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Department of Pediatrics (D.H.G., R.J.A., W.L.M.) Department of Internal Medicine (R.J.A.) and The Metabolic Research Unit (W.L.M.) University of California San Francisco, California 94143-0978
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase and 17,20-lyase activities and hence is a key enzyme
in the production of human glucocorticoids and sex steroids. These two
activities are catalyzed in a single substrate-binding site but are
regulated independently in human physiology. We have recently shown
that cytochrome b5 facilitates 17,20-lyase
activity by allosterically promoting the interaction of P450c17 with
P450 oxidoreductase (OR) and that the human P450c17 mutations, R347H
and R358Q, selectively destroy 17,20-lyase activity while sparing
17-hydroxylase activity. We transfected COS-1 cells with vectors for
these P450c17 mutants and found that an excess of OR and
b5 restored a small amount of 17,20-lyase
activity, suggesting the mutations interfere with electron donation. To
determine whether these mutations selectively interfere with the
interaction of P450c17 and its electron-donating system, we expressed
each P450c17 mutant in yeast with or without OR,
b5, or both, and measured enzyme kinetics in
yeast microsomes using pregnenolone and 17-hydroxypregnenolone as
substrates. The apparent Michaelis-Menten (Km)
values for the R347H mutant with and without coexpressed OR were 0.2
and 0.6 µM, respectively, and for the R358Q
mutant with and without OR they were 0.3 and 0.4
µM, respectively; these values did not differ
significantly from the wild-type values of 0.4 and 0.8
µM with and without OR, respectively.
Furthermore, coincubation with 17-hydroxypregnenolone showed a
competitive mechanism for interference of catalysis. The similar
kinetics and the competitive inhibition prove that the mutations did
not affect the active site. Coexpression of the mutants with OR yielded
insignificant 17,20-lyase activity, but addition of a 30:1 molar excess
cytochrome b5 to these microsomes restored
partial 17,20-lyase activity, with the R358Q mutant achieving twice the
activity of the R347H mutant. These data indicate that both mutations
selectively interfere with 17,20-lyase activity by altering the
interaction of P450c17 with OR, thus proving that the lyase activity
was disrupted by interfering with electron transfer. Furthermore, the
data offer the first evidence that R347 is a crucial component of the
site at which b5 interacts with the
P450c17·OR complex to promote electron transfer. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylation and 17,20-bond scission
(17,20-lyase) reactions (2, 3, 4). P450c17 also has a modest degree of
16-hydroxylase activity (5, 6, 7). There is only a single P450c17
protein moiety in both the adrenal and the gonad, encoded by a single
gene (8) that expresses a single species of mRNA (9), which catalyzes
these three steroidal transformations on a single active site. Sex
steroid production requires both 17-hydroxylase and 17,20-lyase
activities, and deficiencies in one or both actions of P450c17 leads to
ambiguity in male genital formation and lack of progression into
puberty in females (4).
More than 125 patients have been described with clinically
apparent defects in P450c17, most causing complete loss of all P450c17
activity (10). Of the 23 mutant alleles studied at the molecular level
before 1997, all caused defects that affected both the
17-hydroxylase and 17,20-lyase activities of P450c17 in equal
proportion when expressed in transfected cells (11). Seventeen patients
with clinical and hormonal profiles consistent with an isolated
deficiency of 17,20-lyase activity have been described (11) since the
initial description of apparent isolated 17,20-lyase deficiency (12).
However, subsequent molecular genetic and clinical studies showed that
one of the initial patients had both 17-hydroxylase and 17,20-lyase
deficiency (13, 14); thus the existence of patients with isolated
17,20-lyase deficiency was in doubt until recently. We then reported
two new cases of 17,20-lyase deficiency in whom we identified the
P450c17 mutations R347H and R358Q (15). These two mutations
selectively ablated more than 95% of 17,20-lyase activity while
retaining about 65% of 17-hydroxylase activity (15) and
correspond to two site-directed mutants in rat P450c17 that also
preferentially impair 17,20-lyase activity (16). These mutations lie in
a region that appears to be critical for binding P450 oxidoreductase
(OR) (4, 15, 17, 18), which donates electrons to all microsomal
cytochrome P450 enzymes. Thus, these mutations suggested a novel
mechanism for steroidogenic P450 enzyme defects, in which substrate
binding remains intact, while electron coupling and/or transfer within
the P450-electron donor complex is disrupted (15). Initial kinetic
studies in transfected COS-1 cells suggested that
17-hydroxypregnenolone could inhibit the 17-hydroxylation of
pregnenolone by the R347H and R358Q mutants, but this experiment could
not determine whether this inhibition was competitive. A quantitative
demonstration that the mode of inhibition is competitive is needed to
prove that the mutations do not affect the active site. Furthermore,
the endogenous COS-1 cell expression of cytochrome b5 also
precluded studies of the potential mechanism by which these mutations
might alter the interaction of P450c17 with b5.
To investigate rigorously the mechanism by which the R347H and
R358Q mutations selectively disrupt 17,20-lyase activity, it was first
necessary to describe the mechanisms and regulation of 17,20-lyase
activity in the wild-type enzyme. Because the R347H and R358Q mutants
are located in the proposed redox partner-binding domain, the
contributions of the electron donors OR and cytochrome b5
to 17,20-lyase activity are of great significance. By examining the
catalysis of wild-type P450c17 in genetically engineered yeast, we have
recently shown that 1) lyase activity requires OR; 2) human cytochrome
b5 cannot support catalysis by itself; 3) cytochrome
b5 can augment lyase activity, but only in the presence of
OR; 4) b5 promotes lyase activity by an allosteric
mechanism and does not participate detectably in direct electron
transfer; and 5) in vitro, cytochrome b5
modulates lyase activity in a biphasic manner, with inhibition at very
high molar ratios of b5 to P450c17 (19). To delineate the
specific interactions with electron transfer proteins that are
disrupted by the R347H and R358Q mutants, and to quantitate the
affinity of the mutant enzymes for 17-hydroxypregnenolone, we
evaluated the kinetics of these mutants in yeast microsomes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxylase activity but less than 5% of wild-type 17,20-lyase
activity, and our modeling studies indicated that these mutants lie in
the redox-partner binding site of P450c17 (15). Therefore, we
hypothesized that the presence of a substantial excess of the redox
partner, OR, might partially overcome this selective 17,20-lyase
deficiency. Transfection of COS-1 cells confirmed that each mutant
retained substantial 17-hydroxylase activity but lacked detectable
17,20-lyase activity (Fig. 1
).
Coexpression of each mutant with OR appeared to augment the
17-hydroxylase activity, similarly to our previous observations with
wild-type P450c17 (7, 15), but unlike those previous experiments with
wild-type enzyme, the presence of excess OR did not foster significant
17,20-lyase activity (Fig. 1). We recently demonstrated that cytochrome
b5 selectively increases the 17,20-lyase activity of human
P450c17 by allosterically facilitating its interaction with OR (19).
However, cotransfection of cells with P450c17 and b5, in
which the only available OR is that present endogenously in the COS-1
cells, also failed to confer detectable 17,20-lyase activity,
consistent with our recent demonstration that b5 cannot act
as an electron donor to human P450c17 (19). However, when either the
R347H or R358Q mutants are expressed together with vectors
overexpressing both OR and b5, significant 17,20-lyase
activity is restored to each mutant. Thus the R347H and R358Q mutants
appear to lie in the redox partner binding site, so that partial
17,20-lyase activity can be restored by a substantial molar excess of
both OR and b5. To elucidate the molecular mechanisms by
which the combination of OR and b5 achieves the qualitative
results seen in COS-1 cells, we used our recently described (19)
application of a yeast microsome system (20).
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-Hydroxylase Kinetics-hydroxylase and
17,20-lyase activities of P450c17, but the catalytic efficiency
(Vmax/Km) of each reaction is 10-fold higher in
the presence of human OR whether or not the yeast OR is also present
(19). Therefore we used the parental yeast strain W303B doubly
transfected with vector V10 expressing either of the two mutant forms
of P450c17, and with vector cDE2 expressing the cDNA for either human
OR or, in the desired absence of human redox partner, with an equal
mass of empty vector. Microsomal preparations of these transfectants
were then used for kinetic analysis of the Km and
Vmax by Lineweaver-Burk plots (Fig. 2). Yeast transfected with either of the
P450c17 mutants but without human OR are capable of catalyzing
17-hydroxylase activity, indicating that despite the apparent
perturbation in redox partner binding caused by the amino acid
substitutions, some 17-hydroxylase activity can occur utilizing the
endogenous yeast OR. As with wild-type P450c17 (19), the
Vmax for the 17-hydroxylase activity of each mutant is
augmented by the presence of cotransfected human OR, while the apparent
Km is reduced (Table 1). In
yeast microsomes, the apparent Km for pregnenolone is 0.6
µM for R347H and 0.4 µM for R358Q;
coexpression with OR decreases these values to 0.2 µM and
0.3 µM, respectively, similar to the 2-fold effect seen
with the wild-type enzyme (Table 1); this is consistent with the
mutations altering the interaction of P450c17 with OR even though the
mutants retain considerable 17-hydroxylase activity. The
Vmax increases 2- to 3-fold in the presence of the OR,
slightly less than the 4- to 5-fold effect seen when the wild-type
P450c17 is cotransfected with human OR (Table 1). Although these
mutants cause a relatively selective loss of lyase activity, there is
also an effect on 17-hydroxylation, as each mutant retains about
65% of wild-type 17-hydroxylase activity but less than 5% of the
native 17,20-lyase activity in transfected COS-1 cells (15). Similarly,
the Vmax obtained for the hydroxylase activity of each
P450c17 mutant in yeast, with or without coexpressed OR, is only
10–30% of that observed for the wild-type enzyme.
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-hydroxylation of pregnenolone by the wild-type and
both mutant P450c17 proteins (15). Although these data suggested that
neither of the mutations resulted in a major conformational change in
the steroid hormone binding (active) site, the inherent limitations of
kinetic measurements obtained in whole transfected cells precluded our
determining the nature of this inhibition. Therefore, we used the yeast
microsomal system to perform hydroxylase assays in the presence and
absence of unlabeled 17-hydroxypregnenolone at a final
concentration of 5 µM (5- to 10-fold Km). The
Lineweaver-Burk plots demonstrate the competitive nature of this
inhibition for each mutant, as there is no change in Vmax
(Fig. 3). The apparent Km
values of 0.2–0.4 µM pregnenolone and
Ki values of 0.3–0.8 µM
17-hydroxypregnenolone in the presence of OR are remarkably similar
for all three forms of P450c17 (Table 1
) and compare favorably to the
half-maximal inhibition values of 0.3 to 1.0 µM
derived from the qualitative COS-1 cell experiments (15). This
demonstration that 17-hydroxypregnenolone is a competitive inhibitor
of the 17-hydroxylase reaction in both mutants confirms the
hypothesis that substrate binding at the active site is not
significantly impaired by the R347H and R358Q mutations.
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-hydroxylase and 17,20-lyase
activity in environments where OR is limiting (7, 19). By contrast,
cytochrome b5, either coexpressed in yeast with P450c17 and
OR or exogenously added to yeast microsomes containing P450c17 and OR,
will increase 17,20-lyase activity but has no appreciable effect on the
already maximal 17-hydroxylase activity (19). Coexpression of human
OR with the R347H and R358Q mutants of P450c17 increased their
17-hydroxylase activities (Fig. 4A)
similarly to the effect seen with the wild-type enzyme (19), but the
coexpression of OR had no detectable effect on the 17,20-lyase activity
of the mutants despite the presence of endogenous yeast OR (Fig. 4B).
However, unlike the wild-type, exogenous addition of purified human
cytochrome b5 increased both the hydroxylase and lyase
activities of the mutants (Fig. 4). This is consistent with the view
that b5 allosterically fosters the interaction of P450c17
and OR, and that the R347H and R358Q mutations interfere with this
interaction. To test this, we examined the effects of exogenously added
b5 on the kinetics of the 17,20-lyase activity of each
mutant.
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) P450c17, we examined the
effect of varying the ratio of b5 to P450c17 for the
wild-type and each mutant in the presence of OR. When examined over a
range of b5:P450c17 molar ratios of 0.1:1 up to 1000:1, the
peak response of the wild-type enzyme is 2.5- to 6-fold greater than
that seen with either mutant (Fig. 5).
This peak response is shifted to at least 10-fold higher ratios for the
mutants (30 to 100:1) than for wild-type enzyme (1 to 3:1). The two
mutants also respond differently to the presence of excess cytochrome
b5: there is a sharper and more dramatic effect on the
17,20-lyase activity of R358Q than the broader response seen for R347H.
Finally, in the absence of human OR, there is no detectable 17,20-lyase
activity for either mutant, even at b5:P450c17 ratios up to
300:1. This is consistent with our previous observation that OR must be
present for P450c17 to catalyze any 17,20-lyase activity (19). The
differences in the b5 curves (Fig. 5) indicate that even
though the R347H and R358Q mutations alter the charge distribution in
the redox-partner binding site (15), the two mutants can be
distinguished biochemically by the differential effect of
b5 stimulation. These data suggest that R347 is the more
critical residue for the interaction of b5 with the
P450c17·OR complex (19). These differences in the b5
titration curves of the R347H and R358Q mutants provide a more detailed
understanding of the interactions of redox partners with P450c17 during
catalysis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-hydroxyprogesterone to 
4-androstenedione (19),
almost all circulating human sex steroids derive from
dehydroepiandrosterone (DHEA) synthesized by the sequential
17-hydroxylase and 17,20-lyase activities of P450c17. Consequently,
all mutations in P450c17 studied before 1997 reduced both activities
comparably (11, 24, 25). The discovery of patients whose P450c17
mutations abolished almost all lyase activity while preserving most of
the 17-hydroxylase activity provides a unique opportunity to study
the structural requirements for the proper assembly of the catalytic
complex that performs the 17,20-lyase reaction (18). Somewhat
surprisingly, the computational predictions (15) and the direct
evidence presented in this paper demonstrate unequivocally that these
two mutations do not change the enzyme’s affinity for the
17-hydroxypregnenolone intermediate. Instead, mutations R347H and
R358Q impair the ability of these mutants to form productive
interactions with OR and, to a variable extent, with
b5.
The study of the R347H and R358Q mutants shows that these amino acid
replacements do not alter the active site and that 17,20-lyase activity
is more vulnerable to disruptions in redox partner interactions than is
the 17-hydroxylase reaction. While it is theoretically possible that
some hypothetical mutation in the active site of P450c17 could
preferentially reduce affinity for 17-hydroxypregnenolone to cause
isolated 17,20-lyase deficiency, no such mutations have been found,
either by examining mutations found in patients or by site-directed
mutagenesis (17). It is of interest that a third mutation of P450c17,
F417C, was recently reported that also caused selective impairment of
17,20-lyase activity, although with a greater impairment of
17-hydroxylase activity than is found in the R347H or R358Q mutants
(26). While F417 is rather distant in its location in the linear amino
acid sequence, our molecular modeling indicates that F417 lies near
R358 and R347 in three-dimensional space, but unlike the
solvent-exposed arginines, F417 is buried and forms a bulging ridge at
the presumed edge of the redox-partner binding site (18). Thus,
interference with either the electrostatic surface charges or the shape
of the redox-partner binding site can cause selective loss of
17,20-lyase activity.
Although R347 and R358 appear to be close in three-dimensional space, the biochemistry of these two mutants can be distinguished by their activities in the presence of cytochrome b5. Whereas b5 stimulates the wild-type enzyme’s 17,20-lyase activity more than 13-fold (19), b5 stimulates the lyase activity of the R358Q and R347H mutants only 5- and 2.5-fold, respectively. These results suggest that R347 is a critical component of the P450c17-b5 interaction site on P450c17. Our previous studies suggest that the 17,20-lyase reaction occurs in the P450c17·OR complex, with b5 acting on this complex as an allosteric facilitator (19). This model is consistent with the observation that the mutations of the redox-partner binding site eliminate detectable 17,20-lyase activity in the presence of the low levels of OR endogenously provided by the yeast or COS-1 cells. A trace of lyase activity was observed in the presence of human (but not yeast) OR, and we can demonstrate significant lyase activity only in the presence of both human OR and human b5. Thus our genetic, biochemical, and computational studies of wild-type and mutant P450c17 proteins are beginning to delineate the structural details of how this complex is assembled and why it performs this unique oxidative carbon-carbon bond cleavage reaction only with 17-OH-pregnenolone. Furthermore, the differences between the biochemistry of the R347H and R358Q mutants demonstrate that binding of b5 is necessary but not sufficient for maximal lyase activity and that residues that interact preferentially with OR and b5 can be functionally distinguished by specific mutations in P450c17.
The ratio of 17,20-lyase activity to 17-hydroxylase activity is
regulated in normal human adrenal physiology. DHEA concentrations
(reflecting 17, 20-lyase activity) rise dramatically at adrenarche and
then wane during aging (27), while cortisol concentrations (reflecting
17-hydroxylase activity) remain constant. The regulation of this
ratio of activities may be altered in the polycystic ovary
syndrome, which affects 5% of women of reproductive age (28).
These individuals have both ovarian and adrenal hyperandrogenism with
normal cortisol, suggesting a disorder at the level of 17,20-lyase
activity (4, 29). Our studies of mutations causing isolated 17,20-lyase
deficiency show that the surface charges in the redox partner binding
site are crucial for optimizing lyase activity. Obviously, the
physiological regulation of lyase activity will not involve changes in
the amino acid sequence of P450c17, but regulated posttranslational
modification might alter surface charges in the redox partner binding
site, providing a mechanism for the physiological regulation of the
hydroxylase-lyase ratio. Serine/threonine phosphorylation of human
P450c17 by an unidentified cAMP-dependent protein kinase increases
17,20-lyase activity, and dephosphorylation of P450c17 diminishes
17,20-lyase activity without altering its 17-hydroxylase activity
(29). Consistent with this view, a preliminary report indicates that
the P450c17 mutant F419C, found in a patient with isolated 17,20-lyase
deficiency, cannot be phosphorylated normally (30). The similarity of
the dephosphorylation data (29) and our present results with mutations
in the redox partner binding site suggests that specific phosphorylated
serine residues serve to provide surface charges that optimize the
interaction of P450c17 with OR and/or b5 to optimize
electron transfer for the lyase reaction. Thus, understanding the
precise mechanisms contributing to the ratio of 17,20-lyase to
17-hydroxylase activity is of substantial interest and
importance.
Finally, our understanding of how mutations in P450c17 can selectively destroy lyase activity suggests novel approaches to the design of inhibitors of this enzyme that occupies a central role in determining flux through the steroidogenic pathways. We hypothesize that compounds that bind to the redox partner binding site of P450c17 might be highly potent inhibitors of sex steroid biosynthesis, which would be therapeutically useful in polycystic ovary syndrome and in cancers of the breast and prostate. Furthermore, such compounds would not bind to the active site and therefore need not resemble steroids; hence they should not produce side effects often associated with steroids. Therefore, principles deduced from the study of two patients with a rare defect in androgen biosynthesis might yield widespread benefits in the understanding of physiology and treatment of disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) and
the yeast expression vector cDE2 (pYcDE-2) were generous gifts of Drs.
Gregory Petsko and Ira Herskowitz. The yeast expression vector V10
(pYeDP10) was a generous gift from Dr. Denis Pompon (CNRS,
Gif-sur-Yvette, France). Wild-type human P450c17 cDNA (9) was PCR
amplified using primers previously described (19) and digested with
BamHI and EcoRI, facilitating directional cloning
into the complementary ends of
BglII-EcoRI-digested V10 vector. This destroys
the BglII site and places the P450c17 cDNA under the control
of the constitutive pgk promoter, producing the vector
V10-c17. Human OR cDNA was PCR amplified from pECE-OR (7) using primers
previously described (19). The human OR cDNA was then cloned into the
EcoRI site of the cDE2 vector under the control of the
constitutive adc1 promoter, with a trp1
selectable marker. P450c17 cDNAs containing the mutations R347H or
R358Q were PCR amplified from previously constructed pMT2 expression
vectors as templates (15) using pfu polymerase (Stratagene,
La Jolla, CA) and the primers c17S-1 and c17AS-1 (19), and then
sequenced in their entirety to ensure the presence of only the desired
mutation. These mutant cDNAs were then digested with BamHI
and EcoRI and ligated into the V10 vector as with the
wild-type c17 cDNA described above.
Yeast Transformation and Growth
Yeast were transformed using 700 µl of 40% polyethylene
glycol 3350, 0.1 M lithium acetate, 10 mM
Tris-HCl (pH 8), 1 mM EDTA. About 106 yeast
cells were transformed in 100 µl of 0.1 M lithium
acetate, 10 mM Tris-HCl (pH 8), 1 mM EDTA with
1 µg of plasmid DNA, and 50 µg of denatured herring sperm DNA as
carrier (31). Cells were washed in 100 µl of 1 M sorbitol
before final resuspension in 100 µl of 10 mM Tris-HCl (pH
8), 1 mM EDTA, for plating onto selective media. Expression
of mutant or wild-type P450c17 was always under the control of the
constitutive pgk promoter. For microsome preparations,
transformed yeast were cultured in minimal SD media containing 20
g/liter D-glucose, 1.7 g/liter yeast nitrogen base without
amino acids or ammonium sulfate (Difco, Detroit, MI), 5 g/liter
ammonium sulfate, and 45 mg/liter adenine.
Microsome Preparation and Characterization
Yeast cells harvested at a density of 4.5–6 x
107 cells/ml were disrupted by manual breakage with 450- to
600-µm glass beads for 5 min (20), stopping at 1-min intervals, and
icing the cells for 30 sec between disruptions. Three microliters of 1
M ethanolic phenylmethylsulfonyl fluoride were added after
the first minute of breakage. For each 300 ml of culture, the crude
extracts together with the glass beads were washed twice with 5–7 ml
of 50 mM Tris-HCl (pH 8), 1 mM EDTA, 0.4
M sorbitol, and the cellular debris was collected by
centrifugation at 4 C twice for 10 min each at 14,000 x
g. The microsomes were pelleted by centrifugation at 4 C for
60 min at 100,000 x g and were resuspended in 50
mM Tris-HCl (pH 8), 1 mM EDTA, 20% glycerol at
a final concentration of 10–20 µg/µl total protein. Each
preparation was homogenized by shearing the microsomes with passage
through a 27-gauge needle 10 times; aliquots were kept frozen at -70
C. Microsomal protein content was determined colorimetrically.
Microsomal P450 content was measured spectrophotometrically (32) using
either a Cary 3E or a Shimazdu UV 160U spectrophotometer.
P450c17 Enzyme Assay
Microsomes were assayed for hydroxylase activity under
initial rate kinetics by preincubation in 50 mM potassium
phosphate buffer (pH 7.4) with 0.5–5.0 µM steroid (added
in 4 µl of ethanol) in a total volume of 200 µl, at 37 C for 3 min
before the addition of 1 mM NADPH to initiate the reaction.
For 17-hydroxylase assays, each reaction contained 10,000 cpm of
[14C]pregnenolone (55.4 Ci/mol, NEN Life Science
Products, Inc., Boston, MA). For the 17,20-lyase assays, including the
b5 titration experiments, each reaction contained 100,000
cpm of [3H]17-hydroxypregnenolone (21.2 Ci/mmol, NEN
Life Science Products) at a final 17-hydroxypregnenolone
concentration of 50 nM. Steroids were extracted with 400
µl of ethyl acetate-isooctane (1:1, vol/vol), concentrated under
nitrogen, separated by TLC (Whatman PE SIL G/UV silica gel plates,
Maidstone, Kent, UK) using a 3:1 chloroform-ethyl acetate solvent
system, and quantitated as described (15). Purified recombinant human
cytochrome b5 was obtained from PanVera (Madison, WI).
Kinetic behavior was approximated as a Michaelis-Menten system for data
analysis, and all error bars shown represent SDs.
Ki values for 17-OH-pregnenolone were calculated from the
equation K'm = (Km/Ki)[I] +
Km where K'm is the Km in the presence of
inhibitor I at the concentration [I] (33).
COS-1 Cell Transfections
COS-1 cells were grown to 50% confluence in 10% FCS
and at 5% CO2 and transfected with 2–5 µg of cDNA in
the appropriate expression vectors, as previously described (34). Cells
received vectors expressing either mutant P450c17 cDNA alone, mutant
P450c17 plus OR cDNA [in the pECE expression vector, (7)], mutant
P450c17 cDNA plus b5 cDNA (19, 35, 36), or mutant P450c17
plus the vectors for OR and b5. The total amount of
transfected cDNA was standardized by cotransfection with empty pMT2
vector DNA. Transfection efficiency was monitored by cotransfection
with a Rous sarcoma virus-luciferase construct. Transfected cells were
washed with PBS and incubated in fresh medium for 36 h before
addition of either [3H]pregnenolone or
[3H]17-hydroxypregnenolone in 4 ml of medium, at a
final concentration of 0.6 nM. Steroids were extracted and
analyzed as described above.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by the National Cooperative Program for Infertility Research, University of California San Francisco, Grant U54-HD-34449 (to W.L.M.), NIH Grants DK-37922 and DK-42154 (to W.L.M.), and Clinical Investigator Award DK-02387 (to R.J.A.). D.G.H. was supported by Pediatric Endocrinology Training Grant DK-07161 (to W.L.M.) and a grant from the Lawson Wilkins Pediatric Endocrine Society.
Received for publication August 10, 1998. Revision received September 25, 1998. Accepted for publication September 29, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-hydroxylase-C17, 20
lyase). J Biol Chem 256:3871–3876-hydroxylase/C17,20 lyase cytochrome P450. J Biol
Chem 259:3971–397616-C19 steroids by homogeneous cytochrome
P-450 C21 side-chain cleavage from pig testis. Biochem Biophys Res
Commun 132:708–713[CrossRef][Medline]
-hydroxylase activity is catalyzed by human
cytochrome P450 17-hydroxylase. J Clin Endocrinol Metab 77:98–102[Abstract]
-hydroxylase and 17,20 lyase activities of P450c17: contributions
of serine106 and P450 reductase. Endocrinology 132:2498–2506-hydroxylase/17, 20 lyase):
similarity to the gene for P450c21. DNA 6:439–448[Medline]
-hydroxylase/17, 20 lyase): cloning of human adrenal and testis
cDNAs indicates the same gene is expressed in both tissues. Proc Natl
Acad Sci USA 84:407–411-hydroxylase/17,20
lyase deficiency: from clinical investigation to molecular definition.
Endocr Rev 12:91–108-Hydroxylase/17,20 lyase defects. J Steroid
Biochem Mol Biol 53:153–157[CrossRef][Medline]
Cys, Gln(461)Stop) actually cause combined
17-hydroxylase/17,20-lyase deficiency. Biochim Biophys Acta 1139:275–279[Medline]
-hydroxylase
deficiency with age. Acta Endocrinol (Copenh) 127:97–99. Mol Endocrinol 5:1373–1380-hydroxylase
deficiency. J Clin Endocrinol Metab 79:160–164[Abstract]
-hydroxylase/17,20 lyase deficiency in two French Canadian
patients. J Clin Endocrinol Metab 81:264–268[Abstract]
-hydroxylase/17,20-lyase deficiency as a model to investigate
enzyme activity regulation. Program of the 80th Annual Meeting of The
Endocrine Society, New Orleans, LA, 1998, p 177 (Abstract)
Pro causes 17-hydroxylase
deficiency. J Biol Chem 266:15992–15998This article has been cited by other articles:
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  J C. Pattison, D. H Abbott, W. Saltzman, A. J Conley, and I. M Bird Plasticity of the zona reticularis in the adult marmoset adrenal cortex: voyages of discovery in the New World J. Endocrinol., December 1, 2009; 203(3): 313 - 326. [Abstract] [Full Text] [PDF]
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 V. Dhir, N. Reisch, C. M. Bleicken, J. Lebl, C. Kamrath, H.-P. Schwarz, J. Grotzinger, W. G. Sippell, F. G. Riepe, W. Arlt, et al. Steroid 17{alpha}-Hydroxylase Deficiency: Functional Characterization of Four Mutations (A174E, V178D, R440C, L465P) in the CYP17A1 Gene J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 3058 - 3064. [Abstract] [Full Text] [PDF]
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T. Sahakitrungruang, M. K. Tee, P. W. Speiser, and W. L. Miller Novel P450c17 Mutation H373D Causing Combined 17{alpha}-Hydroxylase/17,20-Lyase Deficiency J. Clin. Endocrinol. Metab., August 1, 2009; 94(8): 3089 - 3092. [Abstract] [Full Text] [PDF]
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E. Hershkovitz, R. Parvari, S. A. Wudy, M. F. Hartmann, L. G. Gomes, N. Loewental, and W. L. Miller Homozygous Mutation G539R in the Gene for P450 Oxidoreductase in a Family Previously Diagnosed as Having 17,20-Lyase Deficiency J. Clin. Endocrinol. Metab., September 1, 2008; 93(9): 3584 - 3588. [Abstract] [Full Text] [PDF]
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 M. K. Tee, Q. Dong, and W. L. Miller Pathways Leading to Phosphorylation of P450c17 and to the Posttranslational Regulation of Androgen Biosynthesis Endocrinology, May 1, 2008; 149(5): 2667 - 2677. [Abstract] [Full Text] [PDF]
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 D. Tiosano, C. Knopf, I. Koren, N. Levanon, M. F Hartmann, Z. Hochberg, and S. A Wudy Metabolic evidence for impaired 17{alpha}-hydroxylase activity in a kindred bearing the E305G mutation for isolate 17,20-lyase activity Eur. J. Endocrinol., March 1, 2008; 158(3): 385 - 392. [Abstract] [Full Text] [PDF]
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S. Rosa, C. Duff, M. Meyer, M. Lang-Muritano, G. Balercia, M. Boscaro, A. Kemal Topaloglu, R. Mioni, F. Fallo, L. Zuliani, et al. P450c17 Deficiency: Clinical and Molecular Characterization of Six Patients J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1000 - 1007. [Abstract] [Full Text] [PDF]
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 S. H. Mellon, S. R. Bair, C. Depoix, J.-L. Vigne, N. B. Hecht, and P. B. Brake Translin Coactivates Steroidogenic Factor-1-Stimulated Transcription Mol. Endocrinol., January 1, 2007; 21(1): 89 - 105. [Abstract] [Full Text] [PDF]
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T. Robins, J. Carlsson, M. Sunnerhagen, A. Wedell, and B. Persson Molecular Model of Human CYP21 Based on Mammalian CYP2C5: Structural Features Correlate with Clinical Severity of Mutations Causing Congenital Adrenal Hyperplasia Mol. Endocrinol., November 1, 2006; 20(11): 2946 - 2964. [Abstract] [Full Text] [PDF]
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M. Janner, A. V Pandey, P. E Mullis, and C. E Fluck Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein. Eur. J. Endocrinol., July 1, 2006; 155(1): 143 - 151. [Abstract] [Full Text] [PDF]
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M K Akhtar, S L Kelly, and M A Kaderbhai Cytochrome b5 modulation of 17{alpha} hydroxylase and 17-20 lyase (CYP17) activities in steroidogenesis J. Endocrinol., November 1, 2005; 187(2): 267 - 274. [Abstract] [Full Text] [PDF]
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N. Huang, A. Dardis, and W. L. Miller Regulation of Cytochrome b5 Gene Transcription by Sp3, GATA-6, and Steroidogenic Factor 1 in Human Adrenal NCI-H295A Cells Mol. Endocrinol., August 1, 2005; 19(8): 2020 - 2034. [Abstract] [Full Text] [PDF]
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W. L. Miller Minireview: Regulation of Steroidogenesis by Electron Transfer Endocrinology, June 1, 2005; 146(6): 2544 - 2550. [Abstract] [Full Text] [PDF]
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M. Taniyama, M. Tanabe, H. Saito, Y. Ban, H. Nawata, and T. Yanase Subtle 17{alpha}-Hydroxylase/17,20-Lyase Deficiency with Homozygous Y201N Mutation in an Infertile Woman J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2508 - 2511. [Abstract] [Full Text] [PDF]
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 A. V. Pandey and W. L. Miller Regulation of 17,20 Lyase Activity by Cytochrome b5 and by Serine Phosphorylation of P450c17 J. Biol. Chem., April 8, 2005; 280(14): 13265 - 13271. [Abstract] [Full Text] [PDF]
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W.-H. Yang and S. R Hammes Xenopus laevis CYP17 Regulates Androgen Biosynthesis Independent of the Cofactor Cytochrome b5 J. Biol. Chem., March 18, 2005; 280(11): 10196 - 10201. [Abstract] [Full Text] [PDF]
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 J. R. Wood, V. L. Nelson-Degrave, E. Jansen, J. M. McAllister, S. Mosselman, and J. F. Strauss III Valproate-induced alterations in human theca cell gene expression: clues to the association between valproate use and metabolic side effects Physiol Genomics, February 10, 2005; 20(3): 233 - 243. [Abstract] [Full Text] [PDF]
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 S. Dharia, A. Slane, M. Jian, M. Conner, A. J. Conley, and C. R. Parker Jr Colocalization of P450c17 and Cytochrome b5 in Androgen-Synthesizing Tissues of the Human Biol Reprod, July 1, 2004; 71(1): 83 - 88. [Abstract] [Full Text] [PDF]
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 M. J. ten Kate-Booij, C. Cobbaert, J. W. Koper, and F. H. de Jong Deficiency of 17,20-lyase causing giant ovarian cysts in a girl and a female phenotype in her 46,XY sister: Case report Hum. Reprod., February 1, 2004; 19(2): 456 - 459. [Abstract] [Full Text] [PDF]
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M. Costa-Santos, C. E. Kater, and R. J. Auchus Two Prevalent CYP17 Mutations and Genotype-Phenotype Correlations in 24 Brazilian Patients with 17-Hydroxylase Deficiency J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 49 - 60. [Abstract] [Full Text] [PDF]
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D. P. Sherbet, D. Tiosano, K. M. Kwist, Z. Hochberg, and R. J. Auchus CYP17 Mutation E305G Causes Isolated 17,20-Lyase Deficiency by Selectively Altering Substrate Binding J. Biol. Chem., December 5, 2003; 278(49): 48563 - 48569. [Abstract] [Full Text] [PDF]
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J. R. Wood, V. L. Nelson, C. Ho, E. Jansen, C. Y. Wang, M. Urbanek, J. M. McAllister, S. Mosselman, and J. F. Strauss III The Molecular Phenotype of Polycystic Ovary Syndrome (PCOS) Theca Cells and New Candidate PCOS Genes Defined by Microarray Analysis J. Biol. Chem., July 11, 2003; 278(29): 26380 - 26390. [Abstract] [Full Text] [PDF]
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A. V. Pandey, S. H. Mellon, and W. L. Miller Protein Phosphatase 2A and Phosphoprotein SET Regulate Androgen Production by P450c17 J. Biol. Chem., January 24, 2003; 278(5): 2837 - 2844. [Abstract] [Full Text] [PDF]
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E. L. T. van den Akker, J. W. Koper, A. L. M. Boehmer, A. P. N. Themmen, M. Verhoef-Post, M. A. Timmerman, B. J. Otten, S. L. S. Drop, and F. H. De Jong Differential Inhibition of 17{alpha}-Hydroxylase and 17,20-Lyase Activities by Three Novel Missense CYP17 Mutations Identified in Patients with P450c17 Deficiency J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5714 - 5721. [Abstract] [Full Text] [PDF]
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W. Arlt, J. W. M. Martens, M. Song, J. T. Wang, R. J. Auchus, and W. L. Miller Molecular Evolution of Adrenarche: Structural and Functional Analysis of P450c17 from Four Primate Species Endocrinology, December 1, 2002; 143(12): 4665 - 4672. [Abstract] [Full Text] [PDF]
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M. K. Gupta, D. H. Geller, and R. J. Auchus Pitfalls in Characterizing P450c17 Mutations Associated with Isolated 17,20-Lyase Deficiency J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4416 - 4423. [Abstract] [Full Text] [PDF]
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C. J. Lin, J. W. M. Martens, and W. L. Miller NF-1C, Sp1, and Sp3 Are Essential for Transcription of the Human Gene for P450c17 (Steroid 17{alpha}-hydroxylase/17,20 lyase) in Human Adrenal NCI-H295A Cells Mol. Endocrinol., August 1, 2001; 15(8): 1277 - 1293. [Abstract] [Full Text] [PDF]
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R. Azziz, L. M. Fox, H. A. Zacur, C. R. Parker Jr., and L. R. Boots Adrenocortical Secretion of Dehydroepiandrosterone in Healthy Women: Highly Variable Response to Adrenocorticotropin J. Clin. Endocrinol. Metab., June 1, 2001; 86(6): 2513 - 2517. [Abstract] [Full Text] [PDF]
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 P. C. White and P. W. Speiser Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency Endocr. Rev., June 1, 2000; 21(3): 245 - 291. [Abstract] [Full Text]
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A. Biason-Lauber, M. Zachmann, and E. J. Schoenle Effect of Leptin on CYP17 Enzymatic Activities in Human Adrenal Cells: New Insight in the Onset of Adrenarche Endocrinology, April 1, 2000; 141(4): 1446 - 1454. [Abstract] [Full Text] [PDF]
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A. Biason-Lauber, B. Kempken, E. Werder, M. G. Forest, S. Einaudi, M. B. Ranke, N. Matsuo, V. Brunelli, E. J. Schönle, and M. Zachmann 17{alpha}-Hydroxylase/17,20-Lyase Deficiency as a Model to Study Enzymatic Activity Regulation: Role of Phosphorylation J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1226 - 1231. [Abstract] [Full Text]
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W. L. Miller and R. J. Auchus Role of Cytochrome b5 in the 17,20-Lyase Activity of P450c17 J. Clin. Endocrinol. Metab., March 1, 2000; 85(3): 1346 - 1346. [Full Text]
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R. J. Auchus and W. L. Miller Molecular Modeling of Human P450c17 (17{alpha}-Hydroxylase/ 17,20-Lyase): Insights into Reaction Mechanisms and Effects of Mutations Mol. Endocrinol., July 1, 1999; 13(7): 1169 - 1182. [Abstract] [Full Text]
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T. C. Lee, W. L. Miller, and R. J. Auchus Medroxyprogesterone Acetate and Dexamethasone Are Competitive Inhibitors of Different Human Steroidogenic Enzymes J. Clin. Endocrinol. Metab., June 1, 1999; 84(6): 2104 - 2110. [Abstract] [Full Text]
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W. Arlt, R. J. Auchus, and W. L. Miller Thiazolidinediones but Not Metformin Directly Inhibit the Steroidogenic Enzymes P450c17 and 3beta -Hydroxysteroid Dehydrogenase J. Biol. Chem., May 11, 2001; 276(20): 16767 - 16771. [Abstract] [Full Text] [PDF]
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