Enzymatic activity of Lecithin:retinol acyltransferase: a thermostable and highly active enzyme with a likely mode of interfacial activation.

Lecithin:retinol acyltransferase (LRAT) plays a major role in the vertebrate visual cycle. Indeed, it is responsible for the esterification of all-trans retinol into all-trans retinyl esters, which can then be stored in microsomes or further metabolized to produce the chromophore of rhodopsin. In the present study, a detailed characterization of the enzymatic properties of truncated LRAT (tLRAT) has been achieved using in vitro assay conditions.

Crystal structures of human Delta4-3-ketosteroid 5beta-reductase (AKR1D1) reveal the presence of an alternative binding site responsible for substrate inhibition.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found in Delta4-3-ketosteroids, including steroid hormones and bile acids precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one. In order to elucidate the binding mode and substrate specificity in detail, biochemical and structural studies on human 5beta-reductase (h5beta-red; AKR1D1) have been recently undertaken. The crystal structure of a h5beta-red binary complex provides a complete picture of the NADPH-enzyme interactions involving the flexible loop B, which contributes to the maintenance of the cofactor in its binding site by acting as a "safety belt".

The crystal structure of human Delta4-3-ketosteroid 5beta-reductase defines the functional role of the residues of the catalytic tetrad in the steroid double bond reduction mechanism.

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found into Delta4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu 120, as the major molecular determinant for the unique activity displayed by 5beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5beta-reductase (h5beta-red) have been undertaken.

Secondary structure of a truncated form of lecithin retinol acyltransferase in solution and evidence for its binding and hydrolytic action in monolayers.

Lecithin retinol acyltransferase (LRAT) is a 230 amino acids membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. The enzymatic activity of a truncated form of LRAT (tLRAT) which contains the residues required for catalysis but which is lacking N- and C-terminal hydrophobic segments has been shown to depend on the detergent used for its solubilization. Moreover, it is unknown whether tLRAT can bind membranes in the absence of these hydrophobic segments.

Mouse 17alpha-hydroxysteroid dehydrogenase (AKR1C21) binds steroids differently from other aldo-keto reductases: identification and characterization of amino acid residues critical for substrate binding.

The mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD) is the unique known member of the aldo-keto reductase (AKR) superfamily able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epi-testosterone (epi-T), the 17alpha-epimer of testosterone. Structural and mutagenic studies had already identified one of the residues delineating the steroid-binding cavity, A24, as the major molecular determinant for the stereospecificity of m17alpha-HSD. We report here a ternary complex crystal structure (m17alpha-HSD:NADP(+):epi-T) determined at 1.

Chemical synthesis and biological activities of 16alpha-derivatives of 5alpha-androstane-3alpha,17beta-diol as antiandrogens.

In our efforts to develop compounds with therapeutic potential as antiandrogens, we synthesized a series of 5alpha-androstane-3alpha,17beta-diol derivatives with a fixed side-chain length of 3-methylenes at C-16alpha, but bearing a diversity of functional groups at the end. Among these, the chloride induced the best antiproliferative activity on androgen-sensitive Shionogi cells. Substituting the OH at C-3 by a methoxy group showed the importance of the OH.

Crystal structures of mouse 17alpha-hydroxysteroid dehydrogenase (apoenzyme and enzyme-NADP(H) binary complex): identification of molecular determinants responsible for the unique 17alpha-reductive activity of this enzyme.

Very recently, the mouse 17alpha-hydroxysteroid dehydrogenase (m17alpha-HSD), a member of the aldo-keto reductase (AKR) superfamily, has been characterized and identified as the unique enzyme able to catalyze efficiently and in a stereospecific manner the conversion of androstenedione (Delta4) into epitestosterone (epi-T), the 17alpha-epimer of testosterone. Indeed, the other AKR enzymes that significantly reduce keto groups situated at position C17 of the steroid nucleus, the human type 3 3alpha-HSD (h3alpha-HSD3), the human and mouse type 5 17beta-HSD, and the rabbit 20alpha-HSD, produce only 17beta-hydroxy derivatives, although they possess more than 70% amino acid identity with m17alpha-HSD. Structural comparisons of these highly homologous enzymes thus offer an excellent opportunity of identifying the molecular determinants responsible for their 17alpha/17beta-stereospecificity.

Comparison of crystal structures of human androgen receptor ligand-binding domain complexed with various agonists reveals molecular determinants responsible for binding affinity.

Androgens exert their effects by binding to the highly specific androgen receptor (AR). In addition to natural potent androgens, AR binds a variety of synthetic agonist or antagonist molecules with different affinities. To identify molecular determinants responsible for this selectivity, we have determined the crystal structure of the human androgen receptor ligand-binding domain (hARLBD) in complex with two natural androgens, testosterone (Testo) and dihydrotestosterone (DHT), and with an androgenic steroid used in sport doping, tetrahydrogestrinone (THG), at 1.

Binding of RPE65 fragments to lipid monolayers and identification of its partners by glutathione S-transferase pull-down assays.

RPE65 is the major component of the retinal pigment epithelium (RPE) microsomal membrane, and it plays a critical role in the binding of retinoids involved in the visual cycle. To understand how RPE65 binds to membranes, we have expressed and purified soluble fragments of human RPE65 fused to glutathione S-transferase (GST). The interaction between two fragments of RPE65 (F1 and F2 which include residues 1-125 and 126-250, respectively) and lipid monolayers has been studied by surface pressure, ellipsometry, and surface rheology measurements.

Characterization of 17alpha-hydroxysteroid dehydrogenase activity (17alpha-HSD) and its involvement in the biosynthesis of epitestosterone.

Background: Epi-testosterone (epiT) is the 17alpha-epimer of testosterone. It has been found at similar level as testosterone in human biological fluids. This steroid has thus been used as a natural internal standard for assessing testosterone abuse in sports.

Comparison of crystal structures of human type 3 3alpha-hydroxysteroid dehydrogenase reveals an "induced-fit" mechanism and a conserved basic motif involved in the binding of androgen.

The aldo-keto reductase (AKR) human type 3 3alpha-hydroxysteroid dehydrogenase (h3alpha-HSD3, AKR1C2) plays a crucial role in the regulation of the intracellular concentrations of testosterone and 5alpha-dihydrotestosterone (5alpha-DHT), two steroids directly linked to the etiology and the progression of many prostate diseases and cancer. This enzyme also binds many structurally different molecules such as 4-hydroxynonenal, polycyclic aromatic hydrocarbons, and indanone. To understand the mechanism underlying the plasticity of its substrate-binding site, we solved the binary complex structure of h3alpha-HSD3-NADP(H) at 1.

Structure of primate and rodent orthologs of the prostate cancer susceptibility gene ELAC2.

The human ELAC2 gene was the first candidate prostate cancer susceptibility gene identified by linkage analysis and positional cloning. DNA sequence indicates a protein of 826 amino acids encoded by 24 exons. In the present study, we characterized the coding sequence of chimpanzee and gorilla ELAC2 orthologs by direct sequencing of genomic fragments, and of cynomolgus monkey and rat orthologs by screening cDNA libraries.

Loop relaxation, a mechanism that explains the reduced specificity of rabbit 20alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily.

The aldo-keto reductase rabbit 20alpha-hydroxysteroid dehydrogenase (rb20alpha-HSD; AKR1C5) is less selective than other HSDs, since it exerts its activity both on androgens (C19 steroids) and progestins (C21 steroids). In order to identify the molecular determinants responsible for this reduced selectivity, binary (NADPH) and ternary (NADP(+)/testosterone) complex structures were solved to 1.32A and 2.

The ITIM-bearing CLECSF6 (DCIR) is down-modulated in neutrophils by neutrophil activating agents.

The recently discovered CLECSF6 protein displays the features of a receptor involved in the down-modulation of leukocyte activation. Although CLECSF6 has been the focus of the interest of many researchers lately, a Western blot characterization of the protein is still lacking. This highly reduces our ability to gain full knowledge of the biological relevance of this protein in cell responses.

Human 20alpha-hydroxysteroid dehydrogenase: crystallographic and site-directed mutagenesis studies lead to the identification of an alternative binding site for C21-steroids.

Human 20alpha-hydroxysteroid dehydrogenase (h20alpha-HSD; AKR1C1) catalyzes the transformation of progesterone (Prog) into 20alpha-hydroxy-progesterone (20alpha-OHProg). Although h20alpha-HSD shares 98% sequence identity with human type 3 3alpha-HSD (h3alpha-HSD3, AKR1C2), these two enzymes differ greatly in their activities. In order to explain these differences, we have solved the crystal structure of h20alpha-HSD in a ternary complex with NADP(+) and 20alpha-OHProg at 1.

Expression, crystallization and preliminary X-ray analysis of human and rabbit 20alpha-hydroxysteroid dehydrogenases in complex with NADP(H) and various steroid substrates.

Progesterone plays an essential role in the maintenance of the pregnancy of most mammals. 20alpha-Hydroxysteroid dehydrogenase (20alpha-HSD) catalyses the inactivation of progesterone into its inactive form, 20alpha-hydroxyprogesterone, and could thus be involved in progesterone withdrawal and in the control of gestation. In this report, the purification and crystallization of recombinant human and rabbit 20alpha-HSDs (h20alpha-HSD and rb20alpha-HSD) are described, two highly homologous enzymes possessing, in addition to their common 20alpha-HSD activity, different activities and substrate specificities.