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.

No abstract

The occurence of spontaneous hybridization between Brassica napus (oilseed rape) and Raphanus raphanistrum (wild radish) was investigated under different density conditions in cages and open‐field experiments. Hybrids with wild radish as the seed parent were identified by screening for herbicide resistance belonging to rape. Small seed size and intermediate morphology were used to screen for hybrids with rape as the seed parent. Leaf isozyme patterns and flow cytometry provided confirmation of hybrids. Wild radish in an oilseed rape field produced as many as three interspecific hybrids per 100 plants. This is the first report of such a spontaneous event. The frequency of hybrids is expected to range from 0.006 to 0.2% of the total seed produced, at P = 0.05. Male‐sterile oilseed rape plants surrounded by wild radish can produce up to 37 hybrids per plant. Seed production of the F1 hybrids and their F2 descendants was up to 0.4% and 2%, respectively, of that of wild radish. Gene escape from transgenic oilseed rape to wild related species is discussed.

No abstract

Background: The rapid burgeoning of available protein data makes the use of clustering within families of proteins increasingly important. The challenge is to identify subfamilies of evolutionarily related sequences. This identification reveals phylogenetic relationships, which provide prior knowledge to help researchers understand biological phenomena. A good evolutionary model is essential to achieve a clustering that reflects the biological reality, and an accurate estimate of protein sequence similarity is crucial to the building of such a model. Most existing algorithms estimate this similarity using techniques that are not necessarily biologically plausible, especially for hard-to-align sequences such as proteins with different domain structures, which cause many difficulties for the alignment-dependent algorithms. In this paper, we propose a novel similarity measure based on matching amino acid subsequences. This measure, named SMS for Substitution Matching Similarity, is especially designed for application to non-aligned protein sequences. It allows us to develop a new alignment-free algorithm, named CLUSS, for clustering protein families. To the best of our knowledge, this is the first alignment-free algorithm for clustering protein sequences. Unlike other clustering algorithms, CLUSS is effective on both alignable and non-alignable protein families. In the rest of the paper, we use the term "phylogenetic" in the sense of "relatedness of biological functions".

In mammalian and fish species, P450c17 mediates both 17 alpha-hydroxylase and 17,20-lyase activities in the synthesis of steroid hormones. Previous results have shown that among the adrenal steroid hydroxylase enzymes involved in adrenal C19 steroid and glucocorticoid synthesis, regulation of cytochrome P450c17 is of primary importance because it is localized at the key branch between glucocorticoid and C19 steroid synthesis. A cDNA library from guinea pig adrenal was constructed, and the complete 17 alpha-hydroxylase cytochrome P450 cDNA was isolated. The guinea pig P450c17 cDNA includes the full-length coding region (1,524 nucleotide), the complete 3' untranslated region (169 nucleotide), and 39 bases of the 5' untranslated region. Our clone shares most of the features of the other P450c17 cDNAs; however, in addition, we identified a novel conserved region of 18 amino acids located in exon I between residues 80 and 97. This region presents the highest percentage of identity among the other P450c17 enzymes and is positioned one helixturn upstream of the important Ser106 on the corresponding human form. On Northern blot, the cDNA hybridizes with a major 1.8-kb mRNA and with two other related P450c17 mRNA of about 3 and 4 kb. P450c17 mRNA is equally distributed in male and female gonads and adrenals. Characterization of the enzymatic activity shows that 17 alpha-hydroxylase and 17,20-lyase are carried by a single protein, but in homogenates 17,20-lyase activity is barely detectable. Moreover, we demonstrate in vitro and in vivo that the guinea pig enzyme preferentially has very high levels of 17 alpha-hydroxylase and 17,20-lyase activities only toward delta 4 steroids. Second-messenger cyclic adenosine monophosphate and adrenocorticotropin specifically increased the abundance of P450c17 mRNA levels in guinea pig adrenal cells.

No abstract

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...