Role of cytochrome b5 in the 17,20-lyase activity of P450c17.
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Animals↗
Biomedical subjects
W L Miller
Publications and source records attributed to W L Miller.
Evidence that StAR and MLN64 act on the outer mitochondrial membrane as molten globules.
StAR increases the flow of cholesterol from the outer to inner mitochondrial membrane (OMM to IMM), but its mechanism of action remains unclear. MLN64 is a 445 amino acid protein of unknown function that has four N-terminal transmembrane domains and whose C-terminal domain from 218-445 is 37% identical to StAR. N-62 StAR is as active as wild-type StAR, and N-234 MLN64 has 1/3 to 1/2 of StAR's activity. N-62 StAR lacks a mitochondrial leader and is confined to the cytoplasm, indicating that it acts on the OMM. Bacterially expressed N-62 StAR and N-218 MLN64 are active with isolated MA-10 cell mitochondria, indicating the proteins were properly folded. Far-UV CD spectroscopy, unfolding in urea, and fluorescence spectroscopy indicate that StAR undergoes a pH-dependent transition to a molten globule (retained secondary structure, partially lost tertiary structure) and stabilizes in mildly acid conditions. Far-UV CD data indicate that MLN64 undergoes a much less pronounced transition. Western blotting shows that normal human placenta has abundant N-terminally-cleaved 30 kDa MLN64. Partial proteolysis followed by mass spectrometry shows that the C-termini of StAR and MLN64 are sensitive to proteolysis, indicating looser folding. Our model of StAR action is that the protease-resistant domain unfolds slowly during normal mitochondrial entry, keeping StAR in contact with the OMM longer, increasing activity. The transition to the molten globule may be related to interaction with the OMM. These data are consistent with the recent crystallographic structure of N-216 MLN64 in which MLN64 binds cholesterol one molecule at a time, but are not consistent with the suggestion that StAR/MLN64 must reside in the intramembraneous space to transfer cholesterol form the OMM to the IMM.
Animals↗
Probing structural and functional domains of human P450c17.
Human P450c17 performs at least six chemical transformations, but this spectrum of activity is differentially regulated by structural changes and by redox partner proteins. Furthermore, P450c17 isoforms from different species with approximately 90% amino acid identity exhibit markedly different relative rates for these transformations. Although this phenomenology has been recognized for nearly 20 years, the underlying chemistry and structural basis for these effects are poorly understood. We have constructed a structural model of human P450c17 using computational chemistry to understand informative, naturally occurring human mutations and to provide a rational basis for designing alterations in P450c17 that probe functional domains of the protein. We have mapped with considerable confidence key residues involved in the interaction with redox partner proteins, including K89, R347, and R358, which form positive charges on the "proximal" surface of P450c17. Neutralization of these charges selectively impairs 17, 20-lyase activity without large reductions in 17alpha-hydroxylase activity or 17alpha-hydroxypregnenolone binding. We are now directing our efforts to the identification of key residues in the active site that mediate the substrate specificity and catalytic selectivity of human P450c17.
Adrenal Hyperplasia, Congenital↗
Transcriptional regulatory elements of the human gene for cytochrome P450c21 (steroid 21-hydroxylase) lie within intron 35 of the linked C4B gene.
The CYP21 gene, which encodes P450c21, the adrenal steroid 21-hydroxylase needed for glucocorticoid synthesis, lies in the major histocompatibility locus only 2.3 kilobase pairs (kb) downstream from the C4 gene. A 300-base pair (bp) proximal promoter and two upstream regions within C4 are needed for expression of mouse CYP21; the human gene also has a proximal promoter, but upstream elements have not been studied. To search for upstream regulatory elements in human CYP21B, we examined up to 9 kb of 5'-flanking DNA by transient transfection into human adrenal NCI-H295A cells. The 300-bp proximal promoter had substantial activity, but constructs retaining the DNA between -4.6 and -5.6 kb had increased activity, indicating the presence of distal elements. This region does not correspond to the mouse upstream regions, lying further upstream within intron 35 of C4B, which encompasses the previously described "Z promoter." DNase I footprinting located two elements, F1 and F2, lying -186 to -195 bp and -142 to -151 bp upstream from the Z cap site (-4862 to -4871 and -4818 to -4827 bp upstream of the CYP21B cap site). Each element formed a specific DNA-protein complex and conferred orientation-independent expression to a heterologous promoter. Mutations abolished formation of the DNA-protein complexes but only partially decreased expression. We identified a third site, F3, lying at -33 to -42 bp from Z. Competitive gel mobility supershift assays and co-transfection studies with SF-1 produced in vitro indicate F2 and F3 bind SF-1; BLAST searches and Southwestern blotting suggest that NF-W2 may bind F1. These results indicate that the Z promoter is a component of the CYP21 promoter needed to drive its adrenal-specific expression and that CYP21 transcription elements within C4 have kept these two genes linked during evolution.
Animals↗
Resting heart rate and cardiac function in dilated cardiomyopathy.
We hypothesized that, within the normal range of resting heart rate, heart rate and left ventricular ejection fraction would be inversely correlated and heart rate and left ventricular filling would be correlated in patients with dilated cardiomyopathy and not correlated in patients with normal cardiac function. At rest, heart rate, left ventricular ejection fraction, and three measures of diastolic filling (time to peak filling rate, peak filling rate, and first half filling fraction) were recorded using radionuclide ventriculography in subjects with no cardiac disease, patients with idiopathic dilated cardiomyopathy, and patients with dilated cardiomyopathy associated with ischemic heart disease. Heart rate had significant inverse correlations with left ventricular ejection fraction (r=-0.55, P=0.0007) and time to peak filling rate (r=-0.47, P=0.005) and a positive correlation with peak filling rate (r=0.73, P<0.0001) in patients with idiopathic dilated cardiomyopathy; heart rate was correlated only weakly with these measures in the absence of cardiac disease and essentially was not correlated in dilated cardiomyopathy due to ischemic heart disease. The change in resting heart rate with left ventricular ejection fraction and time to peak filling rate were significantly (P<0.05) different between patients with no cardiac disease and those with idiopathic dilated cardiomyopathy. Thus, resting heart rate correlated significantly with left ventricular ejection fraction and diastolic filling in patients with idiopathic dilated cardiomyopathy.
Adult↗
The active form of the steroidogenic acute regulatory protein, StAR, appears to be a molten globule.
The steroidogenic acute regulatory protein (StAR) increases the movement of cholesterol from the outer to the inner membrane of adrenal and gonadal mitochondria, thus providing the substrate for steroid hormone biosynthesis. Deletion of 62 amino-terminal aa produces a cytoplasmic form of StAR (N-62 StAR) that lacks the mitochondrial leader sequence but retains full activity and appears to act at the outer mitochondrial membrane. At neutral pH the native state of bacterially expressed N-62 StAR protein displays cooperative unfolding under the influence of urea with DeltaGH2O = -4.1 kcal/mol, and it remains correctly folded down to pH 4. Limited proteolysis at different pHs shows that the biologically essential C-terminal region is accessible to solvent, and that the N-terminal domain is compact at pH 8 and partially unfolds below pH 4. Secondary structural analysis of CD curves suggests that the unfolding may coincide with an increase in alpha-helical character at pH 3.5. Fluorescence spectroscopy at pH 3-8 and at 0-6 M urea is consistent with two distinct domains, a compact N-terminal domain containing tryptophans 96 and 147 and a more solvent-accessible C-terminal domain containing tryptophans 241 and 250. These observations suggest that StAR forms a molten globule structure at pH 3.5-4.0. As the mitochondrial proton pump results in an electrochemical gradient, and as StAR must unfold during mitochondrial entry, StAR probably undergoes a similar conformational shift to an extended structure while interacting with the mitochondrial outer membrane, allowing this apparent molten globule form to act as an on/off switch for cholesterol entry into the mitochondria.
Animals↗
Genetic disorders of vitamin D biosynthesis.
The principal hormonal regulator of bone mineralization is vitamin D, which must be activated by a metabolic pathway consisting of a 25-hydroxylase and a 1 alpha-hydroxylase to yield 1,25 dihydroxy vitamin D. The hormonal regulation of vitamin D activation is at the level of the 1 alpha-hydroxylase. This article reviews the biology of vitamin D, and describes the biochemistry of its activation and the molecular biology of the vitamin D-metabolizing enzymes. Recent advances, principally in the authors' laboratories, have resulted in the cloning of the human vitamin D 1 alpha-hydroxylase and the identification of mutations in its gene that cause Vitamin D Dependent Rickets type I.
25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
Genetic causes of rickets.
Dietary deficiency of vitamin D, genetic disorders of its bioactivation to 1,25-dihydroxyvitamin D [1,25(OH)2D], or disorders of vitamin D action can cause rickets. The rate-limiting, hormonally-regulated, biologically activating step in the synthesis of 1,25(OH)2D is the 1 alpha-hydroxylation of 25-hydroxyvitamin D, which occurs in kidney and other tissues and is mediated by a mitochondrial cytochrome P450 enzyme, P450c1 alpha. After many years of effort, the cDNA and gene for this enzyme were cloned in late 1997. Mutations in the P450c1 alpha gene, located on chromosome 12, cause 1 alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type I, an autosomal recessive disease characterized by rickets and impaired growth due to failure of renal synthesis of 1,25(OH)2D. X-linked hypophosphatemic rickets, a dominantly inherited disease, is caused by mutations in the PHEX gene, whose function in regulating renal phosphate and vitamin D metabolism remains to be elucidated.
Avitaminosis↗
The molecular basis of premature adrenarche: an hypothesis.
Adrenarche is characterized by a prepubertal rise in adrenal secretion of dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS) that is independent of the gonads or gonadotropins. Adrenopause is the corresponding diminution in DHEA and DHEAS concentrations in later life. The mechanisms by which adrenarche and adrenopause are induced and regulated are unknown. Early work focused on identifying hypothetical adrenal androgen regulatory hormones that would induce DHEA in much the same way that adrenocorticotropin induces cortisol, but no such factors have been found. Current studies of adrenarche focus on intra-adrenal events, particularly those concerning 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17alpha-hydroxylase/17,20-lyase (P450c17). Molecular data implicate a decrease in 3beta-HSD specifically in the adrenal zona reticularis. However, a decrease in 3beta-HSD is insufficient to explain why the reticularis catalyzes 17,20-lyase activity and hence makes DHEA, rather than catalyzing only 17alpha-hydroxylase activity, as does the zona fasciculata. P450c17 appears to catalyze 17,20-lyase activity only if P450c17 has undergone serine phosphorylation and has access to cytochrome b5 as an allosteric cofactor. Although these two factors have not yet been investigated in adrenarche, it appears that both a zone-specific diminution in 3beta-HSD and a zone-specific induction of 17,20-lyase activity are required to account for the physiological data. Exaggerated premature adrenarche appears to be an early sign of polycystic ovary syndrome (PCOS). Mechanistic considerations of PCOS suggest a key role for serine phosphorylation of P450c17 in both adrenarche and some forms of heritable PCOS.
Adrenal Glands↗
Maturation of lobster stomatogastric ganglion rhythmic activity.
The stomatogastric ganglion of the adult lobster, Homarus americanus generates extremely regular pyloric rhythms with a characteristic period of 0.5-1.5 Hz. To study the changes in the pyloric rhythm during embryonic and larval development, we recorded excitatory junctional potentials evoked by lateral pyloric (LP) neuron activity. Early in development the motor discharge of the LP neuron was often irregular, preventing use of conventional analysis methods that rely on extracting burst times to calculate cycle frequency and its variability. Instead, cycle frequency was determined for the LP neuron from the peak of the power spectrum obtained from the occurrence times of excitatory junctional potentials in the p1 muscle. The ratio of the power in the peak to the power from 0 to 3 Hz was used as a relative measure of the regularity of the rhythm. Throughout embryonic and the first larval stage, LP neuron activity is slow, irregular, and only weakly periodic. The regularity of the rhythm increased during midlarval stages, and both the frequency and regularity increased considerably by the postlarval stage LIV.
Animals↗
Medroxyprogesterone acetate and dexamethasone are competitive inhibitors of different human steroidogenic enzymes.
Medroxyprogesterone acetate (MPA), a widely used progestin, can suppress the hypothalamic-pituitary-gonadal axis but can also directly inhibit gonadal steroidogenesis; the success of MPA as a treatment for gonadotropin-independent sexual precocity derives from its direct action on steroidogenic tissues. Dexamethasone, a widely used glucocorticoid, can suppress the hypothalamic-pituitary-adrenal axis, but its potential effect directly on the adrenal is unclear. Previous reports suggested that these two drugs may act on the initial steps in the rodent steroidogenic pathway; therefore, we investigated their abilities to inhibit the first three human enzymes in steroidogenesis: the cholesterol side-chain cleavage enzyme (P450scc), the 17alpha-hydroxylase/17,20-lyase (P450c17), and type II 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSDII). We found no effect of either drug on P450scc in intact human choriocarcinoma JEG-3 cells. Using microsomes from yeast expressing human P450c17 or microsomes from human adrenals, we found that dexamethasone inhibited P450c17 with a Ki of 87 micromol/L, which is about 1000 times higher than typical therapeutic concentrations, but that MPA has no detectable action on P450c17. Using microsomes from yeast expressing human 3betaHSDII, we found that this enzyme has indistinguishable apparent Km values of 5.2-5.5 micromol/L and similar maximum velocities of 0.34-0.56 pmol steroid/min x microg microsomal protein for the three principal endogenous substrates, pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone. In this system, MPA inhibited 3betaHSDII with a Ki of 3.0 micromol/L, which is near concentrations achieved by high therapeutic doses of 5-20 mg MPA/kg x day. These data establish the mechanism of action of MPA as an inhibitor of human steroidogenesis, and are in contrast with the results of earlier studies indicating that MPA inhibited both P450c17 and 3betaHSD in rat Leydig cells. These studies establish the "humanized yeast" system as a model for studying the actions of drugs on human steroidogenic enzymes and suggest that 3betaHSDII may be an appropriate target for pharmacological interventions in human disorders characterized by androgen excess or sex steroid dependency.
Animals↗
P450c17 mutations R347H and R358Q selectively disrupt 17,20-lyase activity by disrupting interactions with P450 oxidoreductase and cytochrome b5.
Cytochrome P450c17 catalyzes steroid 17alpha-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 17alpha-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 P450cl7 mutant in yeast with or without OR, b5, or both, and measured enzyme kinetics in yeast microsomes using pregnenolone and 17alpha-hydroxypregnenolone as substrates. The apparent Michaelis-Menten (Km) values for the R347H mutant with and without coexpressed OR were 0.2 and 0.6 microM, respectively, and for the R358Q mutant with and without OR they were 0.3 and 0.4 microM, respectively; these values did not differ significantly from the wild-type values of 0.4 and 0.8 microM with and without OR, respectively. Furthermore, coincubation with 17alpha-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 x OR complex to promote electron transfer.
17-alpha-Hydroxypregnenolone↗
Molecular modeling of human P450c17 (17alpha-hydroxylase/17,20-lyase): insights into reaction mechanisms and effects of mutations.
P450c17 (17alpha-hydroxylase/17,20-lyase) catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities in the biosynthesis of androgens and estrogens. These two activities are differentially regulated in a tissue-specific and developmentally programmed manner. To visualize the active site topology of human P450c17 and to study the structural basis of its substrate specificity and catalytic selectivity, we constructed a second-generation computer-graphic model of human P450c17. The energetics of the model are comparable to those of the principal template of the model, P450BMP, as determined from its crystallographic coordinates. The protein structure analysis programs PROCHECK, WHATIF, and SurVol indicate that the predicted P450c17 structure is reasonable. The hydrophobic active site accommodates both delta4 and delta5 steroid substrates in a catalytically favorable orientation. The predicted contributions of positively charged residues to the redox-partner binding site were confirmed by site-directed mutagenesis. Molecular dynamic simulations with pregnenolone, 17-OH-pregnenolone, progesterone, and 17-OH-progesterone docked into the substrate-binding pocket demonstrated that regioselectivity of the hydroxylation reactions is determined both by proximity of hydrogens to the iron-oxo complex and by the stability of the carbon radicals generated after hydrogen abstraction. The model explains the activities of all known naturally occurring and synthetic human P450c17 mutants. The model predicted that mutation of lysine 89 would disrupt 17,20-lyase activity to a greater extent than 17alpha-hydroxylase activity; expression of a test mutant, K89N, in yeast confirmed this prediction. Hydrogen peroxide did not support catalysis of the 17,20-lyase reaction, as would be predicted by mechanisms involving a ferryl peroxide. Our present model and biochemical data suggest that both the hydroxylase and lyase activities proceed from a common steroid-binding geometry by an iron oxene mechanism. This model will facilitate studies of sex steroid synthesis and its disorders and the design of specific inhibitors useful in chemotherapy of sex steroid-dependent cancers.
Amino Acid Sequence↗
Dexamethasone treatment of congenital adrenal hyperplasia in utero: an experimental therapy of unproven safety.
Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency is a common cause of genital virilization in female infants resulting from inappropriate fetal adrenal androgen secretion. Some investigators have advocated treating pregnant women who are at risk for carrying a CAH fetus with dexamethasone to suppress fetal adrenal androgen synthesis. Experience to date shows that this treatment can be effective in ameliorating the genital virilization in female fetuses. However, the doses used are supraphysiological, the mechanism of dexamethasone action in the fetus is unclear and no long-term followup studies have been done. To be effective the treatment would need to be started by week 6 of gestation but the genetic diagnosis cannot be made until week 12. If the mother has had a previous CAH child, only 1 in 4 pregnancies will be affected and only the female fetuses stand to benefit from treatment, thus, 7 of 8 fetuses will be treated needlessly. In view of these and other concerns, the prenatal treatment of CAH remains an experimental therapy and, hence, must only be done with fully informed consent in controlled prospective trials approved by human experimentation committees at centers that see enough of these patients to collect meaningful data.
Adrenal Hyperplasia, Congenital↗
Congenital adrenal hyperplasia in the adult patient.
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3-Hydroxysteroid Dehydrogenases↗
Analysis and modeling of the locomotor central pattern generator as a network of coupled oscillators.
The primary functions of spinal locomotor central pattern generators (CPGs) are to provide oscillatory motor commands to individual joints or segments and to control the precise timing of those commands across all joints or segments for efficient, coordinated locomotor behavior. Our ability to understand the neuronal mechanisms underlying intersegmental coordination has been hampered by the complexity of propriospinal interconnectivity and the paucity of quantitative data on the magnitude and timing of those connections. Theoretical approaches have therefore been employed to discover general rules by which CPG-like oscillator systems must be constructed to produce appropriate coordinated locomotor behavior; the locomotor CPG is represented as a network of oscillators, where each oscillator generates local motor output and interoscillator coupling provides intersegmental coordination. Mathematical analysis of such coupled oscillator systems has provided a number of experimentally testable predictions regarding the link between coupling and coordination. Application of these network-level predictions to the results of electrophysiological experiments has required data analysis methods that can relate the behavior of the in vitro spinal cord to the variables employed by the mathematical model. Hence, our most recent work has focused on developing analytic tools for quantifying the changes in locomotor output that result form experimental manipulations of the propriospinal system in terms of frequency, intersegmental phase, and intersegmental correlation. Results of recent experiments can now be used to put further constraints on the allowable kinds of intersegmental coupling provided by mathematical modeling of the system.
Animals↗
Incorrect folding of steroidogenic acute regulatory protein (StAR) in congenital lipoid adrenal hyperplasia.
Steroidogenic acute regulatory protein (StAR) rapidly stimulates the movement of cholesterol into adrenal and gonadal mitochondria to mediate the acute steroidogenic response; StAR mutations cause potentially lethal congenital lipoid adrenal hyperplasia (lipoid CAH). Bacterially expressed wild-type StAR and four amino acid replacement/deletion mutants that cause lipoid CAH were purified to apparent homogeneity. Sedimentation equilibrium ultracentrifugation showed that all five proteins were monomeric and fit a globular protein model of the correct molecular mass. Circular dichroism (CD) spectra of both the wild-type and mutants showed minima near 208 and 222 nm, confirming the presence of substantial alpha-helical structure. However, subtle differences in the CD signals of the wild-type and mutants in the far-UV and stronger differences in near-UV indicated differences in protein folding. The amide I and II bands in the 1400-1700 cm-1 region of Fourier transform infrared spectra showed that the proteins fell into two groups. The wild-type and a partially active conservative mutant were predominantly alpha-helical with some intramolecular beta-sheet. By contrast, three mutants that lost charged residues retained much of their alpha-helical structure, but also tended to form intermolecular beta-sheets. Urea at 2.0 or 4.0 M had less effect on the CD spectrum of the wild-type than of the mutants, particularly those having lost a charged residue; 50 mM guanidinium hydrochloride did not alter the CD spectrum of the wild-type, but elicited dramatic changes to the secondary structure in all four mutants. Despite this, thermal melting curves of the mutant proteins in 50 mM guanidinium hydrochloride showed surprising stability, even exceeding that of the wild-type protein. These data suggest that the StAR amino acid replacement mutants that cause lipoid CAH are inactive because of fairly gross errors in protein folding, probably due to the loss of salt bridges that stabilize the tertiary structure.
Adrenal Hyperplasia, Congenital↗
Spectral analysis of oscillatory neural circuits.
Oscillatory dynamics are found at all levels of the nervous system. The goal of our current research on the control of rhythmic motor output by the lamprey spinal cord is to determine the features of neuronal coupling that lead to stable oscillatory activity and precisely-controlled intersegmental phase. Since our experimental manipulations can greatly increase the variability of the ventral root bursting pattern, it is important for us to employ a data analysis method which remains valid independent of this variability. Traditional analysis approaches which rely on identification of burst event times do not generally satisfy this requirement. In this paper, we illustrate the application of a straightforward statistically-based method for determining important parameters of oscillatory motor circuits using Fourier spectral analysis of spike trains. The frequency, phase, and their variabilities can be quantified; and the relative strength of coupling between different parts of the circuit can be tested for statistical significance. The approach we adopt is highly convenient for neuroscientists who study oscillatory systems as it operates directly on trains of action potentials stored as lists of event times (point-processes). Basic concepts and practical issues concerning use of Fourier analysis are discussed.
Animals↗