ERRgamma tethers strongly bisphenol A and 4-alpha-cumylphenol in an induced-fit manner

A receptor-binding assay and X-ray crystal structure analysis demonstrated that the endocrine disruptor bisphenol A (BPA) strongly binds to human estrogen-related receptor γ (ERRγ). BPA is well anchored to the ligand-binding pocket, forming hydrogen bonds with its two phenol-hydroxyl groups. In this study, we found that 4-α-cumylphenol lacking one of its phenol-hydroxyl groups also binds to ERRγ very strongly. The 2.0 Å crystal structure of the 4-α-cumylphenol/ERRγ complex clearly revealed that ERRγ’s Leu345-β-isopropyl plays a role in the tight binding of 4-α-cumylphenol and BPA, rotating in a back-and-forth induced-fit manner.     

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty microg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB-treated group produced significantly more ultrafiltration than the non-treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with the non-treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB-treated endothelial cells, compared with the non-treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter.     

Cosynthesis of monofluoroacetate and 4-fluorothreonine by resting cells of blocked mutants of Streptomyces cattleya NRRL8057

Streptomyces cattleya NRRL 8057 produces monofluoroacetate and 4-fluorothreonine from inorganic fluoride. Mutants blocked in fluorometabolite production were prepared by chemical mutagenesis, and cosynthesis experiments with these blocked mutants were carried out by suspending cells of one blocked mutant in the supernatant broth of another blocked mutant. The harvest age of the cells, pH of the buffer, potassium fluoride concentration and glycerol supplementation were optimized for the monofluoroacetate production by a resting-cell suspension of S. cattleya. Successful cosynthesis with pairs of the mutants characterized four distinctive blocked sites in the order N-82, N-44, N-43 and N-47. Additional preparation of blocked mutants by UV irradiation and their cosynthesis assay confirmed that U-303, U-304, U-400 and U-500 were blocked in later steps than N-47. O’Hagan et al. recently proposed that fluoroacetaldehyde, the hypothetical precursor of monofluoroacetate and 4-fluorothreonine, derives from 5′-fluoro-5′-deoxyadenosine, the first fluorinated metabolite synthesized from S-adenosyl- -methionine and inorganic fluoride by the novel enzyme ‘fluorinase’. We were able to detect fluorinase activity in crude extracts of wild type and N-47 mutant strains, but not in the other mutant strains whose blocked steps flanked that of N-47.     

From glycomics to functional glycomics of sugar chains: Identification of target proteins with functional changes using gene targeting mice and knock down cells of FUT8 as examples

Comprehensive analyses of proteins from cells and tissues are the most effective means of elucidating the expression patterns of individual disease-related proteins. On the other hand, the simultaneous separation and characterization of proteins by 1-DE or 2-DE followed by MS analysis are one of the fundamental approaches to proteomic analysis. However, these analyses do not permit the complete structural identification of glycans in glycoproteins or their structural characterization. Over half of all known proteins are glycosylated and glycan analyses of glycoproteins are requisite for fundamental proteomics studies. The analysis of glycan structural alterations in glycoproteins is becoming increasingly important in terms of biomarkers, quality control of glycoprotein drugs, and the development of new drugs. However, usual approach such as proteoglycomics, glycoproteomics and glycomics which characterizes and/or identifies sugar chains, provides some structural information, but it does not provide any information of functionality of sugar chains. Therefore, in order to elucidate the function of glycans, functional glycomics which identifies the target glycoproteins and characterizes functional roles of sugar chains represents a promising approach. In this review, we show examples of functional glycomics technique using alpha 1,6 fucosyltransferase gene (Fut8) in order to identify the target glycoprotein(s). This approach is based on glycan profiling by CE/MS and LC/MS followed by proteomic approaches, including 2-DE/1-DE and lectin blot techniques and identification of functional changes of sugar chains.     

Selective expansion of foxp3-positive regulatory T cells and immunosuppression by suppressors of cytokine signaling 3-deficient dendritic cells

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10(+) Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4(+) T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3(+) Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3(+) T cell-inducing tolerogenic DCs. SOCS3(-/-) DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3(-) effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3(+) Treg cells were selectively expanded by SOCS3(-/-) DCs. Adoptive transfer of SOCS3(-/-) DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3(+) T cell expansion was blocked by anti-TGF-beta Ab, and SOCS3(-/-) DCs produced higher levels of TGF-beta than WT-DCs, suggesting that TGF-beta plays an essential role in the expansion of Foxp3(+) Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs.     

Structural and functional differences in two cyclic bacteriocins with the same sequences produced by lactobacilli

Lactobacillus gasseri LA39 and L. reuteri LA6 isolated from feces of the same human infant were found to produce similar cyclic bacteriocins (named gassericin A and reutericin 6, respectively) that cannot be distinguished by molecular weights or primary amino acid sequences. However, reutericin 6 has a narrower spectrum than gassericin A. In this study, gassericin A inhibited the growth of L. reuteri LA6, but reutericin 6 did not inhibit the growth of L. gasseri LA39. Both bacteriocins caused potassium ion efflux from indicator cells and liposomes, but the amounts of efflux and patterns of action were different. Although circular dichroism spectra of purified bacteriocins revealed that both antibacterial peptides are composed mainly of alpha-helices, the spectra of the bacteriocins did not coincide. The results of D- and L-amino acid composition analysis showed that two residues and one residue of D-Ala were detected among 18 Ala residues of gassericin A and reutericin 6, respectively. These findings suggest that the different D-alanine contents of the bacteriocins may cause the differences in modes of action, amounts of potassium ion efflux, and secondary structures. This is the first report that characteristics of native bacteriocins produced by wild lactobacillus strains having the same structural genes are influenced by a difference in D-amino acid contents in the molecules.     

Identification of difructose dianhydride I synthase/hydrolase from an oral bacterium establishes a novel glycoside hydrolase family

Fructooligosaccharides and their anhydrides are widely used as health-promoting foods and prebiotics. Various enzymes acting on β-D-fructofuranosyl linkages of natural fructan polymers have been used to produce functional compounds. However, enzymes that hydrolyze and form α-D-fructofuranosyl linkages have been less studied. Here, we identified the BBDE_2040 gene product from Bifidobacterium dentium (α-D-fructofuranosidase and difructose dianhydride I synthase/hydrolase from Bifidobacterium dentium [αFFase1]) as an enzyme with α-D-fructofuranosidase and α-D-arabinofuranosidase activities and an anomer-retaining manner. αFFase1 is not homologous with any known enzymes, suggesting that it is a member of a novel glycoside hydrolase family. When caramelized fructose sugar was incubated with αFFase1, conversions of β-D-Frup-(2→1)-α-D-Fruf to α-D-Fruf-1,2′:2,1′-β-D-Frup (diheterolevulosan II) and β-D-Fruf-(2→1)-α-D-Fruf (inulobiose) to α-D-Fruf-1,2′:2,1′-β-D-Fruf (difructose dianhydride I [DFA I]) were observed. The reaction equilibrium between inulobiose and DFA I was biased toward the latter (1:9) to promote the intramolecular dehydrating condensation reaction. Thus, we named this enzyme DFA I synthase/hydrolase. The crystal structures of αFFase1 in complex with β-D-Fruf and β-D-Araf were determined at the resolutions of up to 1.76 Å. Modeling of a DFA I molecule in the active site and mutational analysis also identified critical residues for catalysis and substrate binding. The hexameric structure of αFFase1 revealed the connection of the catalytic pocket to a large internal cavity via a channel. Molecular dynamics analysis implied stable binding of DFA I and inulobiose to the active site with surrounding water molecules. Taken together, these results establish DFA I synthase/hydrolase as a member of a new glycoside hydrolase family (GH172).     

Neuronal cell type–specific alternative splicing is regulated by the KH domain protein SLM1

The unique functional properties and molecular identity of neuronal cell populations rely on cell type–specific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domain–containing splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell type–specific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.