Construction of a ball-and-spike protein supramolecule.
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Amino Acid Sequence↗
Biomedical subjects
Kenji Sugimoto
Publications and source records attributed to Kenji Sugimoto.
Vertebrate Arp6, a novel nuclear actin-related protein, interacts with heterochromatin protein 1.
Actin-related proteins (Arps) were recently shown to contribute to the organization and regulation of chromatin structures. The nuclear functions of Arps have been investigated principally in budding yeast in which six of the ten Arp subfamilies are localized in the nucleus. In vertebrates, only two isoforms of Arp4 have so far been identified as showing localization to the nucleus. Here we show the predominant nuclear localization of another Arp subfamily, Arp6, in vertebrate cells. Vertebrate Arp6 directly interacted with heterochromatin protein 1 (HP1) orthologs and the two proteins colocalized in pericentric heterochromatin. Yeast Arp6 is involved in telomere silencing, while Drosophila Arp6 is localized in the pericentric heterochromatin. Our data strongly suggest that Arp6 has an evolutionarily conserved role in heterochromatin formation and also provide new insights into the molecular organization of heterochromatin.
Actins↗
Glyceraldehyde-3-phosphate dehydrogenase in the extracellular space inhibits cell spreading.
The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.
Animals↗
Proteome analysis of a rat liver nuclear insoluble protein fraction and localization of a novel protein, ISP36, to compartments in the interchromatin space.
A rat liver nuclear insoluble protein fraction was analyzed to investigate candidate proteins participating in nuclear architecture formation. Proteins were subjected to two-dimensional separation by reversed-phase HPLC in 60% formic acid and SDS/PAGE. The method produced good resolution of insoluble proteins. One hundred and thirty-eight proteins were separated, and 28 of these were identified. The identified proteins included one novel protein, seven known nuclear proteins and 12 known nuclear matrix proteins. The novel 36 kDa protein was further investigated for its subnuclear localization. The human ortholog of the protein was expressed in Escherichia coli and antibodies were raised against the recombinant protein. Exclusive localization of the protein to the nuclear insoluble protein fraction was confirmed by cell fractionation followed by immunoblotting. Immunostaining of mouse C3H cells suggested that the 36 kDa protein was a constituent of an insoluble macromolecular complex spread throughout the interchromatin space of the nucleus. The protein was designated 'interchromatin space protein of 36 kDa', ISP36.
Amino Acid Sequence↗
Syntheses of alpha-arbutin-alpha-glycosides and their inhibitory effects on human tyrosinase.
Alpha-arbutin is a tyrosinase inhibitor. We synthesized alpha-arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase from Bacillus macerans using alpha-arbutin and starch as acceptor and donor molecules, respectively. We isolated and characterized two major products from the reaction mixture. The structural analyses using 13C- and 1H-NMR spectroscopy proved that they were 4-hydroxyphenyl alpha-maltoside (alpha-Ab-alpha-G1) and 4-hydroxyphenyl alpha-maltotrioside (alpha-Ab-alpha-G2). Both alpha-Ab-alpha-G1 and alpha-Ab-alpha-G2 exhibited competitive-type inhibition on human tyrosinase as alpha-arbutin does. Their K(i) values were calculated to be 0.6 mM and 2.8 mM, respectively, which is slightly and significantly higher than that of alpha-arbutin (0.2 mM).
Arbutin↗
Direct association with inner centromere protein (INCENP) activates the novel chromosomal passenger protein, Aurora-C.
A family of serine/threonine kinase Aurora constitutes a key regulator in the orchestration of mitotic events. The human Aurora paralogues Aurora-A, Aurora-B, and Aurora-C have a highly conserved catalytic domain. Extensive studies on the role of Aurora-A and Aurora-B have revealed distinct localizations and functions in regulating mitotic processes, whereas little is known about Aurora-C. The present study shows that human Aurora-C is a chromosomal passenger protein that forms complexes with Aurora-B and inner centromere protein (INCENP), which are known passenger proteins. We show that INCENP binds and activates Aurora-C in vivo and in vitro. Furthermore, Aurora-C co-expressed with INCENP elicits the phosphorylation of endogenous histone H3 in mammalian cells, even though this phosphorylation is not sufficient to establish chromosome condensation in interphase cells. We therefore suggest that Aurora-C is a novel chromosomal passenger protein that cooperates with Aurora-B to regulate mitotic chromosome dynamics in mammalian cells.
Animals↗
Palladium-catalyzed ring expansion reaction of (Z)-1-(1,3-butadienyl)cyclobutanols with aryl iodides. stereospecific synthesis of (Z)-2-(3-aryl-1-propenyl)cyclopentanones.
[reaction: see text] A novel type of cascade ring expansion process has been developed by the palladium-catalyzed reaction of (Z)-1-(1,3-butadienyl)cyclobutanols with aryl iodides. The reaction proceeds in a stereospecific manner to produce (Z)-2-(3-aryl-1-propenyl)cyclopentanones. It has also been found that regioselective alpha-arylation of alkenyl cyclopentanones proceeds to afford the alpha-arylated cyclopentanones.
Butanols↗
Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells.
Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.
Animals↗
A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation.
A serine/threonine protein kinase, Cot/Tpl2, is indispensable for extracellular signal-regulated kinase (ERK) activation and production of TNF-alpha and PGE2 in LPS-stimulated macrophages. We show here that Cot/Tpl2 is also activated by other Toll-like receptor (TLR) ligands. Bacterial DNA rich in the dinucleotide CG (CpG-DNA), unlike LPS or synthetic lipopeptide, activated ERK in a Cot/Tpl2-independent manner. Peritoneal macrophages and bone marrow-derived DCs from Cot/Tpl2-/- mice produced significantly more IL-12 in response to CpG-DNA than those from WT mice. Enhanced IL-12 production in Cot/Tpl2-/- macrophages is, at least partly, regulated at the transcriptional level, and the elevated IL-12 mRNA level in Cot/Tpl2-/- macrophages is accompanied by decreased amounts of IL-12 repressors, such as c-musculoaponeurotic fibrosarcoma (c-Maf) and GATA sequence in the IL-12 promoter-binding protein (GA-12-binding protein; GAP-12) in the nucleus. Consistently, Cot/Tpl2-/- mice showed Th1-skewed antigen-specific immune responses upon OVA immunization and Leishmania major infection in vivo. These results indicate that Cot/Tpl2 is an important negative regulator of Th1-type adaptive immunity, that it achieves this regulation by inhibiting IL-12 production from accessory cells, and that it might be a potential target molecule in CpG-DNA-guided vaccination.
Animals↗
Inhibitory effects of alpha-arbutin on melanin synthesis in cultured human melanoma cells and a three-dimensional human skin model.
We studied the inhibitory effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) on melanogenesis in cultured human melanoma cells, HMV-II, and in a three-dimensional cultured human skin model. alpha-Arbutin showed no inhibitory effect on HMV-II cell growth at a concentration below 1.0 mM. Melanin synthesis in cells treated with alpha-arbutin at 0.5 mM decreased to 76% of that in non-treated cells. The cellular tyrosinase activity of HMV-II cells also significantly decreased, while the expression of its mRNA was not affected. Melanin synthesis in a human skin model was also evaluated by the macro- and microscopic observation of its pigmentation as well as by quantitative measurements of melanin. Treatment of the human skin model with 250 microg of alpha-arbutin did not inhibit cell viability, while melanin synthesis was reduced to 40% of that in the control. These results indicate that alpha-arbutin is an effective and safe ingredient for skin-lightening.
Arbutin↗
Visualization of chromosomes and nuclear envelope in living cells for molecular dynamics studies.
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Animals↗
Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCgamma2 in B lymphocytes.
In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cgamma2 (PLCgamma2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCgamma2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCgamma2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCgamma2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCgamma2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCgamma2. Strikingly, Ca2+ entry-activated PLCgamma2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCgamma2 translocation and activation controls the amplification and co-ordination of receptor signalling.
Animals↗
JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation.
Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-kappaB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4-JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs.
Adaptor Proteins, Signal Transducing↗
Functional reassembly of a split PH domain.
The pleckstrin homology (PH) domain forms a structurally conserved protein module of approximately 120 amino acid residues. Several proteins involved in cellular signaling and cytoskeletal organization possess split PH domains while their biological roles and ligand binding activity remain to be clarified. We have designed a split PH domain from a structurally well-characterized PH domain of phospholipase Cdelta(1) by dissecting the PH domain and tethering a coiled coil module to each subunit to ask a question of whether the coiled coil could mediate a functional reassembly of the split PH domain. Isothermal titration microcalorimetry measurements indicated a formation of a thermodynamically stable 1:1 complex of the N-terminal and C-terminal halves of the split PH domain by the coiled coil formation. The reassembled split PH domain binds to IP(3), a target molecule of the parent PLCdelta(1) PH domain, but not to L-IP(3), indicating that the split PH domain maintains a binding selectivity similar to the native PLCdelta(1) PH domain. These results demonstrate that the split PH domain folds into a functional structure when the split halves are brought to close proximity, and suggest that the native split PH domains, such as found in PLCgamma(1), have distinctive functions upon the reassembly.
Amino Acid Sequence↗
Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori (silkworm).
Cry1Aa, an insecticidal protein produced by Bacillus thuringiensis, has been shown to bind to cadherin-like protein, BtR175, in Bombyx mori (silkworm) midgut. We previously reported three variant alleles of BtR175 (BtR175a, b and c). When transiently expressed in COS7 cells, all the three BtR175 variants bound to Cry1Aa. We stably expressed BtR175b in HEK293 cells. These BtR175b-expressing cells swelled and died in the presence of activated Cry1Aa in a dose- and time-dependent manner, showing that BtR175b itself can impart Cry1Aa-susceptibility to mammalian cells. These cells were more susceptible to Cry1Aa than to Cry1Ab and Cry1Ac. Since dispersed B. mori midgut cells were reported to be highly susceptible to Cry1Ac, this result suggested that other Cry1Ac-specific receptor(s) were simultaneously working with BtR175 in the midgut cells. Advantages are also discussed of applying these transfected mammalian cells to toxicity assays of mutant Cry proteins.
Animals↗
Monocyte CD40 expression in severe sepsis.
D40 is a cell surface protein belonging to the tumor necrosis factor (TNF) receptor family. Ligation of monocyte CD40 by the T cell-derived CD40 ligand can trigger the production of various mediators, the transcription and activation of enzymes, and the upregulation of costimulatory molecules involved in the pathogenesis of sepsis. To test the hypothesis that CD40 is expressed on the surface of monocytes during sepsis, we measured CD40 expression by flow cytometry on freshly sampled monocytes from 40 patients with severe sepsis, including 15 patients with bacteremia, and from eight healthy volunteers. Plasma concentrations of interleukin (IL) 6, IL-10, and IL-13 were also measured. We detected CD40 only on monocytes from patients with sepsis (mean 6.5 +/- 0.4 median channel fluorescence). There was an inverse correlation between peak CD40 expression and survival (P = 0.05), particularly in the patients with bacteremia (P = 0.019). In the bacteremic group, there was an inverse correlation between CD40 expression and bilirubin levels (r2 = 0.52, P = 0.004) and plasma IL-6 concentrations (r2 = 0.30, P = 0.04). Our results showed that upregulation of CD40 expression on peripheral blood monocytes is a protective phenomenon during severe sepsis. Monocyte deactivation reflected by low CD40 expression may represent impairment of immune function associated with severity of illness and poor outcome. Further studies on monocyte phenotype and function may help to assess the immune status of patients with sepsis and perhaps be useful to guide immunomodulatory strategy in the future.
Adult↗
Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase.
The effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) and 4-hydroxyphenyl beta-glucopyranoside (arbutin) on the activity of tyrosinase from human malignant melanoma cells were examined. The inhibitory effect of alpha-arbutin on human tyrosinase was stronger than that of arbutin. The K(i) value for alpha-arbutin was calculated to be 1/20 that for arbutin. We then synthesized arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase using arbutin and starch, respectively, as acceptor and donor molecules. The structural analyses using 13C- and 1H-NMR proved that the transglycosylated products were 4-hydroxyphenyl beta-maltoside (beta-Ab-alpha-G1) and 4-hydroxyphenyl beta-maltotrioside (beta-Ab-alpha-G2). These arbutin-alpha-glycosides exhibited competitive type inhibition on human tyrosinase, and their K(i) values were calculated to be 0.7 mM and 0.9 mM, respectively. These arbutin-alpha-glycosides possessed stronger inhibitory activity than arbutin, but less activity than alpha-arbutin. These results suggested that the alpha-glucosidic linkage of hydroquinone-glycosides plays an important role in the inhibitory effect on human tyrosinase.
Arbutin↗
Chemical approaches untangling sequence-specific DNA binding by proteins.
Structure-based design of novel DNA-binding proteins provides an ultimate test of our understanding of protein-DNA interactions. A combination of synthetic, organic, biochemical and molecular biological approaches has been developed to study the principle of molecular recognition associated with the protein-DNA interactions. The strategies enabled a specific formation of noncovalent peptide dimers and determination of the preferential DNA-binding sequence of short peptides.
Amino Acid Sequence↗