Mechanism of metal activation of human hematopoietic prostaglandin D synthase

Here we report the crystal structures of human hematopoietic prostaglandin (PG) D synthase bound to glutathione (GSH) and Ca2+ or Mg2+. Using GSH as a cofactor, prostaglandin D synthase catalyzes the isomerization of PGH2 to PGD2, a mediator for allergy response. The enzyme is a homodimer, and Ca2+ or Mg2+ increases its activity to approximately 150% of the basal level, with half maximum effective concentrations of 400 microM for Ca2+ and 50 microM for Mg2+. In the Mg2+-bound form, the ion is octahedrally coordinated by six water molecules at the dimer interface. The water molecules are surrounded by pairs of Asp93, Asp96 and Asp97 from each subunit. Ca(2+) is coordinated by five water molecules and an Asp96 from one subunit. The Asp96 residue in the Ca2+-bound form makes hydrogen bonds with two guanidium nitrogen atoms of Arg14 in the GSH-binding pocket. Mg2+ alters the coordinating water structure and reduces one hydrogen bond between Asp96 and Arg14, thereby changing the interaction between Arg14 and GSH. This effect explains a four-fold reduction in the K(m) of the enzyme for GSH. The structure provides insights into how Ca2+ or Mg2+ binding activates human hematopoietic PGD synthase.     

The untranslated region of exon 2 of the human neuronal nitric oxide synthase (NOS1) gene exerts regulatory activity

Expressional dysregulation of the human neuronal nitric oxide synthase (NOS1) gene represents an important mechanism in the pathogenesis of certain neuronal disease states. The structure and regulation of the human NOS1 gene is highly complex based on cell type- and stimulus-dependent usage of multiple exon 1 variants. Here we demonstrate that the untranslated region of exon 2 exerts promoter and enhancer functions as well, facilitated in large part by cooperative interaction of two conserved adjacent CREB/AP-1 binding sites. In human neuronal A673 cells, NOS1 expression is stimulated by several compounds which act through these sites, but also stimulate the combined promoter region of exons 1f and 1g. While stimulation of NOS1 expression by dibutyryl-cAMP is mediated by protein kinase A (blocked by H-89), the antiepileptic drug valproic acid is likely to activate phosphatidylinositol-3 kinase (inhibited by LY 294002).     

A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

Negative regulation of human insulin gene expression by the 5'-flanking region in non-pancreatic cells

We have examined the effect of the 5'-flanking region of the human insulin gene on its expression in non-pancreatic cells. The presence of the region containing the insulin gene enhancer (-339 to -169 bp) markedly repressed the promoter activity of the insulin gene. This suppressive phenomenon was restored by the addition of forskolin or dibutyryl cAMP, suggesting that this region alone is not sufficient to repress completely insulin gene expression in the presence of extracellular stimuli which increase the intracellular cAMP level. The hypervariable region (HVR) located at -365 bp also repressed the promoter activity. These results show negative regulation of human insulin gene expression in non-pancreatic cells by these regions.     

Identification of a glucocorticoid response element in the 3'-flanking region of the human Dexras1 gene

The Dexras1 gene responds to glucocorticoids with a rapid and profound induction. A glucocorticoid response element (GRE) was identified in the 3'-flanking region (2.3 kb downstream of poly(A) signal) of the human Dexras1 gene. This element conferred rapid glucocorticoid responsiveness when inserted into a homologous promoter-driven luciferase reporter. A point mutation within the 15-bp GRE abolished this glucocorticoid responsiveness.     

Transcriptional activation of the human claudin-18 gene promoter through two AP-1 motifs in PMA-stimulated MKN45 gastric cancer cells

Claudin-18 (CLDN18), a member of the claudin family of proteins that are structural components of tight junctions, has two alternatively spliced variants, claudin-18a1 and claudin-18a2, which are highly expressed in lung and stomach, respectively. Downregulation of claudin-18a2 is associated with gastric cancers of an intestinal phenotype; however, the mechanisms regulating its expression have not been defined. Here, we found that phorbol 12-myristate 13-acetate (PMA) treatment of MKN45 human gastric cancer cell line increased claudin-18a2 expression. In addition, this study aimed to characterize the human CLDN18a2 promoter. Using reporter gene assays and deletion analysis, we mapped the critical promoter region of the PMA-stimulated claudin-18a2 expression to the -923/-286 region. Electrophoretic mobility shift assays and mutational analyses revealed that two activator protein (AP)-1 binding sites played an important role in the expression of claudin-18a2 in PMA-stimulated MKN45 cells. Protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors suppressed the upregulation of claudin-18a2. These results indicate that the PKC/MAPK/AP-1 dependent pathway regulates claudin-18a2 expression in gastric cells.     

First determination of the inhibitor complex structure of human hematopoietic prostaglandin D synthase

Hematopoietic prostaglandin (PG) D synthase (H-PGDS) is responsible for the production of PGD(2) as an allergy or inflammation mediator in mast and Th2 cells. We determined the X-ray structure of human H-PGDS complexed with an inhibitor, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT) at 1.9 A resolution in the presence of Mg(2+). The styryl group of the inhibitor penetrated to the bottom of the active site cleft, and the tetrazole ring was stabilized by the stacking interaction with Trp104, inducing large movement around the alpha5-helix, which caused the space group of the complex crystal to change from P2(1) to P1 upon binding of BSPT. The phthalhydrazidyl group of BSPT exhibited steric hindrance due to the cofactor, glutathione (GSH), increasing the IC(50) value of BSPT for human H-PGDS from 36.2 micro M to 98.1 micro M upon binding of Mg(2+), because the K(m) value of GSH for human H-PGDS was decreased from 0.60 micro M in the presence of EDTA to 0.14 micro M in the presence of Mg(2+). We have to avoid steric hindrance of the GSH molecule that was stabilized by intracellular Mg(2+) in the mM range in the cytosol for further development of structure-based anti-allergic drugs.