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Original Studies |
-Hydroxylase/17,20-Lyase Deficiency as a Model to Study Enzymatic Activity Regulation: Role of Phosphorylation1
Department of Pediatrics, Divisions of Pediatric Endocrinology and Diabetology and Clinical Chemistry and Biochemistry, University of Zurich (A.B.-L., B.K., E.W., M.Z.), 8032 Zurich, Switzerland; Pathologie Hormonale Moleculaire, Hopital Debrousse (M.G.F.), Lyon, France; Divisione di Endocrinologia, Ospedale Infantile Regina Margherita (S.E.), Turin, Italy; Kinderklinik, Eberhard-Karls-Universität Tübingen (M.B.R.), Tubingen, Germany; Department of Pediatrics, Keio University Hospital (N.M.), Tokyo, Japan; and Clinica Pediatrica III, Centro di Endocrinologia Infantile e dell’Adolescenza, Universitá di Milano (V.B.), Milan, Italy
Address all correspondence and requests for reprints to: Dr. Anna Biason-Lauber, Department of Pediatrics, Divisions of Pediatric Endocrinology/Diabetology and Clinical Chemistry and Biochemistry, University of Zurich, Steinwiesstrasse 75, 8032 Zurich, Switzerland. E-mail: alauber{at}kispi.unizh.ch
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (CYP17) is a single gene-encoded
protein with two activities: 17-hydroxylase and 17,20-lyase. The two
catalytic activities are differentially regulated in health and
disease. We took advantage of naturally occurring human mutations to
understand the molecular bases of this differential regulation. We
identified eight novel mutations in the CYP17 gene,
different in nature and spread throughout the gene. As
posttranslational modifications appear to be important for activity
control, we investigated the phosphorylation state of wild-type and
mutant CYP17 proteins. Although phospholabeled protein was seen when
the wild-type and most mutant proteins were expressed, no
phosphorylation was detected for the F417C mutant. F417C is the only
17,20-lyase deficiency case confirmed at the molecular level and
represents the first phosphorylation CYP17-deficient mutant. In search
of the physiological agents involved in this process, the effect of
cAMP was tested on activity and phosphorylation state of our mutant
CYP17 proteins. cAMP stimulates activity and phosphorylation in all
cases, except in the F417C and R35L mutants. The lack of response to
the physiological second messenger might explain the different
phenotypes. The F417C mutant protein, which is already shown to be
associated with the lack of electron transfer, provides for the first
time a link between the electron transfer system and the
phosphorylation state of the CYP17 enzyme in the control of 17,20-lyase
activity. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase, which is necessary for
the synthesis of cortisol, and 17,20-lyase, which cleaves the
C17–20 carbon bond, converting
C21 compounds to C19
steroids dehydroepiandrosterone (DHEA) and
androstenedione, the precursors of the sex hormones. Humans have only
one gene, CYP17, that encodes only one form of the CYP17
enzyme. Clinical observations have revealed that the two activities are
differentially regulated in a tissue- and time-dependent manner, as
demonstrated by the distinct steroid secretion pattern in adrenals and
gonads and by the adrenarche, the maturation of the adrenal zona
reticularis, respectively. Although 17,20-lyase activity can
be significantly influenced by the abundance and the interaction with
redox partners (1, 2) in different cell types, it seems unlikely that
the amount of reducing agents varies substantially in the same cells in
a hormonally and developmentally regulated fashion, as observed for
17,20-lyase activity at adrenarche. Posttranslational modifications
seem to be a more suitable mechanism for the maturation-dependent
regulation of 17,20-lyase activity. Consistent with this hypothesis,
Zhang et al (3) demonstrated that phosphorylation of CYP17
is necessary for 17,20-lyase, but not for 17-hydroxylase, activity.
Nevertheless, the physiological trigger for adrenarche and/or CYP17
phosphorylation remains unknown. The possibility of analyzing patients
affected by CYP17 deficiency provides a unique chance not only to
clarify the molecular bases of the disease, but also to use a human
knockout model to study the influence of cofactors or posttranslational
changes on 17-hydroxylase and 17,20-lyase activities of CYP17. The
same model can be useful to analyze the role of agents that may be
involved in the developmental regulation of 17,20-lyase activity in the
phosphorylation state and the activity of CYP17. We therefore collected a cohort of patients suffering from CYP17 deficiency, analyzed their CYP17 genes for genetic rearrangements, and used them as a model for studying the role of phosphorylation and its control of CYP17 catalytic activities.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten patients were diagnosed to suffer from CYP17 deficiency.
Based on the clinical and biochemical picture, they were divided into
two groups: 1) patients suffering from complete combined
17-hydroxylase/17,20-lyase deficiency (n = 7), and 2) patients
affected by isolated 17,20-lyase deficiency (n = 3). Although the
diagnosis of complete combined CYP17 deficiency is relatively easy
(female infantile external genitalia independent from genetic sex and
age; hypertension, hypokalemia, and lack of sexual maturation at the
time of expected puberty; lack of response of cortisol,
DHEA, and androstenedione to ACTH; and no increase in sex
hormones in response to exogenous gonadotropins), the more difficult
diagnosis of 17,20-lyase deficiency was made in three genotypically
male patients born with ambiguous genitalia on the basis of the known
criteria (4). Briefly, in the presence of intersexual genitalia, a
stimulation test with hCG showed no significant response of
testosterone, but an increase, or even an exaggerated response of
17-hydroxyprogesterone in the presence of normal cortisol levels.
DHEA and androstenedione levels fail to rise under ACTH
stimulation.
Mutation analysis
Genomic DNA was extracted from peripheral blood leukocytes (PBL) using the QIAGEN DNA blood and cell culture kit (QIAGEN, Hilden, Germany) and was used to perform PCR exonic amplification and direct sequencing using the dideoxy method applied to thermal cycling as previously described (1).
Expression studies
To study the functional implications of the mutations found, we
established a RT-PCR method using CYP17 messenger ribonucleic acid
(RNA) ectopically expressed in PBL of the patients (5). Total RNA was
extracted from 500 µL whole blood using the RNeasy minikit
(QIAGEN). RT was performed on 100 ng total RNA using
Superscript reverse transcriptase (Life Technologies, Inc., Grand Island, NY). PCR amplification of CYP17
complementary DNA (cDNA) was performed using the direct primer
5'-TCTTGCCTGCCCGCACCCAGCCACC-3' and the reverse primer
5'-CCCTAACCCCTGGCTGAATGC-3' at the following cycling conditions: 93 C
for 1 min, 48 C for 2 min, and 68 C for 1 min for 40 cycles. The
primers contained an EcoRI and a BamHI
restriction site at their 5'-ends, respectively, to facilitate
subcloning. The mutated cDNAs were subcloned into a pCMV4 vector and
transiently transfected into confluent COS-1 cells using 50 µg
Lipofectamine and 10 µg DNA on a 10-cm plate (Life Technologies, Inc.). The correctness of the sequence was proven
by sequencing. The transfection efficiency ranged from 30–50%.
Forty-eight hours after transfection, steroidogenic precursors
(pregnenolone and progesterone for 17-hydroxylase activity and
17-hydroxypregnenolone for 17,20-lyase activity) were added at a
concentration of 1 µmol/L after suspension in 1 x phosphate
buffer. Six hours after its addition, supernatant was removed and kept
frozen at -20 C until measured. To standardize the steroid production,
cells were lysed in 1 x PBS, 1.5 mmol/L
MgCl2, 1 mmol/L ethylenediamine tetraacetate, 1%
Triton-X, and 10% glycerol in the presence of protease inhibitors (34
µg/mL phenylmethanesulfonylfluoride, 0.7 µg/mL pepstatin, and 5
µg/mL leupeptin; Roche Molecular Biochemicals), and
protein content was measured using Bio-Rad Laboratories, Inc. (Hercules, CA), protein assay reagents. The secreted
steroids, i.e. 17-hydroxyprogesterone (17-hydroxylase
activity) and DHEA (17,20-lyase activity), were measured
in duplicate by RIA using Diagnostic Product kits (Los
Angeles, CA).
The stimulation experiments were conducted in a similar way, adding 200 µmol/L 8-bromo-cAMP (Sigma, Buchs, Switzerland) 2 h before termination of the experiments. The synthesized steroids were assayed as secreted products in the medium, after 6-h incubation, using RIA kits (17-hydroxypregnenolone, ICN Biochemicals, Inc., Costa Mesa, CA; 17-hydroxyprogesterone and DHEA, Diagnostic Products, Los Angeles, CA). All values are expressed as the mean ± SD and represent the results of three independent experiments.
In vivo metabolic labeling and immunoprecipitation
Cells were metabolically labeled with either [32P]orthophosphate (32Pi; 200 µCi/mL; NEN Life Science Products, Boston, MA) for 1 h in phosphate-free medium 11963–022(11963–022, Life Technologies, Inc.) or with [35S]methionine (100 µCi/mL; NEN Life Science Products, Easytag) for 2 h in methionine-free medium (Life Technologies, Inc.). Labeled cells were lysed in 1 x PBS, 1.5 mmol/L MgCl2, 1 mmol/L ethylenediamine tetraacetate, 1% Triton X, and 10% glycerol in the presence of protease inhibitors (34 µg/mL phenylmethanesulfonylfluoride, 0.7 µg/mL pepstatin, and 5 µg/mL leupeptin; Roche Molecular Biochemical) and phosphatase inhibitors for the phosphorylation experiments (100 mmol/L sodium fluoride, 10 mmol/L sodium pyrophosphate, and 2 mmol/L sodium orthovanadate; Sigma). The lysates were clarified by centrifugation at 15,000 x g for 10 min. The supernatants were then bound to protein A-Sepharose (Pharmacia Biotech) preincubated (at least 20 min at room temperature) with anti-human CYP17 antibodies (a gift from Prof. M. Waterman, Nashville, TN) at a dilution of 1:50.000. The cell lysates were incubated on the protein A-Sepharose/antibody complex overnight at 4 C. The immune complexes were then extensively washed and analyzed on 10% SDS-PAGE.
Human NCI-H295R (CRL-2128, American Type Culture Collection, Manassas, VA) adrenocortical carcinoma cell extract was used as a positive control for CYP17 protein.
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Results |
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Two related patients of Italian origin affected by complete CYP17
deficiency were found to bear a homozygote 24-bp deletion in exon 1,
leading to the predictive loss of eight amino acid residues 70–77. The
mutant protein lost both 17-hydroxylase and 17,20-lyase activities,
as expected given the severe phenotype.
Intriguingly, analysis of the expressed gene in PBL of the parents, who
are obligate heterozygotes, showed only one product corresponding to
the wild type, as the deleted product would not be expressed. The
RT-PCR analysis of RNA derived from PBL of the affected individuals
showed the expected smaller product. When the same analysis was
performed on RNA extracted from the gonads of these two patients, only
patient 2 had the expected product, whereas patient 1 showed
consistently two smaller cDNA products (Fig. 1
).
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F53 protein retains 10% 17-hydroxylase activity and
13% 17,20-lyase activity, with no significant difference between the
two enzymatic activities. This slightly contrasts what was previously
observed (6). In fact, in the previously published work there was a
more striking divergence between the two enzymatic activities of CYP17
(20% for 17-hydroxylase and 5% for 17,20-lyase compared to wild
type). The reason for this discrepancy is at present unclear. The
results of the mutation analysis and expression studies are summarized
in Table 1.
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The F417C mutation has been previously described and has been
included in this work as the only case of isolated 17,20-lyase
deficiency confirmed at the molecular level. The CYP17 gene
of a 46,XY Turkish patient with genital ambiguity and undescended
testes was found to carry two different mutations in the two alleles.
The allele of paternal origin has a G to A transition in exon 8,
leading to a R496H missense mutation in the C-terminal region of the
protein. The allele inherited from the mother bears the missense N177D
rearrangement due to an A to G transition in exon 3. The expression
studies of the mutant proteins demonstrated that the two mutations are
responsible for a dramatic reduction of both enzymatic activities, in
contrast to the clinical phenotype of an isolated 17,20-lyase
deficiency. In particular, the N177D mutant cDNA encodes a protein with
10% 17-hydroxylase and 17,20-lyase activities. The R496H mutant
protein retains 30% 17-hydroxylase activity and 29% 17,20-lyase
activity compared to wild type. The molecular basis of an isolated
17,20-lyase defect in a third genotypically male patient, again of
Turkish ancestry, was found to be a homozygote R35L missense mutation.
The expressed protein retains 38% 17-hydroxylase and 33%
17,20-lyase activity, again in apparent contradiction to the phenotype.
All data are summarized in Table 1
.
In light of the recent findings pointing to the role of phosphorylation
in the regulation of 17,20-lyase activity, we investigated the
phosphorylation state of our mutant proteins. Although labeled
immunoprecipitable protein was seen when wild-type, N177D, R496H, R35L,
and R96W enzymes were expressed in COS-1 cells, no phosphorylated
protein was present when the previously characterized F417C mutant
protein was expressed (Fig. 2). The rate
of phosphate incorporation in the other mutants ranged from 61–107%
of that in the wild type, with no relation to the activity data, except
for the F417C mutant. To explain the apparent discrepancy between the
clinical picture and the genotype, we investigated the role of the
physiological second messenger cAMP on activities and phosphorylation
state of our 17,20-lyase-deficient mutant proteins. The addition of
8-bromo-cAMP induces an increase in 17-hydroxylase activity in all
proteins tested ranging from 1.3- to 15-fold and a comparable rise in
17,20-lyase activity in wild-type, N177D, and R496H proteins (2- to
4.5-fold). The mutants F417C and R35L showed no such change in
17,20-lyase activity upon cAMP treatment (Table 2). Consistent with that, cAMP treatment
induced an increment in phosphorylated protein in the wild-type protein
(Fig. 3) and all of the other mutant
proteins (not shown), with no effect on the amount of
immunoprecipitable F417C and R35L proteins. The changes in labeled
protein are not due to an increase in protein content, as demonstrated
by the comparable amounts of
[35S]methionine-labeled proteins (Fig. 3). The
lack of response of R35L protein to the physiological effector,
although not completely clear, can partly explain the
phenotype-genotype discrepancy. In addition, this experiment confirms
that the kinase involved in CYP17 phosphorylation is cAMP dependent, as
physiologically expected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase and
17,20-lyase. The two activities are differentially regulated in a
tissue- and time-dependent fashion, although the physiological factors
governing these phenomena are still unknown. The study of CYP17
enzymatic activity regulation is of great interest to clarify the
control of gonadal steroidogenesis in health (e.g. puberty)
and disease (e.g. polycystic ovary disease) and to discover
the mechanism underlying the maturation of the adrenal gland
(adrenarche) in children.
We identified the molecular bases of CYP17 deficiency in 7 patients
affected by complete combined 17-hydroxylase/17,20-lyase deficiency
and 3 patients suffering from an isolated 17,20-lyase defect. We
identified 10 mutations, 8 of which were never previously described.
The rearrangements are of different natures, ranging from deletions to
insertions to single base changes, and are spread throughout the gene
(Fig. 4). The expression studies
demonstrated in all complete deficiency cases that both activities were
diminished to the same extent and confirmed that CYP17 enzyme must
retain about one fourth of its catalytic capability to prevent the
onset of mineralocorticoid-dependent hypertension. Among the isolated
deficiencies, only the previously described F417C mutant could be
confirmed at the molecular level. We then took advantage of these human
knockouts to study the possible mechanisms of 17,20-lyase activity
control. With regard to the tissue-dependent differences in 17,20-lyase
activity, the abundance and interaction with the reducing agents have
been shown to be crucial for CYP17 activity regulation (1, 2).
Nevertheless, posttranslational modifications are a more feasible
mechanism to explain the developmental activation of 17,20-lyase at
adrenarche (3). We therefore investigated the phosphorylation state of
our mutant proteins. Although phosphorylated CYP17 proteins were seen
in all cases, no phosphorylated enzyme was present when the previously
characterized F417C mutant protein was expressed. This mutant allowed
us to demonstrate for the first time a connection between the electron
donor system and 17,20-lyase deficiency (1). The same rearrangement now
provides the first example of phosphorylation mutant among
CYP17-deficient patients and confirms the important role of phosphate
incorporation for 17,20-lyase activity. Moreover, our experiments
demonstrate for the first time the presence of a link between the
electron donor system and the phosphorylation state of the enzyme for
the differential regulation of 17,20-lyase vs.
17-hydroxylase activity of the human CYP17 enzyme. In search of the
physiological player(s) governing this process, we tested the influence
of the second messenger cAMP on the enzymatic activities.
Administration of cAMP stimulated both 17-hydroxylase and
17,20-lyase activities (4-fold) and protein phosphorylation (4- to
5-fold) in all cases, with the exception of the F417C and R35L mutant
proteins that did not show any change in either 17,20-lyase or
phosphorylation state under cAMP treatment. These data suggest that the
kinase responsible for the phosphorylation of CYP17 is at least in part
cAMP dependent. The lack of response to the physiological second
messenger cAMP in some mutants might provide an explanation for the
occurrence of different phenotypes and justify the discrepancy between
phenotype and genotype in some cases.
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-hydroxylase
activity in adrenal cells, although it augments the cAMP-stimulated
increase in 17,20-lyase activity in ovarian cells (Biason-Lauber, A.,
unpublished data). This is in agreement with previous observations
demonstrating that activation of the IGF-I receptor enhances
steroidogenesis in human fetal adrenal cells exclusively in the
presence of ACTH (7). IGFs appear to modulate the ACTH signal
transduction pathway at a point distal from the ACTH receptor, as the
same augmentation was seen when the second messenger cAMP was used. As
IGF-I exerts no differential control on the two enzymatic activities of
CYP17, and ACTH does not seem to play a central physiological role in
the onset of adrenarche in children (8), these two factors are probably
not directly involved in the control of 17,20-lyase activity.
In conclusion, we were able to positively diagnose and collect 10 new
cases of complete combined 17-hydroxylase/17,20-lyase deficiency and
isolated 17,20-lyase deficiency, an otherwise rare entity. That allows
us to deal with clinically and biochemically in vivo well
characterized phenotypes. More importantly, the use of mutations whose
clinical consequences are precisely definable provides a reliable
starting point to study the mechanisms involved in the regulation of
17,20-lyase vs. 17-hydroxylase activity. This approach
has proven to be particularly useful in the case of the
17,20-lyase-deficient F417C protein. In fact, F417C not only represents
our unique example of 17,20-lyase deficiency confirmed at the molecular
level and has been linked to lack of electron transfer as a possible
mechanism for the 17,20-lyase defect (1), but is also the first proven
case of a phosphorylation mutant among the CYP17 mutants. Using this
model, we could prove for the first time the existence of a link
between the electron transfer system and the phosphorylation state of
the CYP17 enzyme in the control of 17,20-lyase activity. From the
three-dimensional structural point of view, based on the model recently
described by Auchus et al. (9), F417 appears to be located
just C-terminal to the meander peptide and, although inaccessible to
solvent (and therefore probably not directly involved in redox partner
binding), appears to contribute to hydrophobic interactions that help
to stabilize the flap between the heme-binding domain and the meander
protein. Thus, F417 may help to form an edge of the redox-binding site
pocket. Although F417 is not the target of phosphorylation and is not
part of any known kinase recognition site, the loss of this aromatic
amino acid can cause conformational changes in the CYP17 protein,
impairing the binding of a specific kinase to target Ser/Thr
residues.
Furthermore, the dependency of 17,20-lyase activity and phosphorylation
on cAMP can help to identify new elements responsible for the clinical
phenotype when a phenotype-genotype discrepancy is apparent. An example
of such an event is the R35L mutant protein. Although in basal
conditions R35L protein lacks 17-hydroxylase and 17,20-lyase
activities to a similar degree, only 17,20-lyase activity failed to
respond to cAMP in the presence of the clinical picture of isolated
17,20- lyase deficiency. The implications of such findings for
identification of the agent governing adrenal and gonadal androgen
production are clear. The factor(s) involved in the control of androgen
secretion appears to function via regulation of the phosphorylation
state of CYP17 and to interact with the cAMP-dependent signal
transduction pathway. Therefore, we gained some more insights into the
nature of the still unknown physiological trigger of adrenarche in
children and perhaps into the physiopathological mechanisms underlying
androgen excess, as, for example, in functional ovarian
hyperandrogenism.
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Acknowledgments |
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Footnotes |
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Received June 15, 1999.
Revised November 19, 1999.
Accepted December 7, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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results in partial combined
17-hydroxylase/17,20-lyase deficiency. J Biol Chem. 264:18076–18082.-hydroxylase/17,20-lyase): insights into reaction
mechanisms and effects of mutations. Mol Endocrinol. 13:1169–1182.-hydroxylase/17,20-lyase deficiency due to
a stop codon in the N-terminal region of 17-hydroxylase cytochrome
P450. Mol Cell Endocrinol. 59:249.[CrossRef][Medline]
-hydroxylase/17,20-lyase (P450c17) in Escherichia coli. J Biol Chem. 268:19681–19689.-hydroxylase deficiency. J Biol Chem. 266:15992–15998.-hydroxylase/17,20-lyase deficiency
due to a 7 base pair duplication in the N-terminal region of the
cytochrome P450c17 (CYP17) gene. J Clin Endocrinol Metab. 70:1325.[Abstract]
-hydroxylase/17,20-lyase deficiency in an Italian patient. Mol
Endocrinol. 5:2037–2045.[Abstract]
-hydroxylase/17,20-lyase deficiency caused by
heterozygous stop codons in the cytochrome P450 17-hydroxylase gene. Clin Endocrinol (Oxf). 39:483–485.[Medline]
) gene lead to ambiguous external
genitalia in a male patient with partial combined
17-hydroxylase/17,20-lyase deficiency. J Clin Endocrinol Metab. 74:667–672.[Abstract]
-hydroxylase deficiency. J Biol
Chem. 268:25811–25817.-hydroxylase gene in 17-hydroxylase deficiency. J
Clin Endocrinol Metab. 82:1934–1938.-hydroxylase deficiency. J Clin Endocrinol Metab. 79:160–164.[Abstract]
-hydroxylase deficiency with
one base pair deletion of the cytochrome P450c17 (CYP17) gene. J
Clin Endocrinol Metab. 80:2526–2529.[Abstract]
Cys, Gln 461stop) actually causes
combined 17-hydroxylase/17,20-lyase deficiency. Biochim Biophys
Acta. 1139:275–279.[Medline]
-hydroxylase17,20-lyase deficiency in two Mennonite families. Hum
Genet. 82:285.[CrossRef][Medline]
-hydroxylase deficiency. J Clin Endocrinol Metab. 77:489–493.[Abstract]
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