Biomedical subjects

Petra Lukacik

Publications and source records attributed to Petra Lukacik.

5 recordsLinked to original sources

PubMed2007

Structural and biochemical characterization of human orphan DHRS10 reveals a novel cytosolic enzyme with steroid dehydrogenase activity.

To this day, a significant proportion of the human genome remains devoid of functional characterization. In this study, we present evidence that the previously functionally uncharacterized product of the human DHRS10 gene is endowed with 17beta-HSD (17beta-hydroxysteroid dehydrogenase) activity. 17beta-HSD enzymes are primarily involved in the metabolism of steroids at the C-17 position and also of other substrates such as fatty acids, prostaglandins and xenobiotics. In vitro, DHRS10 converts NAD+ into NADH in the presence of oestradiol, testosterone and 5-androstene-3beta,17beta-diol. Furthermore, the product of oestradiol oxidation, oestrone, was identified in intact cells transfected with a construct plasmid encoding the DHRS10 protein. In situ fluorescence hybridization studies have revealed the cytoplasmic localization of DHRS10. Along with tissue expression data, this suggests a role for DHRS10 in the local inactivation of steroids in the central nervous system and placenta. The crystal structure of the DHRS10 apoenzyme exhibits secondary structure of the SDR (short-chain dehydrogenase/reductase) family: a Rossmann-fold with variable loops surrounding the active site. It also reveals a broad and deep active site cleft into which NAD+ and oestradiol can be docked in a catalytically competent orientation.

17-Hydroxysteroid Dehydrogenases↗

PubMed2006

Structure and function of human 17beta-hydroxysteroid dehydrogenases.

17Beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyze the NAD(P)(H) dependent oxidoreduction at C17 oxo/beta-hydroxyl groups of androgen and estrogen hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands, since the conversion "switches" between the 17beta-OH receptor ligands and their inactive 17-oxo metabolites. At present, 14 mammalian 17beta-HSDs are described, of which at least 11 exist within the human genome, encoded by different genes. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of sex steroid hormone levels. Broad and overlapping substrate specificities with enzymes involved in lipid metabolism suggest interactions of several 17beta-HSDs with other metabolic pathways. Several 17beta-HSDs enzymes constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and possibly immunity.

17-Hydroxysteroid Dehydrogenases↗

PubMed2005

Characterization of human DHRS6, an orphan short chain dehydrogenase/reductase enzyme: a novel, cytosolic type 2 R-beta-hydroxybutyrate dehydrogenase.

Human DHRS6 is a previously uncharacterized member of the short chain dehydrogenases/reductase family and displays significant homologies to bacterial hydroxybutyrate dehydrogenases. Substrate screening reveals sole NAD(+)-dependent conversion of (R)-hydroxybutyrate to acetoacetate with K(m) values of about 10 mm, consistent with plasma levels of circulating ketone bodies in situations of starvation or ketoacidosis. The structure of human DHRS6 was determined at a resolution of 1.8 A in complex with NAD(H) and reveals a tetrameric organization with a short chain dehydrogenases/reductase-typical folding pattern. A highly conserved triad of Arg residues ("triple R" motif consisting of Arg(144), Arg(188), and Arg(205)) was found to bind a sulfate molecule at the active site. Docking analysis of R-beta-hydroxybutyrate into the active site reveals an experimentally consistent model of substrate carboxylate binding and catalytically competent orientation. GFP reporter gene analysis reveals a cytosolic localization upon transfection into mammalian cells. These data establish DHRS6 as a novel, cytosolic type 2 (R)-hydroxybutyrate dehydrogenase, distinct from its well characterized mitochondrial type 1 counterpart. The properties determined for DHRS6 suggest a possible physiological role in cytosolic ketone body utilization, either as a secondary system for energy supply in starvation or to generate precursors for lipid and sterol synthesis.

Amino Acid Motifs↗

PubMed2004

Crystallization and preliminary X-ray diffraction analysis of three EGF domains of EMR2, a 7TM immune-system molecule.

Crystals of three epidermal growth-factor-like (EGF) domains of EMR2 (143 residues) have been grown. EMR2 is a member of the EGF-TM7 family of proteins. Different splice variants exist with between three and five consecutive EGF modules linked to a seven-span transmembrane G-protein-coupled receptor. Although its precise function is unknown, EMR2 is highly expressed in immune tissues and has been shown to weakly bind CD55, a complement-system regulator. Here, crystallization of EMR2 in the presence of Ca(2+), Ba(2+) and Sr(2+) ions is reported. A complete data set has been collected from all three crystal types, all of which belong to space group P2(1). An anomalous Patterson map from the Ba(2+) crystal data reveals three Ba(2+) ions bound within the asymmetric unit.

Animals↗

PubMed2004

Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli.

Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.

Amino Acid Sequence↗