Fusion partners of NTRK3 affect subcellular localization of the fusion kinase and cytomorphology of melanocytes.

A subset of Spitz tumors harbor fusions of NTRK3 with ETV6, MYO5A, and MYH9. We evaluated a series of 22 melanocytic tumors in which an NTRK3 fusion was identified as part of the diagnostic workup. Tumors in which NTRK3 was fused to ETV6 occurred in younger patients were predominantly composed of epithelioid melanocytes and were classified by their histopathologic features as Spitz tumors. In contrast, those in which NTRK3 was fused to MYO5A were predominantly composed of spindled melanocytes arrayed in fascicles with neuroid features such as pseudo-Verocay bodies. To further investigate the effects of the fusion kinases ETV6-NTRK3 and MYO5A-NTRK3 in melanocytes, we expressed them in immortalized melanocytes and determined their subcellular localization by immunofluorescence. ETV6-NTRK3 was localized to the nucleus and diffusely within the cytoplasm and caused melanocytes to adopt an epithelioid cytomorphology. In contrast, MYO5A-NTRK3, appeared excluded from the nucleus of melanocytes, was localized to dendrites, and resulted in a highly dendritic cytomorphology. Our findings indicate that ETV6-NTRK3 and MYO5A-NTRK3 have distinct subcellular localizations and effects on cellular morphology.

Combined activation of MAP kinase pathway and ß-catenin signaling cause deep penetrating nevi.

Deep penetrating nevus (DPN) is characterized by enlarged, pigmented melanocytes that extend through the dermis. DPN can be difficult to distinguish from melanoma but rarely displays aggressive biological behavior. Here, we identify a combination of mutations of the ß-catenin and mitogen-activated protein kinase pathways as characteristic of DPN. Mutations of the ß-catenin pathway change the phenotype of a common nevus with BRAF mutation into that of DPN, with increased pigmentation, cell volume and nuclear cyclin D1 levels. Our results suggest that constitutive ß-catenin pathway activation promotes tumorigenesis by overriding dependencies on the microenvironment that constrain proliferation of common nevi. In melanoma that arose from DPN we find additional oncogenic alterations. We identify DPN as an intermediate stage in the step-wise progression from nevus to melanoma. In summary, we delineate specific genetic alterations and their sequential order, information that can assist in the diagnostic classification and grading of these distinctive neoplasms.Deep penetrating nevi (DPN) are unusual melanocytic neoplasms with unknown genetic drivers. Here the authors show that majority of DPN harbor activating mutations in the ß-catenin and the MAP-kinase pathways; this characteristic can help in the classification and grading of these distinctive neoplasms.

NTRK3 kinase fusions in Spitz tumours.

Oncogenic fusions in TRK family receptor tyrosine kinases have been identified in several cancers and can serve as therapeutic targets. We identified ETV6-NTRK3, MYO5A-NTRK3 and MYH9-NTRK3 fusions in Spitz tumours, and demonstrated that NTRK3 fusions constitutively activate the mitogen-activated protein kinase, phosphoinositide 3-kinase and phospholipase Cγ1 pathways in melanocytes. This signalling was inhibited by DS-6051a, a small-molecule inhibitor of NTRK1/2/3 and ROS1. NTRK3 fusions expand the range of oncogenic kinase fusions in melanocytic neoplasms and offer targets for a small subset of melanomas for which no targeted options currently exist. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alternative splicing of DENND1A, a PCOS candidate gene, generates variant 2.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy characterized by hyperandrogenism and metabolic disorders. The excess androgens may be of both ovarian and adrenal origin. PCOS has a strong genetic component, and genome-wide association studies have identified several candidate genes, notably DENND1A, which encodes connecdenn 1, involved in trafficking of endosomes. DENND1A encodes two principal variants, V1 (1009 amino acids) and V2 (559 amino acids). The androgen-producing ovarian theca cells of PCOS women over-express V2. Knockdown of V2 in these cells reduces androgen production, and overexpression of V2 in normal theca cells confers upon them a PCOS phenotype of increased androgen synthesis. We report that human adrenal NCI-H295A cells express V1 and V2 mRNA and that the V2 isoform is produced by exonization of sequences in intron 20, which generates a unique exon 20A, encoding the C-terminus of V2. As in human theca cells from normal women, forced expression of V2 in NCI-H295A cells resulted in increased abundance of CYP17A1 and CYP11A1 mRNAs. We also found genetic variation in the intronic region 330 bp upstream from exon 20A, which could have the potential to drive the selective expression of V2. There was no clear association with these variants with PCOS when we analyzed genomc DNA from normal women and women with PCOS. Using minigene expression vectors in NCI-H295A cells, this variable region did not consistently favor splicing of the V2 transcript. These findings suggest increased V2 expression in PCOS theca cells is not the result of genomic sequence variation in intron 20.

Potential role of increased oxygenation in altering perinatal adrenal steroidogenesis.

BACKGROUND: At birth, the large fetal adrenal involutes rapidly, and the patterns of steroidogenesis change dramatically; the event(s) triggering these changes remain largely unexplored. Fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen of only ~2 kPa (20-23 mm Hg); perinatal circulatory changes change this to adult values (~20 kPa). We hypothesized that transition from fetal hypoxia to postnatal normoxia participates in altering perinatal steroidogenesis. METHODS: We grew midgestation human fetal adrenal cells and human NCI-H295A adrenocortical carcinoma cells in 2% O2, then transitioned them to 20% O2 and quantitated steroidogenic mRNAs by quantitative PCR and microarrays. RESULTS: Transitioning fetal adrenal cells from hypoxia to normoxia increased mRNAs for 17α-hydroxylase/17,20 lyase (P450c17), 3ß-hydroxysteroid dehydrogenase (3ßHSD2), and steroidogenic acute regulatory protein (StAR). We repeated the protocol with NCI-H295A cells acclimated to hypoxia for 15 d, quantitating 31,255 transcripts by microarray. Using an arbitrary 1.5-fold difference, 1 d of normoxia increased 4 transcripts and decreased 56, whereas 2 d of normoxia increased 62 transcripts and decreased 105. P450c17, 3ßHSD2, and StAR were ranked among the top eight increased transcripts. CONCLUSION: These data suggest that the hypoxic/normoxic transition at birth contributes to perinatal changes in adrenal steroidogenesis.

The post-translational regulation of 17,20 lyase activity.

A single enzyme, microsomal P450c17, catalyzes the 17α-hydroxylase activity needed to make cortisol and the subsequent 17,20 lyase activity needed to produce the 19-carbon precursors of sex steroids. The biochemical decision concerning whether P450c17 stops after 17α-hydroxylation or proceeds to 17,20 lyase activity is largely dependent on three post-translational factors. First, 17,20 lyase activity is especially sensitive to the molar abundance of the electron-transfer protein P450 oxidoreductase (POR). Second, cytochrome b5 strongly promotes 17,20 lyase activity, principally by acting as an allosteric factor promoting the interaction of P450c17 with POR, although a minor role as an alternative electron-transfer protein has not been wholly excluded. Third, the serine/threonine phosphorylation of P450c17 itself promotes 17,20 lyase activity, again apparently by promoting the interaction of P450c17 with POR. The principal kinase that phosphorylates P450c17 to confer 17,20 lyase activity appears to be p38α (MAPK14), which increases the maximum velocity of the 17,20 lyase reaction, while having no effect on the Michaelis constant for 17,20 lyase or any detectable effect on the 17α-hydroxylase reaction. Other kinases can also phosphorylate P450c17, but only p38α has been shown to affect its enzymology. Understanding the mechanisms regulating 17,20 lyase activity is essential for the understanding of hyperandrogenic disorders such as premature, exaggerated adrenarche and the polycystic ovary syndrome, and also for the design of selective 17,20 lyase inhibitors for use in hyperandrogenic states and in sex-steroid dependent cancers.

Quantitation of CYP24A1 enzymatic activity with a simple two-hybrid system.

CONTEXT: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult. OBJECTIVE: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years. METHODS: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system. RESULTS: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012). CONCLUSIONS: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.

Phosphorylation of human cytochrome P450c17 by p38α selectively increases 17,20 lyase activity and androgen biosynthesis.

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17α-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b5 to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17α-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38α or p38ß. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38α, but not p38ß, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38α, but not knockdown of p38ß, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38α in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38α phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.

Varied clinical presentations of seven patients with mutations in CYP11A1 encoding the cholesterol side-chain cleavage enzyme, P450scc.

CONTEXT: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously. OBJECTIVE: We sought to expand clinical and genetic experience with P450scc deficiency. PATIENTS AND METHODS: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel. RESULTS: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity. CONCLUSIONS: Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

Distinguishing deficiencies in the steroidogenic acute regulatory protein and the cholesterol side chain cleavage enzyme causing neonatal adrenal failure.

OBJECTIVES: To determine the genetic basis of disordered steroidogenesis in Kuwaiti siblings. STUDY DESIGN: Two siblings (46,XX and 46,XY) had normal female external genitalia and severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Abdominal ultrasonography showed normal size adrenal glands, suggesting cholesterol side chain cleavage enzyme (P450scc) deficiency. The CYP11A1 gene encoding P450scc and the STAR gene encoding the steroidogenic acute regulatory protein (StAR) were directly sequenced from leukocyte DNA. RESULTS: All exons and intron/exon boundaries of the CYP11A1 gene were normal; the STAR gene was homozygous for a novel 14-base deletion/frameshift in exon 4 (g.4643_4656del), so that no functional protein could be produced. Both parents and an unaffected sibling were heterozygous; zygosity was confirmed with a BsmF1 restriction fragment length polymorphism. CONCLUSIONS: Unlike most patients with StAR deficiency, our patients did not have the massive adrenal hyperplasia typical of congenital lipoid adrenal hyperplasia. The distinction between StAR and P450scc deficiency may require gene sequencing.

Expression of P450c17 in the human fetal nervous system.

P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain. P450c17 was readily detected in the dorsal root ganglia (DRG) and spinal cord. Neural structures were identified with antisera to the cytoskeletal protein neural cell adhesion molecule; DRG were identified with antisera to the neuronal transcription factor BRN3A and neurotrophin receptor tropomyosin-receptor-kinase B. The identification of P450c17 was confirmed using commercial antisera directed against different domains of P450c17 and by using antisera immunodepleted with authentic human P450c17. We also found expression of the P450 cholesterol side-chain cleavage enzyme (P450scc) in the spinal cord and DRG. Expression of P450scc is limited to cell bodies; unlike P450c17, we never detected P450scc in fiber tracts. Catalysis by P450c17 requires electron donation from P450 oxidoreductase (POR). Dual-label immunohistochemistry detected P450c17 and POR colocalized in DRG bundles, but some fibers containing P450c17 lacked POR. These data suggest that neurosteroids synthesized via these two enzymes may act in the developing human nervous system. The expression of P450c17 in structures lacking POR means that P450c17 may not be steroidogenic in those locations, suggesting that P450c17 may have additional functions that do not require POR.

Transcriptional regulation of the human P450 oxidoreductase gene: hormonal regulation and influence of promoter polymorphisms.

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon. Common human POR polymorphisms at -208 and -173 had little influence on transcription, but the polymorphism at -152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between -249 and -261 and binding of thyroid hormone receptor-ß (TRß) at -240/-245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRß, and estrogen receptor-α were bound between -374 and -149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T(3) exerts major tropic effects via TRß, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T(3) also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRß and Smad3/4 in its expression and indicating that the common polymorphism at -152 may play a role in genetic variation in steroid biosynthesis and drug metabolism.

Molecular regulation of human placental growth factor (PlGF) gene expression in placental villi and trophoblast cells is mediated via the protein kinase a pathway.

Cyclic 3',5'-adenosine monophosphate (cAMP) is a critical second messenger for human trophoblasts and regulates the expression of numerous genes. It is known to stimulate in vitro the fusion and differentiation of BeWo choriocarcinoma cells, which acquire characteristics of syncytiotrophoblasts. A DNA microarray analysis of BeWo cells undergoing forskolin-induced syncytialization revealed that among the induced genes, placental growth factor (PlGF) was 10-fold upregulated. We verified this result in two choriocarcinoma cell lines, BeWo and JEG-3, and also in first trimester placental villous explants by quantifying PlGF mRNA (real time PCR) and PlGF protein secreted into the supernatant (ELISA). Similar effects were noted for vascular endothelial growth factor (VEGF) mRNA and protein expression. Treatment with cholera toxin and the use of a specific inhibitor of protein kinase A (PKA) blocked these effects, indicating that the cAMP/PKA pathway is responsible for the cAMP-induced upregulation of PlGF and that one or more G protein coupled receptor(s) was involved. We identified two functional cAMP responsive elements (CRE) in the PlGF promoter and demonstrated that the CRE binding protein, CREB, contributes to the regulation of PlGF gene expression. We speculate that defects in this signaling pathway may lead to abnormal secretion of PlGF protein as observed in the pregnancy-related diseases preeclampsia and intrauterine growth restriction.

Partial defect in the cholesterol side-chain cleavage enzyme P450scc (CYP11A1) resembling nonclassic congenital lipoid adrenal hyperplasia.

CONTEXT: The cholesterol side-chain cleavage enzyme (P450scc), encoded by the CYP11A1 gene, converts cholesterol to pregnenolone to initiate steroidogenesis. Genetic defects in P450scc cause a rare autosomal recessive disorder that is clinically indistinguishable from congenital lipoid adrenal hyperplasia (lipoid CAH). Nonclassic lipoid CAH is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function. OBJECTIVE: We describe two siblings with hormonal findings suggesting nonclassic lipoid CAH, who had a P450scc mutation that retains partial function. PATIENTS AND METHODS: A 46,XY male presented with underdeveloped genitalia and partial adrenal insufficiency; his 46,XX sister presented with adrenal insufficiency. Hormonal studies suggested nonclassic lipoid CAH. Sequencing of the StAR gene was normal, but compound heterozygous mutations were found in the CYP11A1 gene. Mutations were recreated in the F2 plasmid expressing a fusion protein of the cholesterol side-chain cleavage system. P450scc activity was measured as Vmax/Km for pregnenolone production in transfected COS-1 cells. RESULTS: The patients were compound heterozygous for the previously described frameshift mutation 835delA and the novel missense mutation A269V. When expressed in the P450scc moiety of F2, the A269V mutant retained 11% activity of the wild-type F2 protein. CONCLUSIONS: There is a broad clinical spectrum of P450scc deficiency. Partial loss-of-function CYP11A1 mutation can present with a hormonal phenotype indistinguishable from nonclassic lipoid CAH.

Consequences of POR mutations and polymorphisms.

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism.

Functional characterization of vasopressin receptor 2 mutations causing partial and complete congenital nephrogenic diabetes insipidus in Thai families.

BACKGROUND: AVPR2 mutations cause most cases of nephrogenic diabetes insipidus (NDI); 211 AVPR2 mutations have been described, but only 7 are described causing partial NDI. METHODS: Two unrelated Thai boys had polyuria and polydipsia in infancy but had normal electrolytes and serum osmolality at 2 years of age. Patient 1 could not concentrate his urine in response to water deprivation or 1-desamino-8-D-arginine vasopressin (DDAVP); patient 2 could concentrate to approximately 600 mosm/l. The patients' AVPR2 genes were sequenced and the identified mutations were re-created in AVPR2 cDNA expression vectors. AVPR2 activities were measured by stimulating transfected HEK293T cells with arginine vasopressin (AVP) or DDAVP, and assessing the resulting cAMP production by the activation of a luciferase reporter. RESULTS: Patient 1 carried the previously described missense mutation R181C; patient 2 carried the novel missense mutation M311V. When transiently transfected into HEK293T cells, 6.8 x 10(-12) M AVP induced the half-maximal response (EC50) of the wild-type, whereas the EC50 value for R181C was 5.9 x 10(-9) M and for M311V was 2.6 x 10(-10)M. Responses to DDAVP were qualitatively similar but required 10-fold higher concentrations. CONCLUSION: The novel AVPR2 mutation M311V retains partial activity and results in a milder form of NDI.

Human cytochrome p450c17: single step purification and phosphorylation of serine 258 by protein kinase a.

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4xHis tag is replaced by 3xGly6xHis. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258.

Clinical, genetic, and enzymatic characterization of P450 oxidoreductase deficiency in four patients.

CONTEXT: P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility. OBJECTIVE: We describe four patients with POR deficiency and identify and characterize the activities of their mutations. A 46,XY male with micropenis and two 46,XX female infants with genital ambiguity presented with skeletal malformations, and a 46,XX adolescent presented with primary amenorrhea, elevated 17alpha-hydroxyprogesterone, and low sex steroids. METHODS: The coding regions of the POR gene were sequenced, and the identified mutations were recreated in human POR cDNA expression vectors lacking 27 N-terminal residues. POR and human P450c17 were expressed in bacteria. POR activity was measured by four assays: reduction of cytochrome c, oxidation of reduced nicotinamide adenine dinucleotide phosphate, and support of the 17alpha-hydroxylase and 17,20 lyase activities of P450c17. RESULTS: All four patients were compound heterozygotes for POR mutations, including five novel mutations: L577R, N185K, delE217, and frameshift mutations 1363delC and 697-698insGAAC. N185K and delE217 lacked measurable activity in the assays based on P450c17 but retained partial activity in the assays based on cytochrome c. As assessed by V(max)/Km, L577R supported 46% of 17alpha-hydroxylase activity but only 27% of 17,20 lyase activity. Computational modeling of these novel mutants revealed the structural basis for their reduced or absent activities. CONCLUSION: These patients illustrate the broad clinical spectrum of POR deficiency, including amenorrhea and infertility as the sole manifestation. POR assays based on P450c17 correlate well with hormonal and clinical phenotypes.

Novel P450c17 mutation H373D causing combined 17alpha-hydroxylase/17,20-lyase deficiency.

CONTEXT: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare autosomal recessive form of congenital adrenal hyperplasia presenting with hypertension and sexual infantilism. This disorder is caused by defects in P450c17, encoded by the CYP17A1 gene. OBJECTIVE: We describe a 14-yr-old female with clinical and hormonal features of 17alpha-hydroxylase/17,20-lyase deficiency and identify and characterize the activities of her CYP17A1 mutations. METHODS: The coding regions of the CYP17A1 gene were amplified by PCR and sequenced. Mutations were recreated in P450c17 cDNA expression vectors; activities in transfected COS-1 cells were assayed by conversion of radiolabeled precursor steroids. One mutant was also expressed in Escherichia coli, and the reduced adsorption spectrum was measured. RESULTS: The patient carried the previously described mutation R96W and the novel missense mutation H373D. Neither mutant had detectable activity when expressed in COS-1 cells. Membrane preparations from E. coli expressing the H373D mutant vector produced an absorption peak at 420 nm, whereas the wild-type produced a peak at 450 nm, suggesting that the H373D mutation interferes with protein folding. CONCLUSION: The novel P450c17 mutation H373D abolished enzyme activity because of protein misfolding. These data indicate an important role for this residue in P450c17 activity.

Natural and recombinant human glycodelin activate a proapoptotic gene cascade in monocyte cells.

Glycodelin-A (GdA) is a member of the superfamily of lipocalins and the predominant glycoprotein secreted by human and primate endometrium in the secretory and early pregnancy phases. GdA can inhibit NK cell activity, T cell proliferation, and chemotaxis of monocytes. Its physiological function is thought to mediate immunotolerance at the fetomaternal interface. In the present studies, we engineered recombinant Gd (rGd) in yeast and tested its biological effects on monocyte viability. rGd, like the natural, purified endometrial GdA, is glycosylated and secreted, and they both induced apototic changes in monocytic U937 cells and primary human monocytes. Trypan blue exclusion, nucleosome release, DNA laddering, and immunocytochemistry to detect free 3'-OH DNA ends were used to characterize the effects of GdA and rGd. Using U937 cells as a model, cDNA microarray analyses revealed several pro- and antiapoptotic genes that were up- and down-regulated, respectively, in accordance with the kinetics of rGd-induced monocyte cell death. Real-time RT-PCR confirmed that Bad, Bax, and TNF-R1 gene expression were increased, whereas Bcl-2A1 and a proliferation-inducing ligand (APRIL) were reduced by rGd. Transfection assays in U937 cells indicated that the immunomodulatory actions of rGd were associated with NF-kappaB inhibition. Western blotting of U937 and primary monocyte lysates demonstrated that rGd activated caspase-8, -2, and -3 to execute programmed cell death in these cells. We postulate that infiltrating monocytes and potentially other innate immune cells of the decidua might be manipulated by this glycoprotein to enhance embryonic implantation rates or conversely, to develop novel contraceptive strategies.