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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
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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-
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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-,
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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. Characterization of
these and other novel modifications represents an area that merits future attention. In the
next 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 industrially
partnered studentship awarded to Mike Aguiar.
REGULATION OF CYTOCHROME P450
397
REFERENCES
Addya, S., Anandatheerthavarada, H. K., Biswas, G., Bhagwat, S. V., Mullick, J., Avadhani, N. G.
(1997). Targeting of NH2-terminal-processed microsomal protein to mitochondria: a novel
pathway for the biogenesis of hepatic mitochondrial P450MT2. J. Cell. Biol. 139(3):589599.
Amarneh, B., Corbin, C. J., Peterson, J. A., Simpson, E. R., Graham-Lorence, S. (1993). Functional
domains of human aromatase cytochrome P450 characterized by linear alignment and sitedirected mutagenesis. Moi. Endocrinol. 7(12): 1617-1624.
Anandatheerthavarada, H. K., Addya, S., Dwivedi, R. S., Biswas, G., Mullick, J., Avadhani, N. G.
(1997). Localization of multiple forms of inducible cytochromes P450 in rat liver
mitochondria: immunological characteristics and patterns of xenobiotic substrate metabolism. Arch. Biochem. Biophys. 339(1): 136-150.
Anandatheerthavarada, H. K., Biswas, G., Mullick, J., Sepuri, N. B. V., Otvos, L., Pain, D.,
Avadhani, N. G. (1999). Dual targeting of cytochrome P4502B1 to endoplasmic reticulum
and mitochondria involves a novel signal activation by cyclic AMP-dependent phosphorylation at Serl28. EMBO J. 18(20):5494-5504.
Balthazart, J., Baillien, M., Ball, G. F. (2001a). Phosphorylation processes mediate rapid changes of
brain aromatase activity. J. Steroid Biochem. Moi. Biol. 79(1-5):261-277.
Balthazart, J., Baillien, M., Ball, G. F. (2001b). Rapid and reversible inhibition of brain aromatase
activity. J. Neuroendocrinol. 13(l):63-73.
Balthazart, J., Baillien, M., Charlier, T. D., Ball, G. F. (2003). Calcium dependent phosphorylation
processes control brain aromatase in quail. Eur. J. Neurosci. 17(8): 1591-1606.
Banerjee, A., Kocarek, T. A., Novak, R. F. (2000). Identification of a ubiquitination-target/
substrate-interaction domain of cytochrome P-450 (GYP) 2E1. Drug Metab. Dispos.
28(2): 118-124.
Bar-Nun, S., Kreibich, G., Adesnik, M., Alterman, J., Negishi, M., Sabatini, D. D. (1980). Synthesis
and insertion of cytochrome P-450 into endoplasmic reticulum membranes. PNAS
77(2):965-969.
Bellino, F. L., Holben, L. (1989). Placental estrogen synthetase (aromatase): evidence for
phosphatase-dependent inactivation. Biochem. Biophys. Res. Commun. 162(l):498-504.
Berglund, L., Bjorkhem, I., Angelin, B., Einarsson, K. (1986). Evidence against in vitro modulation
of rat liver cholesterol 7 a-hydroxylase activity by phosphorylation-dephosphorylation:
comparison with hydroxymethylglutaryl CoA reductase. Acta Chem. Scand. B 40(6):457461.
Bhagwat, S. V., Mullick, J., Raza, H., Avadhani, N. G. (1999a). Constitutive and inducible
cytochromes P450 in rat lung mitochondria: xenobiotic induction, relative abundance, and
catalytic properties. Toxicoi. Appl. Pharmacol. 156(3):23l-240.
Bhagwat, S. V., Biswas, G., Anandatheerthavarada, H. K., Addya, S., Pandak, W., Avadhani, N. G.
(1999b). Dual targeting property of the N-terminal signal sequence of P4501A1. Targeting of
heterologous proteins to endoplasmic reticulum and mitochondria. J. Biol. Chem.
274(34):24014-24022.
Biason-Lauber, A., Zachmann, M., Schoenle, E. J. (2000). Effect of leptin on CYP17 enzymatic
activities in human adrenal cells: new insight in the onset of adrenarche. Endocrinology
141(4): 1446-1454.
Burgos-Trinidad, M., Brown, A. J., DeLuca, H. F. (1986). Solubilization and reconstitution of
chick renal mitochondrial 25-hydroxyvitamin D3 24-hydroxylase. Biochemistry 25(9):26922696.
Caron, M. G., Goldstein, S., Savard, K., Marsh, J. M. (1975). Protein kinase stimulation of a
reconstituted cholesterol side chain cleavage enzyme system in the bovine corpus luteum.
J. Biol. Chem. 250(13):5137-5143.
398
M. AGUIAR ET AL.
Cohen, P. (2002). Protein kinases—the major drug targets of the twenty-first century? Nat. Rev.
l(4):309-315.
Correia, M. A. (2003). Hepatic cytochrome P450 degradation: mechanistic diversity of the cellular
sanitation brigade. Drug Metab. Rev. 35(2,3): tO7-143.
Correia, M. A., Davoll, S. H., Wrighton, S. A., Thomas, P. E. (1992). Degradation of rat liver
cytochromes P450 3A after their inactivation by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-l,
4-dihydropyridine: characterization of the proteolytic system. Arch. Biochem. Biophys.
297(2)1228-238.
Cross, E., Roselli, C. E. (1999). 17|3-estradiol rapidly facilitates chemoinvestigation and mounting
in castrated male rats. Am. J. PhysioL, Regul. Integr. Comp. Physiol 276(5 pt. 2):R1346R1350.
Cutler, G. B., Glenn, M. Jr., Bush, M., Hodgen, G. D., Graham, C. E., Loriaux, D. L. (1978).
Adrenarche: a survey of rodents, domestic animals, and primates. Endocrinology
103(6):2112-2118.
Daiber, A., Schoneich, V., Schmidt, P., Jung, C, Ullrich, V. (2000a). Autocatalytic nitration of
P450CAM by peroxynitrite. J. Inorg. Biochem. 81(3):213-220.
Daiber, A., Herold, S., Schoneich, C , Namgaladze, D., Peterson, J. A., Ullrich, V. (2000b).
Nitration and inactivation of cytochrome P450BM-3 by peroxynitrite: stopped-flow
measurements prove ferryl intermediates. Eur. J. Biochem. 267(23):6729-6739.
Defaye, G., Monnier, N., Guidicelli, C, Chambaz, E. M. (1982). Phosphorylation of purified
mitochondrial cytochromes P-450 (cholesterol desmolase and 11 P-hydroxylase) from
bovine adrenal cortex. Mol. Cell. Endocrinol. 27(2): 157-168.
Diven, W. E., Sweeney, J., Warty, V., Sanghvi, A. (1988). Regulation of bile acid synthesis.
Isolation and characterization of microsomal phosphatases. Biochem. Biophys. Res.
Commun. 155(1):7-13.
Drewett, J. G., Adams-Hays, R. L., Ho, B. Y.. Hegge, D. J. (2002). Nitric oxide potently inhibits the
rate-limiting enzymatic step in steroidogenesis. Mol. Cell. Endocrinol. 194(1,2):39-50.
Einarsson, K., Angelin, B., Ewerth, S., Nilsell, K., Bjorkhem, I. (1986). Bile acid synthesis in man:
assay of hepatic microsomal cholesterol 7 a-hydroxylase activity by isotope dilution-mass
spectrometry. J. Lipid. Res. 27(l):82-88.
Eliasson, E., Mkrtchian, S., Halpert, J. R., Ingelman-Sundberg, M. (1994). Substrate-regulated,
cAMP-dependent phosphorylation, denaturation, and degradation of glucocorticoid-inducible rat liver cytochrome P450 3A1. J. Biol. Chem. 269(28): 18378-18383.
Epstein, P. M., Curti, M., Jansson, I., Huang, C. K., Schenkman, J. B. (1989). Phosphorylation of
cytochrome P450: regulation by cytochrome b5. Arch. Biochem. Biophys. 271(2):424432.
Estabrook, R. W., Baron, J., Peterson, J., Ishimura, Y. (1972). Oxygenated cytochrome P-450 as an
intermediate in hydroxylation reactions. Biochem. Soc. Symp. 34:159-185.
Evans, R. M. (1988). The steroid and thyroid hormone receptor superfamily. Science
240(4854):889-895.
Freeman, J. E., Wolf, C. R. (1994). Evidence against a role for serine 129 in determining murine
cytochrome P450 Cyp2e-1 protein levels. Biochemistry 33(47): 13963-13966.
Ghazarian, J. G., Yanda, D. M. (1985). Inhibition of 25-hydroxyvitamin D 1 a-hydroxylase by renal
mitochondrial protein kinase-catalyzed phosphorylation. Biochem. Biophys. Res. Commun.
132(3): 1095-1102.
Gilmore, R., Walter, P., Blobel, G. (1982). Protein translocation across the endoplasmic reticulum.
II. Isolation and characterization of the signal recognition particle receptor. J. Cell. Biol. 95
(2 pt. l):470-477.
Gray, R. W., Ghazarian, J. G. (1989). Solubilization and reconstitution of kidney 25hydroxyvitamin D3 1 a- and 24-hydroxylases from vitamin D-replete pigs. Biochem. J.
259(2):561-568.
REGULATION OF CYTOCHROME P450
399
Guengerich, F. P. (2001). Common and uncommon cytochrome P450 reactions related to
metabolism and chemical toxicity. Chem. Res. Toxicoi. 14(6);611-650.
Han, K. K., Martinage, A. (1992). Post-translational chemical modification(s) of proteins. Int. J.
Biochem. 24(1): 19-28.
Haugen, D. A., Coon, M. J. (1976). Properties of electrophoretically homogeneous phenobarbitalinducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.
/ Biol. Chem. 251(24):7929-7939.
Hendy, G. N. (1997). Calcium-regulating hormones. In; Conn, P. M., Melmed, S., eds. Endocrinology: Basic and Clinical Principles. Totowa, NJ: Humana Press, pp. 307-323.
Hink, U., Oelze, M., Kolb, P., Bachschmid, M., Zou, M. H., Daiber, A., Mollnau, H., August, M.,
Baldus, S., Tsilimingas, N., Walter, U., Ullrich, V., Munzel, T. (2003). Role for peroxynitrite
in the inhibition of prostacyclin synthase in nitrate tolerance. J. Am. Coll. Cardiol.
42(10): 1826-1834.
Hiwatashi, A., Ichikawa, Y. (1980). Purification and organ-specific properties of cholecalciferol 25hydroxylase system: cytochrome P-450D25-linked mixed function oxidase system. Biochem.
Biophys. Res. Commun. 97(4): 1443-1449.
Hiwatashi, A., Ichikawa, Y. (1981). Purification and reconstitution of the steroid 21-hydroxylase
system (cytochrome P-450-linked mixed function oxidase system) of bovine adrenocortical
microsomes. Biochim. Biophys. Acta 664(l):33-48.
HIavica, P., Lewis, D. F. V. (2001). Allosteric phenomena in cytochrome P450-catalyzed
monooxygenations. Eur. J. Biochem. 268(18):4817-4832.
Holsztynska, E. J., Waxman, D. J. (1987). Cytochrome P-450 cholesterol 7 a-hydroxylase:
inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and
phosphatase inhibitors. Arch. Biochem. Biophys. 256(2):543-559.
Ichikawa, Y., Hiwatashi, A. (1982). The role of the sugar regions of components of the cytochrome
P-450-linked mixed-function oxidase (monooxygenase) system of bovine adrenocortical
mitochondria. Biochim. Biophys. Acta 705(l):82-91.
Jansson, I., Curti, M., Epstein, P. M., Peterson, J. A., Schenkman, J. B. (1990). Relationship
between phosphorylation and cytochrome P450 destruction. Arch. Biochem. Biophys.
283(2):285-292.
Khatsenko, O. G., Gross, S. S., Rifkind, A. B., Vane, J. R. (1993). Nitric oxide is a mediator of the
decrease in cytochrome P450-dependent metabolism caused by immunostimulants. PNAS
9O(23):11147-1I151.
Koch, J. A., Waxman, D. J. (1989). Posttranslational modification of hepatic cytochrome P-450.
Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIBI) and PB-5 (I1B2) in
isolated rat hepatocytes and in vivo. Biochemistry 28(8):3145-3152.
Korsmeyer, K. K., Davoll, S., Figueiredo-Pereira, M. E., Correia, M. A. (1999). Proteolytic
degradation of heme-modified hepatic cytochromes P450: a role for phosphorylation,
ubiquitination, and the 26S proteasome? Arch. Biochem. Biophys. 365(l):3l-44.
Korth-Schutz, S., Levine, L. S., New, M. I. (1976). Dehydroepiandrosterone sulfate (DS) levels, a
rapid test for abnormal adrenal androgen secretion. / Clin. Endocrinol. Metab. 42(6): 10051013.
Liaudet, L., Soriano, F. G., Szabo, C. (2000). Biology of nitric oxide signaling. Crit. Care. Med.
28(4):N37-N52.
Lin, H. L., Kent, U. M., Zhang, H., Waskell, L., Hollenberg, P. F. (2003). Mutation of tyrosine 190
to alanine eliminates the inactivation of cytochrome P450 2B1 by peroxynitrite. Chem. Res.
Toxicoi. 16(2): 129-136.
Lohr, J. B., Kuhn-Velten, W. N. (1997). Protein phosphorylation changes ligand-binding efficiency
of cytochrome P450cl7 (CYP 17) and accelerates its proteolytic degradation: putative
relevance for hormonal regulation of CYP17 activity. Biochem. Biophys. Res. Commun.
231(2):403-408.
400
M. AGUIAR ET AL.
Lohr, J. W., Willsky, G. R., Acara, M. A. (1998). Renal drug metabolism. Pharmacol. Rev.
50(l);107-142.
Mandel, M. L., Moorthy, B., Ghazarian, J. G. (1990). Reciprocal post-translational regulation of
renal 1 a- and 24-hydroxylases of 25-hydroxyvitamin D3 by phosphorylation of ferredoxin.
mRNA-directed cell-free synthesis and immunoisolation of ferredoxin. Biochem. J.
266(2):385-392.
Masaki, R., Yamamoto, A., Tashiro, Y. (1987). Cytochrome P-450 and NADPH-cytochrome P-450
reductase are degraded in the autolysosomes in rat liver. J. Cell. Biol. 104(5):1207-1215.
Mehl, M., Daiber, A., Herold, S., Shoun, H., Ullrich, V. (1999). Peroxynitdte reaction with heme
proteins. Nitric Oxide 3(2):142-152.
Menez, J. F., Machu, T. K., Song, B. J., Browning, M. D., Deitrich, R. A. (1993). Phosphorylation
of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase
C and cAMP dependent protein kinase. Alcohol Alcohol. 28(4):445-451.
Minamiyama, Y., Takemura, S., Imaoka, S., Funae, Y., Tanimoto, Y., Inoue, M. (1997).
Irreversible inhibition of cytochrome P450 by nitric oxide. J. Pharmacol. Exp. Ther.
283(3): 1479-1485.
Monier, S., Van Luc, P., Kreibich, G., Sabatini, D. D., Adesnik, M. (1988). Signals for the
incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane.
J. Cell. Biol. 107(2):457-470.
Monnier, N., Defaye, G., Chambaz, E. M. (1987). Phosphorylation of bovine adrenodoxin.
Structural study and enzymatic activity. Eur. J. Biochem. 169(1):147-153.
Morgan, E. T. (1997). Regulation of cytochromes P450 during inflammation and infection. Drug
Metab. Rev. 29(3): 1129-1188.
Morgan, E. T. (2001). Regulation of cytochrome P450 by inflammatory mediators: why and how?
Drug Metab. Dispos. 29(4):207-212.
Morgan, E. T., Ullrich, V., Daiber, A., Schmidt, P., Takaya, N., Shoun, H., McGiff, J. C , Oyekan,
A., Hanke, C. J., Campbell, W. B., Park, C , Kang, J., Yi, H., Cha, Y., Mansuy, D., Boucher,
J. (2001). Cytochromes P450 and flavin monooxygenases. Targets and sources of nitric
oxide. Drug Metab. Dispos. 29(11): 1366-1376.
Moslemi, S., Vibet, A., Papadopoulos, V., Camoin, L., Silberzahn, P., Gaillard, J. L. (1997).
Purification and characterization of equine testicular cytochrome P-450 aromatase:
comparison with the human enzyme. Comp. Biochem. PhysioL, B Biochem. Mol. Biol.
118B(l):217-227.
Murray, B. P., Correia, M. A. (2001). Ubiquitin-dependent 26S proteasomal pathway: a role in the
degradation of native human liver CYP3A4 expressed in Saccharomyces cerevisiaei Arch.
Biochem. Biophys. 393(l):106-l 16.
Murray, B. P., Zgoda, V. G., Correia, M. A. (2002). Native CYP2C11: heterologous expression in
Saccharomyces cerevisiae reveals a role for vacuolar proteases rather than the proteasome
system in the degradation of this endoplasmic reticulum. Protein Mol. Pharmacol.
61(5):1146-1153.
Myant, N. B., Mitropoulos, K. A. (1977). Cholesterol 7 a-hydroxylase. / Lipid Res. 18(2): 135153.
Nakajin, S., Hall, P. F. (1981). Microsomal cytochrome P-450 from neonatal pig testis. Purification
and properties of A C21 steroid side-chain cleavage system (17 a-hydroxylase-C 17,20
lyase). J. Biol. Chem. 256(8):3871-3876.
Nakajin, S., Hall, P. F., Onoda, M. (1981). Testicular microsomal cytochrome P-450 for C21
steroid side chain cleavage. Spectral and binding studies. / Biol. Chem. 256(12):61346139.
Narbaitz, R., Stumpf, W. E., Sar, M. (1981). The role of autoradiographic and immunocytochemical
techniques in the clarification of sites of metabolism and action of vitamin D. J. Histochem.
Cytochem. 29(l):91-100.
REGULATION OF CYTOCHROME P450
401
Negishi, M., Jensen, N. M., Garcia, G. S., Nebert, D. W. (1981). Structural gene products of the
murine Ah complex. Differences in ontogenesis and glucosamine incorporation between
liver microsomal cytochromes PI-450 and P-448 induced by polycyclic aromatic
compounds. Eur. J. Biochem. 115(3):585-594.
Nelson, D. R., Koymans, L., Kamataki, T., Stegeman, J. J., Feyereisen, R., Waxman, D. J.,
Waterman, M. R., Gotoh, C , Coon, M. J., Estabrook, R. W., Gunsalus, I. C , Nebert, D. W.
(1996). P450 superfamily: update on new sequences, gene mapping, accession numbers and
nomenclature. Pharmacogenetics 6( 1): 1 -42.
Nelson, D. R., Zeldin, D. C , Hoffman, S. M., Maltais, L. J., Wain, H. M., Nebert, D. W. (2004).
Comparison of cytochrome P450 (CYP) genes from the mouse and human genomes,
including nomenclature recommendations for genes, pseudogenes and alternative-splice
variants. Pharmacogenetics 14(1):1-18.
Neupert, W. (1997). Protein import into mitochondria. Annu. Rev. Biochem. 66:863-917.
Nguyen, L. B., Shefer, S., Salen, G., Chiang, J. Y., Patel, M. (1996). Cholesterol 7ot-hydroxylase
activities from human and rat liver are modulated in vitro posttranslationally by
phosphorylation/dephosphorylation. Hepatology 24(6): 1468-1474.
Oesch-Bartlomowicz, B., Oesch, F. (1990). Phosphorylation of cytochrome P450 isoenzymes in
intact hepatocytes and its importance for their function in metabolic processes. Arch.
Toxicol. 64(4):257-261.
Oesch-Bartlomowicz, B., Padma, P. R., Becker, R., Richter, B., Hengstler, J. G., Freeman, J.
E., Wolf, C. R., Oesch, F. (1998). Differential modulation of CYP2E1 activity by cAMPdependent protein kinase upon Serl29 replacement. Exp. Cell. Res. 242(l):294-302.
Oesch-Bartlomowicz, B., Richter, B., Becker, R., Vogel, S., Padma, P. R., Hengstler, J. G.,
Oesch, F. (2001). cAMP-dependent phosphorylation of CYP2B1 as a functional switch for
cyclophosphamide activation and its hormonal control in vitro and in vivo. Int. J. Cancer
94(5):733-742.
Orentreich, N., Brind, J. L., Rizer, R. L., Vogelman, J. H. (1984). Age changes and sex differences
in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J. Clin.
Endocrinol. Metab. 59(3):551-555.
Osawa, Y., Davila, J. C , Nakatsuka, M., Meyer, C. A., Darbyshire, J. F. (1995). Inhibition of P450
cytochromes by reactive intermediates. Drug Metab. Rev. 27(l,2):61-72.
Parker, L. N., Odell, W. D. (1980). Control of adrenal androgen secretion. Endocr. Rev. 1(4):392410.
Parker, K. L., Schimmer, B. P. (1993). Transcriptional regulation of the adrenal steroidogenic
enzymes. Trends Endocrinol. Metab. 4(2):46-50.
Parker, K. L., Schimmer, B. P. (1996). The roles of the nuclear receptor steroidogenic factor 1 in
endocrine differentiation and development. Trends Endocrinol. Metab. 7(6):203-207.
Parker, K. L., Schimmer, B. P. (1997). Steroidogenic factor 1: a key determinant of endocrine
development and function. Endocr. Rev. 18(3):361-377.
Porter, T. D., Coon, M. J. (1991). Cytochrome P-450. Multiplicity of isoforms, substrates, and
catalytic and regulatory mechanisms. / Biol. Chem. 266(21): 13469-13472.
Poulos, T. L. (2003). Cytochrome P450 flexibility. PNAS 100(23):13121-13122.
Pyerin, W., Taniguchi, H. (1989). Phosphorylation of hepatic phenobarbital-inducible cytochrome
P-450. EMBO J. 8(1O):3OO3-3OIO.
Pyerin, W., Taniguchi, H., Horn, F., Oesch, F., Amelizad, Z., Friedberg, T., Wolf, C. R. (1987).
Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing
enzymes. Biochem. Biophys. Res. Commun. 142(3):885-892.
Roberts, B. J. (1997). Evidence of proteasome-mediated cytochrome P-450 degradation. J. Biol.
Chem. 272(15):9771-9778.
Roberts, B. J., Song, B., Soh, Y., Park, S. S., Shoaf, S. E. (1995). Ethanol induces CYP2E1 by
protein stabilization. J. Biol. Chem. 270(50):29632-29635.
402
M. AGUIAR ET AL.
Roberts, E. S., Lin, H., Crowley, J. R., Vuletich, J. L., Osawa, Y., Hollenberg, P. F. (1998).
Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of
cytochrome P450 2B1. Chem. Res. Toxicol. 11(9): 1067-1074.
Robin, M., Anandatheerthavarada, H. K., Fang, J., Cudic, M., Otvos, L., Avadhani, N. G.
(2001). Mitochondrial targeted cytochrome P450 2E1 (P450 MT5) contains an intact N
terminus and requires mitochondrial specific electron transfer proteins for activity. J.
Biol. Chem. 276(27):24680-24689.
Robin, M., Anandatheerthavarada, H. K., Biswas, G., Sepuri, N. B. V., Gordon, D. M., Pain, D.,
Avadhani, N. G. (2002). Bimodal targeting of microsomal CYP2E1 to mitochondria through
activation of an N-terminal chimeric signal by cAMP-mediated phosphorylation. J. Biol.
Chem. 277(43):40583-40593.
Ronis, M. J., Johansson, I., Hultenby, K., Lagercrantz, J., Glaumann, H., Ingelman-Sundberg, M.
(1991). Acetone-regulated synthesis and degradation of cytochrome P4502E1 and
cytochrome P4502B1 in rat liver. Eur. J. Biochem. 198(2):383-389.
Sakaguchi, M., Mihara, K., Sato, R. (1984). Signal recognition particle is required for
cotranslational insertion of cytochrome P-450 into microsomal membranes. PNAS
81(ll):3361-3364.
Sakaguchi, M., Mihara, K., Sato, R. (1987). A short amino-terminal segment of microsomal
cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence.
EMBOJ. 6(8):2425-2431.
Schmidt, P., Youhnovski, N., Daiber, A., Balan, A., Arsic, M., Bachschmid, M., Przybylski,
M., Ullrich, V. (2003). Specific nitration at tyrosine 430 revealed by high resolution mass
spectrometry as basis for redox regulation of bovine prostacyclin synthase. J. Biol. Chem.
278(15):12813-12819.
Schoch, G. A., Yano, J. K., Wester, M. R., Griffin, K. J., Stout, C. D., Johnson, E. F. (2004).
Structure of human microsomal cytochrome P450 2C8. Evidence for a peripheral fatty acid
binding site. J. Biol. Chem. 279(10):9497-9503.
Scott, E. E., He, Y. A., Wester, M. R., White, M. A., Chin, C. C , Halpert, J. R., Johnson, E. F.,
Stout, C. D. (2003). An open conformation of mammalian cytochrome P450 2B4 at 1.6-A
resolution. PNAS 100(23):13196-13201.
Scott, E. E., White, M. A., He, Y. A., Johnson, E. F., Stout, C. D., Halpert, J. R. (2004). Structure of
mammalian cytochrome P450 2B4 complexed with 4-(4-chlorophenyl)imidazole at 1.9 A
resolution: insight into the range of P450 conformations and coordination of redox partner
binding. / Biol. Chem. 279(26):27294-27301.
Sethumadhavan, K., Bellino, F. L., Thotakura, N. R. (1991). Estrogen synthetase (aromatase). The
cytochrome P-450 component of the human placenta! enzyme is a glycoprotein. Mol. Cell.
Endocrinol. 78(l,2):25-32.
Shimozawa, 0., Sakaguchi, M., Ogawa, H., Harada, N., Mihara, K., Omura, T. (1993). Core
glycosylation of cytochrome P-450 (arom). Evidence for localization of N terminus of
microsomal cytochrome P-450 in the lumen. J. Biol. Chem. 268(28):21399-21402.
Siegel, N., Wongsurawat, N., Armbrecht, H. J. (1986). Parathyroid hormone stimulates
dephosphorylation of the renoredoxin component of the 25-hydroxyvitamin D3-1 ahydroxylase from rat renal cortex. J. Biol. Chem. 261(36): 16998-17003.
Sonoda, Y., Amano, C, Endo, M., Sato, Y., Sekigawa, Y., Fukuhara, M. (1995). Lanosterol 14-ademethylase (cytochrome P-45014DM): modulation of its enzyme activity by cholesterol
feeding. Biol. Pharm. Bull. 18(7): 1009-1011.
Stadler, J., Trockfeld, J., Schmalix, W. A., Brill, T., Siewert, J. R., Greim, H., Doehmer, J. (1994).
Inhibition of cytochromes P4501A by nitric oxide. PNAS 91(9):3559-3563.
Stocco, D. M. (2000). The role of the StAR protein in steroidogenesis: challenges for the future.
J. Endocrinol. 164(3):247-253.
Szczesna-Skorupa, E., Kemper, B. (1989). NH2-terminal substitutions of basic amino acids induce
REGULATION OF CYTOCHROME P450
403
translocation across the microsomal membrane and glycosylation of rabbit cytochrome
P450tIC2. / Cell. Biol. 108(4):1237-1243.
Szczesna-Skorupa, E., Browne, N., Mead, D., Kemper, B. (1988). Positive charges at the NH2
terminus convert the membrane-anchor signal peptide of cytochrome P-450 to a secretory
signal peptide. PNAS 85(3):738-742.
Tang, P. M., Chiang, J. Y. (1986). Modulation of reconstituted cholesterol 7 a-hydroxylase by
phosphatase and protein kinase. Biochem. Biophys. Res. Commun. 134(2):797-802.
Tiemey, D. J., Haas, A. L., Koop, D. R. (1992). Degradation of cytochrome P450 2E1: selective
loss after labilization of the enzyme. Arch. Biochem. Biophys. 293(1):9-16.
Ullrich, V., Bachschmid, M. (2000). Superoxide as a messenger of endothelial function. Biochem.
Biophys. Res. Commun. 278(1): 1-8.
Vilgrain, L, Defaye, G., Chambaz, E. M. (1984). Adrenocortical cytochrome P-450 responsible for
cholesterol side chain cleavage (P-450scc) is phosphorylated by the calcium-activated,
phospholipid-sensitive protein kinase (protein kinase C). Biochem. Biophys. Res. Commun.
125(2):554-561.
Wang, H. F., Figueiredo Pereira, M. E., Correia, M. A. (1999). Cytochrome P450 3A degradation in
isolated rat hepatocytes: 26S proteasome inhibitors as probes. Arch. Biochem. Biophys.
365(l):45-53.
Wang, X., Medzihradszky, K. F., Maltby, D., Correia, M. A. (2001). Phosphorylation of native and
heme-modified CYP3A4 by protein kinase C: a mass spectrometric characterization of the
phosphorylated peptides. Biochemistry 40(38): 11318- 11326.
Weissman, A. M. (2001). Ubiquitin and proteasomes: themes and variations on ubiquitylation. Nat.
Rev. Mol. Cell. Biol. 2(3): 169-178.
Wester, M. R., Johnson, E. F., Marques-Soares, C , Dansette, P. M., Mansuy, D., Stout, C.
D. (2003a). Structure of a substrate complex of mammalian cytochrome P450 2C5 at 2.3 A
resolution: evidence for multiple substrate binding modes. Biochemistry 42(21):63706379.
Wester, M. R., Johnson, E. F., Marques-Soares, C, Dijols, S., Dansette, P. M., Mansuy, D.,
Stout, C. D. (2003b). Structure of mammalian cytochrome P450 2C5 complexed with
diclofenac at 2.1 A resolution: evidence for an induced fit model of substrate binding.
Biochemistry 42(31):9335-9345.
Wester, M. R., Yano, J. K., Schoch, G. A., Yang, C., Griffin, K. J., Stout, C. D., Johnson, E. F.
(2004). The structure of human microsomal cytochrome P450 2C9 complexed with
fiurbiprofen at 2.0 A resolution. J. Biol. Chem. 279(34):35630-35637.
Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., McRee, D. E. (2000). Mammalian
microsomal cytochrome P450 monooxygenase: structural adaptations for membrane binding
and functional diversity. Mol. Cell. 5(1): 121-131.
Williams, P. A., Cosme, J., Ward, A., Angove, H. C, Matak Vinkovic, D., Jhoti, H. (2003).
Crystal structure of human cytochrome P450 2C9 with bound warfarin. Nature 424(6947):
464-468.
Williams, P. A., Cosme, J., Vinkovic, D. M., Ward, A., Angove, H. C , Day, P. J., Vonrhein, C,
Tickle, I. J., Jhoti, H. (2004). Crystal structures of human cytochrome P450 3A4 bound to
metyrapone and progesterone. Science 305(5684):683-686.
Wink, D. A., Osawa, Y., Darbyshire, J. F., Jones, C. R., Eshenaur, S. C , Nims, R. W. (1993).
Inhibition of cytochromes P450 by nitric oxide and a nitric oxide-releasing agent. Arch.
Biochem. Biophys. 3OO(I):115-123.
Yamazaki, H., Shimada, T., Martin, M. V., Guengerich, F. P. (2001). Stimulation of cytochrome
P450 reactions by apo-cytochrome ^5. Evidence against transfer of heme from cytochrome P450 3A4 to apo-cytochrome b^ or heme oxygenase. / Biol. Chem. 276(33):3088530891.
Yang, M. X., Cederbaum, A. 1. (1997). Characterization of cytochrome P4502EI turnover in
404
M. AGUIAR ET AL.
transfected HepG2 cells expressing human CYP2Et. Arch. Biochem. Biophys. 341(1):2533.
Yano, J. K., Griffin, K., Stout, D. C , Johnson, E. F. (2004a). The crystal structure of human
microsomal cytochrome P450 2A6 (CYP2A6): the principle nicotine oxidase, In: Abstracts,
Microsomes and Drug Oxidations, Mainz, Germany, July 4 - 9 , P4-CH-lt.
Yano, J. K., Wester, M. R., Schoch, G. A., Griffin, K. J., Stout, C. D., Jotinson, E. F. (2004b). The
structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography
to 2.05' resolution. J. Biol. Chem. 279(37):38091-38094.
Yue, W., Santen, R. J., Wang, J. P., Hamilton, C. J., Demers, L. M. (1999). Aromatase within the
breast. Endocr. Relat. Cancer 6(2):157-164.
Yue, W., Wang, J. P., Conaway, M. R., Li, Y., Santen, R. J. (2003). Adaptive hypersensitivity
following long-term estrogen deprivation: involvement of multiple signal pathways. J.
Steroid Biochem. Mol. Biol. 86(3-5):265-274.
Zhang, L., Rodriguez, H., Ohno, S., Miller, W. L. (1995). Serine phosphorylation of human
P450cl7 increases t7,20-lyase activity: implications for adrenarche and the polycystic ovary
syndrome. PNAS 92(23): 10619-10623.
Zhukov, A., Werlinder, V., Ingelman-Sundberg, M. (1993). Purification and characterization of two
membrane bound serine proteinases from rat liver microsomes active in degradation of
cytochrome P450. Biochem. Biophys. Res. Commun. t97(t):22t-228.
Zou, M., Martin, C, Ullrich, V. (1997). Tyrosine nitration as a mechanism of selective inactivation
of prostacyclin synthase by peroxynitrite. Biol. Chem. 378(7):707-713.