107 results on '"Yamashita T"'
Search Results
2. Activation of mitogen-activated protein kinase and its activator by ras in intact cells and in a cell-free system.
- Author
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Hattori, S, primary, Fukuda, M, additional, Yamashita, T, additional, Nakamura, S, additional, Gotoh, Y, additional, and Nishida, E, additional
- Published
- 1992
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3. Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells
- Author
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Yamashita, T, primary, Shimizu, T, additional, and Ogata, E, additional
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- 1991
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4. Effects of glycosylation on the secretion and enzyme activity of Mucor rennin, an aspartic proteinase of Mucor pusillus, produced by recombinant yeast.
- Author
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Aikawa, J., primary, Yamashita, T., additional, Nishiyama, M., additional, Horinouchi, S., additional, and Beppu, T., additional
- Published
- 1990
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5. Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin.
- Author
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Ihara, H, Denhardt, D T, Furuya, K, Yamashita, T, Muguruma, Y, Tsuji, K, Hruska, K A, Higashio, K, Enomoto, S, Nifuji, A, Rittling, S R, and Noda, M
- Abstract
Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone.
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- 2001
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6. Cloning and functional expression of a brain peptide/histidine transporter.
- Author
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Yamashita, T, Shimada, S, Guo, W, Sato, K, Kohmura, E, Hayakawa, T, Takagi, T, and Tohyama, M
- Abstract
Here we report the cloning and functional characterization of a rat novel peptide/histidine transporter (PHT1), which was expressed in the brain and the retina. The cDNA encodes the predicted protein of 572 amino acid residues with 12 putative membrane-spanning domains. The amino acid sequence has moderate homology with a nonspecific peptide transporter found in the plant. When expressed in Xenopus laevis oocytes, PHT1 cRNA induced high affinity proton-dependent histidine transport activity. This transport process was inhibited by dipeptides and tripeptides but not by free amino acids such as glutamate, glycine, leucine, methionine, and aspartate. Dipeptide carnosine transport activity was also confirmed by direct uptake measurement. By in situ hybridization analysis, PHT1 mRNA was widely distributed throughout whole brain. Especially, intense hybridization signals were found in the hippocampus, choroid plexus, cerebellum, and pontine nucleus. Signals were located in both the neuronal and small nonneuronal cells in these areas. PHT1 protein could contribute to uptake of oligopeptides, which function as neuromodulators, and clearance of degraded neuropeptides and be a new member in the growing superfamily of proton-coupled peptide and nitrate transporters, although its structure, localization, and pharmacological characteristics are unique among these members.
- Published
- 1997
7. Magnesium(II) is a crucial constituent of the blood coagulation cascade. Potentiation of coagulant activities of factor IX by Mg2+ ions.
- Author
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Sekiya, F, Yoshida, M, Yamashita, T, and Morita, T
- Abstract
We recently showed that not only Ca2+ ions but also Mg2+ ions play a crucial role in stabilizing the native conformation of coagulation factor IX. We here report that Mg2+ ions at physiological concentrations greatly augment the biological activities of factor IX. In clotting assays with dialyzed plasma, addition of Mg2+ ions enhanced the apparent coagulant activity of factor IXa, while that of factor Xa was scarcely affected. Activation of factor X by factor IXa in the presence of factor VIIIa, phospholipids, and Ca2+ ions was accelerated by Mg2+ ions. It appeared that the cation increased the affinity between factor IXa and factor VIIIa, thereby increasing the apparent catalytic efficacy of the enzyme. We also evaluated the effect of Mg2+ ions in the coagulation pathway initiated by tissue factor and found that activation of factor IX by factor VIIa*tissue factor was accelerated by the cation. Consequently, clotting of normal plasma induced by factor VIIa*tissue factor was shortened by the cation, while no such effect was observed in plasma deficient in factor IX or VIII. These results indicate that the previously unrecognized plasma component, Mg2+ ions, plays crucial roles in blood coagulation and, moreover, that contributions of factors IX and VIII in the coagulation cascade have been seriously underestimated in previous investigations.
- Published
- 1996
8. RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region.
- Author
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Yamashita, T, Kaneko, S, Shirota, Y, Qin, W, Nomura, T, Kobayashi, K, and Murakami, S
- Abstract
The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.
- Published
- 1998
9. Isolation of cDNA encoding guinea pig neutrophil cationic antibacterial polypeptide of 11 kDa (CAP11) and evaluation of CAP11 mRNA expression during neutrophil maturation.
- Author
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Nagaoka, I, Tsutsumi-Ishii, Y, Yomogida, S, and Yamashita, T
- Abstract
Neutrophils contain various antibacterial polypeptides and proteins in the granules that contribute to the killing of microorganisms. Recently, we have purified a cationic antibacterial polypeptide of 11 kDa (CAP11) from guinea pig neutrophil granules. CAP11 is a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 joined with one disulfide bond. In this study, to understand the regulation of CAP11 expression, we isolated and analyzed cDNA encoding CAP11. Furthermore, we investigated the expression of CAP11 mRNA during neutrophil maturation and localization of CAP11 among neutrophil granule subsets. Sequence analysis of CAP11 cDNA isolated from guinea pig bone marrow cells using rapid amplification of cDNA ends technique indicated that CAP11 is synthesized as a precursor comprising 178 amino acid residues, which is composed of a signal peptide (N-terminal 29 residues), a propeptide (106 residues), and a C-terminal mature peptide (43 residues). Interestingly, the predicted CAP11 precursor displayed the characteristic features of cathelicidins, a novel protein family of antibacterial polypeptides with a conserved cathelin-like pro-region and a variable C-terminal antibacterial domain. Northern blot and Western blot analyses using neutrophils, macrophages, eosinophils, mononuclear cells, and bone marrow cells revealed that only neutrophils and bone marrow cells expressed CAP11 mRNA and contained CAP11, suggesting that expression of CAP11 is neutrophil lineage-specific. Furthermore, Northern blot analysis using bone marrow cells separated according to their maturation stages showed that CAP11 mRNA was predominantly expressed in the cells at later stages of neutrophil maturation. Consistent with this, in situ hybridization using CAP11-specific cRNA probe demonstrated that CAP11 mRNA was primarily expressed at metamyelocyte stage. In addition, extracellular release assay revealed that CAP11 was readily released from neutrophils accompanied with gelatinase by low concentrations of N-formyl-Met-Leu-Phe without release of specific and azurophil granule components, and CAP11 was found to be exclusively present in the fraction containing gelatinase granules, prepared by Percoll density gradient centrifugation. Together these observations indicate that CAP11 is a member of cathelicidin family and its mRNA is preferentially expressed at the later stage of neutrophil maturation (i.e. metamyelocyte stage). Furthermore, CAP11 may be stored in the granule subset, possibly the gelatinase granule.
- Published
- 1997
10. Coordinate expression of alpha-tropomyosin and caldesmon isoforms in association with phenotypic modulation of smooth muscle cells.
- Author
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Kashiwada, K, Nishida, W, Hayashi, K, Ozawa, K, Yamanaka, Y, Saga, H, Yamashita, T, Tohyama, M, Shimada, S, Sato, K, and Sobue, K
- Abstract
Isoform diversity of tropomyosin is generated from the limited genes by a combination of differential transcription and alternative splicing. In the case of the alpha-tropomyosin (alpha-TM) gene, exon 2a rather than exon 2b is specifically spliced in alpha-TM-SM mRNA, which is one of the major tropomyosin isoforms in smooth muscle cells. Here we demonstrate that expressions of alpha-tropomyosin and caldesmon isoforms are coordinately regulated in association with phenotypic modulation of smooth muscle cells. Molecular cloning and Western and Northern blottings have revealed that in addition to the down-regulation of beta-TM-SM, alpha-TM-SM converted to alpha-TM-F1 and alpha-TM-F2 by a selectional change from exon 2a to exon 2b during dedifferentiation of smooth muscle cells in culture. Simultaneously, a change of caldesmon isoforms from high Mr type to low Mr type was also observed by alternative selection between exons 3b and 4 in the caldesmon gene during this process. In contrast, cultured smooth muscle cells maintaining a differentiated phenotype continued to express alpha-TM-SM, beta-TM-SM, and high Mr caldesmon. In situ hybridization revealed specific coexpression of alpha-TM-SM and high Mr caldesmon in smooth muscle in developing embryos. These results suggest a common splicing mechanism for phenotype-dependent expression of tropomyosin and caldesmon isoforms in both visceral and vascular smooth muscle cells.
- Published
- 1997
11. Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts.
- Author
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Kaibuchi, K, Tsuda, T, Kikuchi, A, Tanimoto, T, Yamashita, T, and Takai, Y
- Abstract
The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.
- Published
- 1986
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12. Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding.
- Author
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Hattori, S, Yamashita, T, Copeland, T D, Oroszlan, S, and Shih, T Y
- Abstract
We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.
- Published
- 1986
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13. Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes.
- Author
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Kikuchi, A, Yamashita, T, Kawata, M, Yamamoto, K, Ikeda, K, Tanimoto, T, and Takai, Y
- Abstract
At least six GTP-binding proteins (G proteins) with Mr values between 20,000 and 25,000 were extracted from the crude membrane fraction of bovine brain by sodium cholate and purified by successive column chromatographies of Ultrogel AcA-44, phenyl-Sepharose CL-4B, hydroxyapatite, and Mono Q HR5/5. One G protein with a Mr of about 24,000 (24K G) was purified to near homogeneity and characterized. 24K G bound maximally about 0.7 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 46 nM. [35S]GTP gamma S binding to this protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. 24K G hydrolyzed GTP to liberate Pi with a rate of about 40 mmol of Pi/mol of protein/min. 24K G did not associate with the beta gamma subunit of Go. 24K G was distinguishable on sodium dodecyl sulfate-polyacrylamide gel electrophoresis from the ras protein (ras p21) and ADP-ribosylation factor, the G protein with a Mr of about 21,000 serving as a cofactor for the cholera toxin-dependent ADP-ribosylation of Gs. 24K G was not recognized by the antibody against ras p21, ADP-ribosylation factor, the alpha subunit each of Gs, Gi, and Go, or tubulin. These results indicate that 24K G is a novel G protein and suggest that there are multiple forms of G proteins with small Mr values as is the case with the G proteins which have the alpha subunits with Mr values of about 40,000.
- Published
- 1988
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14. Possible involvement of cyclic AMP and calcium ion in prostaglandin E1-induced elevation of c-myc mRNA levels in Swiss 3T3 fibroblasts.
- Author
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Yamashita, T, Tsuda, T, Hamamori, Y, and Takai, Y
- Abstract
Prostaglandin E1 (PGE1) caused a rapid and dose-dependent increase in cAMP levels, followed by elevation of c-myc mRNA levels and then increased DNA synthesis in quiescent cultures of Swiss 3T3 fibroblasts. The dose-response curves of PGE1 were nearly the same for each of these three processes. Both 8-bromo-cAMP and forskolin increased c-myc mRNA levels to 40-50% and DNA synthesis to 70-80% of those caused by a maximally effective dose of PGE1. Under the comparable conditions, PGE1 did not stimulate diacylglycerol formation or activate protein kinase C. However, PGE1 did elevate cytoplasmic free Ca2+ concentration as measured with the fluorescent Ca2+ indicator quin 2. 8-Bromo-cAMP and forskolin were inactive in this capacity. The Ca2+ ionophore A23187 increased the level of c-myc mRNA. Diacylglycerol and Ca2+ mediate the elevation of c-myc mRNA levels which is caused by platelet-derived growth factor and fibroblast growth factor (Kaibuchi, K., Tsuda, T., Kikuchi, A., Tanimoto, T., Yamashita, T., and Takai, Y. (1986) J. Biol. Chem. 261, 1187-1192). In contrast, the present results suggest that both cAMP and Ca2+ are involved in this PGE1-induced response in Swiss 3T3 cells.
- Published
- 1986
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15. A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells.
- Author
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Takumi, T, Ishii, T, Horio, Y, Morishige, K, Takahashi, N, Yamada, M, Yamashita, T, Kiyama, H, Sohmiya, K, and Nakanishi, S
- Abstract
We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.
- Published
- 1995
16. Regulation of the tertiary structure and function of coagulation factor IX by magnesium (II) ions.
- Author
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Sekiya, F, Yamashita, T, Atoda, H, Komiyama, Y, and Morita, T
- Abstract
The indispensable role of Ca2+ ions in the maintenance of the functional tertiary structures of vitamin K-dependent coagulation factors has been definitively established but the participation of Mg2+ ions, another alkaline-earth metal that is present abundantly in blood plasma, in such a process is not yet understood. We show here that the Ca(2+)-stabilized conformation of coagulation factor IX undergoes a further conformational change upon binding of Mg2+ ions using three independent structural probes. The probes we used were (i) IX/X-bp, a snake venom anticoagulant that recognizes the Gla domains in coagulation factors IX and X, (ii) conformation-specific polyclonal antibodies against bovine factor IX, and (iii) monoclonal antibodies against the Gla domain of human factor IX. The binding of all these probes had an absolute requirement for Ca2+ ions, and Mg2+ ions alone were ineffective. However, when added together with Ca2+ ions, Mg2+ ions at physiological concentrations greatly augmented the binding of these probes to factor IX; the required concentration of Ca2+ ions was much reduced, and the affinity of each probe for factor IX was increased even in the presence of an excess of Ca2+ ions. These results suggest the presence of a Mg(2+)-specific binding site that does not interact with Ca2+ ions in factor IX. Furthermore, Mg2+ ions potentiated the susceptibility of factor IX to activation by factor XIa, concomitant with their effect on the conformation. Similarly, the required Ca2+ concentration was reduced by Mg2+ ions, and the rate of conversion to factor IXa was increased by Mg2+ ions in the presence of an excess of Ca2+ ions. At a saturating concentration of Ca2+ ions (5 mM), addition of 1 mM Mg2+ reduced the apparent Km value for factor IX from 0.31 to 0.18 microM, and in the presence of a physiological concentration of Ca2+ ions (1 mM), the reduction in Km by Mg2+ ions was far more striking (from 0.91 to 0.24 microM). The apparent Vmax values were hardly affected by Mg2+ ions. Our present data reveal a hitherto novel physiological role of the Mg2+ ions in plasma. Not only Ca2+ ions but also Mg2+ ions are important regulators of the stabilization of the native conformation of factor IX as well as of its efficient activation.
- Published
- 1995
17. Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes.
- Author
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Yamashita, T, Yamamoto, K, Kikuchi, A, Kawata, M, Kondo, J, Hishida, T, Teranishi, Y, Shiku, H, and Takai, Y
- Abstract
In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5′-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.
- Published
- 1988
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18. Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts.
- Author
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Yamashita, T. and Takai, Y.
- Abstract
In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.
- Published
- 1987
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19. Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.
- Author
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Yamashita, T, primary, Arens, M, additional, and Green, M, additional
- Published
- 1975
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20. Adenovirus deoxyribonucleic acid replication. Characterization of the enzyme activities of a soluble replication system.
- Author
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Arens, M., primary, Yamashita, T., additional, Padmanabhan, R., additional, Tsuruo, T., additional, and Green, M., additional
- Published
- 1977
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21. Adenovirus deoxyribonucleic acid replication. Isolation of a soluble replication system and analysis of the in vitro DNA product.
- Author
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Yamashita, T., primary, Arens, M., additional, and Green, M., additional
- Published
- 1977
- Full Text
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22. Convergent mechanism underlying the acquisition of vertebrate scotopic vision.
- Author
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Kojima K, Yanagawa M, Imamoto Y, Yamano Y, Wada A, Shichida Y, and Yamashita T
- Subjects
- Animals, Light, Vertebrates, Cone Opsins chemistry, Cone Opsins metabolism, Evolution, Molecular, Night Vision physiology, Rhodopsin chemistry, Rhodopsin metabolism
- Abstract
High sensitivity of scotopic vision (vision in dim light conditions) is achieved by the rods' low background noise, which is attributed to a much lower thermal activation rate (k
th ) of rhodopsin compared with cone pigments. Frogs and nocturnal geckos uniquely possess atypical rods containing noncanonical cone pigments that exhibit low kth , mimicking rhodopsin. Here, we investigated the convergent mechanism underlying the low kth of rhodopsins and noncanonical cone pigments. Our biochemical analysis revealed that the kth of canonical cone pigments depends on their absorption maximum (λmax ). However, rhodopsin and noncanonical cone pigments showed a substantially lower kth than predicted from the λmax dependency. Given that the λmax is inversely proportional to the activation energy of the pigments in the Hinshelwood distribution-based model, our findings suggest that rhodopsin and noncanonical cone pigments have convergently acquired low frequency of spontaneous-activation attempts, including thermal fluctuations of the protein moiety, in the molecular evolutionary processes from canonical cone pigments, which contributes to highly sensitive scotopic vision., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
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23. eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.
- Author
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Gotoh S, Mori K, Fujino Y, Kawabe Y, Yamashita T, Omi T, Nagata K, Tagami S, Nagai Y, and Ikeda M
- Subjects
- Animals, Dipeptides genetics, Drosophila genetics, Drosophila metabolism, HeLa Cells, Humans, Disease Models, Animal, Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis physiopathology, C9orf72 Protein genetics, DNA Repeat Expansion genetics, Eukaryotic Initiation Factor-5 genetics, Eukaryotic Initiation Factor-5 metabolism, Frontotemporal Lobar Degeneration genetics, Frontotemporal Lobar Degeneration physiopathology
- Abstract
Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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24. Mammalian type opsin 5 preferentially activates G14 in Gq-type G proteins triggering intracellular calcium response.
- Author
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Sato K, Yamashita T, and Ohuchi H
- Subjects
- Humans, Mice, Animals, Calcium metabolism, Signal Transduction, Receptors, G-Protein-Coupled metabolism, Rod Opsins metabolism, Mammals metabolism, Opsins genetics, Opsins metabolism, GTP-Binding Protein alpha Subunits, Gq-G11 genetics, GTP-Binding Protein alpha Subunits, Gq-G11 metabolism
- Abstract
Mammalian type opsin 5 (Opn5m), a UV-sensitive G protein-coupled receptor opsin highly conserved in vertebrates, would provide a common basis for UV sensing from lamprey to humans. However, G protein coupled with Opn5m remains controversial due to variations in assay conditions and the origin of Opn5m across different reports. Here, we examined Opn5m from diverse species using an aequorin luminescence assay and Gα-KO cell line. Beyond the commonly studied major Gα classes, Gαq, Gα11, Gα14, and Gα15 in the Gq class were individually investigated in this study, as they can drive distinct signaling pathways in addition to a canonical calcium response. UV light triggered a calcium response via all the tested Opn5m proteins in 293T cells, which was abolished by Gq-type Gα deletion and rescued by cotransfection with mouse and medaka Gq-type Gα proteins. Opn5m preferentially activated Gα14 and close relatives. Mutational analysis implicated specific regions, including α3-β5 and αG-α4 loops, αG and α4 helices, and the extreme C terminus, in the preferential activation of Gα14 by Opn5m. FISH revealed co-expression of genes encoding Opn5m and Gα14 in the scleral cartilage of medaka and chicken eyes, supporting their physiological coupling. This suggests that the preferential activation of Gα14 by Opn5m is relevant for UV sensing in specific cell types., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the content of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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25. Trophoblast cell surface antigen-2 phosphorylation triggered by binding of galectin-3 drives metastasis through down-regulation of E-cadherin.
- Author
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Iwamoto S, Mori Y, Yamashita T, Ojima K, Akita K, Togano S, Kushiyama S, Yashiro M, Yatera Y, Yamaguchi T, Komiyama A, Sago Y, Itano N, and Nakada H
- Subjects
- Animals, Humans, Mice, Cadherins genetics, Cadherins metabolism, Cell Line, Tumor, Down-Regulation, Epithelial-Mesenchymal Transition genetics, Gene Expression Regulation, Neoplastic, MCF-7 Cells, Mice, Nude, RNA, Messenger genetics, Trophoblasts metabolism, Zinc Finger E-box-Binding Homeobox 1 metabolism, beta Catenin genetics, beta Catenin metabolism, Galectin 3 genetics, Galectin 3 metabolism
- Abstract
The expression of trophoblast cell surface antigen-2 (Trop-2) is enhanced in many tumor tissues and is correlated with increased malignancy and poor survival of patients with cancer. Previously, we demonstrated that the Ser-322 residue of Trop-2 is phosphorylated by protein kinase Cα (PKCα) and PKCδ. Here, we demonstrate that phosphomimetic Trop-2 expressing cells have markedly decreased E-cadherin mRNA and protein levels. Consistently, mRNA and protein of the E-cadherin-repressing transcription factors zinc finger E-Box binding homeobox 1 (ZEB1) were elevated, suggesting transcriptional regulation of E-cadherin expression. The binding of galectin-3 to Trop-2 enhanced the phosphorylation and subsequent cleavage of Trop-2, followed by intracellular signaling by the resultant C-terminal fragment. Binding of β-catenin/transcription factor 4 (TCF4) along with the C-terminal fragment of Trop-2 to the ZEB1 promoter upregulated ZEB1 expression. Of note, siRNA-mediated knockdown of β-catenin and TCF4 increased the expression of E-cadherin through ZEB1 downregulation. Knockdown of Trop-2 in MCF-7 cells and DU145 cells resulted in downregulation of ZEB1 and subsequent upregulation of E-cadherin. Furthermore, wild-type and phosphomimetic Trop-2 but not phosphorylation-blocked Trop-2 were detected in the liver and/or lung of some nude mice bearing primary tumors inoculated intraperitoneally or subcutaneously with wild-type or mutated Trop-2 expressing cells, suggesting that Trop-2 phosphorylation, plays an important role in tumor cell mobility in vivo, too. Together with our previous finding of Trop-2 dependent regulation of claudin-7, we suggest that the Trop-2-mediated cascade involves concurrent derangement of both tight and adherence junctions, which may drive metastasis of epithelial tumor cells., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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26. Diversification processes of teleost intron-less opsin genes.
- Author
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Fujiyabu C, Sato K, Ohuchi H, and Yamashita T
- Subjects
- Animals, Phylogeny, Rhodopsin genetics, Rhodopsin metabolism, Zebrafish genetics, Oryzias genetics, Synteny genetics, Evolution, Molecular, Fishes genetics, Fishes physiology, Introns, Opsins genetics, Opsins metabolism
- Abstract
Opsins are universal photosensitive proteins in animals. Vertebrates have a variety of opsin genes for visual and non-visual photoreceptions. Analysis of the gene structures shows that most opsin genes have introns in their coding regions. However, teleosts exceptionally have several intron-less opsin genes that are presumed to have been duplicated by an RNA-based gene duplication mechanism, retroduplication. Among these retrogenes, we focused on the Opn4 (melanopsin) gene responsible for non-image-forming photoreception. Many teleosts have five Opn4 genes including one intron-less gene, which is speculated to have been formed from a parental intron-containing gene in the Actinopterygii. In this study, to reveal the evolutionary history of Opn4 genes, we analyzed them in teleost (zebrafish and medaka) and non-teleost (bichir, sturgeon, and gar) fishes. Our synteny analysis suggests that the intron-less Opn4 gene emerged by retroduplication after the branching of the bichir lineage. In addition, our biochemical and histochemical analyses showed that, in the teleost lineage, the newly acquired intron-less Opn4 gene became abundantly used without substantial changes in the molecular properties of the Opn4 protein. This stepwise evolutionary model of Opn4 genes is quite similar to that of rhodopsin genes in the Actinopterygii. The unique acquisition of rhodopsin and Opn4 retrogenes would have contributed to the diversification of the opsin gene repertoires in the Actinopterygii and the adaptation of teleosts to various aquatic environments., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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27. The porphyrin TMPyP4 inhibits elongation during the noncanonical translation of the FTLD/ALS-associated GGGGCC repeat in the C9orf72 gene.
- Author
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Mori K, Gotoh S, Yamashita T, Uozumi R, Kawabe Y, Tagami S, Kamp F, Nuscher B, Edbauer D, Haass C, Nagai Y, and Ikeda M
- Subjects
- Amyotrophic Lateral Sclerosis genetics, Amyotrophic Lateral Sclerosis metabolism, C9orf72 Protein genetics, Frontotemporal Lobar Degeneration genetics, Frontotemporal Lobar Degeneration metabolism, HeLa Cells, Humans, Polyribosomes metabolism, C9orf72 Protein biosynthesis, DNA Repeat Expansion, Models, Biological, Peptide Chain Elongation, Translational drug effects, Porphyrins pharmacology
- Abstract
GGGGCC (G
4 C2 ) repeat expansion in the C9orf72 gene has been shown to cause frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Dipeptide repeat proteins produced through repeat-associated non-AUG (RAN) translation are recognized as potential drivers for neurodegeneration. Therefore, selective inhibition of RAN translation could be a therapeutic avenue to treat these neurodegenerative diseases. It was previously known that the porphyrin TMPyP4 binds to G4 C2 repeat RNA. However, the consequences of this interaction have not been well characterized. Here, we confirmed that TMPyP4 inhibits C9orf72 G4 C2 repeat translation in cellular and in in vitro translation systems. An artificial insertion of an AUG codon failed to cancel the translation inhibition, suggesting that TMPyP4 acts downstream of non-AUG translation initiation. Polysome profiling assays also revealed polysome retention on G4 C2 repeat RNA, along with inhibition of translation, indicating that elongating ribosomes stall on G4 C2 repeat RNA. Urea-resistant interaction between G4 C2 repeat RNA and TMPyP4 likely contributes to this ribosome stalling and thus to selective inhibition of RAN translation. Taken together, our data reveal a novel mode of action of TMPyP4 as an inhibitor of G4 C2 repeat translation elongation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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28. A peroxisome deficiency-induced reductive cytosol state up-regulates the brain-derived neurotrophic factor pathway.
- Author
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Abe Y, Honsho M, Kawaguchi R, Matsuzaki T, Ichiki Y, Fujitani M, Fujiwara K, Hirokane M, Oku M, Sakai Y, Yamashita T, and Fujiki Y
- Subjects
- Animals, CHO Cells, Cell Line, Cell Line, Tumor, Cells, Cultured, Cricetinae, Cricetulus, Cytosol metabolism, Fatty Acids metabolism, Hippocampus cytology, Humans, Oxidation-Reduction, Plasmalogens metabolism, Rats, Rats, Wistar, Up-Regulation, Astrocytes metabolism, Brain-Derived Neurotrophic Factor metabolism, Neurons metabolism, Peroxisomal Disorders metabolism, Peroxisomes metabolism
- Abstract
The peroxisome is a subcellular organelle that functions in essential metabolic pathways, including biosynthesis of plasmalogens, fatty acid β-oxidation of very-long-chain fatty acids, and degradation of hydrogen peroxide. Peroxisome biogenesis disorders (PBDs) manifest as severe dysfunction in multiple organs, including the central nervous system (CNS), but the pathogenic mechanisms in PBDs are largely unknown. Because CNS integrity is coordinately established and maintained by neural cell interactions, we here investigated whether cell-cell communication is impaired and responsible for the neurological defects associated with PBDs. Results from a noncontact co-culture system consisting of primary hippocampal neurons with glial cells revealed that a peroxisome-deficient astrocytic cell line secretes increased levels of brain-derived neurotrophic factor (BDNF), resulting in axonal branching of the neurons. Of note, the BDNF expression in astrocytes was not affected by defects in plasmalogen biosynthesis and peroxisomal fatty acid β-oxidation in the astrocytes. Instead, we found that cytosolic reductive states caused by a mislocalized catalase in the peroxisome-deficient cells induce the elevation in BDNF secretion. Our results suggest that peroxisome deficiency dysregulates neuronal axogenesis by causing a cytosolic reductive state in astrocytes. We conclude that astrocytic peroxisomes regulate BDNF expression and thereby support neuronal integrity and function., (© 2020 Abe et al.)
- Published
- 2020
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29. Trophoblast cell surface antigen 2 (Trop-2) phosphorylation by protein kinase C α/δ (PKCα/δ) enhances cell motility.
- Author
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Mori Y, Akita K, Ojima K, Iwamoto S, Yamashita T, Morii E, and Nakada H
- Subjects
- Claudins genetics, Claudins metabolism, HCT116 Cells, Humans, Phosphorylation, Protein Binding, Antigens, Neoplasm metabolism, Cell Adhesion Molecules metabolism, Cell Movement, Protein Kinase C-alpha metabolism, Protein Kinase C-delta metabolism
- Abstract
Dysfunction of tight junctions is a critical step during the initial stage of tumor progression. Trophoblast cell surface antigen 2 (Trop-2) belongs to the family of tumor-associated calcium signal transducer ( TACSTD ) and is required for the stability of claudin-7 and claudin-1, which are often dysregulated or lost in carcinogenesis. Here, we investigated the effects of Trop-2 phosphorylation on cell motility. Analyses using HCT116 cells expressing WT Trop-2 (HCT116/WT) or Trop-2 alanine-substituted at Ser-303 (HCT116/S303A) or Ser-322 (HCT116/S322A) revealed that Trop-2 is phosphorylated at Ser-322. Furthermore, coimmunoprecipitation and Transwell assays indicated that Trop-2 S322A interacted with claudin-7 the strongest, and a phosphomimetic variant, Trop-2 S322E, the weakest and that HCT116/S322E cells have the highest motility and HCT116/S322A cells the lowest. All cell lines had similar levels of claudin-7 mRNA, but levels of claudin-7 protein were markedly decreased in the HCT116/S322E cells, suggesting posttranscriptional control of claudin-7. Moreover, claudin-7 was clearly localized to cell-cell borders in HCT116/S322A cells but was diffusely distributed on the membrane and partially localized in the cytoplasm of HCT116/S322E and HCT116/WT cells. These observations suggested that Trop-2 phosphorylation plays a role in the decrease or mislocalization of claudin-7. Using protein kinase C (PKC) inhibitors and PKC-specific siRNAs, we found that PKCα and PKCδ are responsible for Trop-2 phosphorylation. Of note, chemical PKC inhibition and PKCα- and PKCδ-specific siRNAs reduced motility. In summary, our findings provide evidence that Trop-2 is phosphorylated at Ser-322 by PKCα/δ and that this phosphorylation enhances cell motility and decreases claudin-7 localization to cellular borders., (© 2019 Mori et al.)
- Published
- 2019
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30. A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH.
- Author
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Ueda M, Okada M, Mizuguchi M, Kluve-Beckerman B, Kanenawa K, Isoguchi A, Misumi Y, Tasaki M, Ueda A, Kanai A, Sasaki R, Masuda T, Inoue Y, Nomura T, Shinriki S, Shuto T, Kai H, Yamashita T, Matsui H, Benson MD, and Ando Y
- Subjects
- Amyloid drug effects, Amyloid Neuropathies, Familial metabolism, Apomorphine pharmacology, Cells, Cultured, Drug Repositioning, Humans, Hydrogen-Ion Concentration, Inflammation genetics, Neuroglia metabolism, Neurons metabolism, Prealbumin chemistry, Protein Conformation, Proteolysis, Pyrvinium Compounds pharmacology, Trypsin metabolism, Amyloid metabolism, High-Throughput Screening Assays methods, Prealbumin metabolism
- Abstract
Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro , even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo , and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis., (© 2019 Ueda et al.)
- Published
- 2019
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31. Identification, characterization, and structural analyses of a fungal endo-β-1,2-glucanase reveal a new glycoside hydrolase family.
- Author
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Tanaka N, Nakajima M, Narukawa-Nara M, Matsunaga H, Kamisuki S, Aramasa H, Takahashi Y, Sugimoto N, Abe K, Terada T, Miyanaga A, Yamashita T, Sugawara F, Kamakura T, Komba S, Nakai H, and Taguchi H
- Subjects
- Structure-Activity Relationship, Substrate Specificity, Fungal Proteins chemistry, Glycoside Hydrolases chemistry, Soil Microbiology, Talaromyces enzymology
- Abstract
endo -β-1,2-Glucanase (SGL) is an enzyme that hydrolyzes β-1,2-glucans, which play important physiological roles in some bacteria as a cyclic form. To date, no eukaryotic SGL has been identified. We purified an SGL from Talaromyces funiculosus ( Tf SGL), a soil fungus, to homogeneity and then cloned the complementary DNA encoding the enzyme. Tf SGL shows no significant sequence similarity to any known glycoside hydrolase (GH) families, but shows significant similarity to certain eukaryotic proteins with unknown functions. The recombinant Tf SGL ( Tf SGLr) specifically hydrolyzed linear and cyclic β-1,2-glucans to sophorose (Glc-β-1,2-Glc) as a main product. Tf SGLr hydrolyzed reducing-end-modified β-1,2-gluco-oligosaccharides to release a sophoroside with the modified moiety. These results indicate that Tf SGL is an endo -type enzyme that preferably releases sophorose from the reducing end of substrates. Stereochemical analysis demonstrated that Tf SGL is an inverting enzyme. The overall structure of Tf SGLr includes an (α/α)
6 toroid fold. The substrate-binding mode was revealed by the structure of a Michaelis complex of an inactive Tf SGLr mutant with a β-1,2-glucoheptasaccharide. Mutational analysis and action pattern analysis of β-1,2-gluco-oligosaccharide derivatives revealed an unprecedented catalytic mechanism for substrate hydrolysis. Glu-262 (general acid) indirectly protonates the anomeric oxygen at subsite -1 via the 3-hydroxy group of the Glc moiety at subsite +2, and Asp-446 (general base) activates the nucleophilic water via another water. Tf SGLr is apparently different from a GH144 SGL in the reaction and substrate recognition mechanism based on structural comparison. Overall, we propose that Tf SGL and closely-related enzymes can be classified into a new family, GH162., (© 2019 Tanaka et al.)- Published
- 2019
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32. Biochemical and structural analyses of a bacterial endo -β-1,2-glucanase reveal a new glycoside hydrolase family.
- Author
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Abe K, Nakajima M, Yamashita T, Matsunaga H, Kamisuki S, Nihira T, Takahashi Y, Sugimoto N, Miyanaga A, Nakai H, Arakawa T, Fushinobu S, and Taguchi H
- Subjects
- Bacterial Proteins isolation & purification, Catalysis, Catalytic Domain, Cellulase isolation & purification, Crystallography, X-Ray, Bacterial Proteins chemistry, Bacteroidetes enzymology, Cellulase chemistry
- Abstract
β-1,2-Glucan is an extracellular cyclic or linear polysaccharide from Gram-negative bacteria, with important roles in infection and symbiosis. Despite β-1,2-glucan's importance in bacterial persistence and pathogenesis, only a few reports exist on enzymes acting on both cyclic and linear β-1,2-glucan. To this end, we purified an endo -β-1,2-glucanase to homogeneity from cell extracts of the environmental species Chitinophaga arvensicola , and an endo -β-1,2-glucanase candidate gene ( Cpin_6279 ) was cloned from the related species Chitinophaga pinensis The Cpin_6279 protein specifically hydrolyzed linear β-1,2-glucan with polymerization degrees of ≥5 and a cyclic counterpart, indicating that Cpin_6279 is an endo -β-1,2-glucananase. Stereochemical analysis demonstrated that the Cpin_6279-catalyzed reaction proceeds via an inverting mechanism. Cpin_6279 exhibited no significant sequence similarity with known glycoside hydrolases (GHs), and thus the enzyme defines a novel GH family, GH144. The crystal structures of the ligand-free and complex forms of Cpin_6279 with glucose (Glc) and sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) determined up to 1.7 Å revealed that it has a large cavity appropriate for polysaccharide degradation and adopts an (α/α)
6 -fold slightly similar to that of GH family 15 and 8 enzymes. Mutational analysis indicated that some of the highly conserved acidic residues in the active site are important for catalysis, and the Cpin_6279 active-site architecture provided insights into the substrate recognition by the enzyme. The biochemical characterization and crystal structure of this novel GH may enable discovery of other β-1,2-glucanases and represent a critical advance toward elucidating structure-function relationships of GH enzymes., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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33. Prostacyclin prevents pericyte loss and demyelination induced by lysophosphatidylcholine in the central nervous system.
- Author
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Muramatsu R, Kuroda M, Matoba K, Lin H, Takahashi C, Koyama Y, and Yamashita T
- Subjects
- Animals, Blood-Brain Barrier drug effects, Cell Count, Demyelinating Diseases metabolism, Demyelinating Diseases physiopathology, Disease Progression, Female, Iloprost pharmacology, Mice, Motor Activity drug effects, Proteolysis drug effects, Rats, Signal Transduction drug effects, Tight Junction Proteins metabolism, Demyelinating Diseases chemically induced, Demyelinating Diseases prevention & control, Epoprostenol metabolism, Lysophosphatidylcholines pharmacology, Pericytes cytology, Pericytes drug effects, Spinal Cord pathology
- Abstract
Pericytes play pivotal roles in physiological and pathophysiological conditions in the central nervous system. As pericytes prevent vascular leakage, they can halt neuronal damage stemming from a compromised blood-brain barrier. Therefore, pericytes may be a good target for the treatment of neurodegenerative disorders, although evidence is lacking. In this study, we show that prostacyclin attenuates lysophosphatidylcholine (LPC)-mediated vascular dysfunction through pericyte protection in the adult mouse spinal cord. LPC decreased the number of pericytes in an in vitro blood-brain barrier model, and this decrease was prevented by iloprost treatment, a prostacyclin analog. Intrathecal administration of iloprost attenuated vascular barrier disruption after LPC injection in the mouse spinal cord. Furthermore, iloprost treatment diminished demyelination and motor function deficits in mice injected with LPC. These results support the notion that prostacyclin acts on pericytes to maintain vascular barrier integrity., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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34. Dynamics of the heme-binding bacterial gas-sensing dissimilative nitrate respiration regulator (DNR) and activation barriers for ligand binding and escape.
- Author
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Lobato L, Bouzhir-Sima L, Yamashita T, Wilson MT, Vos MH, and Liebl U
- Subjects
- Animals, Bacterial Proteins physiology, Binding Sites, Carbon Monoxide chemistry, Cytochromes c chemistry, DNA, Bacterial chemistry, Fluorescence Polarization, Gene Expression Regulation, Bacterial, Heme analogs & derivatives, Heme chemistry, Horses, Kinetics, Ligands, Nitric Oxide chemistry, Promoter Regions, Genetic, Protein Binding, Pseudomonas aeruginosa genetics, Transcription Factors physiology, Bacterial Proteins chemistry, Pseudomonas aeruginosa metabolism, Transcription Factors chemistry
- Abstract
DNR (dissimilative nitrate respiration regulator) is a heme-binding transcription factor that is involved in the regulation of denitrification in Pseudomonas aeruginosa. In the ferrous deoxy state, the heme is 6-coordinate; external NO and CO can replace an internal ligand. Using fluorescence anisotropy, we show that high-affinity sequence-specific DNA binding occurs only when the heme is nitrosylated, consistent with the proposed function of DNR as NO sensor and transcriptional activator. This role is moreover supported by the NO "trapping" properties revealed by ultrafast spectroscopy that are similar to those of other heme-based NO sensor proteins. Dissociated CO-heme pairs rebind in an essentially barrierless way. This process competes with migration out of the heme pocket. The latter process is thermally activated (Ea ∼ 7 kJ/mol). This result is compared with other heme proteins, including the homologous CO sensor/transcription factor CooA, variants of the 5-coordinate mycobacterial sensor DosT and the electron transfer protein cytochrome c. This comparison indicates that thermal activation of ligand escape from the heme pocket is specific for systems where an external ligand replaces an internal one. The origin of this finding and possible implications are discussed., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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35. Specific targeting of a naturally presented osteosarcoma antigen, papillomavirus binding factor peptide, using an artificial monoclonal antibody.
- Author
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Tsukahara T, Emori M, Murata K, Hirano T, Muroi N, Kyono M, Toji S, Watanabe K, Torigoe T, Kochin V, Asanuma H, Matsumiya H, Yamashita K, Himi T, Ichimiya S, Wada T, Yamashita T, Hasegawa T, and Sato N
- Subjects
- Amino Acid Sequence, Antigen Presentation, Antigens, Neoplasm genetics, Base Sequence, Bone Neoplasms genetics, Bone Neoplasms therapy, Cancer Vaccines genetics, Cancer Vaccines immunology, Cell Line, Tumor, HLA-A2 Antigen genetics, HLA-A2 Antigen immunology, HLA-B Antigens immunology, Humans, Immunotherapy, Active, Molecular Sequence Data, Osteosarcoma genetics, Osteosarcoma therapy, Papillomaviridae immunology, Peptide Library, Single-Chain Antibodies genetics, Single-Chain Antibodies immunology, T-Lymphocytes, Cytotoxic immunology, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antigens, Neoplasm immunology, Bone Neoplasms immunology, Osteosarcoma immunology
- Abstract
Osteosarcoma is a rare but highly malignant tumor occurring most frequently in adolescents. The prognosis of non-responders to chemotherapy is still poor, and new treatment modalities are needed. To develop peptide-based immunotherapy, we previously identified autologous cytotoxic T lymphocyte-defined osteosarcoma antigen papillomavirus binding factor (PBF) in the context of HLA-B55 and the cytotoxic T lymphocyte epitope (PBF A2.2) presented by HLA-A2. PBF and HLA class I are expressed in ∼90 and 70% of various sarcomas, respectively. However, the expression status of peptide PBF A2.2 presented by HLA-A2 on osteosarcoma cells has remained unknown because it is difficult to generate a specific probe that reacts with the HLA·peptide complex. For detection and qualification of the HLA-A*02:01·PBF A2.2 peptide complex on osteosarcoma cells, we tried to isolate a single chain variable fragment (scFv) antibody directed to the HLA-*A0201·PBF A2.2 complex using a naïve scFv phage display library. As a result, scFv clone D12 with high affinity (KD = 1.53 × 10(-9) M) was isolated. D12 could react with PBF A2.2 peptide-pulsed T2 cells and HLA-A2+PBF+ osteosarcoma cell lines and simultaneously demonstrated that the HLA·peptide complex was expressed on osteosarcoma cells. In conclusion, scFv clone D12 might be useful to select candidate patients for PBF A2.2 peptide-based immunotherapy and develop antibody-based immunotherapy., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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36. Rod visual pigment optimizes active state to achieve efficient G protein activation as compared with cone visual pigments.
- Author
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Kojima K, Imamoto Y, Maeda R, Yamashita T, and Shichida Y
- Subjects
- Animals, Cattle, Chickens, HEK293 Cells, Humans, Kinetics, Light, Mice, Retinal Cone Photoreceptor Cells radiation effects, Retinal Rod Photoreceptor Cells radiation effects, Rhodopsin metabolism, Rod Cell Outer Segment metabolism, Rod Cell Outer Segment radiation effects, Spectrum Analysis, Retinal Cone Photoreceptor Cells metabolism, Retinal Pigments metabolism, Retinal Rod Photoreceptor Cells metabolism, Transducin metabolism
- Abstract
Most vertebrate retinas contain two types of photoreceptor cells, rods and cones, which show different photoresponses to mediate scotopic and photopic vision, respectively. These cells contain different types of visual pigments, rhodopsin and cone visual pigments, respectively, but little is known about the molecular properties of cone visual pigments under physiological conditions, making it difficult to link the molecular properties of rhodopsin and cone visual pigments with the differences in photoresponse between rods and cones. Here we prepared bovine and mouse rhodopsin (bvRh and mRh) and chicken and mouse green-sensitive cone visual pigments (cG and mG) embedded in nanodiscs and applied time-resolved fluorescence spectroscopy to compare their Gt activation efficiencies. Rhodopsin exhibited greater Gt activation efficiencies than cone visual pigments. Especially, the Gt activation efficiency of mRh was about 2.5-fold greater than that of mG at 37 °C, which is consistent with our previous electrophysiological data of knock-in mice. Although the active state (Meta-II) was in equilibrium with inactive states (Meta-I and Meta-III), quantitative determination of Meta-II in the equilibrium showed that the Gt activation efficiency per Meta-II of bvRh was also greater than those of cG and mG. These results indicated that efficient Gt activation by rhodopsin, resulting from an optimized active state of rhodopsin, is one of the causes of the high amplification efficiency of rods.
- Published
- 2014
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37. Evolution of mammalian Opn5 as a specialized UV-absorbing pigment by a single amino acid mutation.
- Author
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Yamashita T, Ono K, Ohuchi H, Yumoto A, Gotoh H, Tomonari S, Sakai K, Fujita H, Imamoto Y, Noji S, Nakamura K, and Shichida Y
- Subjects
- Amino Acid Substitution, Animals, Callithrix, Chick Embryo, Humans, Membrane Proteins genetics, Mice, Mice, Inbred ICR, Opsins genetics, Retinaldehyde genetics, Retinaldehyde metabolism, Xenopus, Zebrafish, cis-trans-Isomerases genetics, cis-trans-Isomerases metabolism, Brain metabolism, Evolution, Molecular, Membrane Proteins metabolism, Mutation, Missense, Opsins metabolism, Retina metabolism, Ultraviolet Rays
- Abstract
Opn5 is one of the recently identified opsin groups that is responsible for nonvisual photoreception in animals. We previously showed that a chicken homolog of mammalian Opn5 (Opn5m) is a Gi-coupled UV sensor having molecular properties typical of bistable pigments. Here we demonstrated that mammalian Opn5m evolved to be a more specialized photosensor by losing one of the characteristics of bistable pigments, direct binding of all-trans-retinal. We first confirmed that Opn5m proteins in zebrafish, Xenopus tropicalis, mouse, and human are also UV-sensitive pigments. Then we found that only mammalian Opn5m proteins lack the ability to directly bind all-trans-retinal. Mutational analysis showed that these characteristics were acquired by a single amino acid replacement at position 168. By comparing the expression patterns of Opn5m between mammals and chicken, we found that, like chicken Opn5m, mammalian Opn5m was localized in the ganglion cell layer and inner nuclear layer of the retina. However, the mouse and primate (common marmoset) opsins were distributed not in the posterior hypothalamus (including the region along the third ventricle) where chicken Opn5m is localized, but in the preoptic hypothalamus. Interestingly, RPE65, an essential enzyme for forming 11-cis-retinal in the visual cycle is expressed near the preoptic hypothalamus of the mouse and common marmoset brain but not near the region of the chicken brain where chicken Opn5m is expressed. Therefore, mammalian Opn5m may work exclusively as a short wavelength sensor in the brain as well as in the retina with the assistance of an 11-cis-retinal-supplying system.
- Published
- 2014
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38. Degradation and synthesis of β-glucans by a Magnaporthe oryzae endotransglucosylase, a member of the glycoside hydrolase 7 family.
- Author
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Takahashi M, Yoshioka K, Imai T, Miyoshi Y, Nakano Y, Yoshida K, Yamashita T, Furuta Y, Watanabe T, Sugiyama J, and Takeda T
- Subjects
- Carbohydrate Conformation, Cell Wall chemistry, Cellulose biosynthesis, Cloning, Molecular, Cotyledon chemistry, Cotyledon cytology, Fungal Proteins genetics, Fungal Proteins metabolism, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Hydrolysis, Oligosaccharides chemistry, Oryza microbiology, Plant Leaves microbiology, Substrate Specificity, Transcription, Genetic, Triticum chemistry, Triticum cytology, Fungal Proteins chemistry, Glycoside Hydrolases chemistry, Magnaporthe enzymology, beta-Glucans metabolism
- Abstract
Background: Plant pathogens secrete enzymes that degrade plant cell walls to enhance infection and nutrient acquisition., Results: A novel endotransglucosylase catalyzes cleavage and transfer of β-glucans and decreases the physical strength of plant cell walls., Conclusion: Endotransglucosylation causes depolymerization and polymerization of β-glucans, depending on substrate molecular size., Significance: Enzymatic degradation of plant cell walls is required for wall loosening, which enhances pathogen invasion. A Magnaporthe oryzae enzyme, which was encoded by the Mocel7B gene, was predicted to act on 1,3-1,4-β-glucan degradation and transglycosylation reaction of cellotriose after partial purification from a culture filtrate of M. oryzae cells, followed by liquid chromatography-tandem mass spectrometry. A recombinant MoCel7B prepared by overexpression in M. oryzae exhibited endo-typical depolymerization of polysaccharides containing β-1,4-linkages, in which 1,3-1,4-β-glucan was the best substrate. When cellooligosaccharides were used as the substrate, the recombinant enzyme generated reaction products with both shorter and longer chain lengths than the substrate. In addition, incorporation of glucose and various oligosaccharides including sulforhodamine-conjugated cellobiose, laminarioligosaccharides, gentiobiose, xylobiose, mannobiose, and xyloglucan nonasaccharide into β-1,4-linked glucans were observed after incubation with the enzyme. These results indicate that the recombinant enzyme acts as an endotransglucosylase (ETG) that cleaves the glycosidic bond of β-1,4-glucan as a donor substrate and transfers the cleaved glucan chain to another molecule functioning as an acceptor substrate. Furthermore, ETG treatment caused greater extension of heat-treated wheat coleoptiles. The result suggests that ETG functions to induce wall loosening by cleaving the 1,3-1,4-β-glucan tethers of plant cell walls. On the other hand, use of cellohexaose as a substrate for ETG resulted in the production of cellulose II with a maximum length (degree of polymerization) of 26 glucose units. Thus, ETG functions to depolymerize and polymerize β-glucans, depending on the size of the acceptor substrate.
- Published
- 2013
- Full Text
- View/download PDF
39. Glutamate acts as a partial inverse agonist to metabotropic glutamate receptor with a single amino acid mutation in the transmembrane domain.
- Author
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Yanagawa M, Yamashita T, and Shichida Y
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Cattle, DNA Mutational Analysis, Fluorescence Resonance Energy Transfer methods, Glutamic Acid chemistry, Glycine chemistry, HEK293 Cells, Humans, Molecular Conformation, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Phenotype, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Proteins chemistry, Rhodopsin genetics, Sequence Homology, Amino Acid, Tyrosine chemistry, Amino Acids chemistry, Glutamic Acid metabolism, Receptors, Metabotropic Glutamate agonists
- Abstract
Metabotropic glutamate receptor (mGluR), a prototypical family 3 G protein-coupled receptor (GPCR), has served as a model for studying GPCR dimerization, and growing evidence has revealed that a glutamate-induced dimeric rearrangement promotes activation of the receptor. However, structural information of the seven-transmembrane domain is severely limited, in contrast to the well studied family 1 GPCRs including rhodopsins and adrenergic receptors. Homology modeling of mGluR8 transmembrane domain with rhodopsin as a template suggested the presence of a conserved water-mediated hydrogen-bonding network between helices VI and VII, which presumably constrains the receptor in an inactive conformation. We therefore conducted a mutational analysis to assess structural similarities between mGluR and family 1 GPCRs. Mutational experiments confirmed that the disruption of the hydrogen-bonding network by T789Y(6.43) mutation induced high constitutive activity. Unexpectedly, this high constitutive activity was suppressed by glutamate, the natural agonist ligand, indicating that glutamate acts as a partial inverse agonist to this mutant. Fluorescence energy transfer analysis of T789Y(6.43) suggested that the glutamate-induced reduction of the activity originated not from the dimeric rearrangement but from conformational changes within each protomer. Double mutational analysis showed that the specific interaction between Tyr-789(6.43) and Gly-831(7.45) in T789Y(6.43) mutant was important for this phenotype. Therefore, the present study is consistent with the notion that the metabotropic glutamate receptor shares a common activation mechanism with family 1 GPCRs, where rearrangement between helices VI and VII causes the active state formation.
- Published
- 2013
- Full Text
- View/download PDF
40. Chondroitin sulfate proteoglycans down-regulate spine formation in cortical neurons by targeting tropomyosin-related kinase B (TrkB) protein.
- Author
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Kurihara D and Yamashita T
- Subjects
- Animals, Brain-Derived Neurotrophic Factor metabolism, Dendritic Spines metabolism, Gene Expression Regulation, Developmental, Humans, Mice, Nerve Growth Factors metabolism, Neurons metabolism, Phosphorylation, Plasmids metabolism, Proteoglycans chemistry, Receptor, Nerve Growth Factor metabolism, Synapses metabolism, Chondroitin Sulfate Proteoglycans chemistry, Down-Regulation, Receptor, trkB metabolism, Tropomyosin chemistry
- Abstract
Chondroitin sulfate proteoglycans (CSPGs) are components of the extracellular matrix that inhibit axonal sprouting and experience-dependent plasticity. Although protein-tyrosine phosphatase σ (PTPσ) has been proven to be a receptor for CSPGs, its downstream signaling has remained a mystery. Here, we show that CSPGs target and dephosphorylate tropomyosin-related kinase B, the receptor of brain-derived neurotrophic factor (BDNF), via PTPσ in embryonic cortical neurons in vitro. Whereas BDNF promoted dendritic spine formation in embryonic cortical neurons, CSPGs abolished the effects of BDNF and eliminated existing dendritic spines when BDNF was present. The latter effect was dependent on the p75 receptor, presumably because BDNF binding to the p75 receptor elicits elimination of dendritic spines. These results suggest that the inhibitory activity of CSPGs on dendritic spine formation operates through the targeting of neurotrophins at the receptor level.
- Published
- 2012
- Full Text
- View/download PDF
41. Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes.
- Author
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Mitsutake S, Zama K, Yokota H, Yoshida T, Tanaka M, Mitsui M, Ikawa M, Okabe M, Tanaka Y, Yamashita T, Takemoto H, Okazaki T, Watanabe K, and Igarashi Y
- Subjects
- Animals, CD36 Antigens genetics, CD36 Antigens metabolism, Caveolae pathology, Caveolin 1 genetics, Caveolin 1 metabolism, Ceramides genetics, Ceramides metabolism, Diabetes Mellitus, Type 2 chemically induced, Diabetes Mellitus, Type 2 genetics, Diabetes Mellitus, Type 2 pathology, Dietary Fats adverse effects, Dietary Fats pharmacology, Fatty Liver chemically induced, Fatty Liver genetics, Fatty Liver pathology, Hep G2 Cells, Humans, Insulin Resistance genetics, Insulin Resistance radiation effects, Membrane Proteins genetics, Membrane Proteins metabolism, Mice, Mice, Knockout, Mice, Obese, Nerve Tissue Proteins genetics, Nerve Tissue Proteins metabolism, Obesity chemically induced, Obesity genetics, Obesity pathology, Sphingomyelins genetics, Transferases (Other Substituted Phosphate Groups) genetics, Transferases (Other Substituted Phosphate Groups) metabolism, Caveolae metabolism, Diabetes Mellitus, Type 2 metabolism, Fatty Liver metabolism, Obesity metabolism, Sphingomyelins metabolism
- Abstract
Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.
- Published
- 2011
- Full Text
- View/download PDF
42. Comparative fluorescence resonance energy transfer analysis of metabotropic glutamate receptors: implications about the dimeric arrangement and rearrangement upon ligand bindings.
- Author
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Yanagawa M, Yamashita T, and Shichida Y
- Subjects
- Fluorescence Resonance Energy Transfer, HEK293 Cells, Humans, Ligands, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Metabotropic Glutamate genetics, Protein Multimerization physiology, Receptors, Metabotropic Glutamate metabolism
- Abstract
Dimerization of G protein-coupled receptors has received much attention as a regulatory system of physiological function. Metabotropic glutamate receptors (mGluRs) are suitable models for studying the physiological significance of G protein-coupled receptor dimers because they form constitutive homodimers and function through dimeric rearrangement of their extracellular ligand binding domains. However, the molecular architecture of the transmembrane domains (TMDs) and their rearrangement upon agonist binding are still largely unknown. Here we show that the two helix Vs are arranged as the closest part in the dimeric TMDs and change their positions through synergistic control by the binding of two glutamates. The possibility that helix V is involved in an inter-protomer communication was first suggested by the finding that constitutively active mutation sites were identified on both sides of helix V. Then, comprehensive fluorescence resonance energy transfer (FRET) analysis using mGluRs whose cytoplasmic loops were labeled with donor and acceptor fluorescent proteins revealed that the third intracellular loop connecting helices V and VI of one protomer was in close proximity to the second and third intracellular loops of the other protomer and that all the intracellular loops became closer during the activation. Furthermore, FRET analysis of heterodimers in which only one protomer had ligand binding ability revealed the synergistic effect of the binding of two glutamates on the dimeric rearrangements of the TMD. Thus, the glutamate-dependent synergistic relocation of the helix Vs in the dimer is important for the signal flow from the extracellular ligand binding domain to the cytoplasmic surface of the mGluR.
- Published
- 2011
- Full Text
- View/download PDF
43. Reaction mechanism of single subunit NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae: evidence for a ternary complex mechanism.
- Author
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Yang Y, Yamashita T, Nakamaru-Ogiso E, Hashimoto T, Murai M, Igarashi J, Miyoshi H, Mori N, Matsuno-Yagi A, Yagi T, and Kosaka H
- Subjects
- Anaerobiosis physiology, Binding Sites, Electron Transport Complex I genetics, Electron Transport Complex I metabolism, Kinetics, Mutation, NAD genetics, NAD metabolism, Oxidation-Reduction, Oxygen metabolism, Protein Structure, Quaternary, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Ubiquinone genetics, Ubiquinone metabolism, Electron Transport Complex I chemistry, NAD chemistry, Oxygen chemistry, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins chemistry, Ubiquinone chemistry
- Abstract
The flavoprotein rotenone-insensitive internal NADH-ubiquinone (UQ) oxidoreductase (Ndi1) is a member of the respiratory chain in Saccharomyces cerevisiae. We reported previously that bound UQ in Ndi1 plays a key role in preventing the generation of reactive oxygen species. Here, to elucidate this mechanism, we investigated biochemical properties of Ndi1 and its mutants in which highly conserved amino acid residues (presumably involved in NADH and/or UQ binding sites) were replaced. We found that wild-type Ndi1 formed a stable charge transfer (CT) complex (around 740 nm) with NADH, but not with NADPH, under anaerobic conditions. The intensity of the CT absorption band was significantly increased by the presence of bound UQ or externally added n-decylbenzoquinone. Interestingly, however, when Ndi1 was exposed to air, the CT band transiently reached the same maximum level regardless of the presence of UQ. This suggests that Ndi1 forms a ternary complex with NADH and UQ, but the role of UQ in withdrawing an electron can be substitutable with oxygen. Proteinase K digestion analysis showed that NADH (but not NADPH) binding induces conformational changes in Ndi1. The kinetic study of wild-type and mutant Ndi1 indicated that there is no overlap between NADH and UQ binding sites. Moreover, we found that the bound UQ can reversibly dissociate from Ndi1 and is thus replaceable with other quinones in the membrane. Taken together, unlike other NAD(P)H-UQ oxidoreductases, the Ndi1 reaction proceeds through a ternary complex (not a ping-pong) mechanism. The bound UQ keeps oxygen away from the reduced flavin.
- Published
- 2011
- Full Text
- View/download PDF
44. Neogenin, a receptor for bone morphogenetic proteins.
- Author
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Hagihara M, Endo M, Hata K, Higuchi C, Takaoka K, Yoshikawa H, and Yamashita T
- Subjects
- Bone Morphogenetic Protein Receptors, Type I genetics, Bone Morphogenetic Protein Receptors, Type I metabolism, Bone Morphogenetic Protein Receptors, Type II genetics, Bone Morphogenetic Protein Receptors, Type II metabolism, Bone Morphogenetic Proteins genetics, Cell Differentiation physiology, Enzyme Activation physiology, HEK293 Cells, Humans, Membrane Proteins genetics, Osteoblasts cytology, Phosphorylation physiology, Protein Binding physiology, Smad Proteins genetics, Smad Proteins metabolism, rhoA GTP-Binding Protein genetics, rhoA GTP-Binding Protein metabolism, Bone Morphogenetic Proteins metabolism, Membrane Proteins metabolism, Osteoblasts metabolism
- Abstract
Bone morphogenetic proteins (BMPs) regulate many mammalian physiologic and pathophysiologic processes. These proteins bind with the kinase receptors BMPR-I and BMPR-II, thereby activating Smad transcription factor. In this study, we demonstrate that neogenin, a receptor for netrins and proteins of the repulsive guidance molecule family, is a receptor for BMPs and modulates Smad signal transduction. Neogenin was found to bind directly with BMP-2, BMP-4, BMP-6, and BMP-7. Knockdown of neogenin in C2C12 cells resulted in the enhancement of the BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1, Smad5, and Smad8. Conversely, overexpression of neogenin in C2C12 cells suppressed these processes. Our results also indicated that BMP-induced activation of RhoA was mediated by neogenin. Inhibition of RhoA promoted BMP-2-induced processes of osteoblastic differentiation and phosphorylation of Smad1/5/8. However, treatment with Y-27632, an inhibitor of Rho-associated protein kinase, did not modulate BMP-induced phosphorylation of Smad1/5/8. Taken together, our findings suggest that neogenin negatively regulates the functions of BMP and that this effect of neogenin is mediated by the activation of RhoA.
- Published
- 2011
- Full Text
- View/download PDF
45. Paired immunoglobulin-like receptor B knockout does not enhance axonal regeneration or locomotor recovery after spinal cord injury.
- Author
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Nakamura Y, Fujita Y, Ueno M, Takai T, and Yamashita T
- Subjects
- Animals, Gene Knockdown Techniques, Humans, Mice, Mice, Knockout, Myelin Proteins genetics, Myelin Proteins metabolism, Myelin Proteins pharmacology, Myelin Sheath genetics, Myelin Sheath metabolism, Neurites pathology, Nogo Proteins, Peptide Fragments pharmacology, Receptors, Immunologic antagonists & inhibitors, Receptors, Immunologic genetics, Spinal Cord Injuries pathology, Motor Activity, Neurites metabolism, Receptors, Immunologic metabolism, Recovery of Function, Regeneration, Spinal Cord Injuries metabolism
- Abstract
Myelin components that inhibit axonal regeneration are believed to contribute significantly to the lack of axonal regeneration noted in the adult central nervous system. Three proteins found in myelin, Nogo, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, inhibit neurite outgrowth in vitro. All of these proteins interact with the same receptors, namely, the Nogo receptor (NgR) and paired immunoglobulin-like receptor B (PIR-B). As per previous reports, corticospinal tract (CST) regeneration is not enhanced in NgR-knock-out mice after spinal cord injury. Therefore, we assessed CST regeneration in PIR-B-knock-out mice. We found that hindlimb motor function, as assessed using the Basso mouse scale, footprint test, inclined plane test, and beam walking test, did not differ between the PIR-B-knock-out and wild-type mice after dorsal hemisection of the spinal cord. Further, tracing of the CST fibers after injury did not reveal enhanced axonal regeneration or sprouting in the CST of the PIR-B-knock-out mice. Systemic administration of NEP1-40, a NgR antagonist, to PIR-B knock-out mice did not enhance the regenerative response. These results indicate that PIR-B knock-out is not sufficient to induce extensive axonal regeneration after spinal cord injury.
- Published
- 2011
- Full Text
- View/download PDF
46. Reduction of brain beta-amyloid (Abeta) by fluvastatin, a hydroxymethylglutaryl-CoA reductase inhibitor, through increase in degradation of amyloid precursor protein C-terminal fragments (APP-CTFs) and Abeta clearance.
- Author
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Shinohara M, Sato N, Kurinami H, Takeuchi D, Takeda S, Shimamura M, Yamashita T, Uchiyama Y, Rakugi H, and Morishita R
- Subjects
- Animals, Blood-Brain Barrier drug effects, Blood-Brain Barrier metabolism, Brain drug effects, Brain enzymology, Endothelial Cells drug effects, Endothelial Cells metabolism, Fatty Acids, Monounsaturated pharmacokinetics, Fluvastatin, Humans, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacokinetics, Indoles pharmacokinetics, Low Density Lipoprotein Receptor-Related Protein-1, Lysosomes drug effects, Lysosomes metabolism, Mice, Mice, Inbred C57BL, Microvessels drug effects, Microvessels pathology, Models, Biological, Monomeric GTP-Binding Proteins metabolism, Protein Structure, Tertiary, Protein Transport drug effects, Receptors, LDL metabolism, Subcellular Fractions drug effects, Subcellular Fractions metabolism, Tissue Distribution drug effects, Tumor Suppressor Proteins metabolism, Amyloid beta-Protein Precursor chemistry, Amyloid beta-Protein Precursor metabolism, Brain metabolism, Fatty Acids, Monounsaturated pharmacology, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Indoles pharmacology, Protein Processing, Post-Translational drug effects
- Abstract
Epidemiological studies suggest that statins (hydroxymethylglutaryl-CoA reductase inhibitors) could reduce the risk of Alzheimer disease. Although one possible explanation is through an effect on beta-amyloid (Abeta) metabolism, its effect remains to be elucidated. Here, we explored the molecular mechanisms of how statins influence Abeta metabolism. Fluvastatin at clinical doses significantly reduced Abeta and amyloid precursor protein C-terminal fragment (APP-CTF) levels among APP metabolites in the brain of C57BL/6 mice. Chronic intracerebroventricular infusion of lysosomal inhibitors blocked these effects, indicating that up-regulation of the lysosomal degradation of endogenous APP-CTFs is involved in reduced Abeta production. Biochemical analysis suggested that this was mediated by enhanced trafficking of APP-CTFs from endosomes to lysosomes, associated with marked changes of Rab proteins, which regulate endosomal function. In primary neurons, fluvastatin enhanced the degradation of APP-CTFs through an isoprenoid-dependent mechanism. Because our previous study suggests additive effects of fluvastatin on Abeta metabolism, we examined Abeta clearance rates by using the brain efflux index method and found its increased rates at high Abeta levels from brain. As LRP1 in brain microvessels was increased, up-regulation of LRP1-mediated Abeta clearance at the blood-brain barrier might be involved. In cultured brain microvessel endothelial cells, fluvastatin increased LRP1 and the uptake of Abeta, which was blocked by LRP1 antagonists, through an isoprenoid-dependent mechanism. Overall, the present study demonstrated that fluvastatin reduced Abeta level by an isoprenoid-dependent mechanism. These results have important implications for the development of disease-modifying therapy for Alzheimer disease as well as understanding of Abeta metabolism.
- Published
- 2010
- Full Text
- View/download PDF
47. Covalent bond between ligand and receptor required for efficient activation in rhodopsin.
- Author
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Matsuyama T, Yamashita T, Imai H, and Shichida Y
- Subjects
- Animals, Cattle, Cells, Cultured, GTP-Binding Proteins metabolism, Humans, Isomerism, Kidney cytology, Ligands, Lysine metabolism, Mutagenesis, Photic Stimulation, Retinaldehyde metabolism, Schiff Bases, Spectrum Analysis, Structure-Activity Relationship, Temperature, Photoreceptor Cells, Vertebrate physiology, Rhodopsin chemistry, Rhodopsin genetics, Rhodopsin metabolism, Vision, Ocular physiology
- Abstract
Rhodopsin is an extensively studied member of the G protein-coupled receptors (GPCRs). Although rhodopsin shares many features with the other GPCRs, it exhibits unique features as a photoreceptor molecule. A hallmark in the molecular structure of rhodopsin is the covalently bound chromophore that regulates the activity of the receptor acting as an agonist or inverse agonist. Here we show the pivotal role of the covalent bond between the retinal chromophore and the lysine residue at position 296 in the activation pathway of bovine rhodopsin, by use of a rhodopsin mutant K296G reconstituted with retinylidene Schiff bases. Our results show that photoreceptive functions of rhodopsin, such as regiospecific photoisomerization of the ligand, and its quantum yield were not affected by the absence of the covalent bond, whereas the activation mechanism triggered by photoisomerization of the retinal was severely affected. Furthermore, our results show that an active state similar to the Meta-II intermediate of wild-type rhodopsin did not form in the bleaching process of this mutant, although it exhibited relatively weak G protein activity after light irradiation because of an increased basal activity of the receptor. We propose that the covalent bond is required for transmitting structural changes from the photoisomerized agonist to the receptor and that the covalent bond forcibly keeps the low affinity agonist in the receptor, resulting in a more efficient G protein activation.
- Published
- 2010
- Full Text
- View/download PDF
48. Heme ligand binding properties and intradimer interactions in the full-length sensor protein dos from Escherichia coli and its isolated heme domain.
- Author
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Lechauve C, Bouzhir-Sima L, Yamashita T, Marden MC, Vos MH, Liebl U, and Kiger L
- Subjects
- Carbon Monoxide metabolism, Escherichia coli Proteins metabolism, Heme metabolism, Ligands, Oxygen metabolism, Phosphoric Diester Hydrolases metabolism, Protein Structure, Quaternary physiology, Protein Structure, Tertiary physiology, Carbon Monoxide chemistry, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Heme chemistry, Oxygen chemistry, Phosphoric Diester Hydrolases chemistry, Protein Multimerization physiology
- Abstract
Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant approximately 1 mum), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O(2)) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated approximately 10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.
- Published
- 2009
- Full Text
- View/download PDF
49. Engulfment of axon debris by microglia requires p38 MAPK activity.
- Author
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Tanaka T, Ueno M, and Yamashita T
- Subjects
- Animals, Brain metabolism, Cell Line, Central Nervous System metabolism, L-Lactate Dehydrogenase metabolism, Mice, Mice, Inbred C57BL, Models, Biological, Phagocytosis, Rats, Rats, Wistar, Spleen metabolism, Axons metabolism, Microglia metabolism, p38 Mitogen-Activated Protein Kinases metabolism
- Abstract
The clearance of debris after injuries to the nervous system is a critical step for restoration of the injured neural network. Microglia are thought to be involved in elimination of degenerating neurons and axons in the central nervous system (CNS), presumably restoring a favorable environment after CNS injuries. However, the mechanism underlying debris clearance remains elusive. Here, we establish an in vitro assay system to estimate phagocytosis of axon debris. We employed a Wallerian degeneration model by cutting axons of the cortical explants. The cortical explants were co-cultured with primary microglia or the MG5 microglial cell line. The cortical neurites were then transected. MG5 cells efficiently phagocytosed the debris, whereas primary microglia showed phagocytic activity only when they were activated by lipopolysaccharide or interferon-beta. When MG5 cells or primary microglia were co-cultured with degenerated axons, p38 mitogen-activated protein kinase (MAPK) was activated in these cells. Engulfment of axon debris was blocked by the p38 MAPK inhibitor SB203580, indicating that p38 MAPK is required for phagocytic activity. Receptors that recognize dying cells appeared not to be involved in the process of phagocytosis of the axon debris. In addition, the axons undergoing Wallerian degeneration did not release lactate dehydrogenase, suggesting that degeneration of the severed axons and apoptosis may represent two distinct self-destruction programs. We observed regrowth of the severed neurites after axon debris was removed. This finding suggests that axon debris, in addition to myelin debris, is an inhibitory factor for axon regeneration.
- Published
- 2009
- Full Text
- View/download PDF
50. Differential dephosphorylation of the FcRgamma immunoreceptor tyrosine-based activation motif tyrosines with dissimilar potential for activating Syk.
- Author
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Yamashita T, Suzuki R, Backlund PS, Yamashita Y, Yergey AL, and Rivera J
- Subjects
- Amino Acid Motifs, Animals, Humans, Mice, Mice, Transgenic, Models, Biological, Mutagenesis, Site-Directed, Mutation, Phosphorylation, Protein Binding, Protein Structure, Tertiary, Signal Transduction, Syk Kinase, Intracellular Signaling Peptides and Proteins metabolism, Protein-Tyrosine Kinases metabolism, Receptors, IgG chemistry, Tyrosine chemistry
- Abstract
The cell surface-expressed gamma chain of the high affinity receptor for IgE (FcepsilonRI) can be phosphorylated on two tyrosine residues of the immunoreceptor tyrosine-based activation motif (ITAM), leading to recruitment and activation of spleen tyrosine kinase (Syk), a kinase that is essential for mast cell signaling and allergic responses. However, it is not known whether preferential phosphorylation or dephosphorylation of the two individual FcRgamma tyrosines (the N-terminal Tyr47 and C-terminal Tyr58) could regulate Syk activation. Herein we report that phosphorylation of only Tyr58 was able to elicit Syk phosphorylation and a weak rise in intracellular calcium, suggesting that Tyr58 phosphorylation may be distinctively important for Syk activation. In vitro and in vivo studies revealed that both Tyr47 and Tyr58 could be similarly phosphorylated. However, mass spectrometric analysis of the phosphorylated FcepsilonRgamma from bone marrow-derived mast cells showed that phosphorylation at Tyr47 was at least 2-fold greater than at Tyr58. This suggested that, once phosphorylated, Tyr58 is preferentially dephosphorylated. In vitro studies demonstrated more efficient dephosphorylation of Tyr58 (by the receptor-associated phosphatases SHP-1 and SHP-2) than of Tyr47. Analysis of Syk binding to wild type and mutant phosphorylated FcepsilonRI revealed that mutation at Tyr58 almost completely ablated Syk binding, whereas mutation at Tyr47 moderately reduced Syk binding. The findings argue for a novel regulatory mechanism, where dephosphorylation of phospho-Tyr58 is likely to promote the down-regulation of Syk activation and suppression of mast cell responses.
- Published
- 2008
- Full Text
- View/download PDF
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