The structure of adrenodoxin reductase of mitochondrial P450 systems: electron transfer for steroid biosynthesis.

Adrenodoxin reductase is a monomeric 51 kDa flavoenzyme that is involved in the biosynthesis of all steroid hormones. The structure of the native bovine enzyme was determined at 2.8 A resolution, and the structure of the respective recombinant enzyme at 1.7 A resolution. Adrenodoxin reductase receives a two-electron package from NADPH and converts it to two single electrons that are transferred via adrenodoxin to all mitochondrial cytochromes P 450. The structure suggests how the observed flavin semiquinone is stabilized. A striking feature is the asymmetric charge distribution, which most likely controls the approach of the electron carrier adrenodoxin. A model for the interaction is proposed. Adrenodoxin reductase shows clear sequence homology to half a dozen proteins identified in genome analysis projects, but neither sequence nor structural homology to established, functionally related electron transferases. Yet, the structure revealed a relationship to the disulfide oxidoreductases, permitting the assignment of the NADP-binding site.

Chaperone-assisted expression of authentic bovine adrenodoxin reductase in Escherichia coli.

Adrenodoxin reductase is an essential component of the mitochondrial monooxygenase systems that are involved in the synthesis of steroid hormones and related compounds. After removing by mutagenesis a secondary ribosome binding site and an mRNA loop formed between the gene and the vector, large amounts of the enzyme could be produced in Escherichia coli by coexpression with the HSP60-chaperone system. The purified protein was homogeneous enough for reproducible crystallization. The crystals diffracted X-rays isotropically beyond 1.7 A resolution permitting a structure analysis.

Gene structure of the human amiloride-sensitive epithelial sodium channel beta subunit.

ENaC functions in the transport of sodium ions across epithelial cells and consequently regulates blood volume and pressure. ENaC complex includes at least three different subunits, alpha, beta, and gamma, which are developmentally regulated and differentially controlled by aldosterone. In this study, we determined the exon-intron organization of the beta ENaC subunit by sequencing genomic DNA from three subjects from three different ethnic groups. The results showed that the coding region of the human betaENaC gene (SCNN1B) extends from exon 2 to exon 13. No polymorphism was observed in the examined subjects, indicating strict conservation of the coding region sequence. The introns of beta subunit gene are located at exactly the same positions as in the alpha and gamma subunits, although these proteins share only 26-32% sequence identity. These results thus elucidate the gene structure of the beta subunit and indicate that exon-intron architecture of the three genes encoding the three subunits of ENaC have remained highly conserved despite the divergence of their sequences.

Antioxidant capacity is correlated with steroidogenic status of the corpus luteum during the bovine estrous cycle.

The reactions of steroid hormone biosynthesis are accompanied by formation of oxygen radicals. We determined the levels of some antioxidants and antioxidative enzymes at different developmental stages of bovine corpora lutea to examine their correlation with steroidogenic status. Plasma progesterone concentrations of estrous cycle synchronized cows increased until day 16, and then decreased rapidly during luteal regression. The levels of steroidogenic cytochrome P450scc and adrenodoxin paralleled the changes in plasma progesterone. Among the antioxidative enzymes examined, the SOD and catalase activities showed patterns most similar to plasma progesterone. Catalase and SOD activities increased 6-8 fold from day 6 to 16 of the estrous cycle and then decreased during the luteal regression. Ascorbate and beta-carotene showed low but significant correlation with P450scc and plasma progesterone levels. The profiles of two lipophilic antioxidants in corpora lutea were very different. beta-carotene concentration increased by approximately 6 fold from day 6 to 16, and decreased in regressive tissue. alpha-tocopherol showed a 3 fold increase between days 6 and 9 followed by a rapid decrease. Thus, at the peak of steroidogenesis at mid-luteal phase alpha-tocopherol levels decreased, but beta-carotene levels increased. The correlation between the levels of some antioxidant enzymes and compounds with progesterone levels indicates that antioxidative mechanisms are activated to cope with steroidogenesis dependent oxyradical formation in the bovine corpus luteum.

In situ localization of ACTH receptor-like mRNA in molluscan and human immunocytes.

Adrenocorticotropin hormone (ACTH) receptor-like messenger RNA was localized in molluscan hemocytes and human peripheral blood mononuclear cells by in situ hybridization using a digoxigenin-labelled bovine complementary DNA probe. These findings suggest that the ACTH receptor gene has been highly conserved during evolution. Moreover, these data represent further support for a relationship between the immune and neuroendocrine systems in invertebrates, as documented in our previous studies.

Pseudohypoaldosteronism due to renal and multisystem resistance to mineralocorticoids respond differently to carbenoxolone.

Type I pseudohypoaldosteronism (PHA) is a hereditary syndrome of salt wasting resulting from unresponsiveness to mineralocorticoids. PHA is manifested in two clinically and genetically distinct forms, affecting either only the kidney or multiple target organs of aldosterone. We examined the mineralocorticoid effect of carbenoxolone (CBX) in young PHA patients with either renal or multisystem resistance to aldosterone to find out whether CBX may help reduce the requirement for a high-salt diet. CBX did not show any significant salt-retaining effect in two patients with multiple PHA, and did not affect the renin-aldosterone system. In contrast, CBX significantly suppressed the renin-aldosterone system in a renal PHA patient for the whole duration of treatment, but without a long-term salt-retaining effect. On CBX treatment, urinary cortisone levels decreased and the cortisol:cortisone ratio increased, indicating that CBX inhibited 11beta-HSD activity that metabolizes cortisol to cortisone. The complete lack of effect of CBX on the renin-aldosterone system in multisystem PHA patients indicates that CBX does not exert an effect via mineralocorticoid (MR) or glucocorticoid receptors. Examination of the structure and expression of the MR gene by Southern blot analysis and polymerase chain reaction (PCR) showed no abnormality. Whereas multiple PHA results from a spectrum of mutations in the mineralocorticoid activated epithelial sodium channel subunits, the genetic basis of renal PHA is still unknown. The response to CBX suggests that there is at least a partly functional MR in renal PHA patients.

Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.

Autosomal recessive pseudohypoaldosteronism type I is a rare life-threatening disease characterized by severe neonatal salt wasting, hyperkalaemia, metabolic acidosis, and unresponsiveness to mineralocorticoid hormones. Investigation of affected offspring of consanguineous union reveals mutations in either the alpha or beta subunits of the amiloride-sensitive epithelial sodium channel in five kindreds. These mutations are homozygous in affected subjects, co-segregate with the disease, and introduce frameshift, premature termination or missense mutations that result in loss of channel activity. These findings demonstrate the molecular basis and explain the pathophysiology of this disease.

Localisation of pseudohypoaldosteronism genes to chromosome 16p12.2-13.11 and 12p13.1-pter by homozygosity mapping.

Pseudohypoaldosteronism type 1 (PHA1, OMIM 264350) is a rare Mendelian disorder characterised by end-organ unresponsiveness to mineralocorticoids. Most steroid hormone insensitivity syndromes arise from mutations in the corresponding receptor, but available genetic evidence is against involvement of the mineralocorticoid receptor gene, MLR, in PHA1. A complete genome scan for PHA1 genes was undertaken using homozygosity mapping in 11 consanguineous families. Conclusive evidence of linkage with heterogeneity was obtained with a maximum two-locus admixture lod score of 9.9. The disease locus mapped to chromosome 16p12.2-13.11 in six families and to 12p13.1-pter in the other five families. The two chromosomal regions harbour genes for subunits of the amiloride-sensitive epithelial sodium channel: SCNN1B and SCNN1G on 16p and SCNN1A on 12p. Liddle's syndrome of hypertension and pseudoaldosteronism has been shown to arise from mutations in SCNN1B and SCNN1G. These results strongly suggest that PHA1 and Liddle's syndrome are allelic variants caused by mutations in genes encoding subunits of this sodium channel. These genes are of broad biological interest both in relation to sodium and water homeostasis in mammals and by virtue of their homology to the mec genes of Caenorhabditis elegans involved in mechanosensitivity and neuronal degeneration.

Tissue- and species-dependent expression of sheep lung microsomal cytochrome P4502B(LgM2).

Expression of sheep lung microsomal cytochrome P4502B(LgM2) isozyme was determined in lung and liver preparations from rabbits, rats, sheep, cows and humans. Several tissues of sheep such as lung, liver, adrenal, kidney, ovary, intestine, muscle, spleen, pancreas and brain were also examined. Tissue homogenates were analyzed by Western-blotting with an antibody raised against purified sheep lung P4502B(LgM2) isozyme in rabbits. This isozyme was detected in lung samples from every species examined. Homologues of cytochrome P4502B(LgM2) were expressed in livers of sheep and rabbits but not in those of humans and cows. Although homologous forms of P4502B(LgM2) were not expressed in sheep brain, ovary, pancreas, spleen and muscle tissues, they were expressed in sheep adrenal, intestine and kidney.

Exclusion of the locus for autosomal recessive pseudohypoaldosteronism type 1 from the mineralocorticoid receptor gene region on human chromosome 4q by linkage analysis.

Pseudohypoaldosteronism type 1 (PHA1) is an uncommon inherited disorder characterized by salt-wasting in infancy arising from target organ unresponsiveness to mineralocorticoids. Clinical expression of the disease varies from severely affected infants who may die to apparently asymptomatic individuals. Inheritance is Mendelian and may be either autosomal dominant or autosomal recessive. A defect in the mineralocorticoid receptor has been implicated as a likely cause of PHA1. The gene for human mineralocorticoid receptor (MLR) has been cloned and physically mapped to human chromosome 4q31.1-31.2. The etiological role of MLR in autosomal recessive PHA1 was investigated by performing linkage analysis between PHA1 and three simple sequence length polymorphisms (D4S192, D4S1548, and D4S413) on chromosome 4q in 10 consanguineous families. Linkage analysis was carried out assuming autosomal recessive inheritance with full penetrance and zero phenocopy rate using the MLINK program for two-point analysis and the HOMOZ program for multipoint analysis. Lod scores of less than -2 were obtained over the whole region from D4S192 to D4S413 encompassing MLR. This provdes evidence against MLR as the site of mutations causing PHA1 in the majority of autosomal recessive families.

Selective increases in adrenal steroidogenic capacity during acute respiratory disease in infants.

To examine steroidogenic responses of the different zones of the adrenal cortex to acute disease we determined the basal and adrenocorticotropin (ACTH)-stimulated levels of cortisol, dehydroepiandrosterone (DHEAS) and aldosterone in 16 infants aged 1-4 months with acute bronchiolitis. Fourteen of the infants were retested after recovery. During illness the mean basal levels of cortisol and DHEAS were twice as high as the levels after recovery (370 vs 180 nmol/l and 2.7 vs 1.3 mumol/l, respectively). The mean peak ACTH-stimulated levels of cortisol and DHEAS during illness were 1.5- and 2.5-fold higher, respectively, than the levels found after recovery. Although aldosterone secretion was stimulated > or = 3-fold by ACTH, illness was not associated with any change in aldosterone secretory capacity. The basal and stimulated levels of both cortisol and DHEAS during illness and after recovery were correlated significantly. Thus, the relative steroidogenic capacities for these two steroids were characteristic of the individual infant and showed constancy over a period of at least several weeks. While the levels of cortisol and aldosterone were not dependent on the age of the infants, both the basal and stimulated levels of DHEAS correlated strongly with age. We conclude that during acute disease the steroidogenic capacity selectively increases in the zones that secrete cortisol and DHEAS (only in infants < 3 months) but not in the zona glomerulosa that secretes aldosterone. The DHEAS response may be related to its putative effects to enhance immune responses.

Electron leakage from the adrenal cortex mitochondrial P450scc and P450c11 systems: NADPH and steroid dependence.

In the present study we examined the coupling of NADPH oxidation to substrate hydroxylation and the effects of steroids on this process in reconstituted P450scc and P450c11 systems. To determine the relative rates of substrate hydroxylation vs electron leakage we assayed both the steroid product and H2O2 in the same sample. For both P450 systems the rates of steroid product and superoxide formation increased as NADPH concentration was increased. However, P450c11 was found to be more leaky. The leakage from the P450scc system was not affected by pregnenolone, the product of cholesterol side chain cleavage. In contrast, corticosterone, the product of P450c11, increased the rate of futile NADPH oxidation by the P450c11 system. We also tested a series of steroids to analyze the stereospecificity of their effects. Relative to the control without steroid, both C-19 and C-21 steroids with 11 alpha-hydroxy groups (11 alpha-OH-testosterone and 11 alpha-OH-cortisol) decreased leakage, and those with 11 beta-OH groups (11 beta-OH-testosterone and cortisol) stimulated both NADPH oxidation and electron leakage as measured by H2O2 formation. The results revealed a correlation between the effects previously observed in living cells and in our reconstituted systems. These findings provide further evidence that mitochondrial P450 systems indeed function as a significant source of oxygen radicals in steroidogenic cells.

Routes and regulation of NADPH production in steroidogenic mitochondria.

The first and rate-limiting step of steroidogenesis is catalyzed by the mitochondrial cholesterol side chain cleavage system that is dependent on NADPH. The pathways of NADPH generation in steroidogenic mitochondria include three major routes catalyzed by: 1. NADP-linked malic enzyme, 2. NADP-linked isocitrate dehydrogenase, and 3. nicotinamide nucleotide transhydrogenase. The main route may differ among cell types and across species. Generally operation of alternative routes, with different substrates is not excluded. The oxidation of NADPH by the mitochondrial P450 systems is not tightly coupled with substrate metabolism, as these systems can reduce O2 by a single electron to produce harmful superoxide radical. To minimize such futile NADPH oxidation, NADPH generation may be regulated by two types of mechanisms: 1. Feedback mechanisms that maintain the ratio of NADPH/NADP+ at a steady-state level by enhancing the rate of NADPH production to keep up with its rate of oxidation, e.g., allosteric regulation of enzymes involved in NADPH production. 2. Hormonal signals that enhance the level of NADPH production in coordination with steroidogenesis. One major hypothesis with experimental evidence is that stimulation of mitochondrial NAD(P)H synthesis is mediated by Ca++ as a second messenger of tropic factors. Tropic stimulation of cells increases the levels of Ca++ in the cytosol and then in the mitochondrial matrix, and the rise in Ca++ activates enzymes involved in NAD(P)H synthesis. These regulatory mechanisms most probably operate in concert adjusted to the steroidogenic activity of the cell.

Cloning of ACTH-regulated genes in the adrenal cortex.

To search for genes that are induced by ACTH in adrenocortical cells, we screened adrenal cortex cDNA libraries by a differential hybridization method using cDNA probes representing mRNAs from cells with or without ACTH stimulation. Forty clones were identified as ACTH induced (yielding a frequency of about 1/2500 plaques screened), and two clones as ACTH repressed. The cDNAs isolated and sequenced include nuclear genes for microsomal steroidogenic enzymes and novel proteins of yet unidentified functions, and mitochondrial genes encoding subunits of oxidative phosphorylation enzymes. Northern blot analysis of RNA from cells stimulated with ACTH confirmed the induction of these genes by ACTH, yet revealed important differences in the relative responses of the respective mRNAs. The time courses showed the major increase in the initial 6 h; and a decline after 24-36 h. The enhancement of the levels of the mRNAs could be ascribed to transcriptional activation. Since the mitochondrial genome is transcribed as a single polycistronic unit, to account for the > 20-fold differences in the levels of the mitochondrial mRNAs it is necessary to invoke differential stabilities of these mRNAs. The synchronous increase in the expression of both the steroidogenic enzymes and the mitochondrial oxidative phosphorylation system subunits, provides evidence for coregulation of steroidogenic and energy producing capacities of adrenal cells to meet the metabolic needs of steroid hormone production. Suppression of beta-actin gene expression may be related to changes in actin polymerization during ACTH-dependent cytoskeletal reorganization.

A fluorimetric assay for hydrogen peroxide, suitable for NAD(P)H-dependent superoxide generating redox systems.

We report a simple and sensitive fluorimetric method for quantitative assay of the production rate of hydrogen peroxide, and indirectly of superoxide, during electron transfer reactions. The assay requires the inclusion of superoxide dismutase, catalase, and 6% methanol in the tested reaction system, to stochiometrically produce formaldehyde per molecule of H2O2 generated. The reaction is terminated by adding 2 vol of Nash reagent and heating at 60 degrees C for 10 min, to convert accumulated formaldehyde to diacetyldihydrolutidine (DDL). The standard curve for formaldehyde, based on the fluorescence of DDL, is highly reproducible and allows measurement of 1 microM amounts in the reaction sample (coefficient of variation < 15%). The excitation and emission wavelengths of DDL at 412 and 505 nm are distant from those of NAD(P)H. Thus, the method can be used in NAD(P)H-dependent enzymatic systems to measure both NAD(P)H oxidation and superoxide production in the same sample. We validated the assay in a mitochondrial P450 system determining the fraction of total electron flow that is channeled to oxy-radical formation. The assay should be useful in the study of this and other superoxide/H2O2 generating systems.

cDNA cloning and sequence analysis of the bovine adrenocorticotropic hormone (ACTH) receptor.

We isolated five independent cDNAs of nearly 3000 bp for the bovine ACTH receptor by screening adrenal cortex cDNA libraries with a PCR cloned cDNA fragment. The deduced receptor sequence includes 297 residues (M(r) = 33,258) with 81% identity with the human ACTH receptor, and shows seven hydrophobic transmembrane domains. The calculated M(r) of the receptor is smaller than the 40-45 kDa observed in crosslinking studies with labeled ACTH. Since the bovine and human receptors have two glycosylation motifs in the N-terminus, the difference may result from glycosylation of the receptor. Analysis of the sequences of both bovine and human receptors revealed a single protein kinase. A phosphorylation motif located in the third intracellular loop (Ser-209) juxtaposed to a protein kinase C phosphorylation motif (Thr-204). Thus, the involvement of protein kinase A and C pathways in ACTH action may be mediated in part by phosphorylation of the ACTH receptor at these motifs. The 3'-untranslated region of the bovine cDNA is > 2000 bp and includes two inverse repeats giving an extensive and strong secondary structure to the ACTH receptor RNA.

Cloning of LL5, a novel protein encoding cDNA from a rat pituitary library.

While screening a rat pituitary cDNA library for a peptide hormone receptor, we identified a cDNA that represents a novel gene. The 3.8 kb cDNA has an open reading frame of 2.3 kb encoding a protein of 781 amino acids (M(r) = 87,507). Southern blot analysis of rat, mouse, bovine and human genomic DNAs revealed that a homologous gene is present in these species probably in a single copy. Northern blot analysis showed that in addition to the pituitary gland, the gene is also expressed in other rat tissues. Scanning of DNA and protein databanks revealed no significant homology to any other sequence. Thus, this gene encodes a heretofore unidentified protein.

Mitochondrial-genome-encoded RNAs: differential regulation by corticotropin in bovine adrenocortical cells.

Differential screening of an adrenal cortex cDNA library for corticotropin (ACTH)-inducible genes led to the isolation of a group of cDNAs representing mitochondrial genes that encode subunits of cytochrome oxidase, ATPase, and NADH dehydrogenase. Northern blot analysis of RNA from cells stimulated by ACTH confirmed the induction of these genes by ACTH yet revealed major differences in the relative responses of the respective mRNAs. The levels of mRNAs for cytochrome oxidase subunit I and ATPase increased 2- to 4-fold and for NADH dehydrogenase subunit 3 increased 20-fold, whereas the levels of the mitochondrial 16S rRNA showed no change within 6 h of ACTH stimulation. These effects of ACTH on mitochondrial mRNA levels probably result from both activation of the H2 transcription unit that encodes mitochondrial mRNAs and alteration of mRNA stability. ACTH also increased the activity of cytochrome oxidase after 12 h of stimulation. Examination of the tissue specificity of expression of five mitochondrial genes showed a wide range of RNA levels among 11 tissues but high correlations between individual RNA levels, consistent with a coordinated expression of the mitochondrial genes, although at different levels in each cell type. Proportionately high levels of mitochondrial mRNAs were found in adrenal cortex, probably reflecting a stimulatory effect of ACTH in vivo. Overall, the results indicate that ACTH enhances the energy-producing capacity of adrenocortical cells.

Electron leakage from the mitochondrial NADPH-adrenodoxin reductase-adrenodoxin-P450scc (cholesterol side chain cleavage) system.

In electron (e-) transfer systems some e- may "leak," reducing O2 to a superoxide radical. This study examined the sites and kinetics of e-leakage from the mitochondrial P450scc system. Adrenodoxin reductase alone oxidized NADPH, reducing O2 to a superoxide radical at a very low rate. However, the reductase-adrenodoxin system reduced O2 at a rapid steady-state rate with Michaelis-Menten dependence on [adrenodoxin](Vmax = 3.5 micro M e-/min). After depletion of NADPH, reduced adrenodoxin was oxidized (autooxidation) with pseudo first order kinetics and the rate of e- transfer decreased 10-fold. Ca2+ (< 1 mM) stimulated e- leakage in both phases. The reductase-adrenodoxin-P450scc system exhibited the highest rate of leakage (Vmax = 7.8 microM e-/min). At low [adrenodoxin] the majority of e-leaked through P450scc and not through adrenodoxin. In the presence of the substrate, cholesterol, leakage drastically decreased to <0.5 microM e-/min. These results indicate that the mitochondrial P450 systems can leak e-, producing O2 derived free radicals. Reduction of leakage during P450scc conversion of cholesterol to pregnenolone provides a clue to understanding physiological mechanisms that control e-leakage. These may include coregulation of NADPH and cholesterol availability to the P450scc system and a system of antioxidants for quenching the oxygen radicals.