Structure of unsaturated rhamnogalacturonyl hydrolase complexed with substrate

Bacillus subtilis strain 168 YteR has been identified as a novel enzyme "unsaturated rhamnogalacturonyl hydrolase" classified in glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) produced from plant cell wall RG type-I treated with RG lyases, releasing unsaturated galacturonic acid (DeltaGalA) from the substrate. The most likely candidate catalytic residue is Asp-143. Here, we show the structure of D143N in complex with unsaturated RG disaccharide (substrate) determined at 1.9A resolution by X-ray crystallography. This structural feature directly contributes to the postulation of the enzyme reaction mechanism. YteR triggers the hydration of vinyl ether group in DeltaGalA, but not of glycoside bond, by using Asp-143 as a general acid and base catalyst. Asp-143 donates proton to the double bond of DeltaGalA as an acid catalyst and also deprotonates a water molecule as a base catalyst. Deprotonated water molecule attacks the C5 atom of DeltaGalA.     

The nuclear receptor hepatocyte nuclear factor 4alpha acts as a morphogen to induce the formation of microvilli

Microvilli are actin-based organelles found on apical plasma membranes that are involved in nutrient uptake and signal transduction. Numerous components, including ezrin/radixin/moesin (ERM) proteins, have been identified that link filamentous actins to transmembrane proteins, but the signals driving microvillus biogenesis are not known. In this study, we show that the conditional and/or ectopic expression of a nuclear receptor, hepatocyte nuclear factor 4alpha (HNF4alpha), triggers microvillus morphogenesis. We also demonstrate that HNF4alpha expression induces ERM-binding phosphoprotein 50 (EBP50) expression and that attenuation of EBP50 using RNA interference inhibits microvillus development. We conclude that HNF4alpha acts as a morphogen to trigger microvillus formation.     

Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralization

Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis.     

Bone morphogenetic protein-2 promotes the haptotactic migration of murine osteoblastic and osteosarcoma cells by enhancing incorporation of integrin beta1 into lipid rafts

Cell migration is essential for both organogenesis and tumor progression. Bone morphogenetic proteins (BMPs) are reported to be critical for not only bone formation but also tumor invasion. Here, we found that treatment with recombinant human BMP-2 (rhBMP-2) enhanced the haptotactic response of murine osteoblastic MC3T3-E1 and osteosarcoma Dunn cells to various extracellular matrix (ECM) components, including fibronectin, type I collagen, and laminin-1. Function-blocking antibody against integrin alpha5beta1 partially inhibited haptotaxis to fibronectin, suggesting that the response was propagated via these integrins. rhBMP-2 slightly increased the expression level of integrin beta1, and enhanced the speed of cell spreading on fibronectin, focal adhesion formation and phosphorylation of focal adhesion kinase (FAK) at Tyr397. By means of sucrose gradient flotation, incorporation of integrin beta1 in fractions of detergent (CHAPS) resistant membrane was increased when the cells were treated with rhBMP-2. Further, treatment with methyl-beta-cyclodextrin to deplete membrane cholesterol abrogated the effect of rhBMP-2 on haptotaxis, and exogenously added cholesterol reversed this inhibitory effect. Collectively, these results provide insights into the mechanism by which BMP signaling enhances cell migration by modulating fibronectin-integrin beta1 signaling via cholesterol enriched membrane microdomains, lipid rafts.     

Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells

Growth factor-mediated stimulation of epithelial cells induces the disassembly of E-cadherin-mediated cell-cell adhesion. We found that overexpression of a disintegrin and metalloprotease 9 (ADAM9) enhanced growth factor-mediated induction of endocytosis and dynamic recycling of E-cadherin in HT29 human colon cancer cells. In addition, ubiquitination and degradation of E-cadherin were reduced in these cells. ADAM9 constitutively interacted with E-cadherin, and the two proteins co-localized at the plasma membrane of HT29 cells. Administration of a metalloprotease inhibitor or overexpression of an ADAM9 mutant lacking metalloprotease activity attenuated growth factor-dependent endocytosis and recycling of E-cadherin as well as scattering of HT29 cells. These results suggest that the metalloprotease activity of ADAM9 mediates growth factor-induced endocytosis and dynamic recycling of E-cadherin and prevents E-cadherin degradation.     

Mutations in the lrpE gene of Ralstonia solanacearum affects Hrp pili production and virulence

The Ralstonia solanacearum hrpB-regulated gene lrpE (hpx5/brg24) encodes a PopC-like leucine-rich repeat (LRR) protein that carries 11 tandem LRR in the central region. Defects in the lrpE gene slightly reduced the virulence of R. solanacearum on host plants and changed the bacterial morphology leading to the formation of large aggregates in a minimal medium. The aggregation in the deltalrpE background required the presence of a functional Hrp type III secretion system. In wild-type R. solanacearum, Hrp pili disappeared from the bacterial surface at the end of the exponential growth phase, when the pili form into long bundles. However, even in the late growth phase, bundled Hrp pili were still observed on the cell surface of the deltalrpE mutant. Such bundles were entangled and anchored the mutant cells in the aggregates. In contrast to PopC, LrpE accumulated in bacterial cells and did not translocate into plant cells as an effector protein. The expression levels of hrp genes increased three- to fivefold in the deltalrpE background compared with those in the wild type. We propose that LrpE may negatively regulate the production of Hrp pili on the cell surface of R. solanacearum to disperse bacterial cells from aggregates. In turn, dispersal may contribute to the movement of the pathogen in the plant vascular system and, as a consequence, the pathogenicity of R. solanacearum.     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.