Aspergillus niger DLFCC-90 rhamnoside hydrolase, a new type of flavonoid glycoside hydrolase

A novel rutin-α-L-rhamnosidase hydrolyzing α-L-rhamnoside of rutin, naringin, and hesperidin was purified and characterized from Aspergillus niger DLFCC-90, and the gene encoding this enzyme, which is highly homologous to the α-amylase gene, was cloned and expressed in Pichia pastoris GS115. The novel enzyme was classified in glycoside-hydrolase (GH) family 13.     

Autopolyploidy differentially influences body size in plants, but facilitates enhanced accumulation of secondary metabolites, causing increased cytosine methylation

Whole genome duplication leads to autopolyploidy and brings about an increase in cell size, concentration of secondary metabolites and enhanced cytosine methylation. The increased cell size offers a positive advantage to polyploids for cell-surface-related activities, but there is a differential response to change in body size across species and taxonomic groups. Although polyploidy has been very extensively studied, having genetic, ecological and evolutionary implications, there is no report that underscores the significance of native secondary metabolites vis-à-vis body size with ploidy change. To address this problem we targeted unique diploid-autotetraploid paired sets of eight diverse clones of six species of Cymbopogon- a species complex of aromatic grasses that accumulate qualitatively different monoterpene essential oils (secondary metabolite) in their vegetative biomass. Based on the qualitative composition of essential oils and the plant body size relationship between the diploid versus autotetraploid paired sets, we show that polyploidy brings about enhanced accumulation of secondary metabolites in all cases, but exerts differential effects on body size in various species. It is observed that the accumulation of alcohol-type metabolites (e.g. geraniol) does not inhibit increase in body size with ploidy change from 2× to 4× (r = 0.854, P < 0.01), but aldehyde-type metabolites (e.g. citral) appear to drastically impede body development (r = -0.895). Such a differential response may be correlated to the metabolic steps involved in the synthesis of essential oil components. When changed to tetraploidy, the progenitor diploids requiring longer metabolic steps in production of their secondary metabolites are stressed, and those having shorter metabolite routes better utilize their resources for growth and vigour. In situ immunodetection of 5-methylcytosine sites reveals enhanced DNA methylation in autopolyploids. It is underpinned that the qualitative composition of secondary metabolites found in the vegetative biomass of the progenitor diploid has a decisive bearing on the body size of the derived autotetraploids and brings about an enhancement in genome-wide cytosine methylation.     

Structural basis for extracellular interactions between calcitonin receptor-like receptor and receptor activity-modifying protein 2 for adrenomedullin-specific binding

The calcitonin receptor-like receptor (CRLR), a class B GPCR, forms a heterodimer with receptor activity-modifying protein 2 (RAMP2), and serves as the adrenomedullin (AM) receptor to control neovascularization, while CRLR and RAMP1 form the calcitonin gene-related peptide (CGRP) receptor. Here, we report the crystal structures of the RAMP2 extracellular domain alone and in the complex with the CRLR extracellular domain. The CRLR-RAMP2 complex exhibits several intermolecular interactions that were not observed in the previously reported CRLR-RAMP1 complex, and thus the shape of the putative ligand-binding pocket of CRLR-RAMP2 is distinct from that of CRLR-RAMP1. The CRLR-RAMP2 interactions were confirmed for the full-length proteins on the cell surface by site-specific photo-crosslinking. Mutagenesis revealed that AM binding requires RAMP2 residues that are not conserved in RAMP1. Therefore, the differences in both the shapes and the key residues of the binding pocket are essential for the ligand specificity.     

AWP1/ZFAND6 functions in Pex5 export by interacting with cys-monoubiquitinated Pex5 and Pex6 AAA ATPase

During biogenesis of the peroxisome, a subcellular organelle, the peroxisomal-targeting signal 1 (PTS1) receptor Pex5 functions as a shuttling receptor for PTS1-containing peroxisomal matrix proteins. However, the precise mechanism of receptor shuttling between peroxisomes and cytosol remains elusive despite the identification of numerous peroxins involved in this process. Herein, a new factor was isolated by a combination of biochemical fractionation and an in vitro Pex5 export assay, and was identified as AWP1/ZFAND6, a ubiquitin-binding NF-κB modulator. In the in vitro Pex5 export assay, recombinant AWP1 stimulated Pex5 export and an anti-AWP1 antibody interfered with Pex5 export. AWP1 interacted with Pex6 AAA ATPase, but not with Pex1-Pex6 complexes. Preferential binding of AWP1 to the cysteine-ubiquitinated form of Pex5 rather than to unmodified Pex5 was mediated by the AWP1 A20 zinc-finger domain. Inhibition of AWP1 by RNA interference had a significant effect on PTS1-protein import into peroxisomes. Furthermore, in AWP1 knock-down cells, Pex5 stability was decreased, similar to fibroblasts from patients defective in Pex1, Pex6 and Pex26, all of which are required for Pex5 export. Taken together, these results identify AWP1 as a novel cofactor of Pex6 involved in the regulation of Pex5 export during peroxisome biogenesis.     

Tetrameric interaction of the ectoenzyme CD38 on the cell surface enables its catalytic and raft-association activities

The leukocyte cell-surface antigen CD38 is the major nicotinamide adenide dinucleotide glycohydrolase in mammals, and its ectoenzyme activity is involved in calcium mobilization. CD38 is also a raft-dependent signaling molecule. CD38 forms a tetramer on the cell surface, but the structural basis and the functional significance of tetramerization have remained unexplored. We identified the interfaces contributing to the homophilic interaction of mouse CD38 by site-specific crosslinking on the cell surface with an expanded genetic code, based on a crystallographic analysis. A combination of the three interfaces enables CD38 to tetramerize: one interface involving the juxtamembrane α-helix is responsible for the formation of the core dimer, which is further dimerized via the other two interfaces. This dimerization of dimers is required for the catalytic activity and the localization of CD38 in membrane rafts. The glycosylation prevents further self-association of the tetramer. Accordingly, the tetrameric interaction underlies the multifaceted actions of CD38.     

Dimerization of DOCK2 Is Essential for DOCK2-Mediated Rac Activation and Lymphocyte Migration

The migratory properties of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain the Dbl homology domain typically found in guanine nucleotide exchange factors (GEFs), DOCK2 mediates the GTP-GDP exchange reaction for Rac via its DOCK homology region (DHR)-2 (also known as CZH2 or Docker) domain. DOCK2 DHR-2 domain is composed of three lobes, and Rac binding site and catalytic center are generated entirely from lobes B and C. On the other hand, lobe A has been implicated in dimer formation, yet its physiological significance remains unknown. Here, we report that lobe A-mediated DOCK2 dimerization is crucial for Rac activation and lymphocyte migration. We found that unlike wild-type DOCK2, DOCK2 mutant lacking lobe A failed to restore motility and polarity when expressed in thymoma cells and primary T cells lacking endogenous expression of DOCK2. Similar results were obtained with the DOCK2 point mutant having a defect in dimerization. Deletion of lobe A from the DHR-2 domain did not affect Rac GEF activity in vitro. However, fluorescence resonance energy transfer analyses revealed that lobe A is required for DOCK2 to activate Rac effectively during cell migration. Our results thus indicate that DOCK2 dimerization is functionally important under the physiological condition where only limited amounts of DOCK2 and Rac are localized to the plasma membrane.     

A novel GIP receptor splice variant influences GIP sensitivity of pancreatic beta-cells in obese mice

Gastric inhibitory polypeptide (GIP) is an incretin that potentiates insulin secretion from pancreatic beta-cells by binding to GIP receptor (GIPR) and subsequently increasing the level of intracellular adenosine 3',5'-cyclic monophosphate (cAMP). We have identified a novel GIPR splice variant in mouse beta-cells that retains intron 8, resulting in a COOH-terminal truncated form (truncated GIPR). This isoform was coexpressed with full-length GIPR (wild-type GIPR) in normal GIPR-expressing tissues. In an experiment using cells transfected with both GIPRs, truncated GIPR did not lead to cAMP production induced by GIP but inhibited GIP-induced cAMP production through wild-type GIPR (n = 3-4, P < 0.05). Wild-type GIPR was normally located on the cell surface, but its expression was decreased in the presence of truncated GIPR, suggesting a dominant negative effect of truncated GIPR against wild-type GIPR. The functional relevance of truncated GIPR in vivo was investigated. In high-fat diet-fed obese mice (HFD mice), blood glucose levels were maintained by compensatory increased insulin secretion (n = 8, P < 0.05), and cAMP production (n = 6, P < 0.01) and insulin secretion (n = 10, P < 0.05) induced by GIP were significantly increased in isolated islets, suggesting hypersensitivity of the GIPR. Total GIPR mRNA expression was not increased in the islets of HFD mice, but the expression ratio of truncated GIPR to total GIPR was reduced by 32% compared with that of control mice (n = 6, P < 0.05). These results indicate that a relative reduction of truncated GIPR expression may be involved in hypersensitivity of GIPR and hyperinsulinemia in diet-induced obese mice.     

A comparison of the tube forming potentials of early and late endothelial progenitor cells

The identification of circulating endothelial progenitor cells (EPCs) has revolutionized approaches to cell-based therapy for injured and ischemic tissues. However, the mechanisms by which EPCs promote the formation of new vessels remain unclear. In this study, we obtained early EPCs from human peripheral blood and late EPCs from umbilical cord blood. Human umbilical vascular endothelial cells (HUVECs) were also used. Cells were evaluated for their tube-forming potential using our novel in vitro assay system. Cells were seeded linearly along a 60 μm wide path generated by photolithographic methods. After cells had established a linear pattern on the substrate, they were transferred onto Matrigel. Late EPCs formed tubular structures similar to those of HUVECs, whereas early EPCs randomly migrated and failed to form tubular structures. Moreover, late EPCs participate in tubule formation with HUVECs. Interestingly, late EPCs in Matrigel migrated toward pre-existing tubular structures constructed by HUVECs, after which they were incorporated into the tubules. In contrast, early EPCs promote sprouting of HUVECs from tubular structures. The phenomena were also observed in the in vivo model. These observations suggest that early EPCs cause the disorganization of pre-existing vessels, whereas late EPCs constitute and orchestrate vascular tube formation.