The genetic and functional basis of isolated 17,20-lyase deficiency.

Human male sexual differentiation requires production of fetal testicular testosterone, whose biosynthesis requires steroid 17,20-lyase activity. Patients with putative isolated 17,20-lyase deficiency have been reported. The existence of true isolated 17,20-lyase deficiency, however, has been questioned because 17 alpha-hydroxylase and 17,20-lyase activities are catalyzed by a single enzyme, microsomal cytochrome P450c17, and because the index case of apparent isolated 17,20-lyase deficiency had combined deficiencies of both activities. We studied two patients with clinical and hormonal findings suggestive of isolated 17,20-lyase deficiency. We found two patients homozygous for substitution mutations in CYP17, the gene encoding P450c17. When expressed in COS-1 cells, the mutants retained 17 alpha-hydroxylase activity but had minimal 17,20-lyase activity. Substrate competition experiments suggested that the mutations did not alter the enzyme's substrate-binding capacity, but co-transfection of cells with P450 oxidoreductase, the electron donor used by P450c17, indicated that the mutants had a diminished ability to interact with redox partners. Computer-graphic modelling of P450c17 suggests that both mutations lie in or near the redox-partner binding site, on the opposite side of the haem from the substrate-binding pocket. These mutations alter electrostatic charge distribution in the redox-partner binding site, so that electron transfer for the 17,20-lyase reaction is selectively lost or diverted to uncoupling reactions. These are the first proven cases of isolated 17,20-lyase deficiency, and they demonstrate a novel mechanism for loss of enzymatic activity.

Tenascin-X deficiency is associated with Ehlers-Danlos syndrome.

The tenascins are a family of large extracellular matrix proteins with at least three members: tenascin-X (TNX), tenascin-C (TNC, or cytotactin) and tenascin-R (TN-R, or restrictin). Although the tenascins have been implicated in a number of important cellular processes, no function has been clearly established for any tenascin. We describe a new contiguous-gene syndrome, involving the CYP21B and TNX genes, that results in 21-hydroxylase deficiency and a connective-tissue disorder consisting of skin and joint hyperextensibility, vascular fragility and poor wound healing. The connective tissue findings are typical of the Ehlers-Danlos syndrome (EDS). The abundant expression of TNX in connective tissues is consistent with a role in EDS, and our patient's skin fibroblasts do not synthesize TNX protein in vitro or in vivo. His paternal allele carries a novel deletion arising from recombination between TNX and its partial duplicate gene, XA, which precludes TNX synthesis. Absence of TNX mRNA and protein in the proband, mapping of the TNX gene and HLA typing of this family suggest recessive inheritance of TNX deficiency and connective-tissue disease. Although the precise role of TNX in the pathogenesis of EDS is uncertain, this patient's findings suggest a unique and essential role for TNX in connective-tissue structure and function.

Evaluation of mosquito responses to pyrethroid insecticides topically applied to sheep.

A rise in the incidence of mosquito-transmitted Cache Valley virus (CVV) in lambs in 2011 prompted a study to evaluate on-animal pyrethroid insecticides to reduce mosquito attacks on sheep. Using enclosure traps for 1 night per wk for 6 wk, we compared engorgement rates of mosquitoes given the opportunity to feed on untreated sheep and sheep treated with 1 Python insecticide ear tag (containing 10% zeta-cypermethrin and 20% piperonyl butoxide) per animal or 2 synergized permethrin body spray treatments (containing 2.5% permethrin and 2.5% piperonyl butoxide). During the 6-wk study, 18,920 mosquitoes were collected in the animal-baited enclosure traps. Thirteen species were identified from these collections with the floodwater species Aedes increpitus and Ae. idahoensis making up 68% of the total. Potential CVV vector species, making up 25% of the samples, included Ae. vexans, Ae. dorsalis, Culex tarsalis, and Culiseta inornata. Traps baited with untreated sheep collected 9,701 mosquitoes with 65% of these engorged. Traps baited with sheep treated with Python ear tags or permethrin spray collected 4,034 and 4,555, respectively, with engorgement rates of 23% and 35%. Blood feeding on ear-tagged sheep was significantly reduced by as much as 90% compared to the untreated sheep, and protection lasted 4 wk or longer. Permethrin spray treatments were most effective within 24 h after application and provided better protection against Ae. dorsalis than the Python tag. Effectiveness of the permethrin spray diminished 1 wk after the 2nd application was made. The effect of these treatments appeared to be repellency because negligible mosquito mortality was observed at the time of collection. Further evaluation of these insecticides under conditions of natural exposure to a mosquito-borne pathogen is warranted.

Tenascin-X: a novel extracellular matrix protein encoded by the human XB gene overlapping P450c21B.

A human gene termed XB overlaps the P450c21B gene encoding steroid 21-hydroxylase and encodes a protein that closely resembles extracellular matrix proteins. Sequencing of phage and cosmid clones and of cDNA fragments shows that the XB gene spans 65 kb of DNA, consisting of 39 exons that encode a 12-kb mRNA. The predicted protein of over 400 kD consists of five distinct domains: a signal peptide, a hydrophobic domain containing three heptad repeats, a series of 18.5 EGF-like repeats, 29 fibronectin type III repeats, and a carboxy-terminal fibrinogen-like domain. Because the structure of the protein encoded by the XB gene closely resembles tenascin, we term this protein tenascin-X (TN-X), and propose a simplified nomenclature system for the family of tenascins. RNase protection experiments show that the TN-X transcript is expressed ubiquitously in human fetal tissues, with the greatest expression in the fetal testis and in fetal skeletal, cardiac, and smooth muscle. Two adrenal-specific transcripts, P450c21B (steroid 21-hydroxylase) and Y (an untranslated transcript) overlap the XB gene on the complementary strand of DNA, yielding a unique array of overlapping transcripts: a "polygene." In situ hybridization histochemistry experiments show that the TN-X transcript and the P450c21 and Y transcripts encoded on the complementary DNA strand are all expressed in the same cells of the human adrenal cortex. Genetic data suggest that TN-X may be essential for life.

Role of steroidogenic acute regulatory protein in adrenal and gonadal steroidogenesis.

Congenital lipoid adrenal hyperplasia is an autosomal recessive disorder that is characterized by impaired synthesis of all adrenal and gonadal steroid hormones. In three unrelated individuals with this disorder, steroidogenic acute regulatory protein, which enhances the mitochondrial conversion of cholesterol into pregnenolone, was mutated and nonfunctional, providing genetic evidence that this protein is indispensable normal adrenal and gonadal steroidogenesis.

Extraadrenal steroid 21-hydroxylation is not mediated by P450c21.

The 21-hydroxylation of progesterone to deoxycorticosterone (DOC) and of 17-hydroxyprogesterone to 11-deoxycortisol in the human adrenal cortex is mediated by a single enzyme termed P450c21. Extraadrenal tissues can clear circulating progesterone and progesterone sulfate by 21-hydroxylation to DOC and DOC-sulfate. It has previously been established that such extraadrenal 21-hydroxylase activity is widely distributed in adult and fetal tissues, but it has not been known if extra-adrenal 21-hydroxylation is mediated by the same P450c21 enzyme found in the adrenal. We examined human RNA from fetal adrenal, liver, kidney, lung, brain, heart, skin, spleen, testis, and placenta by solution hybridization to human P450c21 probes transcribed from cloned human P450c21 cDNA, followed by nuclease protection and acrylamide gel electrophoresis. No P450c21 mRNA was detectable in any extraadrenal tissue. The sensitivity of the assay would have detected P450c21 mRNA at 0.01% of its abundance in the human fetal adrenal. Similar experiments in rats showed no P450c21 mRNA in brain, heart, kidney, liver, lung, testis, ovary, or uterus. These results clearly demonstrate that one or more enzymes other than the classical adrenal 21-hydroxylase are responsible for human and rat extraadrenal 21-hydroxylation.

Human chorionic gonadotropin and 8-bromo cyclic adenosine monophosphate promote an acute increase in cytochrome P450scc and adrenodoxin messenger RNAs in cultured human granulosa cells by a cycloheximide-insensitive mechanism.

Treatment of human granulosa cells with human chorionic gonadotropin (hCG) or an analogue of its second messenger, cyclic AMP (cAMP), promotes a rapid accumulation of the messenger RNAs (mRNAs) for cytochrome P450 side-chain cleavage (scc) and adrenodoxin. A twofold increase in the cellular content of these mRNAs was observed within 4 h of exposure to 8-bromo-cAMP, and was maintained for up to 48 h. Inhibition of protein synthesis by cycloheximide did not prevent the hCG- or 8-bromo-cAMP-stimulated accumulation of either cytochrome P450scc or adrenodoxin mRNAs. We conclude that human granulosa cells respond rapidly to hCG and cAMP analogues with a coordinate increase in levels of the mRNAs encoding two key proteins of the steroidogenic machinery, and that this stimulation does not require synthesis of a protein intermediate.

Integrated cardiac, renal, and endocrine actions of endothelin.

Endothelin, a newly discovered endothelial-derived peptide, has been demonstrated in vitro to have potent vasocontractile properties and has been speculated to play a role in vivo in arterial pressure-volume homeostasis. The present studies in anesthetized dogs were designed to determine the action of endothelin on cardiovascular-renal and endocrine function in vivo as in acute arterial pressure-volume regulation. Intravenous infusion of endothelin (50 ng/kg per min) increases arterial pressure by increasing peripheral vascular resistance but in association with an increase in coronary vascular resistance and decreases in cardiac output. Renal blood flow and glomerular filtration rate were markedly reduced in association with a sustained reduction in sodium excretion and an increase in plasma renin activity. Atrial natriuretic factor, vasopressin, and aldosterone were also elevated. These results indicate that endothelin is a potent vasoconstrictor that elevates systemic blood pressure in association with marked decreases in cardiovascular and renal function. This peptide may function as a counterregulatory hormone to the effects of endothelial-derived vasodilator agent(s).

Role of endogenous atrial natriuretic factor in acute congestive heart failure.

The current studies were designed to investigate the functional significance of elevated endogenous atrial natriuretic factor (ANF) in acute congestive heart failure (CHF). Integrated cardiorenal and endocrine function were measured in three models of acute low-output congestive heart failure with comparably reduced cardiac output (CO) and mean arterial pressure (MAP). Acute CHF was produced by rapid right ventricular pacing (group I, n = 5) which decreases CO and increases atrial pressures and plasma ANF. In group II, n = 5, thoracic inferior vena caval constriction (TIVCC) was produced to decrease venous return and CO but without increases in atrial pressure or plasma ANF. In group III, n = 5, TIVCC was performed and exogenous ANF infused to achieve plasma concentrations observed in acute CHF. In acute CHF with increases in endogenous ANF, sodium excretion (UNaV), renal blood flow (RBF), plasma renin activity (PRA), and plasma aldosterone (PA) were maintained despite decreases in CO and MAP. In contrast, TIVCC with similar reductions in CO and MAP but without increases in ANF resulted in decreases in UNaV and RBF and increases in PRA and PA. Exogenous administration of ANF in TIVCC to mimic levels in acute CHF prevented sodium retention, renal vasoconstriction, and activation of renin and aldosterone. These studies demonstrate that endogenous ANF serves as an important physiologic volume regulator in acute CHF to maintain sodium excretion and possibly participate in the suppression of activation of the renin-angiotensin-aldosterone system despite the stimulus of arterial hypotension.

Normal genes for the cholesterol side chain cleavage enzyme, P450scc, in congenital lipoid adrenal hyperplasia.

Congenital lipoid adrenal hyperplasia is the most severe form of congenital adrenal hyperplasia. Affected individuals can synthesize no steroid hormones, and hence are all phenotypic females with a severe salt-losing syndrome that is fatal if not treated in early infancy. All previous studies have suggested that the disorder is in the cholesterol side chain cleavage enzyme (P450scc), which converts cholesterol to pregnenolone. A newborn patient was diagnosed by the lack of significant concentrations of adrenal or gonadal steroids either before or after stimulation with corticotropin (ACTH) or gonadotropin (hCG). The P450scc gene in this patient and in a previously described patient were grossly intact, as evidenced by Southern blotting patterns. Enzymatic (polymerase chain reaction) amplification and sequencing of the coding regions of their P450scc genes showed these were identical to the previously cloned human P450scc cDNA and gene sequences. Undetected compound heterozygosity was ruled out in the new patient by sequencing P450scc cDNA enzymatically amplified from gonadal RNA. Northern blots of gonadal RNA from this patient contained normal sized mRNAs for P450scc and also for adrenodoxin reductase, adrenodoxin, sterol carrier protein 2, endozepine, and GRP-78 (the precursor to steroidogenesis activator peptide). These studies show that lipoid CAH is not caused by lesions in the P450scc gene, and suggest that another unidentified factor is required for the conversion of cholesterol to pregnenolone, and is disordered in congenital lipoid adrenal hyperplasia.

Rearrangements and point mutations of P450c21 genes are distinguished by five restriction endonuclease haplotypes identified by a new probing strategy in 57 families with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) is caused by disorders of the P450c21B gene, which, with the P450c21A pseudogene, lies in the HLA locus on chromosome 6. The near identity of nucleotide sequences and endonuclease cleavage sites in these A and B loci makes genetic analysis of this disease difficult. We used a genomic DNA probe that detects the P450c21 genes (A pseudogene, 3.2 kb; B gene, 3.7 kb in Taq I digests) and the 3' flanking DNA not detected with cDNA probes (A pseudogene, 2.4 kb; B gene, 2.5 kb) to examine Southern blots of genomic DNA from 68 patients and 165 unaffected family members in 57 families with CAH. Of 116 CAH-bearing chromosomes, 114 could be sorted into five easily distinguished haplotypes based on blots of DNA digested with Taq I and Bgl II. Haplotype I (76 of 116, 65.6%) was indistinguishable from normal and therefore bore very small lesions, presumably point mutations. Haplotype II (4 of 116, 3.4%) and haplotype III (8 of 116, 6.9%) had deletions and duplications of the P450c21A pseudogene but had structurally intact P450c21B genes presumably bearing point mutations; point mutation thus was the genetic defect in 88 of 116 chromosomes (75.9%). Haplotypes IV and V lack the 3.7-kb Taq I band normally associated with the P450c21B gene. Haplotype IV (13 of 116, 11.2%) retains all other bands, indicating that the P450c21B gene has undergone a gene conversion event, so that it is now also associated with a 3.2-kb band. Haplotype V (13 of 116, 11.2%) lacks the 2.4-kb Taq I fragment and the 12-kb Bgl II fragments normally associated with the P450c21A pseudogene, as well as lacking the 3.7-kb Taq I fragment, indicating deletion of approximately 30 kb of DNA, resulting in a single hybrid P450c21A/B gene. Most (114 of 116, 98%) CAH alleles thus can easily be classified with this new probing strategy, eliminating many ambiguities resulting from probing with cDNA.

Mechanism and consequences of the duplication of the human C4/P450c21/gene X locus.

The adjacent C4 and P450c21 genes encode the fourth component of serum complement and steroid 21-hydroxylase respectively, and are tandemly duplicated in the human, murine, and bovine genomes. We recently cloned a cDNA for another duplicated gene, operationally termed X, which overlaps the 3' end of human P450c21 and has the opposite transcriptional orientation. Thus, the organization of the locus is 5'-C4A-21A-XA-C4B-21B-XB-3' (Y. Morel, J. Bristow, S. E. Gitelman, and W. L. Miller, Proc. Natl. Acad. Sci. USA 86:6582-6586, 1989). To determine how this locus was duplicated, we sequenced the DNA at the duplication boundaries and the 7 kb between P450c21A and C4B comprising the XA locus. The sequences located the duplication boundaries precisely and indicate that the duplication occurred by nonhomologous recombination. The boundaries are substantially different from those of the corresponding duplication in the mouse genome, suggesting that similar gene duplications may have occurred independently in ancestors of rodents and primates after mammalian speciation. Compared with XB, the XA gene is truncated at its 5' end and bears a 121-bp intragenic deletion causing a frameshift and premature translational stop signal. Nevertheless, XA is transcribed into a stable 2.6-kb polyadenylated RNA that is expressed uniquely in the adrenal gland.

Human P450scc gene transcription is induced by cyclic AMP and repressed by 12-O-tetradecanoylphorbol-13-acetate and A23187 through independent cis elements.

Long-term regulation of mammalian steroid hormone synthesis occurs principally by transcriptional regulation of the gene for the rate-limiting cholesterol side-chain cleavage enzyme P450scc. Adrenal steroidogenesis is regulated primarily by two hormones: adrenocorticotropin, which works via cyclic AMP (cAMP) and protein kinase A, and angiotensin II, which works via Ca2+ and protein kinase C. Forskolin and 8-bromo-cAMP stimulated, while prolonged treatment with a phorbol ester (12-O-tetradecanoylphorbol-13-acetate [TPA]) and a calcium ionophore (A23187) additively suppressed accumulation of endogenous P450scc mRNA in transformed murine adrenal Y1 cells. In Y1 cells transfected with 2,327 base pairs of the human P450scc promoter fused to the bacterial gene for chloramphenicol acetyltransferase (CAT), forskolin increased CAT activity 900% while combined TPA plus A23187 reduced CAT activity to 15% of the control level. Forskolin induced the P450scc promoter as rapidly as a promoter containing two cAMP-responsive elements fused to a simian virus 40 promoter, a system known to respond directly to cAMP. Basal expression was increased by sequences between -89 and -152 and was increased further by sequences between -605 and -2327. This upstream region also conferred inducibility by cAMP. TPA plus A23187 transiently increased CAT activity before repressing it, reflecting the complex actions of angiotensin II in vivo. Repression by prolonged treatment with TPA plus A23187 was mediated by multiple elements between -89 and -343. Induction of CAT activity by forskolin was not diminished by treatment with TPA plus A23187, nor were the regions of the promoter responsible for regulation by the two pathways coisolated. Thus, the human gene for P450scc is repressed by TPA plus A23187 by mechanisms and sequences independent of those that mediate induction by cAMP.

Prevalence of thiamine deficiency in a stable heart failure outpatient cohort on standard loop diuretic therapy.

BACKGROUND & AIMS: The prevalence of thiamine deficiency in heart failure (HF) patients has been reported to be as high as 50% in outpatient settings and has been found to be as high as 96% in the inpatient setting. Results from previous studies, however, have been inconsistent and further investigation is needed to clarify the true prevalence of thiamine deficiency in patients with chronic HF. The aim of this study was to determine the prevalence of thiamine deficiency in a random sample of stable HF outpatients receiving standard of care loop diuretic therapy. METHODS: A cross-sectional study was conducted in 30 HF patients scheduled for regular follow-up visits in the Mayo Heart Failure Clinic. Whole-blood thiamine diphosphate was measured using high-performance liquid chromatography. Additional clinical and demographic features were collected through review of electronic medical records. RESULTS: The estimated prevalence of thiamine deficiency in stable HF patients was calculated to be <11.6%. There was no correlation between diuretic dose and thiamine levels (r = 0.02, P = 0.93) and there was no correlation found between left-ventricular ejection fraction (LVEF) and thiamine levels (r = 0.147, p = 0.44). CONCLUSION: Our findings suggest that the prevalence of thiamine deficiency, based on standard normal values, in a stable outpatient HF cohort on standard loop diuretic therapy is very low. Previous work has demonstrated improvements in myocardial function with high-dose thiamine supplementation regardless of thiamine blood levels, however, suggesting that thiamine may become conditionally essential with HF. Therefore, we suggest that a disease-specific reference range be determined to accurately identify HF patients that would benefit from thiamine supplementation.

Indomethacin attenuates the constriction of canine epicardial coronary arteries to acetylcholine in the absence of endothelium: contribution of platelets to vasoconstriction in vivo.

This study was designed to evaluate the in vivo effect of acetylcholine on endothelial-damaged canine epicardial coronary arteries and the potential contribution of platelets to those acetylcholine-induced responses. Changes in left anterior descending artery cross-sectional area were determined by quantitative angiography in the closed chest anesthetized dog. Baseline cross-sectional area of the left anterior descending artery was not changed by removal of the endothelium by balloon-tipped catheter. Increased constrictor tone produced by prostaglandin F2 alpha was comparable in endothelium-intact and endothelium-removed vessels, supporting an endothelium-independent mechanism for prostaglandin F2 alpha in vivo. Acetylcholine produced anterior descending artery vasodilation with the endothelium intact; a comparable maximal dilator response was also obtained in the presence of increased constrictor tone (prostaglandin F2 alpha). In contrast, acetylcholine produced vasoconstriction of the anterior descending artery when the endothelium was removed. To evaluate the mechanism of acetylcholine-induced vasoconstriction in endothelium-removed vessels, the same protocol was completed in the presence of the platelet inhibitor indomethacin. Indomethacin did not alter baseline cross-sectional area or the dilator response to acetylcholine in endothelium-intact vessels. In contrast, the constrictor response in endothelium-removed vessels was antagonized, and a dilator response comparable with that in endothelium-intact vessels was produced by acetylcholine. The results of this study provide an experimental basis for the observations in human studies in which apparently atherosclerotic vessels constrict in response to acetylcholine. Removal of the endothelium in vivo abolishes the dilator response to acetylcholine and converts the acetylcholine response to vasoconstriction or vasospasm.(ABSTRACT TRUNCATED AT 250 WORDS)

C-type natriuretic peptide-mediated coronary vasodilation: role of the coronary nitric oxide and particulate guanylate cyclase systems.

OBJECTIVES: We tested the hypothesis that C-type natriuretic peptide (CNP) mediates coronary vasodilation through activation of cyclic guanosine monophosphate (cGMP) by way of particulate guanylate cyclase. BACKGROUND: CNP has known peripheral vasodilator properties, and preliminary data have suggested that it can function as a coronary vasodilator. METHODS: The actions of CNP were studied in instrumented dogs and in organ chamber rings in the presence and absence of a known antagonist to particulate guanylate cyclase, HS-142-1. Additionally, the actions of HS-142-1 were tested on acetylcholine-mediated coronary vasodilation, and immunohistochemical staining was utilized to localize the presence of CNP in the coronary endothelium. RESULTS: CNP relaxed isolated coronary arteries with (mean +/- SEM 45.9 +/- 7%*) and without (72.0 +/- 7%*) an endothelium (*p < 0.05 for CNP effect alone, p < 0.05 for endothelium vs. no endothelium with CNP). Intracoronary infusions increased coronary blood flow (baseline, 64.6 +/- 5.1 ml/min; CNP-5, 79.9 +/- 6.1*; CNP-20, 103.3 +/- 13.6* [*p < 0.05 vs. baseline value]) and reduced coronary vascular resistance (baseline, 1.6 +/- 0.3 mm Hg/ml per min; CNP-5, 1.4 +/- 0.3*; CNP-20, 1.2 +/- 0.3*). Intracoronary injections increased coronary blood flow (delta baseline coronary flow, 30 +/- 9* ml/min [*p < 0.05]). HS-142-1 significantly attenuated these increases (delta coronary flow, 30 +/- 9* ml/min [CNP] to 14 +/- 6 [CNP + HS-142-1] [p < 0.05 CNP vs. CNP + HS-142-1]) and the relaxation of organ chamber rings (56 +/- 7% [CNP] to 18 +/- 6% [HS-142-1 + CNP]). Finally, CNP was localized to the coronary endothelium and smooth muscle by immunohistochemical staining. CONCLUSIONS: CNP functions as a coronary vasodilator through activation of cGMP by way of particulate guanylate cyclase. CNP-mediated coronary vasodilation is attenuated by intracoronary HS-142-1. Intracoronary HS-142-1 does not affect acetylcholine-mediated coronary vasodilation. These observations support a role for exogenous CNP as a potent coronary vasodilator.

Prenatal treatment of congenital adrenal hyperplasia: a promising experimental therapy of unproven safety.

The long-term safety of the prenatal treatment of congenital adrenal hyperplasia (CAH) has not been established, and it remains an experimental therapy that raises unique ethical questions. Prenatal treatment should be performed only according to controlled, prospective, peer-reviewed protocols carried out according to strict scientific and ethical standards. Such studies must incorporate detailed, decades-long follow-up to permit accurate appraisal of its efficacy and safety, especially among the seven of eight fetuses who receive unneeded treatment.

Vitamin D 1 alpha-hydroxylase.

The rate-limiting, hormonally regulated step in the bioactivation of vitamin D is renal 1 alpha-hydroxylation by P450c1 alpha. In late 1997, we reported the cloning of the human cDNA and gene from keratinocytes, and established that P450c1 alpha mutations cause vitamin D-dependent rickets, type I, while three other groups reported the cloning of the rodent enzyme. The genetics of P450c1 alpha are well established, with studies of over 30 patients, but the molecular mechanisms for the hormonal regulation of P450c1 alpha are still under investigation.

The regulation of human P450c17 activity: relationship to premature adrenarche, insulin resistance and the polycystic ovary syndrome.

The polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism and insulin resistance, but the connection between these two features has been unclear. Androgen synthesis is regulated in part by the ratio of the 17alpha-hydroxylase and 17,20 lyase activities of P450c17. Three separate lines of evidence show that the ratio of lyase to hydroxylase activity is regulated by electron flow from P450 oxidoreductase. Lyase activity and androgen synthesis are particularly dependent on the serine phosphorylation of P450c17. Serine phosphorylation of the insulin receptor beta chain causes insulin resistance, and some PCOS women have hyperphosphorylated receptors. We hypothesize that an overactive serine/threonine kinase hyperphosphorylates both the insulin receptor and P450c17 in PCOS, accounting for the characteristic insulin resistance and hyperandrogenism of this disease.

Homologous sequences in steroidogenic enzymes, steroid receptors and a steroid binding protein suggest a consensus steroid-binding sequence.

The amino acid sequences of two steroidogenic enzymes, P450c17 (steroid 17 alpha-hydroxylase/17,20 lyse) and P450c21 (steroid 21-hydroxylase), are only 28.9% identical. However, these proteins share a region of 21 amino acids bearing 17 identical residues, which we previously suggested may represent the steroid binding site. We assembled a sequence database of known steroid-binding proteins and searched this with the sequence of this 21 amino acid region. The steroidogenic enzymes, P450c17, P450c21, P450scc (the cholesterol side-chain cleavage enzyme), and P450c11 (steroid 11 beta/18-hydroxylase) share a subregion of 17 amino acids having at least 15 identical residues. Related sequences were identified in a computerized search of the available sequences of steroid hormone receptors and binding proteins. These sequences were invariably found within larger domains previously associated with steroid binding. From these we propose a more general consensus sequence of LPLLL +/- 000KDRE0LKRL +/- PV, where +/- refers to any charged amino acid, and 0 refers to an uncharged amino acid. This consensus sequence predicts 147 or 187 total amino acids in 11 human proteins examined (78.6%). An equivalent degree of sequence identity, 178 of 221 amino acids (80.5%) was found among 13 animal homologs of these human proteins. The ability of this consensus sequence to predict 325 of 408 amino acids (79.7%) strongly suggests this sequence is necessary, if not sufficient, for a steroid binding site in many proteins. Lecithin-cholesterol acetyl transferase, cholesterol ester transfer protein, and steroid sulfatase did not have sequences similar to our consensus sequence.