REGULATION OF CYTOCHROME P450 BY …Applied R&D, MDS Pharma Services, St. Laurent (Montreal),...

Drug Metabolism Reviews, 37:379^04, 2005Copyright © Taylor & Francis Inc.ISSN: 0360-2532 print / 1097-9883 onlineDOI: 10.1081/DMR-200046136

I Taylor & FrancisTaylor 6. Francis Croup

REGULATION OF CYTOCHROME P450 BYPOSTTRANSLATIONAL MODIFICATION

Mike AguiarApplied R&D, MDS Pharma Services, St. Laurent (Montreal), Quebec, Canada

Department of Medicine, McGill University, Montreal, Quebec, Canada

Robert MasseApplied R&D, MDS Pharma Services, St. Laurent (Montreal), Quebec, Canada

Bernard F. GibbsApplied 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 inmany species. Over 3,000 enzymes have been identified in nature. Humans express 57putatively functional enzymes with a variety of critical physiological roles. They areinvolved in the metabolic oxidation, peroxidation, and reduction of many endogenous andexogenous 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, genemapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA CellBiol. 12(1):I—51.] The development of numerous diseases and disorders including cancerand cardiovascular and endocrine dysfunction has been linked to P450s.

Several levels of regulation, including transcription, translation, and posttranslationalmodification, participate in maintaining the proper function of P450s. Modifications incl-uding phosphorylation, glycosylation, nitration, and ubiquitination have been described forP450s. Their physiological significance includes modulation of enzyme activity, targeting tospecific cellular compartments, and tagging for proteasomal degradation. Knowledge ofP450 posttranslational regulation is derived from studies with relatively few enzymes. Inmany cases, there is only enough evidence to suggest the occurrence and a possible role forthe modification. Thus, many P450 enzymes have not been fully characterized. With theintroduction of current proteomics tools, we are primed to answer many important questionsregarding regulation of P450 in response to a posttranslational modification. This reviewconsiders regulation ofP450 in a context that describes the potential role and physiologicalsignificance 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]

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380 M. AGUIAR ET AL.

INTRODUCTION

Background Information on P450

Since the introduction of modern molecular biology techniques allowing for thesequencing of entire genomes, researchers have identified and continue to discover manyP450 enzymes in a wide variety of organisms. In order to systematically identify andcategorize this growing family of enzymes, a leading group of researchers in the fieldestablished the current system of nomenclature (Nelson et al., 1996). P450s are namedwith the prefix CYP or P450 followed by an Arabic number defining the family, anuppercase letter defining the subfamily, and another Arabic number defining anindividual enzyme (e.g., P450 3A4). In cases where only a single member exists in agiven 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 andlinks to related sites.

All mammalian tissues examined express some P450 enzyme system (Porter andCoon, 1991). In addition, mammals express multiple enzymes simultaneously in a varietyof 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 variousenzymes are found not only in different cell and tissue types, but also in differentsubcellular compartments, such as the outer nuclear membrane, endoplasmic reticulum(ER), mitochondria, golgi, peroxisome, and plasma membrane. Certain enzymes arefound in several different subcellular compartments simultaneously (Guengerich, 2001).

All cytochrome P450s, with the exception of bacterial enzymes, are membranebound. Microsomal enzymes are tethered to the membrane through a hydrophobictransmembrane helix at the N-terminus of the protein, which also serves as a targetingsequence for the signal recognition particle dependent cotranslational incorporation of anascent 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 theP450 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 solubilizingmembrane-bound P450s, all native mammalian P450s have evaded crystallization andx-ray crystallographic study. To avoid this problem, researchers have modified severalP450s resulting in soluble mutants that can be crystallized and that have structures thatwere 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 differentsubstrates (Wester et al., 2003a,b). Interestingly, one of the two substrates binds in twodifferent orientations (Wester et al., 2003a). These studies of P450 2C5 offer insightfulinformation on the fiexibility of the enzyme and shed light on the ability of P450s toaccommodate a variety of substrates with different shapes and sizes. The structure ofhuman P450 2C8 was recently reported, revealing an active site volume twice that ofP450 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 knownanticoagulant substrate (Williams et al., 2003). The structure reveals a new bindingpocket 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 astructure for P450 2C9 complexed with flurbiprofen (Wester et al,, 2004), The structurewas obtained without modifications to the catalytic domain, revealing some significantconformational differences. The more recent structure helps explain some experimentalobservations in terms of the substrate selectivity of the enzyme, which were not easilyexplained with the earlier structure, A crystal structure for rabbit P450 2B4, one of thefirst P450s to be purified and studied in detail, has been reported in a wide openconformation (Scott et al,, 2003) and in a closed form complexed with the specificinhibitor 4-(4-chlorophenyl)-imidazole (Scott et al,, 2004), These structures may help tounderstand how mammalian P450s "open/close," allowing substrate to access theburied active site (Poulos, 2003), Two groups have recently reported crystal structuresfor human P450 3A4: one unliganded structure from each group and two structureshound to different substrates (Williams et al, 2004; Yano et al,, 2004a), The collectionof structures should provide a better understanding of the substrate selectivity andunusual kinetics of this important enzyme. Finally, a structure for P450 2A6 has beenreported 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 fromfamilies 1-3, This group demonstrates wide substrate specificity, with certain enzymesacting on a large number of structurally varied substrates such as in the case of humanP450 3A4,

The second group demonstrates high substrate specificity, catalyzing thebiosynthesis 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 ^ ^ * DehydroepiandrosteroneP450 11AI P45017A1 P450 17A1

J P450 7A1 I 3P HSD J 3P HSD i 3P HSD

7-OH Cholesterol Progesterone ^ ^ ^ 17-OH Progesterone ^ ^ * Androstenedione ^ ^ * EstroneP450 17A1 P450 17A1 P45019A1

Bile Acids 11-Deoxycorticosterone 11-Deoxycortisol Testosterone ^ ^ ^ ^ Estradiol

P450 19AI

| P 4 5 O 1 1 B I JP45O11B1

Corticosterone Cortisol

Aldosterone

|P45O11B2

18-OHCorticosterone

Figure 1. Cholesterol, bile acid, and steroid hormone biosynthesis: phosphorylated P450 enzymes are indicatedin boldface.


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 hacterialsystems. Reducing equivalents from NADPH or NADH are transferred two electrons at atime to redoxin reductase, which carries a flavin adenine dinucleotide (FAD) prostheticgroup. The isoalloxazine ring of FAD may exist in several oxidation states, which allowsfor the subsequent transfer of electrons individually to a mobile Fe2S2-containing proteincalled redoxin. Reduced redoxin is thought to shuttle electrons to the P450, returning tothe reductase in the oxidized form for additional cycles.

Class II P450s are those that reside in the ER and receive reducing equivalents fromNADPH 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 mono-nucleotide (FMN) prosthetic group that also has an isoalloxazine ring with propertiessimilar to FAD, An electron pair from NADPH is received by FAD, which relays theelectrons to FMN, finally transferring electrons in single file to the P450, In certainreactions, the first electron is transferred from P450 reductase, while the second electron istransferred from cytochrome b^. Cytochrome b^ may be reduced either by P450 reductase,or cytochrome b^ reductase. Interestingly, apo-^s (cytochrome ^5 devoid of heme), whichcannot transfer electrons, is required for optimal activity in a number of P450 enzymes andonly with certain substrates (Hlavica and Lewis, 2001; Yamazaki et al,, 2001),

Class III P450s are isomerases instead of monooxygenases. They do not require theparticipation of any redox partners or donors of reducing equivalents. Water is notproduced, and therefore, molecular oxygen is not required for catalysis. Substrates of classIII P450s are simply rearranged into products. Examples of P450s from this class includeP450 8A1 [prostacyclin [PGI2] synthase] and P450 5 [thromboxane [TXA2] synthase].

POSTTRANSLATIOIMAL MODIFICATION

A posttranslational modification may be defined as "any difference between afunctional protein and the linear polypeptide sequence encoded between the initiation andthe termination codons of its structural gene" (Han and Martinage, 1992), Examples ofnoncovalent modifications include incorporation of cofactors such as heme, proteinfolding, and the association of subunits to form an oligomeric protein, Allostericphenomena manifested as deviations from Michaelis-Menten kinetics have beendemonstrated for numerous P450 enzymes. Various components known to interact withP450, including substrates, inhibitors, membrane lipids, and redox partners (such as thepreviously mentioned cytochrome ^5), have been shown to act as homotropic andheterotropic 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 ofdisulfide bonds, and an array of modifications to amino acid residues, includingphosphorylation, nitration, glycosylation, methylation, sulfation, acetylation, andprenylation, provide another means of posttranslational modification. The remainder ofthe present article is dedicated to the identification and characterization of covalent P450posttranslational modifications with an emphasis placed on describing the physiologicalrole of each modification.


PHOSPHORYLATIOIM

An article by Cohen (2002), opens with the following statement, "Proteinphosphorylation regulates most aspects of cell life, whereas abnormal phosphorylation isa cause or consequence of disease." Cascades that activate the production of cyclicadenosine monophosphate (cAMP), leading to activation of protein kinase A (PKA) ineukaryotic cells illustrate a remarkable example. Once activated, PKA can phosphorylatemany target proteins, resulting in a wide variety of cellular responses, includingregulation of gene transcription, modulation of enzyme activity, targeting of protein fordegradation, 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 overtwenty P450 enzymes in microsomes, intact hepatocytes, cell culture, and in vivo(Table 1). Whereas some studies demonstrate stimulation of phosphorylation by additionof hormones and intracellular second messengers, other reports correlate phosphorylationwith modulation of enzyme activity (Koch and Waxman, 1989; Oesch-Bartlomowiczet al., 1998, 2001; Pyerin and Taniguchi, 1989). Much of the work has focused onmembers of family 1-3 P450s. Unfortunately, studies regarding regulation byposttranslational modification of the major drug metabolizing P450s, with the exceptionof 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 involvedspecifically in cholesterol and steroid homeostasis has been reported.

PHOSPHORYLATION OF STEROID HORMONESYNTHESIZING P450 ENZYMES

Steroid hormones play an essential role in stress response, water and electrolytebalance, sexual differentiation and reproduction, bone and tissue homeostasis, cognitivefunction, and numerous other key physiological processes (Evans, 1988; Parker andSchimmer, 1993). Androgens and estrogens are known to participate in the developmentof breast and prostate cancer. Given their potent effects, steroid hormone levels must bevery carefully regulated. The adrenal cortex and the gonads are the key sites ofglucocorticoid, mineralocorticoid, and sex steroid biosynthesis. Synthesis of steroidsrequires the concerted action of a number of cytochrome P450 steroid hydroxylases(Fig. 1). Transcdptional regulation of the genes encoding these steroid hydroxylasesoccurs in response to trophic hormones and transcription factors such as adrenocorti-cotropin (ACTH) and steroidogenic factor 1 (SF-1), with selective expression insteroidogenic cells (Parker and Schimmer, 1993, 1996, 1997; Stocco, 2000). In additionto transcdptional regulation, they are regulated by posttranslational modificationincluding phosphorylation. The underlying theme involves signal transduction pathwaysresulting in the activation of cyclic nucleotide and other second messengermolecules ultimately activating kinase/phosphatases that act on P450s as well asferredoxin/adrenodoxin.


Table 1 Phosphorylated P450 enzymes

P450

P450 1A2

P450 2B1

P450 2B2

P450 2B4P450 2C6

P450 2C7P450 2C11P450 2C12P450 2E1

P450 3A1

P450 3A4P450 3A6P450 7A1

P450 llAl

P450 UBl

P450 17A1

P450 19A1

P450 51P450 27A1**

Species

RatRabbitRat

Rat

RabbitRat

RatRatRatMouse

Rat

Rat

HumanRabbitRat

HumanBovine

Bovine

Human

RatHumanHuman*

Quail

RatChicken

Source

Liver microsomesLiver microsomesLiver microsomesHepatocytesLiver (in vivo)Liver microsomesHepatocytes

Liver {in vivo)Liver microsomesLiver microsomesLiver {in vivo)Liver microsomesLiver microsomesLiver microsomesCOS 7V79Liver microsomesHepatocytesLiver {in vivo)Hepatocytes, Liver microsomesE-coli expressedLiver microsomesLiver microsomesE-coli expressedE-coli expressedCorpus luteum mitochondriaAdrenal cortex mitochondria!

inner membraneAdrenal cortex mitochondrial

inner membraneNCI-H295, COS-1, Kin 8 expressed,

Adrenal mierosomesTestis microsomesNCI-H295R, NCI-H295R expressedPlacentaMCF-7 cellsBrain

Liver microsomesKidney mitochondria

Reference

Pyerin et al., 1987Pyerin et al., 1987Jansson et al., 1990Oesch-Bartlomowicz et al., 2001Oesch-Bartlomowicz et al., 2001Pyerin et al., 1987Oesch-Bartlomowicz and

Oesch, 1990Koch and Waxman, 1989Epstein et al., 1989Epstein et al., 1989Koch and Waxman, 1989Epstein et al., 1989Pyerin et al., 1987Epstein et al., 1989Freeman and Wolf, 1994Oesch-Bartlomowicz et al., 1998Menez et al., 1993Oesch-Bartlomowicz et al., 1998Koch and Waxman, 1989Eliasson et al., 1994Wang et al., 2001Pyerin et al, 1987Tang and Chiang, 1986Nguyen et al., 1996Nguyen et al., 1996Caron et al., 1975Vilgrain et al., 1984

Dcfaye et al., 1982

Zhang et al., 1995

Lohr and Kuhn-Velten, 1997Biason-Lauber, 2000Bellino and Holben, 1989Yue et al., 2003Balthazart et al., 2001a;

Balthazart et al., 2001bSonoda et al., 1995Ghazarian 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 cholesterolfeedback mechanisms on a number of different enzymes, including acetoacetyl-CoAreductase, HMG-CoA synthase, HMG-CoA reductase, prenyl transferase, squalene


synthetase, squalene epoxidase, and low-density lipoprotein (LDL) receptor (Sonodaet al,, 1995), When dietary cholesterol is low, various organisms including humanssynthesize cholesterol entirely from Acetyl-CoA, One of the biosynthetic steps leading upto cholesterol involves the removal of the ]4a-methyl group from lanosterol and 24,25-dihydroxylanosterol (DHL), Sterol 14a-demethylase (P450 51) is the microsomalcytochrome P450 that catalyzes this reaction. The human analog of this enzyme isexpressed in testis, ovary, adrenal, prostate, liver, kidney, and lung.

Experiments performed with preparations of purified rat liver P450 51 havedemonstrated that enzyme activity increases when dephosphorylated. When purifiedenzyme is pretreated with type III bacterial alkaline phosphatase, followed by recon-stitution with the remaining system components (NADPH-P450 reductase, NADPH, and24,25-dihydroxylanosterol), the result is an increase in enzyme activity relative to acontrol (preparation that was not treated with phosphatase). Thus, it has been proposedthat phosphorylation-dephosphorylation of P450 51 may be involved in regulation of theenzyme activity (Sonoda et al,, 1995), and consequently, may represent an importantregulatory mechanism of cholesterol biosynthesis and potentially a molecular target forcholesterol-lowering drugs.

Cholesterol Metabolism and the Synthesis of Bile Acids

Cholesterol is the precursor of all steroids, hence its requirement for maintainingproper endocrine function. However, excessive blood cholesterol levels may lead tomedical disorders such as gallstones. The conversion of cholesterol to hydrophilic bileacids in the liver provides an important pathway for its elimination. The rate-limiting stepin 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), Inaddition, literature has appeared mostly arguing for, but with a few reports against, themodulation 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 (Nguyenet al,, 1996) was that the findings were all based on indirect evidence, relatingperturbations to the enzyme preparation with kinases, phosphatases, phosphataseinhibitors, etc, to changes in enzyme activity without directly observing correspondingchanges to the phosphorylation state of P450 7AI, The study employing Escherichiacoll {E. coll) expressed rat and human P450 7A1 has demonstrated modulation ofenzyme activity in vitro by phosphorylation and dephosphorylation with a directcorresponding change in phosphorylation state of P450 7A1 as demonstrated hyincorporation of •' P phosphate into purified enzyme applied to sodium dodecyl-sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) (Nguyen et al,, 1996), Taken together,these results demonstrate that phosphorylation and dephosphorylation are reversiblemodes of regulation of P450 7A1 activity in vitro. The physiological significance of thesephenomena remains to be established, as no reports have appeared demonstrating theimportance of this mode of regulation in whole cells or in vivo.


Biosynthesis of Pregnenolone, a Checkpoint inSteroid Hormone Biosynthesis

The first step in the conversion of cholesterol to steroids is the synthesis ofpregnenolone. The reaction involves three separate hydroxylation steps, with threeequivalents of O2 and NADPH. The enzyme that catalyzes this reaction (P450 l lAl) isexpressed in all tissues that synthesize steroids from cholesterol. P450 11 Al resides in theinner mitochondrial membrane. Transcriptional regulation of P450 l lAl and othersteroidogenic 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 lAl activity by phosphorylation wasreported several years ago (Caron et al., 1975). A crude preparation of P450 l lAl wasisolated from bovine corpus luteum mitochondria. Limiting P450 was reconstituted withexcess redoxin and redoxin reductase, and the reconstituted system was subjected totreatment with PKA, cAMP, and ATP. The resulting system demonstrated increasedP450 l lAl activity. Decisive evidence for the posttranslational phosphorylation ofP450 l lAl came nearly a decade later (Vilgrain et al., 1984). A demonstration withpurified bovine adrenocortical P450 l lA l showed that the P450 is efficientlyphosphorylated by Ca^"^-activated phospholipid-sensitive protein kinase C (PKC). Fourmoles of phosphate were incorporated per mole of P450 11 Al, with serine and threonineas the target amino acids phosphorylated in a ratio of 1:1 as revealed by amino acidanalysis. Interestingly, PKC activity is also found to be associated with bovineadrenocortical inner mitochondrial membrane (Vilgrain et al., 1984). Thus, it appears thatphosphorylation of P450 l lAl in a reconstituted system results in increased enzymeactivity. Additional experiments would be required to demonstrate the physiologicalrelevance 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 glucocorticoidsand androgens (Fig. 1). Much interest has been focused on developing inhibitors of P45017A1, because activity of this one enzyme can direct the course of an androgen-dependentmalignancy, such as prostate cancer. Understanding all factors, including posttransla-tional modification, that regulate P450 17A1 activity is therefore of great importance.

Two consecutive reactions in steroid biosynthesis are catalyzed by this enzyme; the17a-hydroxylation of pregnenolone and progesterone and the subsequent cleavage of the17-20 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 bifunctionalactive site (Nakajin et al., 1981). The two catalytic steps may be coupled (17-hydroxylation followed immediately by 17-20 lyase activity), or catalysis may stop afterthe initial hydroxylation step, resulting in the formation of androgens and precursors ofglucocorticoids, respectively.

The adrenals of children between one and eight years of age secrete cortisol (a C21steroid) but very little sex steroid precursors (C19 steroids). This is due to the adrenal 17a-hydroxylation activity of P450 17A1 with a lack of lyase activity. Between seven andnine years of age, the adrenals begin to produce and secrete increasing levels of C19


Steroids (Fig, 2) without a corresponding increase in levels of cortisol or adrenocorti-cotropin 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, afterwhich time they begin to drop gradually until they return to childhood levels at 70-80years of age (Orentreich et al,, 1984), This programmed development of adrenal P45017A1 17,20-lyase activity is known as adrenarche.

In an effort to explain this phenomenon, experiments were performed to determineif the change in lyase activity could be associated with an increase in posttranslationalmodification of P450 17A1, Human P450 17A1 is phosphorylated on serine andthreonine residues by PKA, Phosphorylation increases 17,20-lyase activity, whiledephosphorylation has the opposite effect (Zhang et al,, 1995), Phosphorylation of rattesticular P450 17A1 increases the ligand-binding efficiency of the enzyme for its naturalligand (progesterone) as well as the rate of P450 17A1 proteolytic degradation (Lohr andKuhn-Velten, 1997), These findings have led researchers to propose that pituitaryhormones (corticotropin, lutropin, or human chorionic gonadotropin) known to activatethe G protein-coupled receptor (GPCR)/adenylate cyclase/PKA signal transductionpathway, 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 lyaseactivity of P450 17A1 in intact human adrenocortical carcinoma cells in a mannerconsistent with the development of adrenarche. As may he observed in Fig, 3,physiological levels of leptin, acting through its receptor and the downstream signal

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Figure 2. Serum DHEAS concentration in normal children. Points represent single entries; lines repre-sent serial entries. The period between vertical lines represents average age of puberty onset in girls. Theshaded period represents the average age of puberty onset in boys, [Adapted from Korth-Schutz et al,, 1976with permission,]


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Figure 3. Effect of leptin and dephosphorylation on P450 17A1 enzyme activity. Short-term treatment with30 pM leptin of human adrenal NCI-H295R cells (leptin receptor+ B ; leptin receptor- A)- A 17a-hydroxylaseand B 17,20-lyase activity was analyzed in intact cells. Bottom right: Phosphate removal with alkalinephosphatase (AP) selectively abolishes DHEA production. [Adapted from Biason-Lauber et al., 2000with permission.]

transduction pathway, stimulate the phosphorylation of P450 17A1 leading to an acuteand long-term stimulation of P450 17A1 lyase activity in the human adrenal cell (Biason-Lauber et al., 2000).

Biosynthesis of Glucocortlcoid and Mineralocorticoid

P450 l lBl , a Class I P450 expressed only in the adrenal cortex, catalyzes twoadditional steps in steroid hormone biosynthesis. These steps are the oxidation of 11-deoxycorticosterone to corticosterone (a mineralocorticoid precursor) and oxidation of11-deoxycortisol to cortisol (a glucocorticoid).

Studies with bovine P450 l lBl purified from mitochondrial adrenal cortex havedemonstrated that P450 l lBl is phosphorylated by skeletal muscle PKA. Enzymeactivity of P450 l lBl does not change as a result of phosphorylation if the system isreconstituted with excess adrenodoxin. However, kinetic studies demonstrate that P450l lBl phosphorylation strikingly increased the affinity between P450 l lBl andadrenodoxin. As limiting adrenodoxin is the normal state in adrenocortieal mitochondria,phosphorylation may be a physiologically relevant factor in stimulating P450 l lBlactivity (Defaye et al., 1982; Estabrook et al., 1972). Additional studies would berequired 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


androgens, P450 19A1 is also commonly referred to as aromatase. P450 19A1 isexpressed in tissues associated with primary steroid synthesis, as well as a variety of otherperipheral tissues, including adipose and bone. In addition to well-estahlishedtranscdptional regulation, evidence supporting the regulation of P450 19A1 byposttranslational phosphorylation has been reported (Balthazart et al., 2001a,b, 2003;Bellino and Holben, 1989; Yue et al., 2003). Initial evidence came from a studyemploying microsomes isolated from human term placenta, where it was demonstratedthat microsomal P450 19A1 activity could be maintained in phosphate buffer or by theinhibition 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 P45019A1 activity (Bellino and Holben, 1989). P450 19A1 phosphorylation has beenassociated with several different physiological phenomena.

Reports in recent years have demonstrated the neuromodulatory effects ofestrogenic metabolites [referenced in Balthazart et al. (2001a)]. A telling examplecomes from a study with castrated sexually experienced male rats that illustrates thatestrogen administration rapidly activates male sexual behavior (within minutes),presumably by a nongenomic mechanism, as a genomic mechanism would require hoursto days for an observable effect (Cross and Roselli, 1999). Rapid and pronounced changesin P450 19A1 activity of quail hypothalamic homogenates by pharmacological studiesemploying kinase activators and inhibitors have been described (Balthazart et al.,2001a,b). Stimulation of kinase by addition of nonnal intracellular concentrations ofCa " , ATP, and Mg " resulted in significant decreases in P450 19A1 activity. Thedecrease in activity could be completely abolished by the addition of ethylene glycol-fc«(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, thusthe possibility remained that intermediary proteins are phosphorylated that interact withP450 19A1 to modulate its activity. Conclusive experiments confirming P450 19A1phosphorylation have been reported (Balthazart et al, 2003). Aromatase from quailpreoptic area homogenates was immunopurified, and phosphorylated Ser, Thr, and Tyrresidues were detected by Western analysis employing phospho-amino acid specificantibodies. Together, these results demonstrate that the local production of estrogens inquail brain can be rapidly altered by calcium-dependent P450 19A1 phosphorylation.

P450 19A1 activity is also a key factor in the progression of estrogen-dependentdiseases such as breast, endometrial, and ovarian cancers. Like P450 17A1, P450 19A1represents an important target in the treatment of such diseases. Hormone-dependentbreast cancer can be treated by surgical removal of the affected area or by endocrinetreatment 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 thepatients who relapse, suggesting an adaptation to therapy that involves aromatase.Several mechanisms may account for the adaptation. It has been demonstrated that long-term estrogen deprivation causes hypersensitivity of cultured MCF-7 breast cancer cellsto the mitogenic effects of estradiol, with an associated activation of the MAP kinase andPI3 kinase pathways. It was also discovered that aromatase activity is elevated in long-term estrogen deprivation (Yue et al., 1999). In order to establish a link between thekinase cascades and activation of aromatase, MCF-7 cells deprived of estrogens weretreated with inhibitors of the kinase pathways. A significant decrease in aromatase


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 thatmore detailed studies would be required to understand the mechanisms of the kinasepathway 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 ofelectrons from redoxin reductase. Several reports describe the phosphorylation ofredoxin. We will consider the significance of redoxin modification in modulatingthe activity of several P450 enzymes, including adrenal P450 l lAl , P450 l lB l , andrenal la-hydroxylase.

Support for a role of adrenodoxin phosphorylation comes from in vitro studieswith P450 l lAl and P450 UBl systems reconstituted with purified components.Purified bovine adrenocortieal adrenodoxin can be selectively phosphorylated byincubation with purified cAMP-dependent protein kinase. Modification resulted in anaverage twofold decrease in the K^ values for the interaction between the phospho-adrenodoxin 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 lB l phosphorylation,which also decreases the K^ and K^ between phospho-P450 and adrenodoxin. Wepreviously discussed that such an increase in affinity would be physiologically relevantgiven the normal limiting cellular concentration of adrenodoxin. It may well turn outthat phosphorylation of adrenodoxin is critical in modulating the activity of P450 l lAland P450 l lB l .

Vitamin D and Calcium Regulation

Vitamin D is a naturally occurring hormone involved in the regulation of calciumand phosphorus metabolism, affecting bone development. Requirements for vitamin Dmay be met by dietary intake or through biosynthesis. The first step in vitamin Dbiosynthesis is the photoactivation (by exposure of skin to sunlight) of 7-dehydro-cholesterol to vitamin D3 by a nonenzymatic process. Hepatic vitamin D3 25-hydro-xylase (P450 27A1) then acts on vitamin D3 to produce 25-hydroxycholecalciferol(25[OH]D3). Further metabolism of vitamin D involves two renal mitochondrial P450enzymes. The active form of vitamin D (la,25-dihydroxyvitamin D3) is preferentiallyformed under conditions of calcium deficiency. Biosynthesis of la,25-dihydroxyvita-min 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 homioneto oxidation and side-chain cleavage, providing a pathway for the degradation ofthe hormone.

When circulating levels of calcium become low, parathyroid hormone secretion isincreased. This stimulates an increase in the biosynthesis of [1,25[OH]2D], whichtogether with parathyroid hormone, signals the gastrointestinal absorption of calciumuntil bone requirements for calcium are met and circulating calcium levels return to


normal. This closes the endocrine loop by a feedback mechanism decreasing parathyroidhormone secretion (Hendy, 1997; Narbaitz et al., 1981). The mechanisms by whichparathyroid 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 phosphory-lated, and that the activity of the enzyme in vitro is not affected when reconstituted withnative ferredoxin and ferredoxin reductase. However, if la-hydroxylase and ferredoxinare both subject to phosphorylation and reconstituted with native ferredoxin reductase,the system fails to catalyze product formation (Ghazarian and Yanda, 1985). This resultsuggested that phosphorylation of ferredoxin might modulate la-hydroxylase activity.Parathyroid stimulation of intact renal cells favors renal la-hydroxylase over 24-hydroxylase activity with a concurrent decrease in phosphorylation of ferredoxin (Siegelet al., 1986). It was also demonstrated that dephosphorylated ferredoxin can support bothla-hydroxylase and 24-hydroxylase activity (Burgos-Trinidad et al., 1986; Gray andGhazarian, 1989; Mandel et al., 1990). Thus, it appears that phosphorylation offerredoxin significantly diminishes its interaction with la-hydroxylase without a similarchange toward 24-hydroxylase. The overall result is more efficient electron transfer to24-hydroxylase and, hence, greater 24-hydroxylase activity when ferredoxin isphosphorylated. As previously discussed, increased 24-hydroxylase activity translatesinto increased Vitamin D metabolism.

DUAL TARGETTING OF P450 ENZYMES TO ENDOPLASMICRETICULUM AND MITOCHONDRIA

The cellular destination of a protein is dictated by primary amino acid signalsequences. Proteins targeted to mitochondria are translated by cytosolic ribosomes andtransported to the organelle with the aid of mitochondrial translocase complexes thatrecognize the mitochondrial targeting sequence (Neupert, 1997). Proteins destined for theER 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). Incertain 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 andmitochondria (Addya et al, 1997; Anandatheerthavarada et al., 1997, 1999; Bhagwatet 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 beenrelated to cleavage of an N-terminal segment of the protein, which activates a crypticmitochondrial targeting sequence (Addya et al., 1997). Other reports demonstrate thatphosphorylation 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 beenreported (Pyerin et al., 1987). It was demonstrated that the P450 was a substrate for bothPKA and Ca^ "^-phospholipid-dependent kinase. The significance of the modification hasbeen related to two different regulatory mechanisms. Several reports have demonstratedthat modification of P450 2B1 results in a modulation of enzyme activity (Oesch-Bartlomowicz et al., 2001). Interestingly, the dual targeting of P450 2B1 to the ER and


mitochondria is also dependent on phosphorylation of Ser'^* (Anandatheerthavaradaet al., 1999). The authors postulated that a conformational shift is induced by phos-phorylation at Ser'^^, which exposes a cryptic mitochondrial-targeting signal encodedwithin amino acids 21 -36 of the protein. To rule out the possibility that targeting of P4502B1 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 2B1isolated 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 targetingsequence or an alternatively translated mRNA transcript. Furthermore, incubation ofhepatocytes without any inducers of PKA activity resulted in a greatly decreased level ofP450 2B1 targeted to the mitochondria. Unpublished results in the same referencesuggested that mitochondrial targeting of a number of P450s, including P450 2E1, P4503A1, P450 3A2, P450 2D6, P450 2C12, etc., are also regulated by a similar PKA-dependent mechanism. Indeed, P450 2E1 provides an additional example of a P450 thatmay be regulated by two mechanisms as a result of phosphorylation. Catalytic activityhas been found to depend on modification of Ser'^' (Oesch-Bartlomowicz et al., 1998). Inaddition, the dual targeting of P450 2E1 to mitochondria and ER has been postulated tooccur by a mechanism similar to that proposed for P450 2B1 (Robin et al., 2001). Proteinisolated from the ER and mitochondria consisted of the same primary sequence,demonstrating that proteolytic cleavage of an N-terminus is not the determinant ofcellular localization. P450 isolated from the mitochondria was phosphorylated at a higherlevel as compared to the ER fraction. In a recent report by the same group, it wasdemonstrated that Ser'^'-phosphorylated P450 2E1 is efficiently targeted to themitochondria both in vitro and in vivo through activation of an N-terminal chimericsignal 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 inprotein degradation, accounting for the bulk of protein catabolism in the cell. The role of theproteasome is varied, regulating many cellular processes, including cell cycle, organellebiogenesis, apoptosis, cell differentiation and proliferation, protein transport, inflamma-tion, antigen processing, DNA repair, stress response, and catabolism of abnormal ordamaged protein (Weissman, 2001). Because of the importance of this system, its activityis carefully regulated. Failure to maintain precise control can result in disease.

A protein designated for degradation is normally labeled with ubiquitin, a 76 aminoacid polypeptide. Several enzymes are involved in the ubiquitination process. Theubiquitin activating enzyme (El) is an ATP-dependent enzyme that activates ubiquitinand links it to the ubiquitin-conjugating enzyme E2. A third enzyme E3 is a ubiquitinligase, which links the ubiquitin to the target protein. This process is repeated until theprotein is polyubiquitinated. Once recognized by the proteasome, the polyubiquitinatedprotein is unfolded, the ubiquitin subunits are cleaved to be recycled, and the targetprotein is degraded into short peptides (Weissman, 2001). Several types of proteolyticactivities are associated with the 26S proteasome: chymotrypsin-like activity withcleavage after hydrophobic residues, trypsin-like activity cleaving after basic residues,and a caspase-like activity with cleavage after acid residues.


Ubiquitination and Proteasomal Degradation of P450

Normal protein turnover, as well as the degradation of chemically modified orstructurally 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; Roniset al, 1991; Zhukov et al., 1993). As may be observed in Table 2, a number of P450enzymes are substrates for ubiquitinination, leading to degradation by the 26Sproteasomal pathway. Studies undertaken by two groups (Correia et al., 1992; Tierneyet al., 1992) revealed that in intact animals, hepatic P450 3A1, P450 3A2, and P450 2E1are ubiquitinated and proteolytically degraded after a drug-induced mechanism-basedsuicide 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). Additionalexperiments will be required to determine if phosphorylation of these enzymes happensconcurrently with or is a crucial step in the degradation of inactivated P450 by theubiquitin-dependent 26S-proteasomal system. Recently, it was also demonstrated thatnative P450 3A4 expressed in yeast (Saccharomyces cerevisiae) is also degraded by the26S-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 insignal transduction pathways of the cardiovascular and nervous systems (Liaudet et al.,2000). During infection and inflammation, P450 mRNA and protein levels aredownregulated in rat and human liver or hepatocytes (Morgan, 2001). Downregulationis dependent on a simultaneous increase in NO production in hepatocytes and Kupffercells. In addition to translational regulation, recent studies have described NO-dependentposttranslational regulation of P450 (Morgan et al., 2001).

NO is synthesized endogenously from arginine by three different forms of nitricoxide synthase. NO is known to form reversible but stable nitrosyl complexes with

Table 2 Ubiquitinated P450 enzymes

P450

P450 2B1P450 2E1

P450 3A1

P450 3A2

P450 3A4

Species

RatHumanMouseRat

Rat

Rat

Human

Source

Liver microsomesHepG2 expressedLiver (m vivo)Liver microsomesIn vitro mRNA transcribedLiver (in vivo)HepatocytesLiver microsomesLiver (in vivo)HepatocytesLiver microsomesE-coli expressedSaccharomyces cerevisiae expressed

Reference

Korsmeyer et al., 1999Yang and Cederbaum, 1997Tierney et al.. 1992Banerjee et al., 2000Banerjee et al., 2000Correia et al., 1992Wang et al., 1999Korsmeyer et al., 1999Correia et al., 1992Wang et al.. 1999Korsmeyer et al., 1999Korsmeyer et al., 1999Murray and Correia. 2001


Table 3 Tyrosine nitrated P450 enzymes

P450

P450 2B1

P450 8A1

P450 55A1P450 101P450 102A1

Species

Rat

BovineHumanFusarium oxysporumPseudomonas putidaBacillus megaterium

Source

Liver microsomesE-coli expressedAortic microsomesEaHy926 expressedEusarium oxysporumE-coli expressedBacillus megaterium

Reference

Roberts et al., 1998Lin et al., 2003Ullrich and Bachschmid, 2000Zou et al., 1997Morgan et al., 2001Daiber et al., 2000aDaiber et al., 2000b

ferrous iron of hemoproteins. Formation of such nitrosyl complexes inhibits the catalyticactivities 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 modificationof P450 in the form of tyrosine nitration. Several reports have demonstrated that PNreacts with P450 8A1, also known as prostacyclin (PGI2) synthase, to inhibit enzymeactivity (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 werealso 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 (Mehlet 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

.O-N.0 O

"0=N

o" o

2N0,- + O2 + ^

Figure 4. Peroxynitrile reaction with heme proteins. (Adapted from Mehl et al., (1993) with permission.)


associated with oxidative phosphorylation. Superoxide reacts with NO at nearly thediffusion-controlled rate to produce peroxynitrile (PN). Oxidation of heme Fe^^ by PNgenerates a ferryl complex (Fe'^ = O). The ferryl complex is then reduced back to ferriciron by tyrosine, forming a phenoxy radical. In the final step, phenoxy radical reacts withthe NO2 radical formed during the initial oxidation of ferric iron by PN, resulting intyrosine nitration.

The identification and characterization of pre- and posttranslational regulatoryeffects of NO on P450 represent interesting regulatory mechanisms. Additionalexperiments will be required to reveal their physiological importance.

GLYCOSYLATION OF P450

Protein glycosylation is an important posttranslational modification involved in celladhesion, protein targeting, and protection from proteolytic attack. As illustrated inTable 4, a number of P450s have been identified as glycoproteins. The majority of theseenzymes have not been characterized for the modification, and little is known about theunderlying significance of P450 glycosylation. Two enzymes, P450 llAl and P45019A1, have been studied in an attempt to define the relationship between glycosylationand modulation of enzyme activity (Amameh et al., 1993; Ichikawa and Hiwatashi, 1982;Sethumadhavan et al., 1991). Activity of one enzyme is significantly affected, while theother is not.

P450 1 lAl demonstrates that glycosylation can modulate the catalytic activity of aP450. Ichikawa and Hiwatashi (1982) have reported that bovine adrenal P450 11 Al is aglyeoprotein. Treatment of the protein with neuramidase resulted in the inability of theprotein to be reduced by the reducing system. The authors concluded that the sugarmoiety of glycosylated P450 l lAl was essential for electron transport from re-duced redoxin.

P450 19A1 glycosylation has received attention in several reports and representsone of the better-characterized enzymes for this modification. Human placental P45019A1 is a glyeoprotein (Sethumadhavan et al., 1991). Its carbohydrate side chain can be

Table 4 Glycosylated P450 enzymes

P450

P450 1A2*P450 2B2P450 2B4P450 IIAI

P450 17A1P450 I9A1

P450 21AIP450 27B1**

Species

MouseRabbitRabbitBovine

PigHumanHumanHorseBovineBovine

Tissue

Liver {in vivo)Liver {in vivo)Liver {in vivo)Adrenal mitochondrial

cortex {in vivo)Testis {in vivo)Placenta {in vivo)COSl expressedTestis {in vivo)Adrenal cortex {in vivo)Liver {in vivo)

Reference

Negishi et al., 1981Haugen and Coon, 1976Haugen and Coon, 1976Ichikawa and Hiwatashi, 1982

Nakajin and Hall, 1981Shimozawa et al., 1993Amameh et al., 1993Moslemi et al., 1997Hiwatashi and Ichikawa, 1981Hiwatashi and Ichikawa, 1980

*Putatively glycosylated.**Bovine vitamin D3 25-hydroxylase sequence not yet reported.


selectively cleaved by endoglycosidase F and endoglycosidase H, which are known tohydrolyze N-glycans bound to asparagine (Asn) in a canonical N-X-S/T sequence. Thesite of glycosylation was determined as Asn'^, which is consistent with coreglycosylation (glycosylation occurring in the lumen of the ER) of an N-terminal portionof the protein (Amarneh et al., 1993; Shimozawa et al., 1993). The hydrophobic N-terminal region of P450s comprises a signal recognition particle-dependent signal thatdirects insertion of P450 into the membrane of the ER (Bar-Nun et al., 1980; Sakaguchiet al., 1984). Unfortunately, the significance of the core glycosylation has not beenclarified. In terms of an activity modulation, glycosylation of P450 19A1 does notsignificantly alter catalytic activity (Amarneh et al., 1993).

CONCLUSIONS

Cytochromes P450 comprise a family of important enzymes involved in themetabolism of a wide variety of endogenous and exogenous compounds. They areregulated transcriptionally, translationally, and posttranslationally by several differ-ent modifications.

Phosphorylation has been reported for a number of P450 enzymes (Table 1), withevidence of enzyme regulation by a variety of mechanisms, including modulation ofcatalytic activity, substrate binding, binding of redox partners, and substrate specificity.Several P450 enzymes are directed to the mitochondrial inner membrane in responseto phosphorylation.

Ubiquitination of P450 has been associated with the turnover of native anddamaged P450 by the 26S-proteasomal system. As demonstrated in Table 2, this pathwaymay be involved in regulating physiological levels of several enzymes.

In the last decade, progress has been made in defining the regulation of P450 bynitric oxide at the translational and posttranslational regulatory level (Morgan et al.,2001). The posttranslational mechanisms can be of a reversible nature by the interactionof NO with the P450, or via an irreversible mechanism involving tyrosine nitration byperoxynitrile (Table 3). Few mammalian enzymes have been associated with thesemechanisms, and this promises to be an interesting area of future discovery.

Thus far, glycosylation has been identified in eight P450s, with the first twoglycosylated enzymes identified over 25 years ago (Haugen and Coon, 1976). Despitethese discoveries, relatively little is known concerning the physiological role of thismodification. Characterizations of glycosylated P450s represent an area that deservesmore attention. Table 4 lists several enzymes that have been identified as glycoproteins.

Throughout this review, we considered the identification and physiologicalrelevance of P450 posttranslational modification. Despite all the modifications reportedin the literature, many of these have largely not been characterized. Characterization ofthese and other novel modifications represents an area that merits future attention. In thenext few years, we will hopefully see significant progress in this area of research.

ACKNOWLEDGMENTS

This review was made possible by the support of a CIHR Rx&D industriallypartnered studentship awarded to Mike Aguiar.


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