Journal of Biochemistry - recent issues

Matrix Metalloproteinase-9 Associated with Heparan Sulphate Chains of GPI-Anchored Cell Surface Proteoglycans Mediates Motility of Murine Colon Adenocarcinoma Cells

2008-05-06

Using murine colon adenocarcinoma-derived clones with different metastatic potentials, the cellular localization of matrix metalloporteinase-9 (MMP-9) and its role in the cell motility were examined. Highly metastatic LuM1 clone aggressively invaded into adjacent tissue in vivo, but low metastatic NM11 clone did not. As compared with the NM11 clone, the LuM1 clone expressed and secreted a remarkably large amount of MMP-9, and exhibited higher abilities of cell migration and invasion in vitro, which were suppressed by MMP-2/MMP-9 inhibitor IV. MMP-9, exhibiting high affinity to heparin, was demonstrated to be condensed on tips of cellular podia. Treatment of the cells with heparitinase-I or heparin resulted in release of MMP-9 from the cell surface, which caused concomitant suppression of their motility to a similar level to that with the MMP inhibitor. Immunoprecipitation of a LuM1 cell lysate with an anti-MMP-9 antibody resulted in co-precipitation of phosphatidylinositol-specific phospholipase C-susceptible heparan sulphate proteoglycans having 66 and 64 kDa core proteins. Taken together, the present results demonstrate that secreted MMP-9 associates with glypican-like proteoglycans through their heparan sulphate chains, and plays a crucial role in cell motility of LuM1 cells.

Ig L-chain Shuffling for Affinity Maturation of Phage Library-derived Human Anti-human MCP-1 Antibody Blocking its Chemotactic Activity

2008-05-06

Monocyte chemotactic protein-1 (MCP-1, CC-chemokine ligand 2; CCL2) is involved in the development of various forms of chronic inflammations. Employing the naive human single-chain Fv displaying phage library, we established seven MCP-1-specific scFvs. The MC8 and MC32 clones exhibited blocking activity for the MCP-1-induced chemotaxis of THP-1 cells, in spite of their monovalency. The analysis of V gene usage showed that all clones bore the identical Vh1 gene, IGHV1-24*01, with variable DJ joining sequences, while their Vl usage was relatively varied, suggesting the preferential contribution of the Vh gene. Based on these findings, to minimize the deteriorative influences on the MCP-1 specificity of MC32, we aimed to achieve the affinity maturation of MC32 using MC32 L-chain shuffling library and select MC32 variants. Most MC32 variants increased their affinity by reducing the k_off value with no influence of the antigen specificity. MC32 variants #22 or #56 showed ~15-fold higher affinity than MC32, indicating that the L-chain shuffling library is useful if the Vh is dominantly involved in the determination of the antigen specificity.

Gene Identification and Characterization of the Pyridoxine Degradative Enzyme 4-Pyridoxic Acid Dehydrogenase from the Nitrogen-fixing Symbiotic Bacterium Mesorhizobium loti MAFF303099

Ge, F., Yokochi, N., Yoshikane, Y., Ohnishi, K., Yagi, T. — 2008-05-06

The gene encoding 4-pyridoxic acid dehydrogenase was identified as mlr6792 in a chromosome of a nitrogen-fixing symbiotic bacterium Mesorhizobium loti MAFF303099. The enzyme is the fourth enzyme in the vitamin B₆ (pyridoxine)-degradation pathway I. The recombinant enzyme with a his-tag over-expressed in Escherichia coli cells was a membrane-bound protein, and purified to homogeneity. The enzyme was a monomeric protein with a molecular weight of 59,000, and a flavoprotein containing one mole of FAD per mole of subunit. The optimum pH and temperature, and K_m for 4-pyridoxic acid were pH 8.5 and 30°C, and 29 µM, respectively. The enzyme was a glucose-methanol-choline (GMC) family protein with two signature patterns, FAD-binding residues, a putative active site histidine residue and a probable transmembrane segment.

Reactivity of Astrocytes to Fibroblast Growth Factor-1 for Biogenesis of Apolipoprotein E-High Density Lipoprotein is Down-regulated by Long-time Secondary Culture

Nagayasu, Y., Ito, J.-i., Nishida, T., Yokoyama, S. — 2008-05-06

We previously showed that astrocytes produce and release fibroblast growth fator-1 (FGF-1) upon 1-month primary and 1-week secondary culture (M/W cells) and stimulate themselves by an autocrine manner to produce apoE-high-density lipoproteins (HDL), closely associated with their generation of apoE-HDL in brain injury. Astrocytes prepared by 1-week primary and 1-month secondary culture (W/M cells), however, expressed FGF-1 as much as M/W cells but produce apoE-HDL much less. The W/M cells conditioned medium in fact contained FGF-1 activity to stimulate astrocytes prepared by 1-week primary and 1-week-secondary culture (W/W cells). FGF-1 did not stimulate W/M cells for apoE-HDL biogenesis while it stimulated W/W cells. Phosphorylation of Akt, ERK and MEK were induced by FGF-1 in W/W cells but not in W/M cells. Finally, fibroblast growth factor receptor-1 in the membrane decreased in W/M cells in comparison to W/W cells. Interestingly, the reactivity of astrocytes to FGF-1 was recovered when W/M cells were transferred to the tertiary culture of 1 week. We concluded that astrocytes decrease their reactivity to FGF-1 for apoE-HDL biogenesis in certain conditions. The findings indicate astrocyte FGF-1 enhances biogenesis of apoE-HDL also by a paracrine mechanism.

Characterization of Dihydrodipicolinate Reductase from Thermotoga maritima Reveals Evolution of Substrate Binding Kinetics

Pearce, F. G., Sprissler, C., Gerrard, J. A. — 2008-05-06

In lysine biosynthesis, dihydrodipicolinate reductase (DHDPR) catalyses the formation of tetrahydrodipicolinate. Unlike DHDPR enzymes from Escherichia coli and Mycobacterium tuberculosis, which have dual specificity for both NADH and NADPH as co-factors, the enzyme from Thermotoga maritima has a significantly greater affinity for NADPH. Despite low sequence identity with the E. coli and M. tuberculosis DHDPR enzymes, DHDPR from T. maritima has a similar catalytic site, with many conserved residues involved in interactions with substrates. This suggests that as the enzyme evolved, the co-factor specificity was relaxed. Kinetic studies show that the T. maritima DHDPR enzyme is inhibited by high concentrations of its substrate, DHDP, and that at high concentrations NADH also acts as an inhibitor of the enzyme, suggesting a novel method of regulation for the lysine biosynthetic pathway. Increased thermal stability of the T. maritima DHDPR enzyme may be associated with the lack of C-terminal and N-terminal loops that are present in the E. coli DHDPR enzyme.

Role of Disulphide Bonds in a Thermophilic Serine Protease Aqualysin I from Thermus aquaticus YT-1

2008-05-06

A thermophilic serine protease, Aqualysin I, from Thermus aquaticus YT-1 has two disulphide bonds, which are also found in a psychrophilic serine protease from Vibrio sp. PA-44 and a proteinase K-like enzyme from Serratia sp. at corresponding positions. To understand the significance of these disulphide bonds in aqualysin I, we prepared mutants C99S, C194S and C99S/C194S (WSS), in which Cys69-Cys99, Cys163-Cys194 and both of these disulphide bonds, respectively, were disrupted by replacing Cys residues with Ser residues. All mutants were expressed stably in Escherichia coli. The C99S mutant was 68% as active as the wild-type enzyme at 40°C in terms of k_cat value, while C194S and WSS were only 6 and 3%, respectively, as active, indicating that disulphide bond Cys163-Cys194 is critically important for maintaining proper catalytic site conformation. Mutants C194S and WSS were less thermostable than wild-type enzyme, with a half-life at 90°C of 10 min as compared to 45 min of the latter and with transition temperatures on differential scanning calorimetry of 86.7°C and 86.9°C, respectively. Mutant C99S was almost as stable as the wild-type aqualysin I. These results indicate that the disulphide bond Cys163-Cys194 is more important for catalytic activity and conformational stability of aqualysin I than Cys67-Cys99.

Characterization of the Molecular Interaction between Tropoelastin and DANCE/Fibulin-5

2008-05-06

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca²⁺, Ba²⁺ and Mg²⁺ significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2–15 and exons 16–36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Identification of Amino Acid Positions Involved in HLA-E Expression

Matsunami, K., Okura, E., Uchida, H., Otsuka, H., Fukuzawa, M., Miyagawa, S. — 2008-05-06

The cell surface expression of HLA-E molecules by transfection is faint in xenogeneic cells. Therefore, this study was done for the aim of better expression of HLA-E molecules on the surface of pig cells in order to overcome xenograft rejection mediated by human natural killer (NK) cells. The importance of the loading peptide sequence for HLA-E expression has been studied extensively, but much less information is available concerning the HLA-E heavy chain sequence. In our previous study, we developed the S147C substitution of HLA-E as a useful gene tool for xenotransplantation. In this study, a more extensive substitution analysis throughout the entire region led to the identification of nine amino acid positions, positions-9, 11, 25, 40, 66, 67, 74, 99 and 174, that are significantly involved in the cell surface expression of HLA-E molecules. In view of xenotransplantation usage, double and triple point substitutions, HLA-Ev(11,147) and HLA-Ev(11,66,147), were constructed. These constructs led to a high expression on the xenogeneic cell surface and possessed inhibitory functions against human NK cell-mediated cytolysis in an in vitro pig to human xenotransplantation model system.

Contribution of Peroxisome-specific Isoform of Lon Protease in Sorting PTS1 Proteins to Peroxisomes

Omi, S., Nakata, R., Okamura-Ikeda, K., Konishi, H., Taniguchi, H. — 2008-05-06

Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in β-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the β-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology.

Vertebrate Genomes Code Excess Proteins with Charge Periodicity of 28 Residues

Ke, R., Sakiyama, N., Sawada, R., Sonoyama, M., Mitaku, S. — 2008-05-06

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.

Yeast-Based Fluorescence Reporter Assay of G Protein-coupled Receptor Signalling for Flow Cytometric Screening: FAR1-Disruption Recovers Loss of Episomal Plasmid Caused by Signalling in Yeast

2008-05-06

Here, we describe a yeast-based fluorescence reporter assay for G protein-coupled receptor (GPCR) signalling using a flow cytometer (FCM). The enhanced green fluorescent protein (EGFP) gene was integrated into the FUS1 locus as a reporter gene. The engineered yeast was able to express the EGFP in response to ligand stimulation. Gene-disrupted yeast strains were constructed to evaluate the suitability of the yeast-based fluorescence screening system for heterologous GPCR. When receptor was expressed by episomal plasmid, the proportion of the signalling-activated cells in response to ligand stimulation decreased significantly. The GPCR-signalling-activated and non-activated cell clusters were individually isolated by analysing the fluorescence intensity at the single-cell level with FCM, and it was found that the plasmid retention rate decays markedly in the non-activated cell cluster. We attributed the loss of plasmid to G1 arrest in response to signalling, and successfully improved the plasmid retention rate by disrupting the FAR1 gene and avoiding cell cycle arrest. Our system will be a powerful tool for the quantitative and high-throughput GPCR screening of yeast-based combinatorial libraries using FCM.

Transcriptional Regulation of the Human PNRC Promoter by NFY in HepG2 Cells

Zhang, Y., Chen, B., Li, Y., Chen, J., Lou, G., Chen, M., Zhou, D. — 2008-05-06

PNRC (proline-rich nuclear receptor co-activator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel co-activator for multiple nuclear receptors. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the –123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY protein to the human PNRC promoter. Transient transfection and luciferase assays further revealed that over-expression of NFY represses promoter activity of PNRC gene in a dose-dependent manner. These results indicate that the transcription factor NFY specifically binds to promoter region of PNRC and negatively regulates the transcription of the human PNRC gene.

Interaction of Glutathione and Sodium Selenite In vitro Investigated by Electrospray Ionization Tandem Mass Spectrometry

Cui, S.-Y., Jin, H., Kim, S.-J., Kumar, A. P., Lee, Y.-I. — 2008-05-06

Selenite has been found to be an active catalyst for the oxidation of sulphhydryl compounds, such as glutathione (GSH). Considering the biological importance of GSH oxidation and the implication of sulphhydryl compounds in selenium poisoning and other biological activities, more information on selenite oxidation of GSH in enzyme-free conditions is desirable. Herein, we describe glutathione and sodium selenite simply mixed in aqueous solutions. The interaction products and transient intermediate are identified and characterized using electrospray ionization (ESI) tandem mass spectrometry. In the first step, GSH directly reacts to form diglutathione (GSSG) and unstable selenodiglutathione (GS-Se-SG). Then selenodiglutathione further reacted with remaining GSH to form diglutathione and elemental selenium, Se⁰. As the amount of GSSG significantly increased or acidity of the solution increased, the redox potential of glutathione [E^0'(GSSG/2GSH) –250 mV (NHE)] significantly shifted to the positive direction. This makes the GSSG react with elemental selenium formed in the solution, which can be demonstrated by another unstable intermediate ion identified at m/z 418 by mass spectrometry with the elemental composition of [GSS-Se]^–. The reaction mechanism between GSH and sodium selenite has been proposed according to the ESI-MS, NMR and UV-vis spectrometric measurements.

Physical and Functional Interactions between STAT5 and Runx Transcription Factors

Ogawa, S., Satake, M., Ikuta, K. — 2008-05-06

The signal transducers and activators of transcription (STAT) and the Runt-related (Runx) are two of major transcription factor families that play essential roles in lymphocyte development. Although the interaction of Runx2 with STAT1 and STAT3 has been reported before, the interaction between STAT5 and Runx family proteins has not been characterized. In this study, we first showed that STAT5 physically interacts with Runx1, Runx2 and Runx3 by co-immunoprecipitation experiments. The Runt domain of Runx proteins and the DNA-binding domain and -helix loop structure of STAT5 are responsible for the interaction. When expressed in CHO cells, STAT5 inhibits the nuclear localization of Runx proteins and retains them in the cytoplasm. In addition, we showed by reporter assay that the interaction between STAT5 and Runx proteins mutually inhibits their transcriptional activity. Furthermore, Runx proteins inhibit the DNA-binding activity of STAT5. Finally, we found that Runx proteins suppress the transcription of an endogenous STAT5 target gene, cytokine-inducible SH2 protein-1, in an interleukin-3-dependent pro-B cell line, Ba/F3. These results collectively suggested that STAT5 and Runx proteins physically and functionally interact to mutually inhibit their transcriptional activity. Thus, this study implies a potential role of the STAT5–Runx interaction in lymphocyte development.

Cloning, Expression, Purification and Characterization of an Isotype of Clytin, a Calcium-Binding Photoprotein from the Luminous Hydromedusa Clytia gregarium

Inouye, S. — 2008-05-06

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca²⁺ was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity.

The Structure and Functions of NPM1/Nucleophsmin/B23, a Multifunctional Nucleolar Acidic Protein

Okuwaki, M. — 2008-03-27

NPM1/Nucleophosmin/B23, also termed NO38 or numatrin, is an acidic nucleolar protein that plays multiple roles in cell growth and proliferation. In general, the expression level of B23 is proportional to the cell growth rate, suggesting that it plays a positive role(s) in cell growth and proliferation. It is important to note that the deletion of the B23 gene and expression of an aberrant type of this gene—caused by gene conversion via translocation or reading-frame shift via nucleotides insertion—have been observed in diverse haematopoietic malignancies. Thus, it is important to understand the function of B23 in the regulation of cell growth and proliferation. In addition, B23 has been reported to undergo a variety of post-translational modifications such as phosphorylation, ubiquitination, SUMOylation, acetylation and poly-(ADP-ribosyl)ation. In this review, the basic structure and functions of B23 as well as the regulation of these functions are summarized.

An RNAi Screen for Mitochondrial Proteins Required to Maintain the Morphology of the Organelle in Caenorhabditis elegans

Ichishita, R., Tanaka, K., Sugiura, Y., Sayano, T., Mihara, K., Oka, T. — 2008-03-27

Mitochondria are dynamic organelles that frequently divide and fuse together, resulting in the formation of intracellular tubular networks. In yeast and mammals, several factors including Drp1/Dnm1 and Mfn/Fzo1 are known to regulate mitochondrial morphology by controlling membrane fission or fusion. Here, we report the systematic screening of Caenorhabditis elegans mitochondrial proteins required to maintain the morphology of the organelle using an RNA interference feeding library. In C. elegans body wall muscle cells, mitochondria usually formed tubular structures and were severely fragmented by the mutation in fzo-1 gene, indicating that the body wall muscle cells are suitable for monitoring changes in mitochondrial morphology due to gene silencing. Of 719 genes predicted to code for most of mitochondrial proteins, knockdown of >80% of them caused abnormal mitochondrial morphology, including fragmentation and elongation. These findings indicate that most fundamental mitochondrial functions, including metabolism and oxidative phosphorylation, are necessary for maintenance of the tubular networks as well as membrane fission and fusion. This is the first evidence that known mitochondrial activities are prerequisite for regulating the morphology of the organelle. Furthermore, 88 uncharacterized or poorly characterized genes were found in the screening to be implicated in mitochondrial morphology.

Analysis of Origin Recognition Complex in Saccharomyces cerevisiae by Use of Degron Mutants

2008-03-27

Origin recognition complex (ORC), a six-protein complex (Orc1p-Orc6p), may deeply involve in initiation of chromosomal DNA replication. However, since most temperature-sensitive orc mutants of Saccharomyces cerevisiae show the accumulation of cells with nearly 2C DNA content, the exact stage at which ORC acts is not fully understood. In this study, we constructed a heat-inducible degron mutant for each ORC subunit. As well as each targeted subunit, other subunits of ORC were also rapidly degraded under non-permissive conditions. In the orc5 degron mutant, incubation under the non-permissive conditions caused accumulation of cells with nearly 2C DNA content, and phosphorylation of Rad53p. When Orc5p (ORC) is depleted, this inhibits G1/S transition and formation of a pre-replicative complex (pre-RC). For pre-RC to form, and G1/S transition to proceed, Orc5p (ORC) must be present in late G1, rather than early G1, or G2/M. Block and release experiments revealed that Orc5p (ORC) is not necessary for S and G2/M phase progression. We therefore propose that ORC is necessary for the G1/S transition and pre-RC formation, and accumulation of cells with nearly 2C DNA content seen in various orc mutants is due to inefficient pre-RC formation, and/or induction of checkpoint systems.

Selenite Assimilation into Formate Dehydrogenase H Depends on Thioredoxin Reductase in Escherichia coli

2008-03-27

Escherichia coli growing under anaerobic conditions produce H₂ and CO₂ by the enzymatic cleavage of formate that is produced from pyruvate at the end of glycolysis. Selenium is an integral part of formate dehydrogenase H (FDH_H), which catalyses the first step in the formate hydrogen lyase (FHL) system. The genes of FHL system are transcribed only under anaerobic conditions, in the presence of a ⁵⁴-dependent transcriptional activator FhlA that binds formate as an effector molecule. Although the formate addition to the nutrient media has been an established procedure for inducing high FDH_H activity, we have identified a low-salt nutrient medium containing <0.1% NaCl enabled constitutive, high expression of FDH_H even without formate and d-glucose added to the medium. The novel conditions allowed us to study the effects of disrupting genes like trxB (thioredoxin reductase) or gor (glutathione reductase) on the production of FDH_H activity and also reductive assimilation of selenite ($${\hbox{ SeO }}_{3}^{2-}$$) into the selenoprotein. Despite the widely accepted hypothesis that selenite is reduced by glutathione reductase-dependent system, it was demonstrated that trxB gene was essential for FDH_H production and for labelling the FDH_H polypeptide with ⁷⁵Se-selenite. Our present study reports for the first time the physiological involvement of thioredoxin reductase in the reductive assimilation of selenite in E. coli.

Immunoreactivity of Phage Library-derived Human Single-Chain Antibodies to Amyloid Beta Conformers In Vitro

2008-03-27

The pathogenesis of Alzheimer's disease involves conformational changes of Aβ. A series of antibodies recognizing a distinct conformation of Aβ (snapshot antibody) is useful for both understanding the mechanism of molecular conversion and identifying diagnostic and therapeutic reagents. As Aβ with various conformations can be prepared in vitro under varying physicochemical conditions, snapshot antibodies can be isolated by directly binding to target molecules with antibody-displaying phages. We tested the feasibility of this idea. We show a feature of several Aβ-reactive antibodies isolated from our human single-chain Fv antibody-phage library and particularly report the characteristics of an scFv clone, B6, selected from the fibrillar Aβ_1–42-coated biopanning. B6 bound to fibrillar Aβ_1–42 as well as globulomer Aβ_1–42 but not to soluble Aβ_1–42 or Aβ_1–40. B6 inhibited Aβ_1–42 fibril formation with 600 nM IC₅₀ in spite of being the monovalent scFv form. Epitope analysis suggested that the binding site might be located at the β2 sheet of the C-terminus of Aβ_1–42. Although it is believed that N-terminus-recognizing antibodies tend to show the capability to inhibit Aβ_1–42 fibrillation, B6 is the first human inhibitory antibody recognizing the C-terminus of Aβ_1–42.

Role of the Loop Structure of the Catalytic Domain in Rice Class I Chitinase

2008-03-27

In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only towards (GlcNAc)₆ but also towards polysaccharide substrates. Similar results were obtained for k_cat/K_m values determined for substrate reduced-(GlcNAc)₅. The two mutations shifted the splitting positions in (GlcNAc)₆ to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from –2 kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.

Induction of Efficient Differentiation and Survival of Porcine Neonatal Pancreatic Cell Clusters Using an EBV-based Plasmid Expressing HGF

Kim, M. S., Kim, J.-W., Sun, C., Oh, S. T., Yoon, K. H., Lee, S. K. — 2008-03-27

Porcine neonatal pancreatic cell clusters (NPCCs) have been actively studied as a source of pancreatic stem cell transplantation for the treatment of diabetes. In this study, the hepatocyte growth factor (HGF) gene was cloned in an Epstein–Barr virus (EBV)-based plasmid vector (pEBVHGF) and the effects of the HGF expression on the survival and differentiation of NPCCs were analysed. For comparison, pHGF was constructed by deleting EBNA-1 and OriP from pEBVHGF. The expression of HGF, as measured by ELISA, lasted longer when pEBVHGF was used than when pHGF was used. C-Met phosphorylation co-related with the expression of HGF in the transfected NPCCs. Immunocytochemistry experiments showed that NPCCs showed a higher and longer expression of insulin when they were transfected with pEBVHGF than with pHGF. Moreover, a greater number of NPCCs survived for a longer period after they were transfected with pEBVHGF than when they were transfected with pHGF. Taken together, these results indicate that transfecting NPCCs with the HGF gene using an EBV-based plasmid is a more effective method of inducing differentiation to beta cells and enhancing survival than using a conventional plasmid. Therefore, it may be possible to use EBV-based plasmids to modify pancreatic stem cells for xenotransplantation.

Functional Characterization of the Recombinant Group II Chaperonin {alpha} from Thermoplasma acidophilum

Hirai, H., Noi, K., Hongo, K., Mizobata, T., Kawata, Y. — 2008-03-27

The functional characteristics of group II chaperonins, especially those from archaea, have not been elucidated extensively. Here, we performed a detailed functional characterization of recombinant chaperonin subunits (16-mer) (Ta-cpn ) from the thermophilic archaea Thermoplasma acidophilum as a model protein of archaeal group II chaperonins. Recombinant Ta-cpn formed an oligomeric ring structure similar to that of native protein, and displayed an ATP hydrolysis activity (optimal temperature: 60°C) in the presence of either magnesium, manganese or cobalt ions. Ta-cpn was able to bind refolding intermediates of Thermus MDH and GFP in the absence of ATP, and to promote the refolding of Thermus MDH at 50°C in the presence of Mg²⁺-, Mn²⁺-, or Co²⁺-ATP. Ta-cpn also prevented thermal aggregation of rhodanese and luciferase at 50°C. Interestingly, Ta-cpn in the presence of Mn²⁺ ion showed an increased hydrophobicity, which correlated with an increased efficiency in substrate protein binding. Our finding that Ta-cpn chaperonin system displays folding assistance ability with ATP-dependent substrate release may provide a detailed look at the potential functional capabilities of archaeal chaperonins.

Involvement of MDR1 Function in Proliferation of Tumour Cells

Katoh, S.-Y., Ueno, M., Takakura, N. — 2008-03-27

Mdr1 is a multi-drug-resistance protein, a member of the adenosine triphosphate-binding cassette family of drug transporters. Mdr1 is expressed in wide variety of cells and limits absorption of toxicants into the body or tissue; however, it is also expressed in many cancer cells and can render tumour cells resistant to many anti-cancer drugs. Mdr1 is well studied as a multi-drug resistance transporter, but little is known regarding its other role in tumour cells. In the present study, we investigated mdr1 function in tumour cell proliferation. We silenced the mdr1 gene in tumour cells by using an RNA interference method that employed short hairpin RNA. The result showed that knockdown of mdr1 gene suppressed tumour cell proliferation in vitro, and induced the passage of the cell cycle into the G1/G0 phase. Furthermore, in a mice xenograft tumour formation assay, mdr1 knockdown of tumour cells inhibited tumour expansion. These results suggest that Mdr1 plays a role in regulation of tumour cells proliferation.

Residues on the Dimer Interface of SARS Coronavirus 3C-like Protease: Dimer Stability Characterization and Enzyme Catalytic Activity Analysis

Chen, S., Zhang, J., Hu, T., Chen, K., Jiang, H., Shen, X. — 2008-03-27

3C-like protease (3CL^pro) plays pivotal roles in the life cycle of severe acute respiratory syndrome coronavirus (SARS-CoV) and only the dimeric protease is proposed as the functional form. Guided by the crystal structure and molecular dynamics simulations, we performed systematic mutation analyses to identify residues critical for 3CL^pro dimerization and activity in this study. Seven residues on the dimer interface were selected for evaluating their contributions to dimer stability and catalytic activity by biophysical and biochemical methods. These residues are involved in dimerization through hydrogen bonding and broadly located in the N-terminal finger, the -helix A' of domain I, and the oxyanion loop near the S1 substrate-binding subsite in domain II. We revealed that all seven single mutated proteases still have the dimeric species but the monomer–dimer equilibria of these mutants vary from each other, implying that these residues might contribute differently to the dimer stability. Such a conclusion could be further verified by the results that the proteolytic activities of these mutants also decrease to varying degrees. The present study would help us better understand the dimerization-activity relationship of SARS-CoV 3CL^pro and afford potential information for designing anti-viral compounds targeting the dimer interface of the protease.

Annexin A3 Expression Increases in Hepatocytes and is Regulated by Hepatocyte Growth Factor in Rat Liver Regeneration

2008-03-27

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl₄) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl₄ administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.

Identification of {beta}1,4GalT II as a Target Gene of p53-mediated HeLa Cell Apoptosis

Zhou, J., Wei, Y., Liu, D., Ge, X., Zhou, F., Yun, X., Jiang, J., Gu, J. — 2008-03-27

β1,4-galactosyltransferase II (β1,4GalT II) is one of the enzymes transferring galactose to the terminal N-acetylglucosamine of complex-type N-glycans. Previously, we have reported that β1,4GalT II overexpression increased cisplatin-induced HeLa cell apoptosis. However, the mechanisms of its expression regulation have been rarely investigated. Here, we cloned the 1.8-kb 5'-flanking region of the β1,4GalT II gene and analysed its promoter activity. The transcriptional activity and mRNA expression level of β1,4GalT II were dramatically induced by p53 transcription factor in HeLa cells. In response to DNA damage agent adriamycin, the mRNA expression and promoter activity of β1,4GalT II were significantly up-regulated and the binding of p53 to β1,4GalT II promoter was obviously increased. Furthermore, decreasing the expression of β1,4GalT II using RNA interference inhibited p53-mediated HeLa cell apoptosis induced by adriamycin. Collectively, these results suggested that β1,4GalT II might serve as a target gene of p53 transcription factor during adriamycin-induced HeLa cell apoptosis, which elucidated a new mechanism of p53-mediated cell apoptosis.

O-Mannosylation is Required for Degradation of the Endoplasmic Reticulum-associated Degradation Substrate Gas1*p via the Ubiquitin/Proteasome Pathway in Saccharomyces cerevisiae

Hirayama, H., Fujita, M., Yoko-o, T., Jigami, Y. — 2008-03-27

In Saccharomyces cerevisiae, protein O-mannosylation, which is executed by protein O-mannosyltransferases, is essential for a variety of biological processes as well as for conferring solubility to misfolded proteins. To determine if O-mannosylation plays an essential role in endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins, we used a model misfolded protein, Gas1*p. The O-mannose content of Gas1*p, which is transferred by protein O-mannosyltransferases, was higher than that of Gas1p. Both Pmt1p and Pmt2p, which do not transfer O-mannose to correctly folded Gas1p, participated in the O-mannosylation of Gas1*p. Furthermore, in a pmt1 pmt2 double-mutant background, degradation of Gas1*p is altered from a primarily proteasome dependent to a vacuolar protease-dependent pathway. This process is in a manner dependent on a Golgi-to-endosome sorting function of the VPS30 complex II. Collectively, our data suggest that O-mannosylation plays an important role for proteasome-dependent degradation of Gas1*p via the ERAD pathway and when O-mannosylation is insufficient, Gas1*p is degraded in the vacuole. Thus, we propose that O-mannosylation by Pmt1p and Pmt2p might be a key step in the targeting of some misfolded proteins for degradation via the proteasome-dependent ERAD pathway.

Cleavage of the ER-Targeting Signal Sequence of Parathyroid Hormone-related Protein is Cell-Type-Specific and Regulated in Cis by its Nuclear Localization Signal

Amaya, Y., Nakai, T., Komaru, K., Tsuneki, M., Miura, S. — 2008-03-27

Prepro-parathyroid hormone-related protein (ppPTHrP) has two targeting signals, an N-terminal signal sequence and a nuclear localization signal (NLS). In fact, the protein is not only secreted from the cell but also found in the nucleus and/or nucleolus. In order to understand the function of the PTHrP signal sequence for the dual localization, the signal sequence cleavage of a series of ppPTHrP deletion mutants fused to Escherichia coli leader peptidase was analysed in vitro and in several cell lines. Efficiency of the PTHrP signal sequence cleavage was intrinsically low in the in vitro reconstitution system. In cultured cells, cleavage efficiency of the PTHrP signal sequence varied significantly, being lowest in COS-1 cells, but rising in HeLa, HEK293 and CV-1 cells. However, virtually complete signal sequence cleavage was observed in CHO cells. In addition, the NLS of PTHrP had a negative effect on its own signal sequence cleavage, which could be enhanced by deletion of the spacer sequence between the signal sequence and the NLS. There was a roughly inverse relationship between the signal sequence cleavage and the nuclear localization of PTHrP. Thus, the final destination of PTHrP could be regulated at the ER membrane.

Capturing Epigenetic Dynamics During Pre-implantation Development Using Live Cell Imaging

Yamagata, K. — 2008-02-29

During mammalian fertilization and pre-implantation development, the highly differentiated gametes revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in male and female gamete chromatin structure, in global epigenetic modifications and in nuclear architecture. We have developed a live cell imaging technique for oocytes and early embryos to understand these series of phenomena. Using this technique, we were able to observe dynamic changes in DNA methylation status in living embryos. Furthermore, epigenetic abnormalities were detected in reconstructed embryos generated by round spermatid injection or by somatic cell nuclear transfer. In this review, I will discuss the usefulness and possibilities of this imaging technique in studies on nuclear dynamics during fertilization and pre-implantation development.

Cracking the Enigmatic Linker Histone Code

Godde, J. S., Ura, K. — 2008-02-29

Recently, the existence of a ‘histone code’ has been proposed to explain the link between the covalent chemical modification of histone proteins and the epigenetic regulation of gene activity. Although the role of the four ‘core’ histones has been extensively studied, little is known about the involvement of the linker histone, histone H1 and its variants, in this code. For many years, few sites of chemical modification had been mapped in linker histones, but this has changed recently with the use of functional proteomic techniques, principally mass spectrometry, to characterize these modifications. The functionality of many of these sites, however, remains to be determined.

IRAK-4-dependent Degradation of IRAK-1 is a Negative Feedback Signal for TLR-mediated NF-{kappa}B Activation

Kubo-Murai, M., Hazeki, K., Nigorikawa, K., Omoto, T., Inoue, N., Hazeki, O. — 2008-02-29

The activation of interleukin 1 receptor-associated kinase (IRAK)-1 is a key event in the transmission of signals from Toll-like receptors (TLRs). The catalytic activity of the protein kinase is not essential for its ability to activate nuclear factor (NF) B, because transfection of a kinase-dead mutant of IRAK-1 (IRAK-1KD) is able to activate NF-B in HEK293T cells. In the present study, we observed that the effect of IRAK-1KD was impaired by simultaneous expression of IRAK-4. The effect of IRAK-4 was accompanied by the phosphorylation and degradation of IRAK-1KD. Expression of IRAK-4KD instead of IRAK-4 did not cause these events. In IRAK-4-deficient Raw264.7 macrophages that were prepared by introducing short-hairpin RNA probes, the basal level of IRAK-1 was increased markedly. Stimulation of these cells with TLR ligands did not cause the degradation of IRAK-1, which was clearly observed in the parent cells. These results suggested that the expression of IRAK-4 alone is sufficient to cause the degradation of IRAK-1; the autophosphorylation of IRAK-1 is not necessary to terminate the TLR-induced activation of NF-B. IRAK-4 has an ability to induce the degradation of IRAK-1 in addition to its role as an activator of IRAK-1.

A Strong Exonic Splicing Enhancer in Dystrophin Exon 19 Achieve Proper Splicing Without an Upstream Polypyrimidine Tract

2008-02-29

Proper splicing is known to proceed under the control of conserved cis-elements located at exon–intron boundaries. Recently, it was shown that additional elements, such as exonic splicing enhancers (ESEs), are essential for the proper splicing of certain exons, in addition to the splice donor and acceptor site sequences; however, the relationship between these cis-elements is still unclear. In this report, we utilize dystrophin exon 19 to analyse the relationship between the ESE and its upstream acceptor site sequences. Dystrophin exon 19, which maintains adequate splicing donor and acceptor consensus sequences, encodes exonic splicing enhancer (dys-ESE19) sequences. Splice pattern analysis, using a minigene reporter expressed in HeLa cells, showed that either a strong polypyrimidine tract (PPT) or a fully active dys-ESE19 is sufficient for proper splicing. Each of these two cis-elements has enough activity for proper exon 19 splicing suggesting that the PPT, which is believed to be an essential cis-element for splicing, is dispensable when the downstream exon contains a strong ESE. This compensation was only seen in living cells but not in ‘in vitro splicing’. This suggests the possibility that the previous splicing experiments using an in vitro splicing system could underestimate the activity of ESEs.

Induction of Cell Adhesion by Galectin-8 and its Target Molecules in Jurkat T-Cells

2008-02-29

We previously showed that tandem-repeat type galectin-8, which has two covalently linked carbohydrate recognition domains (CRDs), induces neutrophil-adhesion through binding to integrin M. Here, we analysed the function of galectin-8 in Jurkat T-cells. Galectin-8, as well as tandem-repeat galectin-9, and several multivalent plant lectins, induced Jurkat T-cell adhesion to a culture plate, whereas single-CRD galectins-1 and -3 did not. Galectin-8 also induced the adhesion of peripheral blood leucocytes to human umbilical vein endothelial cells. These results suggest that the di- or multi-valent structure of galectin-8 is essential for the induction of cell adhesion and that this ability exhibits broad specificity for leucocytes. The galectin-8-induced cell adhesion was accompanied by stress fibre formation, which suggests that intracellular signalling is required. We have identified integrin 4 as one of the candidate target molecules associated with the induction of cell adhesion. Indeed, inhibition of the function of integrin 4 by treating cells with a blocking-antibody reduced the sensitivity to galectin-8. Also, the phosphorylation of Pyk and ERK1/2, indicators of integrin-mediated signalling, was up-regulated on treatment with galectin-8. Thus, a primary target of galectin-8 must be the sugar chains on members of the integrin family, which are abundantly expressed on the surface of leucocytic cells.

Anti-Peptide Antibodies for Examining the Conformation, Molecular Assembly and Localization of an Intracellular Protein, Ribosomal Protein S6, In vivo

Nakagawa, M., Ohmido, N., Ishikawa, K., Uchiyama, S., Fukui, K., Azuma, T. — 2008-02-29

Ribosomal protein S6 (rpS6) is known to relate to cell proliferation. Our recent proteome analysis of human metaphase chromosomes revealed the enrichment of rpS6 during mitosis. Here, structure, localization and molecular assembly in vitro and in vivo of a human rpS6, were examined using antibodies (Abs) prepared by immunizing rabbits with synthetic peptides. Five peptides, Ser6-Asp20 (S6-1), Ile52-Gly66 (S6-2), Asp103-Gly117 (S6-3), Asn146-Lys160 (S6-4) and Arg178-Ile192 (S6-5) were chosen as epitopes of human rpS6. These peptides except for S6-3 induced strong Ab production, and with an enzyme-linked immunosorbent assay, anti-S6-2, anti-S6-4 and anti-S6-5, showed high reactivity to recombinant rpS6 (r-rpS6), while anti-S6-1 did not, suggesting that S6-2, S6-4 and S6-5 were exposed on the r-rpS6 surface, while S6-1 was less exposed or possessed a different conformation. The immunostaining of HeLa cells as well as isolated chromosomes suggested that rpS6 occurs in endoplasmic reticulum (ER) but less possible on chromosomes since no Abs showed localization of rpS6 to chromosomes. In addition, the immunostaining suggested that only S6-4 is exposed on the protein surface, while S6-2 and S6-5 are buried by the interaction with other macromolecules in HeLa cells. Present our result shows new possibility of antibodies as tools for structure-oriented cell biology.

Copper(II) Inhibits In vitro Conformational Conversion of Ovine Prion Protein Triggered by Low pH

Liu, M.-l., Li, Y.-x., Zhou, X.-m., Zhao, D.-m. — 2008-02-29

To gain insight into the conformational conversion of ovine prion protein (OvPrP^C) at different pH values and/or in the presence of CuCl₂, the secondary structure of OvPrP^C was analysed by circular dichroism (CD) spectroscopy. Copper treatment of OvPrP^C under moderately acidic conditions (pH ~5.0–6.0) as well as physiological conditions (pH 7.4) also makes OvPrP^C adopt protease-resistant and β-sheet-rich conformation. However, under lower pH conditions (2.0–4.5) with copper treatment, OvPrP^C gained higher -helix structure. This study demonstrated that Cu²⁺ can significantly modulate conformational conversion triggered by acidic pH, and this will provide therapeutic intervention approaches for prion diseases.

Recognition of a Bulged RNA by Peptides Derived from the Influenza NS1 Protein

Someya, T., Hosono, K., Morimura, K., Takaku, H., Kawai, G. — 2008-02-29

A competition assay for RNA binding by the influenza virus NS1 protein using model RNAs, U6–45, corresponding to U6 snRNA revealed that deletion of each of the three bulged-out parts reduced the NS1 protein binding and, in contrast, by deleting all three of the bulged-out parts, simultaneously, and thus producing a double-stranded RNA, the binding was recovered. A common feature of target RNAs of the NS1 protein, U6 snRNA, poly(A) and viral RNA, is the stretch of ‘bulged-out’ A residues. Thus, the NS1 protein was found to recognize either the stretch of ‘bulged-out’ A residues or dsRNA which is also a target of the NS1 protein. Furthermore, a basic peptide, NS1–2, derived from the helix-2 of the RNA binding site of NS1 protein was designed and its binding to the U6 snRNA was analysed by using a model RNA for U6 snRNA, U6–34. The NMR signals due to H8/H6 and H1' of U6–34 were assigned and their changes upon binding of NS1–2 were analysed. It was indicated that NS1–2 interacts with the residues in the bulge-out region of U6–34. These results suggest that NS1–2 recognizes the U6 snRNA in a similar manner to NS1 protein.

Nuclear Localization Signal and Phosphorylation of Serine350 Specify Intracellular Localization of DRAK2

Kuwahara, H., Nishizaki, M., Kanazawa, H. — 2008-02-29

DAP kinase-related apoptosis-inducing kinase 2 (DRAK2) is a serine/threonine kinase of the death-associated protein kinase family. DRAK2 mediates apoptosis induced by extracellular stimuli, including UV irradiation and interleukin-2, and also regulates T-cell receptor sensitivity in developing thymocytes. During these events, the subcellular localization of DRAK2 changes between the nucleus and cytoplasm. We found that DRAK2 has a putative nuclear-localization signal (NLS) sequence. Mutations in this sequence interfered with DRAK2 localization to the nucleus. Furthermore, green fluorescence protein fused to the putative NLS accumulated in the nucleus, indicating that the putative sequence functions as an NLS. We also found that the function of the NLS was regulated by phosphorylation. Phorbol myristate acetate (PMA) induced the accumulation of DRAK2 in the cytoplasm of NIH3T3 cells, whereas in the absence of PMA, DRAK2 was localized to the nucleus. Ectopic expression of PKC- induced cytoplasmic localization of DRAK2 and PKC- phosphorylated Ser350 flanking the NLS. DRAK2, but not the Ser350Asp mutant, accumulated in the nuclei of ACL-15 cells in response to UV-irradiation. These results suggest that phosphorylation of Ser350 plays an essential role in regulating translocation of DRAK2 to the nucleus from the cytoplasm, possibly by affecting the activity of the NLS.

Biochemical Characterization of Phospholipids, Sulfatide and Heparin as Potent Stimulators for Autophosphorylation of GSK-3{beta} and the GSK-3{beta}-Mediated Phosphorylation of Myelin Basic Protein In vitro

Kawakami, F., Yamaguchi, A., Suzuki, K., Yamamoto, T., Ohtsuki, K. — 2008-02-29

The stimulatory effects of SH (sulfatide and heparin) and two phospholipids (PI and PS) on autophosphorylation of GSK-3β and the GSK-3β-mediated phosphorylation of myelin basic protein (MBP) and two synthetic MBP peptides (M86 and M156) were comparatively examined in vitro. It was found that (i) both PI and SH highly stimulated the GSK-3β-mediated phosphorylation of MBP, but not glycogen synthase, and two MBP peptides through their direct binding to these substrates and (ii) both PI and heparin, as compared with sulfatide, highly stimulated autophosphorylation of GSK-3β. The K_m value of MBP for GSK-3β was highly reduced and the V_max value was significantly increased in the presence of these acidic modulators, which augmented further phosphorylation of MBP by the kinase. Under our experimental condition, similar stimulatory effects of PI and heparin were observed with the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro. These results presented here suggest that these two phospholipids and SH may function as effective stimulators for autophosphorylation of GSK-3β and for the GSK-3β-mediated high phosphorylation of SH-binding proteins, including MBP and TP, in the highly accumulated levels of these acidic and sulfated modulators in the brain.

Expression of a Novel 90-kDa Protein, Lsd90, Involved in the Metabolism of Very Long-chain Fatty Acid-containing Phospholipids in a Mitosis-defective Fission Yeast Mutant

2008-02-29

The fission yeast lsd1/fas2 strain carries a temperature-sensitive mutation of the fatty-acid-synthase -subunit, exhibiting an aberrant mitosis lsd phenotype, with accumulation of very-long-chain fatty-acid-containing phospholipid (VLCFA-PL). A novel 90-kDa protein, Lsd90 (SPBC16E9.16c), was found to be newly expressed in small particle-like structures in lsd1/fas2 cells under restrictive conditions. Two mismatches leading to a double frame shift were found between the sequences of the lsd90⁺ gene registered in the genomic database and the sequences determined experimentally at the amino acid, cDNA and genomic DNA levels. Unexpectedly, overexpression and disruption of the lsd90⁺ gene in either lsd1/fas2 or wild-type cells did not affect either cell growth or expression of the lsd phenotype. The amounts of VLCFA-PL that accumulated in lsd90-overexpressing lsd1/fas2 cells were significantly lower than those in lsd1/fas2 cells, suggesting the involvement of Lsd90 in the metabolism of VLCFA-PL.

Quantitative Analysis of CUG-BP1 Binding to RNA Repeats

Mori, D., Sasagawa, N., Kino, Y., Ishiura, S. — 2008-02-29

CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-3-like factors (CELF) family of RNA-binding proteins, and is involved in myotonic dystrophy type 1 (DM1). Several mRNA targets of CUG-BP1 have been identified, including the insulin receptor, muscle chloride channel, and cardiac troponin T. On the other hand, CUG-BP1 has only a weak affinity for CUG repeats. We conducted quantitative-binding assays to assess CUG-BP1 affinities for several repeat RNAs by surface plasmon resonance (SPR). Although we detected interactions between CUG-BP1 and CUG repeats, other UG-rich sequences actually showed stronger interactions. Binding constants of CUG-BP1 for RNAs indicated that the affinity for UG repeats was far stronger than for CUG repeats. We also found that N-terminal deletion mutant of CUG-BP1 has UG repeat-binding activity in a yeast three-hybrid system, although C-terminal deletion mutant does not. Our data indicates that CUG-BP1 specifically recognized UG repeats, probably through cooperative binding of RNA recognition motifs at both ends of the protein. This is the first report of a binding constant for CUG-BP1 calculated in vitro.

Impaired Basal Thermal Homeostasis in Rats Lacking Capsaicin-sensitive Peripheral Small Sensory Neurons

Yamashita, H., Wang, Z., Wang, Y., Furuyama, T., Kontani, Y., Sato, Y., Mori, N. — 2008-02-29

We studied the effects of selective loss of capsaicin-sensitive primary sensory neurons on thermosensation and thermoregulation in rats. Neonatal capsaicin treatment in rats caused a remarkable decrease in the number of small-diameter neurons in the dorsal root ganglion (DRG) compared with their number in the control rats. Gene expression analysis for various thermo-sensitive transient receptor potential (TRP) channels indicated marked reductions in the mRNA levels of TRPV1 (70%), TRPM8 (46%) and TRPA1 (64%), but not of TRPV2, in the DRG of capsaicin-treated rats compared with those in the control rats. In addition to the heat and cold insensitivity, capsaicin-treated rats showed lower rectal core temperature, higher skin temperature and decreased sensitivity to ambient temperature alteration under normal housing at room temperature, suggesting impaired thermosensation and change in thermoregulation in the rats. Uncoupling protein 1 (UCP1) expression and the thermogenic ability in brown adipose tissues were attenuated in the capsaicin-treated rats. These results indicate a critical role of capsaicin-sensitive sensory neurons in both heat and cool sensation and hence in basal thermal homeostasis, which is balanced by heat release and production including UCP1 thermogenesis, following sensation of the ambient temperature.

Target Specificities of Estrogen Receptor-Related Receptors: Analysis of Binding Sequences and Identification of Rb1-Inducible Coiled-Coil 1 (Rb1cc1) as a Target Gene

Akter, Mst. H., Chano, T., Okabe, H., Yamaguchi, T., Hirose, F., Osumi, T. — 2008-02-29

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily. A single AGGTCA sequence element preceded by three conserved nucleotides has been identified as a specific recognition motif of ERRs. Here we performed systematic analyses of target sequences on all three ERR subtypes, , β and . In electrophoretic gel-mobility shift assay and transcriptional reporter assays, they exhibited similar patterns of recognition specificities, showing extremely broad ranges of target sequences. We searched a mouse promoter database for a gene carrying possible ERR-binding sequences. The Rb-1 inducible coiled-coil 1 (Rb1cc1) gene was found to contain two putative ERR binding elements, named response element (RE)-1 and RE-2, in the promoter region. In gene reporter assays, RE-2, but not RE-1, functioned as an effective cis-regulatory element for transactivation by ERR in the presence of a coactivator, peroxisome proliferator-activated receptor coactivator-1. Mutational analyses suggested that RE-2 is recognized by ERR partly as a monovalent element, but also as a direct repeat motif separated by four spacer nucleotides. In vivo binding of ERR to the Rb1cc1 promoter region was confirmed by the chromatin immunoprecipitation assay. Thus, Rb1cc1 is a target gene of ERR, driven by a novel type of recognition sequence.

The Medaka FoxP2, a Homologue of Human Language Gene FOXP2, has a Diverged Structure and Function

2008-02-29

Forkhead box (Fox) genes are involved in organogenesis and cell differentiation. A mutation of FOXP2 was discovered in patients with severe defects in speech and language. The medaka FoxP2 was cloned in order to clarify the molecular evolution and difference in the protein structure and function by comparing human/mouse and medaka genes. The result showed that medaka FoxP2 had a 73.7% homology to the human and mouse counterparts, and its zinc finger, leucine zipper and forkhead domain structures were conserved. However, medaka FoxP2 lacked a long polyglutamine repeat and had two insertions of unique amino acid sequences. FoxP2 expression was found in the epiphysis and retina, in addition to the midbrain and cerebellum. The transcriptional assay revealed that medaka FoxP2 showed a very weak repressive activity to the CC10 promoter while mouse Foxp2 exhibited a strong repressive activity. Mutational analyses of medaka FoxP2 showed that the three amino acids of forkhead domain were responsible for the weak repressive activity. These results suggest that medaka FoxP2 may play a different function in the development of the medaka fish.

Staphylococcus aureus MurC Participates in L-Alanine Recognition via Histidine 343, a Conserved Motif in the Shallow Hydrophobic Pocket

2008-02-29

UDP-N-acetylmuramic acid:l-alanine ligase that is encoded by the murC gene, is indispensable for bacterial peptidoglycan biosynthesis and an important target for the development of antibacterial agents. Structure of MurC ligase with substrates has been described, however, little validation via studying the effects of mutations on the structure of MurC has been performed. In this study, we carried out a functional in vitro and in vivo characterization of Staphylococcus aureus MurCH343Y protein that has a temperature-sensitive mutation of a conserved residue in the predicted shallow hydrophobic pocket that holds a short l-alanine side chain. Purified H343Y and wild-type MurC had K_m values for l-alanine of 3.2 and 0.44 mM, respectively, whereas there was no significant difference in their K_m values for ATP and UDP-N-acetylmuramic acid, suggesting the specific alteration of l-alanine recognition in MurCH343Y protein. In a synthetic medium that excluded l-alanine, S. aureus murCH343Y mutant cells showed an allele-specific slow growth phenotype that was suppressed by addition of l-alanine. These results suggest that His343 of S. aureus MurC is essential for high-affinity binding to l-alanine both in vitro and in vivo and provide experimental evidence supporting the structural information of MurC ligase.

Role of HPF (Hibernation Promoting Factor) in Translational Activity in Escherichia coli

Ueta, M., Ohniwa, R. L., Yoshida, H., Maki, Y., Wada, C., Wada, A. — 2008-02-29

During the stationary phase of growth in Escherichia coli, ribosome modulation factor (RMF) and hibernation promoting factor (HPF) dimerize most 70S ribosomes to form 100S ribosomes. The process of 100S formation has been termed ‘ribosomal hibernation’. Here, the contributions of HPF to 100S formation and translation were analysed in vitro. HPF bound to, but did not dimerize the 70S ribosome. RMF dimerized and formed immature 90S ribosomes. Binding of both HPF and RMF converted 90S ribosomes to mature 100S ribosomes, which is consistent with the in vivo data. The role of HPF in in vitro translation also was investigated. In an artificial mRNA poly (U)-dependent phenylalanine incorporation assay, HPF bound to ribosomal particles and inhibited translation. In contrast, in a natural MS2 mRNA-dependent leucine incorporation assay, bound HPF was removed and hardly inhibited normal translation. Multiple alignment and phylogenetic analyses indicates that the hibernation system mediated by the HPF homologue, RMF and 100S ribosome formation may be specific to the proteobacteria gamma group. In contrast, most bacteria have at least one HPF homologue, and these homologues can be classified into three types, long HPF, short HPF and YfiA.

Donor Substrate Specificity of 4-{alpha}-Glucanotransferase of Porcine Liver Glycogen Debranching Enzyme and Complementary Action to Glycogen Phosphorylase on Debranching

Watanabe, Y., Makino, Y., Omichi, K. — 2008-02-29

Glycogen debranching enzyme (GDE) has both 4--glucanotransferase and amylo--1,6-glucosidase activities. Here, we examined 4--glucanotransferase action of porcine liver GDE on four 6⁴-O--maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6⁴-O--glucosyl-PA-maltooctaose (B4/81) was liberated from 6⁴-O--maltopentaosyl-PA-maltooctaose (B4/85), 6⁴-O--maltotetraosyl-PA-maltooctaose (B4/84) and 6⁴-O--maltotriosyl-PA-maltooctaose (B4/83), whereas 6⁴-O--maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo--1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4--glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4--glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4--glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the -1,6-linked glucosyl residue is retained.