The steroidogenic acute regulatory protein (StAR) is the major entrance for cholesterol in mitochondria under acute stimulation. Under such circumstances, dysfunctional StAR activity can ultimately lead to lipoid congenital adrenal hyperplasia (LCAH). A complete understanding of the StAR's molecular structure and mechanism is essential to comprehend LCAH. Thus far, there is no mechanistic model that can explain experimental results at the molecular level. This is partly due to the lack of the molecular structure of StAR. The closest approximation to the StAR molecular structure is the human MLN64 which has a similar activity to StAR, has a highly homologous primary structure and for which an X-ray structure is known. In this context, we have modeled the structure of StAR through standard homology modeling procedures based on the MLN64 structure. Our StAR model shows the presence of a hydrophobic cavity of 783·9 Å 2 in surface area, large enough to fit one molecule of cholesterol. In addition, we have identified a unique charged pair, as in MLN64, lining the surface of the cavity and which could play a key role in the binding of cholesterol through the formation of an H-bond with its OH moiety. This suggests that the cholesterol-binding site of StAR is located inside this cavity. Taking into account that internal cavities are destabilizing to native protein structures and that the lining of the cavity has to become accessible in order to allow cholesterol binding, we have explored the possibility that StAR could exist in equilibrium with partially unfolded states. Using a structure-based thermodynamics approach, we show that partially folded states (with an unfolded C-terminal α-helix, and an open cavity) can be significantly populated at equilibrium and therefore allow cholesterol binding. These results are supported by recent experiments that show a loss of StAR helical character upon binding of an analog of cholesterol. Moreover, we show that the replacement of the residues involved in the charged-pair located in the binding site results in the loss of StAR activity, supporting a key role for these residues. Taken together, our results are applicable to StAR functioning both in the mitochondrial intermembrane space as well as outside the mitochondria.

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The purpose of this study was to evaluate the effects of acute (a single injection) and chronic stimulation (twice daily injection for 9 days) by ACTH on changes occurring in the temporal expression of steroidogenic enzymes in the rat adrenal in vivo. Under acute ACTH stimulation, the level of steroidogenic acute regulatory protein (StAR) messenger RNA (mRNA) was increased within 0.5 h in both zona glomerulosa (ZG) and zona fasciculata-reticularis (ZFR), with maximal increases of 220 -370% and 300 -350% in the ZG and ZFR, respectively. Increases in the levels of StAR protein in homogenates were also found in the ZG (700%) and the ZFR (300%), but were delayed compared with those of their mRNA. Furthermore, the increase in mitochondrial StAR protein was concomitant with that in the homogenate, indicating that the entry of StAR into mitochondria might not be necessary to increase steroidogenesis during the early stimulatory phase. The levels of c-jun, c-fos, junB, and fosB mRNA in ZG and ZFR were also rapidly maximally elevated within 0.5-1 h after ACTH administration and fell to near control levels 5 h posttreatment. The levels of c-jun protein were already increased in both zones at 1 h, reached 200% at 3 h, and remained elevated 5 h post-ACTH treatment. The levels of c-Fos protein were maximally increased by 240% in both zones after 1 h and decreased thereafter to control values at 5 h. Few changes were observed in the adrenal protein contents of cholesterol side-chain cleavage cytochrome P450 (P450scc), cytochrome P450 11␤-hydroxylase (P450C11), cytochrome P450 21-hydroxylase (P450C21), and 3␤-hydroxysteroid dehydrogenase (3␤HSD). Under chronic stimulation by ACTH, we observed elevations in the levels of plasma corticosteroids and changes in the mRNA and protein levels of many adrenal steroidogenic enzymes in both zones. In the ZG, administration of ACTH for 9 days provoked an increase in the level of StAR mRNA (210 -270%) and a decrease in the levels of 3␤HSD, cytochrome P450 aldosterone synthase (P450aldo), and AT 1 receptor mRNA (by 40%, 70%, and 90%, respectively), whereas the levels of P450scc and P450C21 mRNA did not differ significantly from the control values. Western blotting analysis showed that the adrenal ZG protein levels of StAR and P450scc were increased (150%), 3␤HSD was not changed, and P450C21 was decreased by 70%. In the ZFR, the levels of P450scc and StAR mRNAs were increased (260% and 570 -870%, respectively). The levels of 3␤HSD, P450C21, and P450C11 mRNA did not differ from control values in that zone. Western blotting analysis showed that the ZFR protein level of 3␤HSD was not changed, P450scc and P450C21 were decreased by 40% and 60%, respectively, and StAR was increased by 160%. Although c-fos and fosB mRNAs were undetectable after 9 days of chronic ACTH treatment, c-jun mRNA and its protein were still detectable, suggesting a basic role for this protooncogene in maintaining the integrity and function of the adrenal cortex. When dexamethasone was administered to rats for 5 days...

The hamster, like the human produces cortisol as its major glucocorticoid, rather than corticosterone, typical of most enzyme rodents. It is not known, however, if the hamster cytochrome P450C17 (P450C17), a key enzyme for cortisol formation, also exhibits 17,20-lyase activity and if it catalyzes the formation of dehydroepiandrosterone (DHEA) at the adrenal level. To study this, we isolated the cDNA of P450C17 from a hamster adrenal library. This cDNA was sequenced and was found to have an open reading frame for a protein of 511 amino acids, as compared to the human P450C17, which contains 508 amino acids. The hamster P450C17 cDNA, in the coding region, is 76% homologous with the human P450C17 cDNA. The cDNA was then cloned in the expression vector pSV-SPORT 1, which was transiently transfected into COS 1 cells. The transfected cells were used for temporal studies on the transformation of radiolabeled C21-delta5- and C21-delta4-precursors. When transfected cells were incubated with [14C]pregnenolone, rapid formation of [14C]DHEA occurred. The intermediate 17alpha-hydroxypregnenolone accumulated initially with subsequent metabolism to DHEA. Likewise, when incubated with C21-delta4-steroids, [14C]progesterone and [3H]17alpha-hydroxyprogesterone, the 17,20-lyase product androstenedione was produced efficiently. In these studies, with respect to the delta5 pathway, the expressed hamster P450C17 gave similar results to bovine P450C17 cDNA inserted in the same expression vector. However, in contrast to the bovine enzyme, which converted low amounts of progesterone to androstenedione, the expressed hamster P450C17 enzyme showed an active metabolism via the delta4 pathway. Northern blot analysis, using the complete alpha-32P labeled hamster P450C17 cDNA as the probe, demonstrated a strong presence of P450C17 mRNA in hamster adrenals, a weaker presence in testes and ovaries, and no detectable species in brain, mesentery, and kidney. Immunoblotting analysis using an anti-rat P450C17 antibody demonstrated the presence of P450C17 protein in hamster adrenals, testes, and ovaries. Hamster adrenal cell suspensions and microsomal preparations were used to demonstrate the biosynthesis of [14C]17alpha-hydroxypregnenolone and [14C]DHEA from [14C]pregnenolone; both metabolites were formed during incubations. However, the ratio of [14C]DHEA/[14C]17alpha-hydroxypregnenolone was much lower in adrenal cells than in transfected COS 1 cells, indicating the presence of putative factors in hamster adrenal cells, favoring the 17alpha-hydroxylase activity rather than that of the 17,20-lyase. In conclusion, these studies demonstrate that the hamster adrenal is both a DHEA and a cortisol producer, and, therefore, this animal could be a suitable small animal model for the study of the role of DHEA in relation to human biochemistry and physiology.

We are reporting the case of two sisters born to nonrelated French Canadian parents. Patient A is of female phenotype with 46,xy, and patient B with 46,xx. The children had severe manifestations of mineralocorticoid deficiency at the age of 11 and 4.5 months, respectively. Residual cortisol secretion seemed present until the age of 3 years for patient A and until 15 months in the case of her sister. Both patients responded to glucocorticoid and Florinef treatment. Patient A did not show any androgen secretion and gonadectomy was performed at the age of 13.4 years; estrogen therapy was started at the age of 14 years resulting in a good breast development and an increase of growth velocity. In patient B, a progressive development of secondary sex characters occurred at 11.6 years of age followed at 14 years by menarche associated with a normal secretion of LH, FSH and estradiol; regular menstruations continued up to her last visit at the age of 25 years. We identified a homozygous L275P mutation on the StAR gene of both patients and a heterozygous L275P mutation on that of their mother and father. In transfection analysis in COS-1 cells, the mutant L275P was well-expressed, but its StAR activity was 87% impaired. The remaining activity of the L275P StAR mutant is consistent with the moderate severity of clinical onset of manifestations.