Publications (34)77.74 Total impact
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Article: Acetyl-L-carnitine suppresses thyroid hormone-induced and spontaneous anuran tadpole tail shortening.
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ABSTRACT: Mitochondrial membrane permeability transition (MPT) plays a crucial role in apoptotic tail shortening during anuran metamor phosis. L-carnitine is known to shuttle free fatty acids (FFAs) from the cytosol into mitochondria matrix for β-oxidation and energy production, and in a previous study we found that treatment with L-carnitine suppresses 3, 3', 5-triiodothyronine (T3 ) and FFA-induced MPT by reducing the level of FFAs. In the present study we focus on acetyl-L-carnitine, which is also involved in fatty acid oxidation, to determine its effect on T3 -induced tail regression in Rana rugosa tadpoles and spontaneous tail regression in Xenopus laevis tadpoles. The ladder-like DNA profile and increases in caspase-3 and caspase-9 indicative of apoptosis in the tails of T3 -treated tadpoles were found to be suppressed by the addition of acetyl-L-carnitine. Likewise, acetyl-L-carnitine was found to inhibit thyroid hormone regulated spontaneous metamorphosis in X. laevis tadpoles, accompanied by decreases in caspase and phospholipase A2 activity, as well as non-ladder-like DNA profiles. These findings support our previous conclusion that elevated levels of FFAs initiate MPT and activate the signaling pathway controlling apoptotic cell death in tadpole tails during anuran metamorphosis.Hereditas 02/2013; 150(1):1-9. · 0.79 Impact Factor -
Article: Protein N-myristoylation is required for cellular morphological changes induced by two formin family proteins, FMNL2 and FMNL3.
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ABSTRACT: The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs coding for the proteins revealed that most of them were associated with the plasma membrane or the membranes of intracellular compartments, and did not affect cellular morphology. However, two proteins, formin-like2 (FMNL2) and formin-like3 (FMNL3), both of them are members of the formin family of proteins, were associated mainly with the plasma membrane and induced significant cellular morphological changes. Inhibition of protein N-myristoylation by replacement of Gly2 with Ala or by the use of N-myristoylation inhibitor significantly inhibited membrane localization and the induction of cellular morphological changes, indicating that protein N-myristoylation plays critical roles in the cellular morphological changes induced by FMNL2 and FMNL3.Bioscience Biotechnology and Biochemistry 06/2012; 76(6):1201-9. · 1.28 Impact Factor -
Article: Mitochondrial Localization of ABC Transporter ABCG2 and Its Function in 5-Aminolevulinic Acid-Mediated Protoporphyrin IX Accumulation.
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ABSTRACT: Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.PLoS ONE 01/2012; 7(11):e50082. · 4.09 Impact Factor -
Article: Serum-dependent export of protoporphyrin IX by ATP-binding cassette transporter G2 in T24 cells.
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ABSTRACT: Accumulation of protoporphyrin IX (PpIX) in cancer cells is a basis of 5-aminolevulinic acid (ALA)-induced photodymanic therapy. We studied factors that affect PpIX accumulation in human urothelial carcinoma cell line T24, with particular emphasis on ATP-binding cassette transporter G2 (ABCG2) and serum in the medium. When the medium had no fetal bovine serum (FBS), ALA induced PpIX accumulation in a time- and ALA concentration-dependent manner. Inhibition of heme-synthesizing enzyme, ferrochelatase, by nitric oxide donor (Noc18) or deferoxamine resulted in a substantial increase in the cellular PpIX accumulation, whereas ABCG2 inhibition by fumitremorgin C or verapamil induced a slight PpIX increase. When the medium was added with FBS, cellular accumulation of PpIX stopped at a lower level with an increase of PpIX in the medium, which suggested PpIX efflux. ABCG2 inhibitors restored the cellular PpIX level to that of FBS(-) samples, whereas ferrochelatase inhibitors had little effects. Bovine serum albumin showed similar effects to FBS. Fluorescence microscopic observation revealed that inhibitors of ABC transporter affected the intracellular distribution of PpIX. These results indicated that ABCG2-mediated PpIX efflux was a major factor that prevented PpIX accumulation in cancer cells in the presence of serum. Inhibition of ABCG2 transporter system could be a new target for the improvement of photodynamic therapy.Molecular and Cellular Biochemistry 07/2011; 358(1-2):297-307. · 2.06 Impact Factor -
Article: [Comprehensive analysis of lipid modifications of proteins using cell-free protein synthesis system].
Seikagaku. The Journal of Japanese Biochemical Society 09/2010; 82(9):799-813. · 0.04 Impact Factor -
Article: Strategy for comprehensive identification of human N-myristoylated proteins using an insect cell-free protein synthesis system.
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ABSTRACT: To establish a strategy for the comprehensive identification of human N-myristoylated proteins, the susceptibility of human cDNA clones to protein N-myristoylation was evaluated by metabolic labeling and MS analyses of proteins expressed in an insect cell-free protein synthesis system. One-hundred-and-forty-one cDNA clones with N-terminal Met-Gly motifs were selected as potential candidates from approximately 2000 Kazusa ORFeome project human cDNA clones, and their susceptibility to protein N-myristoylation was evaluated using fusion proteins, in which the N-terminal ten amino acid residues were fused to an epitope-tagged model protein. As a result, the products of 29 out of 141 cDNA clones were found to be effectively N-myristoylated. The metabolic labeling experiments both in an insect cell-free protein synthesis system and in the transfected COS-1 cells using full-length cDNA revealed that 27 out of 29 proteins were in fact N-myristoylated. Database searches with these 27 cDNA clones revealed that 18 out of 27 proteins are novel N-myristoylated proteins that have not been reported previously to be N-myristoylated, indicating that this strategy is useful for the comprehensive identification of human N-myristoylated proteins from human cDNA resources.Proteomics 03/2010; 10(9):1780-93. · 4.43 Impact Factor -
Article: Development of an insect cell-free system.
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ABSTRACT: Cell-free protein synthesis systems offer production of native proteins with high speed, even for the proteins that are toxic to cells. Among cell-free systems, the system derived from insect cells has the potential to carry out post-translational modifications that are specific to eukaryotic organisms, as occurs in the rabbit reticulocyte system. In this review, we describe development of this insect cell-free system and its applications.Current pharmaceutical biotechnology 02/2010; 11(3):279-84. · 3.40 Impact Factor -
Article: Preparation of ubiquitin-conjugated proteins using an insect cell-free protein synthesis system.
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ABSTRACT: Ubiquitination is one of the most significant posttranslational modifications (PTMs). To evaluate the ability of an insect cell-free protein synthesis system to carry out ubiquitin (Ub) conjugation to in vitro translated proteins, poly-Ub chain formation was studied in an insect cell-free protein synthesis system. Poly-Ub was generated in the presence of Ub aldehyde (UA), a de-ubiquitinating enzyme inhibitor. In vitro ubiquitination of the p53 tumor suppressor protein was also analyzed, and p53 was poly-ubiquitinated when Ub, UA, and Mdm2, an E3 Ub ligase (E3) for p53, were added to the in vitro reaction mixture. These results suggest that the insect cell-free protein synthesis system contains enzymatic activities capable of carrying out ubiquitination. CBB-detectable ubiquitinated p53 was easily purified from the insect cell-free protein synthesis system, allowing analysis of the Ub-conjugated proteins by mass spectrometry (MS). Lys 305 of p53 was identified as one of the Ub acceptor sites using this strategy. Thus, we conclude that the insect cell-free protein synthesis system is a powerful tool for studying various PTMs of eukaryotic proteins including ubiqutination presented here.Journal of biotechnology 10/2009; 145(1):73-8. · 2.88 Impact Factor -
Article: Mechanism of cell death by 5-aminolevulinic acid-based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937.
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ABSTRACT: Photodynamic therapy (PDT) for tumors is based on the tumor-selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5-aminolevulinic acid (ALA)-based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl-xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase-3 activation, phosphatidylserine (PS) externalization. PDT-induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA-based-PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA.Cell Biochemistry and Function 09/2009; 27(8):503-15. · 1.77 Impact Factor -
Article: Alpha-tocopheryl succinate induces rapid and reversible phosphatidylserine externalization in histiocytic lymphoma through the caspase-independent pathway.
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ABSTRACT: Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer alpha-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca(2+)-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.Molecular and Cellular Biochemistry 08/2009; 333(1-2):137-49. · 2.06 Impact Factor -
Article: Dysfunction of Mouse Liver Mitochondria Induced by 2,2′-Azobis-(2-Amidinopropane) Dihydrochloride, A Radical Initiator, in Vitro And in Vivo
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ABSTRACT: Mouse liver mitochondria were uncoupled in a time dependent by intraperitoneal injection of a radical initiator, 2,2′-azobis-(2-amidinopropane) dihydrochloride (AAPH) (100mg/kg). State 3 respiration, ADP/O ratio and respiratory control ratio (RCR) were decreased 30 min after injection but there was no effect on state 4 respiration. Lipid peroxidation was increased and oxidative phosphorylation was uncoupled at one hr after drug injection but gradually recovered to normal levels after 14 hr in vivo. State 3 respiration, RCR and ADP/O ratio but not state 4 respiration of isolated mouse mitochondria were inhibited by short term incubation with AAPH in vitro. This inhibitory action was concentration dependent (ID50 = 5 mM) but was not prevented by α-tocopherol. AAPH had no effect on electron transport or the membrane potential of these isolated mitochondria. However, mitochondria were uncoupled via lipid peroxidation and swelling by long term incubation with AAPH. These inhibitory effects of AAPH were reduced by its spontaneous degradation not only in vitro but also in vivo. Thus AAPH induces mitochondrial dysfunction by direct action in the early period of treatment and free radicals produced from AAPH mediate mitochondrial swelling via lipid peroxidation in the late period. From these findings, it is concluded that mitochondrial phosphorylation plays an important role in the pathogenesis of liver injury induced by AAPH and that radicals generated by AAPH might be a source of liver injury and mitochondrial dysfunction in vivo.Free Radical Research 07/2009; 21(4):223-234. · 2.88 Impact Factor -
Article: Pyridalyl inhibits cellular protein synthesis in insect, but not mammalian, cell lines.
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ABSTRACT: To gain insight into the mechanism of action and selectivity of the insecticidal activity of pyridalyl, the cytotoxicity of pyridalyl against various insect and mammalian cell lines was characterized by measuring the inhibition of cellular protein synthesis. When the effect of pyridalyl on the cellular protein synthesis in Sf9 cells was evaluated by measuring the incorporation of [(3)H]leucine, rapid and significant inhibition of protein synthesis was observed. However, pyridalyl did not inhibit protein synthesis in a cell-free protein synthesis system, indicating that pyridalyl does not directly inhibit protein synthesis. No obvious cytotoxicity was observed against any of the mammalian cell lines tested. In the case of insect cell lines, remarkable differences in the cytotoxicity of pyridalyl were observed: the highest cytotoxicity (IC50 mM) was found against Sf9 cells derived from Spodoptera frugiperda, whereas no obvious cytotoxicity was observed against BmN4 cells derived from Bombyx mori. Measurements of the insecticidal activity of pyridalyl against Spodoptera litura and B. mori revealed a correlation between the cytotoxicity against cultured cell lines and the insecticidal activity. From these observations, it was concluded that the selective inhibition of cellular protein synthesis by pyridalyl might contribute significantly to the insecticidal activity and the selectivity of this compound.Archives of Insect Biochemistry and Physiology 06/2008; 69(1):22-31. · 1.36 Impact Factor -
Article: Alpha-lipoic acid suppresses 6-hydroxydopamine-induced ROS generation and apoptosis through the stimulation of glutathione synthesis but not by the expression of heme oxygenase-1.
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ABSTRACT: We previously reported that the generation of reactive oxygen species (ROS) is the initial event in cell death induced by 6-hydroxydopamine (6-OHDA), an experimental model of Parkinsonism. Since recent studies suggested the important role of antioxidant activity of alpha-lipoic acid (LA) in the suppression of apoptosis of various types, we studied the effect on 6-OHDA-induced apoptosis of PC12 cells. Biochemical analysis revealed that LA suppressed the 6-OHDA-induced ROS generation, increase of caspase-like activity and chromatin condensation. The suppression of 6-OHDA-induced apoptosis by LA required pre-incubation of PC12 cells with LA for 12-24 h. LA increased the intracellular levels of heme oxygenase-1 (HO-1) and glutathione (GSH) and stimulated the expression of GSH synthesis-related genes such as cystine/glutamate antiporter and gamma-glutamylcysteine synthetase (gamma-GCS). However, Sn-mesoporphyrin IX, an inhibitor of HO-1, did not attenuate the LA-induced suppression of apoptosis. In contrast, buthionine sulfoximine, an inhibitor of gamma-GCS, attenuated the LA-induced suppression of ROS generation and chromatin condensation. In addition, a transcription factor Nrf2, which regulates the expression of antioxidant enzymes such as gamma-GCS, translocated to the nucleus by LA. These results suggested that LA suppressed the 6-OHDA induced-apoptosis by the increase in cellular glutathione through stimulation of the GSH synthesis system but not by the expression of HO-1.Brain Research 05/2008; 1206:1-12. · 2.73 Impact Factor -
Article: Protein prenylation in an insect cell-free protein synthesis system and identification of products by mass spectrometry.
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ABSTRACT: To evaluate the ability of an insect cell-free protein synthesis system to carry out proper protein prenylation, several CAIX (X indicates any C-terminal amino acid) sequences were introduced into the C-terminus of truncated human gelsolin (tGelsolin). Tryptic digests of these mutant proteins were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The results indicated that the insect cell-free protein synthesis system possesses both farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) I, as is the case of the rabbit reticulocyte lysate system. The C-terminal amino acid sequence requirements for protein prenylation in this system showed high similarity to those observed in rat prenyltransferases. In the case of rhoC, which is a natural geranylgeranylated protein, it was found that it could serve as a substrate for both prenyltransferases in the presence of either farnesyl or geranylgeranyl pyrophosphate, whereas geranylgeranylation was only observed when both prenyl pyrophosphates were added to the in vitro translation reaction mixture. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein prenylation.PROTEOMICS 07/2007; 7(12):1942-50. · 4.51 Impact Factor -
Article: Detection of co- and posttranslational protein N-myristoylation by metabolic labeling in an insect cell-free protein synthesis system.
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ABSTRACT: To establish a simple and sensitive method to detect protein N-myristoylation, the usefulness of a newly developed cell-free protein synthesis system derived from insect cells for detecting protein N-myristoylation by in vitro metabolic labeling was examined. The results showed that in vitro translation of cDNA coding for N-myristoylated protein in the presence of [(3)H]myristic acid followed by SDS-PAGE and fluorography is a useful method for rapid detection of protein N-myristoylation. Differential labeling of N-myristoylated model proteins with [(3)H]leucine, [(3)H]myristic acid, and [(35)S]methionine revealed that the removal of the initiating Met during the N-myristoylation reaction could be detected using this system. Analysis of the N-myristoylation of a series of model proteins with mutated N-myristoylation motifs revealed that the amino acid sequence requirements for the N-myristoylation reaction in this system are quite similar to those observed in the rabbit reticulocyte lysate system. N-myristoylation of tBid (a posttranslationally N-myristoylated cytotoxic protein that could not be expressed in transfected cells) was successfully detected in this assay system. Thus, metabolic labeling in an insect cell-free protein synthesis system is an effective strategy to detect co- and posttranslational protein N-myristoylation irrespective of the cytotoxicity of the protein.Analytical Biochemistry 04/2007; 362(2):236-44. · 3.00 Impact Factor -
Article: Preparation of N-acylated proteins modified with fatty acids having a specific chain length using an insect cell-free protein synthesis system.
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ABSTRACT: To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.Bioscience Biotechnology and Biochemistry 02/2007; 71(1):261-4. · 1.28 Impact Factor -
Article: N-Terminal protein modifications in an insect cell-free protein synthesis system and their identification by mass spectrometry.
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ABSTRACT: To evaluate the ability of an insect cell-free protein synthesis system to generate proper N-terminal cotranslational protein modifications such as removal of the initiating Met, N-acetylation, and N-myristoylation, several mutants were constructed using truncated human gelsolin (tGelsolin) as a model protein. Tryptic digests of these mutants were analyzed by MALDI-TOF MS and MALDI-quadrupole-IT-TOF MS. The wild-type tGelsolin, which is an N-myristoylated protein, was found to be N-myristoylated when myristoyl-CoA was added to the in vitro translation reaction mixture. N-myristoylation did not occur on the Gly-2 to Ala mutant, in which the N-myristoylation motif was disrupted, whereas this mutant was found to be N-acetylated after removal of the initiating Met. Analyses of Gly-2 to His and Leu-3 to Asp mutants revealed that the amino acids at positions 2 and 3 strongly affect the susceptibility of the nascent peptide chain to removal of the initiating Met and to N-acetylation, respectively. These results suggest that N-terminal modifications occurring in the insect cell-free protein synthesis system are quite similar to those observed in the mammalian protein synthesis system. Thus, a combination of the cell-free protein synthesis system with MS is an effective strategy to analyze protein modifications.PROTEOMICS 09/2006; 6(16):4486-95. · 4.51 Impact Factor -
Article: Posttranslational N-myristoylation is required for the anti-apoptotic activity of human tGelsolin, the C-terminal caspase cleavage product of human gelsolin.
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ABSTRACT: Protein N-myristoylation has been recognized as a cotranslational protein modification. Recently, it was demonstrated that protein N-myristoylation could occur posttranslationally, as in the case of the pro-apoptotic protein BID and cytoskeletal actin. Our previous study showed that the N-terminal nine residues of the C-terminal caspase cleavage product of human gelsolin, an actin-regulatory protein, efficiently direct the protein N-myristoylation. In this study, to analyze the posttranslational N-myristoylation of gelsolin during apoptosis, metabolic labeling of gelsolin and its caspase cleavage products expressed in COS-1 cells with [3H]myristic acid was performed. It was found that the C-terminal caspase cleavage product of human gelsolin (tGelsolin) was efficiently N-myristoylated. When COS-1 cells transiently transfected with gelsolin cDNA were treated with etoposide or staurosporine, apoptosis-inducing agents, N-myristoylated tGelsolin was generated, as demonstrated by in vivo metabolic labeling. The generation of posttranslationally N-myristoylated tGelsolin during apoptosis was also observed on endogenous gelsolin expressed in HeLa cells. Immunofluorescence staining and subcellular fractionation experiment revealed that exogenously expressed tGelsolin did not localize to mitochondria but rather was diffusely distributed in the cytoplasm. To study the role of this modification in the anti-apoptotic activity of tGelsolin, we constructed the bicistronic expression plasmid tGelsolin-IRES-EGFP capable of overexpressing tGelsolin concomitantly with EGFP. Overexpression of N-myristoylated tGelsolin in COS-1 cells using this plasmid significantly inhibited etoposide-induced apoptosis, whereas overexpression of the non-myristoylated tGelsolinG2A mutant did not cause resistance to apoptosis. These results indicate that posttranslational N-myristoylation of tGelsolin does not direct mitochondrial targeting, but this modification is involved in the anti-apoptotic activity of tGelsolin.Journal of Biological Chemistry 06/2006; 281(20):14288-95. · 4.77 Impact Factor -
Article: Involvement of Ras/extracellular signal-regulated kinase, but not Akt pathway in risedronate-induced apoptosis of U937 cells and its suppression by cytochalasin B.
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ABSTRACT: Although risedronate, a nitrogen containing bisphosphonate (BPs), strongly inhibits bone resorption by enhanced apoptosis of osteoclasts, its mechanism remained unclear. In this study, we investigated the molecular mechanism of risedronate-induced apoptosis of U937 cells, with a focus on extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt) pathways, mitochondria-mediated apoptosis, and the effect of disruption of the actin cytoskeleton. Risedronate facilitated the relocation of Ras from membrane to cytosol through inhibited isoprenylation. Accordingly, risedronate suppressed the phosphorylation of ERK 1/2, a downstream survival signaling kinase of Ras, affected the intracellular distribution of Bcl-xL, and induced the mitochondrial membrane depolarization, cytochrome c release, activated caspase cascade and DNA fragmentation. The risedronate-induced apoptosis was effectively suppressed with cyclosporine A plus trifluoperazine, potent inhibitors of mitochondrial membrane permeability transition (MPT). The risedronate-induced apoptosis was independent of Akt, another cAMP-dependent survival signaling kinase. Risedronate facilitated dephosphorylation of Bad at Ser112, an ERK phosphorylation site, but not at Ser136, an Akt phosphorylation site. All of these apoptosis-related changes induced by risedronate were strongly suppressed by cytochalasin B, an inhibitor of actin filament polymerization. These results indicate that risedronate-induced apoptosis in U937 cells involves Ras/ERK, but not Akt signaling pathway, and is dependent on MPT, and that disruption of the actin cytoskeleton inhibits the risedronate-induced apoptosis at its early step.Biochemical Pharmacology 07/2005; 69(12):1773-84. · 4.70 Impact Factor -
Article: The N-terminus of B96Bom, a Bombyx mori G-protein-coupled receptor, is N-myristoylated and translocated across the membrane.
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ABSTRACT: In eukaryotic cellular proteins, protein N-myristoylation has been recognized as a protein modification that occurs mainly on cytoplasmic or nucleoplasmic proteins. In this study, to search for a eukaryotic N-myristoylated transmembrane protein, the susceptibility of the N-terminus of several G-protein-coupled receptors (GPCRs) to protein N-myristoylation was evaluated by in vitro and in vivo metabolic labeling. It was found that the N-terminal 10 residues of B96Bom, a Bombyx mori GPCR, efficiently directed the protein N-myristoylation. Analysis of a tumor necrosis factor (TNF) fusion protein with the N-terminal 90 residues of B96Bom at its N-terminus revealed that (a) transmembrane domain 1 of B96Bom functioned as a type I signal anchor sequence, (b) the N-myristoylated N-terminal domain (58 residues) was translocated across the membrane, and (c) two N-glycosylation motifs located in this domain were efficiently N-glycosylated. In addition, when Ala4 in the N-myristoylation motif of B96Bom90-TNF, Met-Gly-Gln-Ala-Ala-Thr(1-6), was replaced with Asn to generate a new N-glycosylation motif, Asn-Ala-Thr(4-6), efficient N-glycosylation was observed on this newly introduced N-glycosylation site in the expressed protein. These results indicate that the N-myristoylated N-terminus of B96Bom is translocated across the membrane and exposed to the extracellular surface. To our knowledge, this is the first report showing that a eukaryotic transmembrane protein can be N-myristoylated and that the N-myristoylated N-terminus of the protein can be translocated across the membrane.FEBS Journal 02/2005; 272(2):472-81. · 3.79 Impact Factor
Top co-authors
Top Journals
Institutions
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2000–2012
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Yamaguchi University
- • Graduate School of Medicine
- • Department of Biological Chemistry
Yamaguchi-shi, Yamaguchi-ken, Japan
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2008–2011
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Okayama University
- • Department of Pathology and Experimental Medicine
- • Department of Cytology and Histology
Okayama-shi, Okayama-ken, Japan
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2010
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Shimizu Corporation
Toyama-shi, Toyama-ken, Japan
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2002–2005
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Kurashiki Central Hospital
Kurashiki, Okayama-ken, Japan
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