as a variety of other peripheral tissues, including adipose and bone. In addition to well-estahlished transcdptional regulation, evidence supporting the regulation of P450 19A1 by posttranslational phosphorylation has been reported (Balthazart et al., 2001a,b, 2003; Bellino and Holben, 1989; Yue et al., 2003). Initial evidence came from a study employing microsomes isolated from human term placenta, where it was demonstrated that microsomal P450 19A1 activity could be maintained in phosphate buffer or by the inhibition of phosphatase activity with tartaric acid or EDTA in a phosphate-free buffer. The authors hypothesized that phosphorylation may play a role in the regulation of P450 19A1 activity (Bellino and Holben, 1989). P450 19A1 phosphorylation has been associated with several different physiological phenomena. Reports in recent years have demonstrated the neuromodulatory effects of estrogenic metabolites [referenced in Balthazart et al. (2001a)]. A telling example comes from a study with castrated sexually experienced male rats that illustrates that estrogen administration rapidly activates male sexual behavior (within minutes), presumably by a nongenomic mechanism, as a genomic mechanism would require hours to days for an observable effect (Cross and Roselli, 1999). Rapid and pronounced changes in P450 19A1 activity of quail hypothalamic homogenates by pharmacological studies employing kinase activators and inhibitors have been described (Balthazart et al., 2001a,b). Stimulation of kinase by addition of nonnal intracellular concentrations of Ca^"^, ATP, and Mg^"^ resulted in significant decreases in P450 19A1 activity. The decrease in activity could be completely abolished by the addition of ethylene glycolfc«(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), which chelates free Ca^"^. The authors noted, however, that the studies were performed with total homogenates, thus the possibility remained that intermediary proteins are phosphorylated that interact with P450 19A1 to modulate its activity. Conclusive experiments confirming P450 19A1 phosphorylation have been reported (Balthazart et al, 2003). Aromatase from quail preoptic area homogenates was immunopurified, and phosphorylated Ser, Thr, and Tyr residues were detected by Western analysis employing phospho-amino acid specific antibodies. Together, these results demonstrate that the local production of estrogens in quail brain can be rapidly altered by calcium-dependent P450 19A1 phosphorylation. P450 19A1 activity is also a key factor in the progression of estrogen-dependent diseases such as breast, endometrial, and ovarian cancers. Like P450 17A1, P450 19A1 represents an important target in the treatment of such diseases. Hormone-dependent breast cancer can be treated by surgical removal of the affected area or by endocrine treatment with a selective estrogen receptor modulator such as tamoxifen. Unfortunately, disease progression is only delayed by 12-18 months with endocrine treatment. Subsequent treatment with an aromatase inhibitor blocks disease progression in half the patients who relapse, suggesting an adaptation to therapy that involves aromatase. Several mechanisms may account for the adaptation. It has been demonstrated that longterm estrogen deprivation causes hypersensitivity of cultured MCF-7 breast cancer cells to the mitogenic effects of estradiol, with an associated activation of the MAP kinase and PI3 kinase pathways. It was also discovered that aromatase activity is elevated in longterm estrogen deprivation (Yue et al., 1999). In order to establish a link between the kinase cascades and activation of aromatase, MCF-7 cells deprived of estrogens were treated with inhibitors of the kinase pathways. A significant decrease in aromatase 390 M. AGUIAR ET AL. activity was observed in just 2 hours, suggesting a nongenomic regulation of aromatase, possibly by P450 19A1 dephosphorylation (Yue et al., 2003). The authors concluded that more detailed studies would be required to understand the mechanisms of the kinase pathway inhibitors. REDOXIN PHOSPHORYLATION An important component of mitochondrial hydroxylase systems (Class I) is redoxin (ferredoxin, adrenodoxin), an iron sulfur protein that acts as a shuttle for the transfer of electrons from redoxin reductase. Several reports describe the phosphorylation of redoxin. We will consider the significance of redoxin modification in modulating the activity of several P450 enzymes, including adrenal P450 l l A l , P450 l l B l , and renal la-hydroxylase. Support for a role of adrenodoxin phosphorylation comes from in vitro studies with P450 l l A l and P450 UBl systems reconstituted with purified components. Purified bovine adrenocortieal adrenodoxin can be selectively phosphorylated by incubation with purified cAMP-dependent protein kinase. Modification resulted in an average twofold decrease in the K^ values for the interaction between the phosphoadrenodoxin and the two P450 enzymes without a detectable change to the V^nax (Monnier et al., 1987). This effect is similar to that of P450 l l B l phosphorylation, which also decreases the K^ and K^ between phospho-P450 and adrenodoxin. We previously discussed that such an increase in affinity would be physiologically relevant given the normal limiting cellular concentration of adrenodoxin. It may well turn out that phosphorylation of adrenodoxin is critical in modulating the activity of P450 l l A l and P450 l l B l . Vitamin D and Calcium Regulation Vitamin D is a naturally occurring hormone involved in the regulation of calcium and phosphorus metabolism, affecting bone development. Requirements for vitamin D may be met by dietary intake or through biosynthesis. The first step in vitamin D biosynthesis is the photoactivation (by exposure of skin to sunlight) of 7-dehydrocholesterol to vitamin D3 by a nonenzymatic process. Hepatic vitamin D3 25-hydroxylase (P450 27A1) then acts on vitamin D3 to produce 25-hydroxycholecalciferol (25[OH]D3). Further metabolism of vitamin D involves two renal mitochondrial P450 enzymes. The active form of vitamin D (la,25-dihydroxyvitamin D3) is preferentially formed under conditions of calcium deficiency. Biosynthesis of la,25-dihydroxyvitamin D3 is catalyzed by renal la-hydroxylase (P450 27B1). A second renal enzyme (P450 24) catalyzes the 24-hydroxylation of 25(OH)D3 as well as 1,25(OH)2D3. Vitamin D 24-hydroxylation results in increased susceptibility of the homione to oxidation and side-chain cleavage, providing a pathway for the degradation of the hormone. When circulating levels of calcium become low, parathyroid hormone secretion is increased. This stimulates an increase in the biosynthesis of [1,25[OH]2D], which together with parathyroid hormone, signals the gastrointestinal absorption of calcium until bone requirements for calcium are met and circulating calcium levels return to REGULATION OF CYTOCHROME P450 391 normal. This closes the endocrine loop by a feedback mechanism decreasing parathyroid hormone secretion (Hendy, 1997; Narbaitz et al., 1981). The mechanisms by which parathyroid hormone stimulates the biosynthesis of [1,25[OH]2D] are coming into focus. The emerging picture suggests a role for the reversible phosphorylation of ferredoxin. It has been demonstrated that chick kidney la-hydroxylase may be phosphorylated, and that the activity of the enzyme in vitro is not affected when reconstituted with native ferredoxin and ferredoxin reductase. However, if la-hydroxylase and ferredoxin are both subject to phosphorylation and reconstituted with native ferredoxin reductase, the system fails to catalyze product formation (Ghazarian and Yanda, 1985). This result suggested that phosphorylation of ferredoxin might modulate la-hydroxylase activity. Parathyroid stimulation of intact renal cells favors renal la-hydroxylase over 24hydroxylase activity with a concurrent decrease in phosphorylation of ferredoxin (Siegel et al., 1986). It was also demonstrated that dephosphorylated ferredoxin can support both la-hydroxylase and 24-hydroxylase activity (Burgos-Trinidad et al., 1986; Gray and Ghazarian, 1989; Mandel et al., 1990). Thus, it appears that phosphorylation of ferredoxin significantly diminishes its interaction with la-hydroxylase without a similar change toward 24-hydroxylase. The overall result is more efficient electron transfer to 24-hydroxylase and, hence, greater 24-hydroxylase activity when ferredoxin is phosphorylated. As previously discussed, increased 24-hydroxylase activity translates into increased Vitamin D metabolism. DUAL TARGETTING OF P450 ENZYMES TO ENDOPLASMIC RETICULUM AND MITOCHONDRIA The cellular destination of a protein is dictated by primary amino acid signal sequences. Proteins targeted to mitochondria are translated by cytosolic ribosomes and transported to the organelle with the aid of mitochondrial translocase complexes that recognize the mitochondrial targeting sequence (Neupert, 1997). Proteins destined for the ER encode an N-terminal targeting sequence recognized by a signal recognition particle (SRP), which directs the emerging nascent protein into the ER (Gilmore et al., 1982). In certain cases, a protein may be directed to several cellular compartments simultaneously. Among such proteins are a number of P450s (P450 lAl, P450 2B1, P450 2B2, P450 2E1, P450 3A1, P450 3A2, P450 2D6, and P450 2C12), which are dually targeted to ER and mitochondria (Addya et al, 1997; Anandatheerthavarada et al., 1997, 1999; Bhagwat et al., 1999b). Although the mechanisms accounting for the dual targeting are not clear, efforts are beginning to shed light on the issue. Dual targeting of P450 lAl has been related to cleavage of an N-terminal segment of the protein, which activates a cryptic mitochondrial targeting sequence (Addya et al., 1997). Other reports demonstrate that phosphorylation provides a signal for targeting to the ER (Anandatheerthavarada et al., 1999; Robin et al., 2001, 2002). Evidence for the phosphorylation of P450 2B] isolated from rat liver has been reported (Pyerin et al., 1987). It was demonstrated that the P450 was a substrate for both PKA and Ca^ "^-phospholipid-dependent kinase. The significance of the modification has been related to two different regulatory mechanisms. Several reports have demonstrated that modification of P450 2B1 results in a modulation of enzyme activity (OeschBartlomowicz et al., 2001). Interestingly, the dual targeting of P450 2B1 to the ER and 392 M. AGUIAR ET AL. mitochondria is also dependent on phosphorylation of Ser'^* (Anandatheerthavarada et al., 1999). The authors postulated that a conformational shift is induced by phosphorylation at Ser'^^, which exposes a cryptic mitochondrial-targeting signal encoded within amino acids 21 -36 of the protein. To rule out the possibility that targeting of P450 2B1 was dependent on cleavage of the N-terminus or any other part of the intact protein, PAGE was employed. The position of PAGE protein bands revealed that P450 2B1 isolated from mitochondria and microsomes were of the same molecular weight. Thus, the dual targeting of P450 2B1 was deemed not dependent on cleavage of a targeting sequence or an alternatively translated mRNA transcript. Furthermore, incubation of hepatocytes without any inducers of PKA activity resulted in a greatly decreased level of P450 2B1 targeted to the mitochondria. Unpublished results in the same reference suggested that mitochondrial targeting of a number of P450s, including P450 2E1, P450 3A1, P450 3A2, P450 2D6, P450 2C12, etc., are also regulated by a similar PKAdependent mechanism. Indeed, P450 2E1 provides an additional example of a P450 that may be regulated by two mechanisms as a result of phosphorylation. Catalytic activity has been found to depend on modification of Ser'^' (Oesch-Bartlomowicz et al., 1998). In addition, the dual targeting of P450 2E1 to mitochondria and ER has been postulated to occur by a mechanism similar to that proposed for P450 2B1 (Robin et al., 2001). Protein isolated from the ER and mitochondria consisted of the same primary sequence, demonstrating that proteolytic cleavage of an N-terminus is not the determinant of cellular localization. P450 isolated from the mitochondria was phosphorylated at a higher level as compared to the ER fraction. In a recent report by the same group, it was demonstrated that Ser'^'-phosphorylated P450 2E1 is efficiently targeted to the mitochondria both in vitro and in vivo through activation of an N-terminal chimeric signal by a cAMP-dependent process (Robin et al., 2002). 26S PROTEASOMAL DEGRADATION The 26S proteasome is a large multienzyme protein complex that plays a key role in protein degradation, accounting for the bulk of protein catabolism in the cell. The role of the proteasome is varied, regulating many cellular processes, including cell cycle, organelle biogenesis, apoptosis, cell differentiation and proliferation, protein transport, inflammation, antigen processing, DNA repair, stress response, and catabolism of abnormal or damaged protein (Weissman, 2001). Because of the importance of this system, its activity is carefully regulated. Failure to maintain precise control can result in disease. A protein designated for degradation is normally labeled with ubiquitin, a 76 amino acid polypeptide. Several enzymes are involved in the ubiquitination process. The ubiquitin activating enzyme (El) is an ATP-dependent enzyme that activates ubiquitin and links it to the ubiquitin-conjugating enzyme E2. A third enzyme E3 is a ubiquitin ligase, which links the ubiquitin to the target protein. This process is repeated until the protein is polyubiquitinated. Once recognized by the proteasome, the polyubiquitinated protein is unfolded, the ubiquitin subunits are cleaved to be recycled, and the target protein is degraded into short peptides (Weissman, 2001). Several types of proteolytic activities are associated with the 26S proteasome: chymotrypsin-like activity with cleavage after hydrophobic residues, trypsin-like activity cleaving after basic residues, and a caspase-like activity with cleavage after acid residues. REGULATION OF CYTOCHROME P450 393 Ubiquitination and Proteasomal Degradation of P450 Normal protein turnover, as well as the degradation of chemically modified or structurally damaged P450, is processed by several proteolytic pathways including ER, lysosomal, and 20S and the 26S-ubiquitin system (Banerjee et al., 2000; Correia, 2003; Masaki et al., 1987; Murray and Correia, 2001; Murray et al., 2002; Roberts, 1997; Ronis et al, 1991; Zhukov et al., 1993). As may be observed in Table 2, a number of P450 enzymes are substrates for ubiquitinination, leading to degradation by the 26S proteasomal pathway. Studies undertaken by two groups (Correia et al., 1992; Tierney et al., 1992) revealed that in intact animals, hepatic P450 3A1, P450 3A2, and P450 2E1 are ubiquitinated and proteolytically degraded after a drug-induced mechanism-based suicide inactivation. Subsequent studies revealed that rat liver P450 3A1, P450 3A2, P450 2B1, as well as recombinant human P450 3A4 are phosphorylated, ubiquitinated, and finally degraded by the 26S-proteasome (Korsmeyer et al., 1999). Additional experiments will be required to determine if phosphorylation of these enzymes happens concurrently with or is a crucial step in the degradation of inactivated P450 by the ubiquitin-dependent 26S-proteasomal system. Recently, it was also demonstrated that native P450 3A4 expressed in yeast (Saccharomyces cerevisiae) is also degraded by the 26S-proteasomal pathway in a manner similar to that for suicidally inactivated protein (Murray and Correia, 2001). REGULATION OF P450 BY NITRIC OXIDE Nitric oxide (NO) is a short-lived free-radical gas that plays an important role in signal transduction pathways of the cardiovascular and nervous systems (Liaudet et al., 2000). During infection and inflammation, P450 mRNA and protein levels are downregulated in rat and human liver or hepatocytes (Morgan, 2001). Downregulation is dependent on a simultaneous increase in NO production in hepatocytes and Kupffer cells. In addition to translational regulation, recent studies have described NO-dependent posttranslational regulation of P450 (Morgan et al., 2001). NO is synthesized endogenously from arginine by three different forms of nitric oxide synthase. NO is known to form reversible but stable nitrosyl complexes with Table 2 Ubiquitinated P450 enzymes P450 Species Source Reference P450 2B1 P450 2E1 Rat Human Mouse Rat P450 3A1 Rat P450 3A2 Rat P450 3A4 Human Liver microsomes HepG2 expressed Liver (m vivo) Liver microsomes In vitro mRNA transcribed Liver (in vivo) Hepatocytes Liver microsomes Liver (in vivo) Hepatocytes Liver microsomes E-coli expressed Saccharomyces cerevisiae expressed Korsmeyer et al., 1999 Yang and Cederbaum, 1997 Tierney et al.. 1992 Banerjee et al., 2000 Banerjee et al., 2000 Correia et al., 1992 Wang et al., 1999 Korsmeyer et al., 1999 Correia et al., 1992 Wang et al.. 1999 Korsmeyer et al., 1999 Korsmeyer et al., 1999 Murray and Correia. 2001 394 M. AGUIAR ET AL. Table 3 Tyrosine nitrated P450 enzymes P450 Species Source Reference P450 2B1 Rat P450 8A1 Bovine Human Fusarium oxysporum Pseudomonas putida Bacillus megaterium Liver microsomes E-coli expressed Aortic microsomes EaHy926 expressed Eusarium oxysporum E-coli expressed Bacillus megaterium Roberts et al., 1998 Lin et al., 2003 Ullrich and Bachschmid, 2000 Zou et al., 1997 Morgan et al., 2001 Daiber et al., 2000a Daiber et al., 2000b P450 55A1 P450 101 P450 102A1 ferrous iron of hemoproteins. Formation of such nitrosyl complexes inhibits the catalytic activities of hepatic microsomal and purified P450 enzymes (Drewett et al., 2002; Khatsenko et al., 1993; Minamiyama et al., 1997; Morgan, 1997; Osawa et al., 1995; Stadler et al., 1994; Wink et al., 1993). In addition to reversible inhibition by NO, generation of peroxynitrile (PN) from NO and superoxide leads to covalent modification of P450 in the form of tyrosine nitration. Several reports have demonstrated that PN reacts with P450 8A1, also known as prostacyclin (PGI2) synthase, to inhibit enzyme activity (Hink et al., 2003; Schmidt et al., 2003; Ullrich and Bachschmid, 2000; Zou et al., 1997). In addition to P450 8A1, tyrosine nitration and enzyme inactivation by PN were also described for a few other P450 enzymes (Table 3) (Daiber et al., 2000a,b; Lin et al., 2003; Morgan et al., 2001; Roberts et al., 1998). The mechanism by which NO leads to tyrosine nitration (illustrated in Fig. 4), involves the formation of PN followed by heme-catalyzed nitration of tyrosine (Mehl et al., 1999). Superoxide (02~) is produced by a variety of enzyme-catalyzed reactions, including those catalyzed by P450, nitric oxide synthase, or other mitochondrial reactions 9^2 -H2O 0 .O-N. O 0=N o" " 2N0,- + O2 + o ^ Figure 4. Peroxynitrile reaction with heme proteins. (Adapted from Mehl et al., (1993) with permission.) REGULATION OF CYTOCHROME P450 395 associated with oxidative phosphorylation. Superoxide reacts with NO at nearly the diffusion-controlled rate to produce peroxynitrile (PN). Oxidation of heme Fe^^ by PN generates a ferryl complex (Fe'^ = O). The ferryl complex is then reduced back to ferric iron by tyrosine, forming a phenoxy radical. In the final step, phenoxy radical reacts with the NO2 radical formed during the initial oxidation of ferric iron by PN, resulting in tyrosine nitration. The identification and characterization of pre- and posttranslational regulatory effects of NO on P450 represent interesting regulatory mechanisms. Additional experiments will be required to reveal their physiological importance. GLYCOSYLATION OF P450 Protein glycosylation is an important posttranslational modification involved in cell adhesion, protein targeting, and protection from proteolytic attack. As illustrated in Table 4, a number of P450s have been identified as glycoproteins. The majority of these enzymes have not been characterized for the modification, and little is known about the underlying significance of P450 glycosylation. Two enzymes, P450 llAl and P450 19A1, have been studied in an attempt to define the relationship between glycosylation and modulation of enzyme activity (Amameh et al., 1993; Ichikawa and Hiwatashi, 1982; Sethumadhavan et al., 1991). Activity of one enzyme is significantly affected, while the other is not. P450 1 lAl demonstrates that glycosylation can modulate the catalytic activity of a P450. Ichikawa and Hiwatashi (1982) have reported that bovine adrenal P450 11 Al is a glyeoprotein. Treatment of the protein with neuramidase resulted in the inability of the protein to be reduced by the reducing system. The authors concluded that the sugar moiety of glycosylated P450 l l A l was essential for electron transport from reduced redoxin. P450 19A1 glycosylation has received attention in several reports and represents one of the better-characterized enzymes for this modification. Human placental P450 19A1 is a glyeoprotein (Sethumadhavan et al., 1991). Its carbohydrate side chain can be Table 4 Glycosylated P450 enzymes P450 Species Tissue Reference 1A2* 2B2 2B4 IIAI Mouse Rabbit Rabbit Bovine Negishi et al., 1981 Haugen and Coon, 1976 Haugen and Coon, 1976 Ichikawa and Hiwatashi, 1982 P450 17A1 P450 I9A1 Pig Human Human Horse Bovine Bovine Liver {in vivo) Liver {in vivo) Liver {in vivo) Adrenal mitochondrial cortex {in vivo) Testis {in vivo) Placenta {in vivo) COSl expressed Testis {in vivo) Adrenal cortex {in vivo) Liver {in vivo) P450 P450 P450 P450 P450 21AI P450 27B1** *Putatively glycosylated. **Bovine vitamin D3 25-hydroxylase sequence not yet reported. Nakajin and Hall, 1981 Shimozawa et al., 1993 Amameh et al., 1993 Moslemi et al., 1997 Hiwatashi and Ichikawa, 1981 Hiwatashi and Ichikawa, 1980 396 M. AGUIAR ET AL. selectively cleaved by endoglycosidase F and endoglycosidase H, which are known to hydrolyze N-glycans bound to asparagine (Asn) in a canonical N-X-S/T sequence. The site of glycosylation was determined as Asn'^, which is consistent with core glycosylation (glycosylation occurring in the lumen of the ER) of an N-terminal portion of the protein (Amarneh et al., 1993; Shimozawa et al., 1993). The hydrophobic Nterminal region of P450s comprises a signal recognition particle-dependent signal that directs insertion of P450 into the membrane of the ER (Bar-Nun et al., 1980; Sakaguchi et al., 1984). Unfortunately, the significance of the core glycosylation has not been clarified. In terms of an activity modulation, glycosylation of P450 19A1 does not significantly alter catalytic activity (Amarneh et al., 1993). CONCLUSIONS Cytochromes P450 comprise a family of important enzymes involved in the metabolism of a wide variety of endogenous and exogenous compounds. They are regulated transcriptionally, translationally, and posttranslationally by several different modifications. Phosphorylation has been reported for a number of P450 enzymes (Table 1), with evidence of enzyme regulation by a variety of mechanisms, including modulation of catalytic activity, substrate binding, binding of redox partners, and substrate specificity. Several P450 enzymes are directed to the mitochondrial inner membrane in response to phosphorylation. Ubiquitination of P450 has been associated with the turnover of native and damaged P450 by the 26S-proteasomal system. As demonstrated in Table 2, this pathway may be involved in regulating physiological levels of several enzymes. In the last decade, progress has been made in defining the regulation of P450 by nitric oxide at the translational and posttranslational regulatory level (Morgan et al., 2001). The posttranslational mechanisms can be of a reversible nature by the interaction of NO with the P450, or via an irreversible mechanism involving tyrosine nitration by peroxynitrile (Table 3). Few mammalian enzymes have been associated with these mechanisms, and this promises to be an interesting area of future discovery. Thus far, glycosylation has been identified in eight P450s, with the first two glycosylated enzymes identified over 25 years ago (Haugen and Coon, 1976). Despite these discoveries, relatively little is known concerning the physiological role of this modification. Characterizations of glycosylated P450s represent an area that deserves more attention. Table 4 lists several enzymes that have been identified as glycoproteins. Throughout this review, we considered the identification and physiological relevance of P450 posttranslational modification. Despite all the modifications reported in the literature, many of these have largely not been characterized. 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