34 results on '"Shibata H"'
Search Results
2. Effects of okadaic acid on insulin-sensitive cAMP phosphodiesterase in rat adipocytes. Evidence that insulin may stimulate the enzyme by phosphorylation.
- Author
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Shibata, H., primary, Robinson, F.W., additional, Soderling, T.R., additional, and Kono, T., additional
- Published
- 1991
- Full Text
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3. Aldosterone synthase cytochrome P-450 expressed in the adrenals of patients with primary aldosteronism
- Author
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Ogishima, T., primary, Shibata, H., additional, Shimada, H., additional, Mitani, F., additional, Suzuki, H., additional, Saruta, T., additional, and Ishimura, Y., additional
- Published
- 1991
- Full Text
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4. Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.
- Author
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Takahashi, M, Shibata, H, Shimakawa, M, Miyamoto, M, Mukai, H, and Ono, Y
- Abstract
A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.
- Published
- 1999
5. Interaction of the small G protein RhoA with the C terminus of human phospholipase D1.
- Author
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Yamazaki, M, Zhang, Y, Watanabe, H, Yokozeki, T, Ohno, S, Kaibuchi, K, Shibata, H, Mukai, H, Ono, Y, Frohman, M A, and Kanaho, Y
- Abstract
Mammalian phosphatidylcholine-specific phospholipase D1 (PLD1) is a signal transduction-activated enzyme thought to function in multiple cell biological settings including the regulation of membrane vesicular trafficking. PLD1 is activated by the small G proteins, ADP-ribosylation factor (ARF) and RhoA, and by protein kinase C-alpha (PKC-alpha). This stimulation has been proposed to involve direct interaction and to take place at a distinct site in PLD1 for each activator. In the present study, we employed the yeast two-hybrid system to attempt to identify these sites. Successful interaction of ARF and PKC-alpha with PLD1 was not achieved, but a C-terminal fragment of human PLD1 (denoted "D4") interacted with the active mutant of RhoA, RhoAVal-14. Deletion of the CAAX box from RhoAVal-14 decreased the strength of the interaction, suggesting that lipid modification of RhoA is important for efficient binding to PLD1. The specificity of the interaction was validated by showing that the PLD1 D4 fragment interacts with glutathione S-transferase-RhoA in vitro in a GTP-dependent manner and that it associates with RhoAVal-14 in COS-7 cells, whereas the N-terminal two-thirds of PLD1 does not. Finally, we show that recombinant D4 peptide inhibits RhoA-stimulated PLD1 activation but not ARF- or PKC-alpha-stimulated PLD1 activation. These results conclusively demonstrate that the C-terminal region of PLD1 contains the RhoA-binding site and suggest that the ARF and PKC interactions occur elsewhere in the protein.
- Published
- 1999
6. PKN associates and phosphorylates the head-rod domain of neurofilament protein.
- Author
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Mukai, H, Toshimori, M, Shibata, H, Kitagawa, M, Shimakawa, M, Miyahara, M, Sunakawa, H, and Ono, Y
- Abstract
PKN is a fatty acid-activated serine/threonine kinase that has a catalytic domain highly homologous to that of protein kinase C in the carboxyl terminus and a unique regulatory region in the amino terminus. Recently, we reported that the small GTP-binding protein Rho binds to the amino-terminal region of PKN and activates PKN in a GTP-dependent manner, and we suggested that PKN is located on the downstream of Rho in the signal transduction pathway (Amano, M., Mukai, H., Ono, Y., Chihara, K., Matsui, T., Hamajima, Y., Okawa, K., Iwamatsu, A., and Kaibuchi, K. (1996) Science 271, 648-650; Watanabe, G., Saito, Y., Madaule, P., Ishizaki, T., Fujisawa, K., Morii, N., Mukai, H., Ono, Y. Kakizuka, A., and Narumiya, S. (1996) Science 271, 645-648). To identify other components of the PKN pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. By this screening, a clone encoding the neurofilament L protein, a subunit of neuron-specific intermediate filament, was isolated. The amino-terminal regulatory region of PKN was shown to associate with the head-rod domains of other subunits of neurofilament (neurofilament proteins M and H) as well as neurofilament L protein in yeast cells. The direct binding between PKN and each subunit of neurofilament was confirmed by using the in vitro translated amino-terminal region of PKN and glutathione S-transferase fusion protein containing the head-rod domain of each subunit of neurofilament. PKN purified from rat testis phosphorylated each subunit of the native neurofilament purified from bovine spinal cord and the bacterially synthesized head-rod domain of each subunit of neurofilament. Polymerization of neurofilament L protein in vitro was inhibited by phosphorylation of neurofilament L protein by PKN. The identification and characterization of the novel interaction with PKN may contribute toward the elucidation of mechanisms regulating the function of neurofilament.
- Published
- 1996
7. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain inhibits insulin action on glucose transport in rat adipocytes.
- Author
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Shibata, H, Omata, W, Suzuki, Y, Tanaka, S, and Kojima, I
- Abstract
The present study was conducted to examine the involvement of Rab4, a low molecular weight GTP-binding protein, in the action of insulin on glucose transport. A synthetic peptide corresponding to the Rab4 hypervariable carboxyl-terminal domain, Rab4-(191-210), was successfully transferred into rat adipocytes by electroporation and inhibited insulin-stimulated glucose transport by about 50% without affecting the basal transport activity. In contrast, synthetic peptides corresponding to the Rab3C and Rab3D carboxyl-terminal hypervariable domain had little effect on insulin action on glucose transport. The Rab4-(191-210) peptide also reduced insulin-induced GLUT4 translocation from the intracellular pool to the plasma membrane. Furthermore, the Rab4-(191-210) peptide reduced both insulin-induced glucose transport and GLUT4 translocation in the presence of a major histocompatibility complex class I antigen-derived peptide, D(k)-(62-85), which is a potent inhibitor of GLUT4 internalization, suggesting that the peptide inhibited exocytotic recruitment of GLUT4-containing vesicles. The Rab4-(191-210) peptide also inhibited GTP gamma S-stimulated glucose transport. In addition, insulin-stimulated glucose transport was inhibited by the addition of anti-Rab4 antibody. These results suggest that Rab4 protein plays a crucial role in insulin action on GLUT4 translocation, especially in exocytotic recruitment by the hormone of the glucose transporter to the plasma membrane from the intracellular retention pool.
- Published
- 1996
8. Insulin stimulates guanine nucleotide exchange on Rab4 via a wortmannin-sensitive signaling pathway in rat adipocytes.
- Author
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Shibata, H, Omata, W, and Kojima, I
- Abstract
Rab4, a member of the Rab family of Ras-related small GTP-binding proteins, has been shown to be associated with GLUT4-containing vesicles and implicated in the insulin action on glucose transport in rat adipocytes. In the present study, we investigated the insulin effects on the guanine nucleotide exchange on Rab4. In electrically permeabilized rat adipocytes, the amount of [35S]guanosine 5'-O-(3-thiotrisphosphate) (GTPgammaS) bound to Rab4 increased in a time-dependent manner during 45 min of the incubation period. Addition of insulin resulted in about a 2-fold stimulation of the binding of [35S]GTPgammaS to Rab4, indicating that insulin stimulated the guanine nucleotide exchange on the GTPase. Pretreatment of the cells with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, completely abolished the stimulatory effect of insulin on [35S]GTPgammaS binding to Rab4. Wortmannin also attenuated the nucleotide binding to Rab4 in the basal cells, suggesting that phosphatidylinositol 3-kinase activity may be essential for regulation of guanine nucleotide exchange on the GTPase and insulin may up-regulate the exchange activity by stimulating the lipid kinase. Insulin-induced subcellular redistribution of Rab4 from the microsomal fraction to the soluble fraction was also inhibited by wortmannin. These results suggest that insulin stimulates the guanine nucleotide exchange on Rab4 via a phosphatidylinositol 3-kinase-dependent signaling pathway and that Rab4 is one of possible targets of insulin action on intracellular vesicle traffic in rat adipocytes.
- Published
- 1997
9. Cyclic GMP-dependent Stimulation of the Membrane-bound Insulin-sensitive cAMP Phosphodiesterase from Rat Adipocytes*
- Author
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Robinson, F W, Smith, C J, Flanagan, J E, Shibata, H, and Kono, T
- Abstract
The insulin-sensitive cAMP phosphodiesterase (phosphodiesterase) in rat adipocytes is a membrane-bound low Kmenzyme that can be recovered in a crude microsomal fraction (Fraction P-2). The action of this enzyme to hydrolyze cAMP is known to be inhibited by cGMP; nevertheless, it was found in our present study that under selected conditions, the enzyme can also be stimulated by cGMP as well as some other nucleotide derivatives. The maximum cGMP-dependent stimulation was observed when the enzyme in Fraction P-2 was incubated with 10 µMcGMP for 5–20 min at 37 °C in the presence of Mg2+, washed, and then assayed in the absence of added cGMP. The level of this stimulation was close to, but less than, that achieved by insulin in intact cells. The actions of the cGMP- and insulin-stimulated enzymes to hydrolyze labeled cAMP were inhibited in an identical manner by cilostamide (Ki= 0.10 µM), griseolic acid (Ki= 0.19 µm), unlabeled cAMP (Km= 0.20 µM), and cGMP (Ki= 0.16 µm), all added to the assay system. Also, the basal, insulin-stimulated, and cGMP-activated enzymes were identically inhibited by a polyclonal antibody raised against a purified membrane-bound low Kmphosphodiesterase from bovine adipose tissue. When the same antibody was used for the Western blot analysis of Fraction P-2, it immunoreacted with a single band of protein (165 kDa). These observations indicate that the insulin-sensitive phosphodiesterase in rat adipocytes can be stimulated with 10 µmcGMP and that this stimulation is detectable only after the nucleotide has been eliminated since the enzyme would be strongly inhibited by the nucleotide if the latter exists in the assay system. It is proposed that the insulin-sensitive phosphodiesterase, which is often referred to as a Type IV enzyme, is functionally similar to the Type II enzymes that are known to be stimulated by a low concentration of cGMP and inhibited by higher concentrations of the same nucleotide.
- Published
- 1989
- Full Text
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10. Time-dependent restoration of the trigger pool of calcium after termination of angiotensin II action in adrenal glomerulosa cells.
- Author
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Kojima, I., Shibata, H., and Ogata, E.
- Abstract
In adrenal glomerulosa cells, angiotensin II causes an immediate release of calcium from an intracellular trigger pool (Kojima, I., Kojima, K., and Rasmussen, H. (1985) Am. J. Physiol. 247, E36-E43). The present study was conducted to determine how the trigger pool of calcium is restored after cessation of the agonist action. Upon termination of angiotensin II action, calcium influx rate decreased immediately while total cell calcium increased rapidly. The increase in total cell calcium is not affected by 1 microM nitrendipine, which blocks angiotensin II-stimulated calcium influx without inhibiting basal influx of calcium. In contrast, total cell calcium did not increase in medium containing 1 microM calcium, in which basal calcium influx is negligible. A rapid increase in total cell calcium after an addition of the antagonist was not accompanied by changes in cytoplasmic free calcium concentration. A second stimulation of cells with either angiotensin II or carbachol did not cause calcium release when the interval of two stimulations was shorter than 20 min. The longer the interval, the greater the magnitude of calcium release in response to the second stimulator. The maximum response was obtained when the interval was 40 min or more. When exogenous arachidonic acid, which mobilized calcium by acting directly on the inositol trisphosphate-sensitive pool, was employed as a second stimulator, the magnitude of the decrease in total cell calcium was also dependent on the interval. These results suggest that, upon termination of angiotensin II action, calcium is rapidly accumulated first in an intracellular pool which is insensitive to either inositol 1,4,5-trisphosphate or arachidonic acid and that the trigger pool is restored gradually thereafter.
- Published
- 1987
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11. Gene expression of subunit c(P1), subunit c(P2), and oligomycin sensitivity-conferring protein may play a key role in biogenesis of H+-ATP synthase in various rat tissues.
- Author
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Sangawa, H, Himeda, T, Shibata, H, and Higuti, T
- Abstract
Mammalian H+-ATP synthase is a supramolecule composed of at least 14 subunits that have a constant stoichiometry. Nevertheless the coordinate regulation of the gene expressions of various subunits remains obscure. To clarify the coordinate transcriptional regulatory system of mammalian H+-ATP synthase, we determined the absolute amount of nine species of mRNAs for eight nuclear-encoded subunits of H+-ATP synthase in different tissues of 8-week-old rats by use of the synthetic mRNAs and 32P-labeled DNA probes for each mRNA. Our quantitative analyses of the transcripts of H+-ATP synthase revealed that nine species of the subunits in different tissues of 8-week-old rats were divisible into two groups: a high transcript gene (HTG) group (beta-subunit, subunit b, subunit d, subunit e, and Factor 6) and a low transcript gene (LTG) group (subunit c(P1), subunit c(P2), IF1, and oligomycin sensitivity-conferring protein). The transcription step of LTG could constitute a bottleneck in the biogenesis of H+-ATP synthase. Thus, the transcriptional regulatory system of the LTG may play a key role in the biogenesis of mammalian H+-ATP synthase. The HTG were transcribed in a tissue-specific manner that corresponds with energy demand in the tissues. However, there was no tissue specificity in subunit c(P2). Furthermore, the tissue specificity of the transcript of IF1 differed substantially from that of HTG, suggesting that it could be crucial in the protection of mitochondrial membrane under abnormal conditions.
- Published
- 1997
12. Activation of a calcium-permeable cation channel CD20 expressed in Balb/c 3T3 cells by insulin-like growth factor-I.
- Author
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Kanzaki, M, Nie, L, Shibata, H, and Kojima, I
- Abstract
CD20 functions as a calcium-permeable cation channel. When expressed in Balb/c 3T3 cells, CD20 accelerates the G1 progression induced by insulin-like growth factor-I (IGF-I). To further characterize how CD20 modulates the action of IGF-I, we investigated whether the activity of CD20 channel was affected by IGF-I. In quiescent cells expressing CD20, IGF-I increased cytoplasmic free calcium concentration, [Ca2+]c, which was reversed by the removal of extracellular calcium. In contrast, IGF-I did not increase [Ca2+]c in cells that did not express CD20. In perforated patch clamp recordings, addition of IGF-I to the bath solution augmented the Ca2+ permeability, which was reversed by anti-CD20 antibody. In cell-attached patch, calcium-permeable channel activity with unitary conductance of 7 picosiemens was detected, which was abolished by anti-CD20 antibody. The single channel activities were markedly enhanced when IGF-I was included in the pipette solution, whereas IGF-I added to the bath solution was ineffective. When cells were first exposed to pertussis toxin, activation of the channel by IGF-I was blocked. Transfection of cDNA for Gip2, a constitutive active form of alphai2, activated the CD20 channel. These results indicate that the CD20 channel is regulated by the IGF-I receptor by a mechanism involving pertussis toxin-sensitive G protein.
- Published
- 1997
13. Interaction of PKN with alpha-actinin.
- Author
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Mukai, H, Toshimori, M, Shibata, H, Takanaga, H, Kitagawa, M, Miyahara, M, Shimakawa, M, and Ono, Y
- Abstract
PKN is a fatty acid- and Rho-activated serine/threonine protein kinase, having a catalytic domain homologous to protein kinase C family. To identify components of the PKN-signaling pathway such as substrates and regulatory proteins of PKN, the yeast two-hybrid strategy was employed. Using the N-terminal region of PKN as a bait, cDNAs encoding actin cross-linking protein alpha-actinin, which lacked the N-terminal actin-binding domain, were isolated from human brain cDNA library. The responsible region for interaction between PKN and alpha-actinin was determined by in vitro binding analysis using the various truncated mutants of these proteins. The N-terminal region of PKN outside the RhoA-binding domain was sufficiently shown to associate with alpha-actinin. PKN bound to the third spectrin-like repeats of both skeletal and non-skeletal muscle type alpha-actinin. PKN also bound to the region containing EF-hand-like motifs of non-skeletal muscle type alpha-actinin in a Ca2+-sensitive manner and bound to that of skeletal muscle type alpha-actinin in a Ca2+-insensitive manner. alpha-Actinin was co-immunoprecipitated with PKN from the lysate of COS7 cells transfected with both expression constructs for PKN and alpha-actinin lacking the actin-binding domain. In vitro translated full-length alpha-actinin containing the actin-binding site hardly bound to PKN, but the addition of phosphatidylinositol 4, 5-bisphosphate, which is implicated in actin reorganization, stimulated the binding activity of the full-length alpha-actinin with PKN. We therefore propose that PKN is linked to the cytoskeletal network via a direct association between PKN and alpha-actinin.
- Published
- 1997
14. Dissection of GLUT4 recycling pathway into exocytosis and endocytosis in rat adipocytes. Evidence that GTP-binding proteins are involved in both processes.
- Author
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Shibata, H, Suzuki, Y, Omata, W, Tanaka, S, and Kojima, I
- Abstract
The effects of guanine nucleotides on either exocytosis or endocytosis of GLUT4 were examined in electrically permeabilized rat adipocytes by using Dk-(62-85), a major histocompatibility complex class I-derived peptide. Reversal of glucose transport activity that had been stimulated with insulin was completely blocked by Dk-(62-85). Likewise, endocytosis of the trypsin-cleaved 35-kDa fragment of GLUT4 was almost completely inhibited by the peptide. Insulin-stimulated glucose transport activity was enhanced about 50% by Dk-(62-85), whereas the basal transport activity was stimulated only slightly. Although guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) augmented glucose transport to the same extent as insulin in the absence of the peptide, glucose transport stimulated by GTP gamma S was only 60% of the insulin effect in the presence of the peptide; the effect of insulin was markedly enhanced by Dk-(62-85), whereas GTP gamma S-induced glucose transport was not affected, suggesting that GTP gamma S has an effect similar to that of the peptide. In fact, endocytosis of the 35-kDa fragment of GLUT4 was markedly inhibited by GTP gamma S. Additionally, GLUT4 endocytosis was accelerated by GTP but was inhibited by guanosine 5'-O-(2-thiodiphosphate). These results indicate that GTP gamma S induces translocation of GLUT4 by both stimulating exocytosis and inhibiting endocytosis. With respect to the dependence on GTP hydrolysis, distinct types of GTP-binding proteins are involved in exocytosis and endocytosis of GLUT4.
- Published
- 1995
15. Expression of calcium-permeable cation channel CD20 accelerates progression through the G1 phase in Balb/c 3T3 cells.
- Author
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Kanzaki, M, Shibata, H, Mogami, H, and Kojima, I
- Abstract
CD20 is a transmembrane protein that functions as a Ca(2+)-permeable cation channel (Bubien, J. K., Zhou, L. J., Bell, P. D., Frizzel, R. A., and Tedder, T. F. (1993) J. Cell Biol. 121, 1121-1132) and is involved in growth regulation of B lymphocytes. In order to further investigate the role of calcium entry in cell cycle progression, we introduced the cDNA encoding a Ca(2+)-permeable cation channel, CD20, into Balb/c 3T3 cells. Balb/c 3T3 cells transfected with a vector containing cDNA encoding CD20 expressed the CD20 protein, which was detected by assaying the binding of a monoclonal antibody against CD20. Calcium-permeable cation channel activity was detected in CD20-expressing cells by whole cell patch clamp recording and microfluorometric determination of the cytoplasmic Ca2+ concentration using fura-2. The expression of CD20 induced significant alterations in the responses of the cells to insulin-like growth factor-I (IGF-I). IGF-I induced DNA synthesis by control cells only when they had been pretreated with both platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). In contrast, DNA synthesis by 30% of the quiescent CD20-expressing cells was initiated in response to IGF-I in the absence of priming with PDGF and EGF. When control quiescent cells were primed with PDGF and EGF, the addition of IGF-I led to the initiation of DNA synthesis after 14 h or more, whereas it induced DNA synthesis by CD20-expressing cells primed with PDGF and EGF 4 h earlier. The IGF-induced DNA synthesis was dependent on extracellular Ca2+, and expression of CD20 reduced the concentration of extracellular Ca2+ required for it. Furthermore, DNA synthesis by approximately 25% of the CD20-expressing cells was initiated after priming with PDGF and EGF, even in the absence of the progression factor IGF-I. These results indicate that CD20 expressed in Balb/c 3T3 cells functions as a constitutively active Ca(2+)-permeable cation channel and that expression of CD20 accelerates G1 progression in a Ca(2+)-dependent manner.
- Published
- 1995
16. The primary nucleotide sequence of nuclear U-2 ribonucleic acid. The 5'-terminal portion of the molecule.
- Author
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Shibata, H, primary, Ro-Choi, TS, additional, Reddy, R, additional, Choi, YC, additional, Henning, D, additional, and Busch, H, additional
- Published
- 1975
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17. N-acetylglucosaminyltransferase-V requires a specific noncatalytic luminal domain for its activity toward glycoprotein substrates.
- Author
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Osuka RF, Hirata T, Nagae M, Nakano M, Shibata H, Okamoto R, and Kizuka Y
- Subjects
- Glycosylation, Humans, Oligosaccharides metabolism, Polysaccharides metabolism, Glycoproteins metabolism, N-Acetylglucosaminyltransferases metabolism
- Abstract
N-acetylglucosaminyltransferase-V (GnT-V or MGAT5) catalyzes the formation of an N-glycan β1,6-GlcNAc branch on selective target proteins in the Golgi apparatus and is involved in cancer malignancy and autoimmune disease etiology. Several three-dimensional structures of GnT-V were recently solved, and the recognition mechanism of the oligosaccharide substrate was clarified. However, it is still unclear how GnT-V selectively acts on glycoprotein substrates. In this study, we focused on an uncharacterized domain at the N-terminal side of the luminal region (N domain) of GnT-V, which was previously identified in a crystal structure, and aimed to reveal its role in GnT-V action. Using lectin blotting and fluorescence assisted cell sorting analysis, we found that a GnT-VΔN mutant lacking the N domain showed impaired biosynthetic activity in cells, indicating that the N domain is required for efficient glycosylation. To clarify this mechanism, we measured the in vitro activity of purified GnT-VΔN toward various kinds of substrates (oligosaccharide, glycohexapeptide, and glycoprotein) using HPLC and a UDP-Glo assay. Surprisingly, GnT-VΔN showed substantially reduced activity toward the glycoprotein substrates, whereas it almost fully maintained its activity toward the oligosaccharides and the glycopeptide substrates. Finally, docking models of GnT-V with substrate glycoproteins suggested that the N domain could interact with the substrate polypeptide directly. Our findings suggest that the N domain of GnT-V plays a critical role in the recognition of glycoprotein substrates, providing new insights into the mechanism of substrate-selective biosynthesis of N-glycans., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
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18. The calcium-binding protein ALG-2 regulates protein secretion and trafficking via interactions with MISSL and MAP1B proteins.
- Author
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Takahara T, Inoue K, Arai Y, Kuwata K, Shibata H, and Maki M
- Subjects
- Apoptosis Regulatory Proteins genetics, Calcium metabolism, Calcium-Binding Proteins genetics, Collagen Type I genetics, Endoplasmic Reticulum genetics, Golgi Apparatus genetics, HeLa Cells, Humans, Microtubule-Associated Proteins genetics, Protein Precursors genetics, Protein Transport physiology, Apoptosis Regulatory Proteins metabolism, Calcium-Binding Proteins metabolism, Collagen Type I metabolism, Endoplasmic Reticulum metabolism, Golgi Apparatus metabolism, Microtubule-Associated Proteins metabolism, Protein Precursors metabolism
- Abstract
Mobilization of intracellular calcium is essential for a wide range of cellular processes, including signal transduction, apoptosis, and vesicular trafficking. Several lines of evidence have suggested that apoptosis-linked gene 2 (ALG-2, also known as PDCD6 ), a calcium-binding protein, acts as a calcium sensor linking calcium levels with efficient vesicular trafficking, especially at the endoplasmic reticulum (ER)-to-Golgi transport step. However, how ALG-2 regulates these processes remains largely unclear. Here, we report that M APK1- i nteracting and s pindle- s tabilizing (MISS)- l ike (MISSL), a previously uncharacterized protein, interacts with ALG-2 in a calcium-dependent manner. Live-cell imaging revealed that upon a rise in intracellular calcium levels, GFP-tagged MISSL (GFP-MISSL) dynamically relocalizes in a punctate pattern and colocalizes with ALG-2. MISSL knockdown caused disorganization of the components of the ER exit site, the ER-Golgi intermediate compartment, and Golgi. Importantly, knockdown of either MISSL or ALG-2 attenuated the secretion of se creted a lkaline p hosphatase (SEAP), a model secreted cargo protein, with similar reductions in secretion by single- and double-protein knockdowns, suggesting that MISSL and ALG-2 act in the same pathway to regulate the secretion process. Furthermore, ALG-2 or MISSL knockdown delayed ER-to-Golgi transport of procollagen type I. We also found that ALG-2 and MISSL interact with microtubule-associated protein 1B (MAP1B) and that MAP1B knockdown reverts the reduced secretion of SEAP caused by MISSL or ALG-2 depletion. These results suggest that a change in the intracellular calcium level plays a role in regulation of the secretory pathway via interaction of ALG-2 with MISSL and MAP1B., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
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19. A new role for annexin A11 in the early secretory pathway via stabilizing Sec31A protein at the endoplasmic reticulum exit sites (ERES).
- Author
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Shibata H, Kanadome T, Sugiura H, Yokoyama T, Yamamuro M, Moss SE, and Maki M
- Subjects
- Annexins genetics, Apoptosis Regulatory Proteins genetics, Apoptosis Regulatory Proteins metabolism, Biological Transport, Active physiology, COP-Coated Vesicles genetics, COP-Coated Vesicles metabolism, Calcium metabolism, Calcium-Binding Proteins genetics, Calcium-Binding Proteins metabolism, Endoplasmic Reticulum genetics, Golgi Apparatus genetics, Golgi Apparatus metabolism, HEK293 Cells, Humans, Protein Stability, Vesicular Transport Proteins genetics, Annexins metabolism, Endoplasmic Reticulum metabolism, Vesicular Transport Proteins metabolism
- Abstract
Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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20. Prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer inhibition by a protein kinase CK2-dependent mechanism in 3T3-L1 adipocytes.
- Author
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Ma J, Nakagawa Y, Kojima I, and Shibata H
- Subjects
- 3T3 Cells, Adaptor Proteins, Vesicular Transport genetics, Adaptor Proteins, Vesicular Transport metabolism, Adipocytes cytology, Animals, Casein Kinase II genetics, Down-Regulation drug effects, Gene Knockdown Techniques, Glucose Transporter Type 4 genetics, Hypoglycemic Agents metabolism, Hypoglycemic Agents pharmacology, Insulin pharmacology, Intracellular Membranes metabolism, Liposomes metabolism, Mice, Microsomes metabolism, Mitogen-Activated Protein Kinase 3 genetics, Mitogen-Activated Protein Kinase 3 metabolism, Oxidative Stress drug effects, Phosphatidylinositol 3-Kinases genetics, Phosphatidylinositol 3-Kinases metabolism, Phosphorylation drug effects, Phosphorylation physiology, Protein Transport drug effects, Protein Transport physiology, Proteolysis drug effects, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Adipocytes metabolism, Casein Kinase II metabolism, Down-Regulation physiology, Glucose Transporter Type 4 metabolism, Insulin metabolism, Oxidative Stress physiology
- Abstract
Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.
- Published
- 2014
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21. Nuclear ALG-2 protein interacts with Ca2+ homeostasis endoplasmic reticulum protein (CHERP) Ca2+-dependently and participates in regulation of alternative splicing of inositol trisphosphate receptor type 1 (IP3R1) pre-mRNA.
- Author
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Sasaki-Osugi K, Imoto C, Takahara T, Shibata H, and Maki M
- Subjects
- Apoptosis Regulatory Proteins genetics, Calcium-Binding Proteins genetics, Cell Nucleus genetics, DNA-Binding Proteins genetics, Gene Knockdown Techniques, HeLa Cells, Humans, Inositol 1,4,5-Trisphosphate Receptors genetics, Membrane Proteins genetics, Phosphorylation physiology, Protein Structure, Tertiary, RNA Polymerase II genetics, RNA Polymerase II metabolism, RNA Precursors genetics, RNA-Binding Proteins genetics, Alternative Splicing physiology, Apoptosis Regulatory Proteins metabolism, Calcium metabolism, Calcium Signaling physiology, Calcium-Binding Proteins metabolism, Cell Nucleus metabolism, DNA-Binding Proteins metabolism, Inositol 1,4,5-Trisphosphate Receptors biosynthesis, Membrane Proteins metabolism, RNA Precursors metabolism, RNA-Binding Proteins metabolism
- Abstract
The intracellular Ca(2+) signaling pathway is important for the control of broad cellular processes from fertilization to cell death. ALG-2 is a Ca(2+)-binding protein that contains five serially repeated EF-hand motifs and interacts with various proteins in a Ca(2+)-dependent manner. Although ALG-2 is present both in the cytoplasm and in the nucleus, little is known about its nuclear function. Ca(2+) homeostasis endoplasmic reticulum protein (CHERP) was first identified as an endoplasmic reticulum protein that regulates intracellular Ca(2+) mobilization in human cells, but recent proteomics data suggest an association between CHERP and spliceosomes. Here, we report that CHERP, containing a Pro-rich region and a phosphorylated Ser/Arg-rich RS-like domain, is a novel Ca(2+)-dependent ALG-2-interactive target in the nucleus. Immunofluorescence microscopic analysis revealed localization of CHERP to the nucleoplasm with prominent accumulation at nuclear speckles, which are the sites of storage and modification for pre-mRNA splicing factors. Live cell time-lapse imaging showed that nuclear ALG-2 was recruited to the CHERP-localizing speckles upon Ca(2+) mobilization. Results of co-immunoprecipitation assays revealed binding of CHERP to a phosphorylated form of RNA polymerase II. Knockdown of CHERP or ALG-2 in HT1080 cells resulted in generation of alternatively spliced isoforms of the inositol 1,4,5-trisphosphate receptor 1 (IP3R1) pre-mRNA that included exons 41 and 42 in addition to the major isoform lacking exons 40-42. Furthermore, binding between CHERP and IP3R1 RNA was detected by an RNA immunoprecipitation assay using a polyclonal antibody against CHERP. These results indicate that CHERP and ALG-2 participate in regulation of alternative splicing of IP3R1 pre-mRNA and provide new insights into post-transcriptional regulation of splicing variants in Ca(2+) signaling pathways.
- Published
- 2013
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22. Three-dimensional structure of nylon hydrolase and mechanism of nylon-6 hydrolysis.
- Author
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Negoro S, Shibata N, Tanaka Y, Yasuhira K, Shibata H, Hashimoto H, Lee YH, Oshima S, Santa R, Oshima S, Mochiji K, Goto Y, Ikegami T, Nagai K, Kato D, Takeo M, and Higuchi Y
- Subjects
- Actinomycetales genetics, Amidohydrolases genetics, Amidohydrolases metabolism, Amino Acid Substitution, Bacterial Proteins genetics, Bacterial Proteins metabolism, Caprolactam chemistry, Hydrolysis, Mutation, Missense, Protein Structure, Quaternary, Protein Structure, Tertiary, Actinomycetales enzymology, Amidohydrolases chemistry, Bacterial Proteins chemistry, Caprolactam analogs & derivatives, Polymers chemistry, Protein Multimerization
- Abstract
We performed x-ray crystallographic analyses of the 6-aminohexanoate oligomer hydrolase (NylC) from Agromyces sp. at 2.0 Å-resolution. This enzyme is a member of the N-terminal nucleophile hydrolase superfamily that is responsible for the degradation of the nylon-6 industry byproduct. We observed four identical heterodimers (27 kDa + 9 kDa), which resulted from the autoprocessing of the precursor protein (36 kDa) and which constitute the doughnut-shaped quaternary structure. The catalytic residue of NylC was identified as the N-terminal Thr-267 of the 9-kDa subunit. Furthermore, each heterodimer is folded into a single domain, generating a stacked αββα core structure. Amino acid mutations at subunit interfaces of the tetramer were observed to drastically alter the thermostability of the protein. In particular, four mutations (D122G/H130Y/D36A/E263Q) of wild-type NylC from Arthrobacter sp. (plasmid pOAD2-encoding enzyme), with a heat denaturation temperature of T(m) = 52 °C, enhanced the protein thermostability by 36 °C (T(m) = 88 °C), whereas a single mutation (G111S or L137A) decreased the stability by ∼10 °C. We examined the enzymatic hydrolysis of nylon-6 by the thermostable NylC mutant. Argon cluster secondary ion mass spectrometry analyses of the reaction products revealed that the major peak of nylon-6 (m/z 10,000-25,000) shifted to a smaller range, producing a new peak corresponding to m/z 1500-3000 after the enzyme treatment at 60 °C. In addition, smaller fragments in the soluble fraction were successively hydrolyzed to dimers and monomers. Based on these data, we propose that NylC should be designated as nylon hydrolase (or nylonase). Three potential uses of NylC for industrial and environmental applications are also discussed.
- Published
- 2012
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23. NF-YC functions as a corepressor of agonist-bound mineralocorticoid receptor.
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Murai-Takeda A, Shibata H, Kurihara I, Kobayashi S, Yokota K, Suda N, Mitsuishi Y, Jo R, Kitagawa H, Kato S, Saruta T, and Itoh H
- Subjects
- Aldosterone pharmacology, Animals, COS Cells, Cells, Cultured, Chlorocebus aethiops, Dose-Response Relationship, Drug, Epithelial Sodium Channels metabolism, Histone Deacetylases metabolism, Humans, Hydrocortisone pharmacology, Immunohistochemistry, Kidney Tubules, Collecting cytology, Male, Mice, Promoter Regions, Genetic physiology, Protein Structure, Tertiary, Receptors, Androgen metabolism, Receptors, Glucocorticoid metabolism, Receptors, Mineralocorticoid agonists, Receptors, Mineralocorticoid chemistry, Receptors, Progesterone metabolism, Two-Hybrid System Techniques, Aldosterone metabolism, CCAAT-Binding Factor metabolism, Hydrocortisone metabolism, Kidney Tubules, Collecting metabolism, Receptors, Mineralocorticoid metabolism
- Abstract
The role of aldosterone has been implicated in the metabolic syndrome and cardiovascular diseases. The biological actions of aldosterone are mediated through mineralocorticoid receptor (MR). Nuclear receptor-mediated gene expression is regulated by dynamic and coordinated recruitment of coactivators and corepressors. To identify new coregulators of the MR, full-length MR was used as bait in yeast two-hybrid screening. We isolated NF-YC, one of the subunits of heterotrimeric transcription factor NF-Y. Specific interaction between MR and NF-YC was confirmed by yeast two-hybrid, mammalian two-hybrid, coimmunoprecipitation assays, and fluorescence subcellular imaging. Transient transfection experiments in COS-7 cells demonstrated that NF-YC repressed MR transactivation in a hormone-sensitive manner. Moreover, reduction of NF-YC protein levels by small interfering RNA potentiated hormonal activation of endogenous target genes in stably MR-expressing cells, indicating that NF-YC functions as an agonist-dependent MR corepressor. The corepressor function of NF-YC is selective for MR, because overexpression of NF-YC did not affect transcriptional activity mediated by androgen, progesterone, or glucocorticoid receptors. Chromatin immunoprecipitation experiments showed that endogenous MR and steroid receptor coactivator-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner, and endogenous NF-YC was sequentially recruited to the same element. Immunohistochemistry showed that endogenous MR and NF-YC were colocalized within the mouse kidney. Although aldosterone induces interaction of the N and C termini of MR, NF-YC inhibited the N/C interaction. These findings indicate that NF-YC functions as a new corepressor of agonist-bound MR via alteration of aldosterone-induced MR conformation.
- Published
- 2010
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24. The pathway via D-galacturonate/L-galactonate is significant for ascorbate biosynthesis in Euglena gracilis: identification and functional characterization of aldonolactonase.
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Ishikawa T, Nishikawa H, Gao Y, Sawa Y, Shibata H, Yabuta Y, Maruta T, and Shigeoka S
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- Algal Proteins genetics, Amino Acid Sequence, Animals, Ascorbic Acid genetics, Carboxylic Ester Hydrolases genetics, Escherichia coli enzymology, Escherichia coli genetics, Euglena gracilis genetics, Molecular Sequence Data, Protozoan Proteins genetics, Rats, Recombinant Proteins genetics, Recombinant Proteins metabolism, Algal Proteins metabolism, Ascorbic Acid biosynthesis, Carboxylic Ester Hydrolases metabolism, Euglena gracilis enzymology, Hexuronic Acids metabolism, Protozoan Proteins metabolism, Sugar Acids metabolism
- Abstract
We have previously proposed that Euglena gracilis possesses a pathway for the production of ascorbate (AsA) through d-galacturonate/L-galactonate as representative intermediates ( Shigeoka, S., Nakano, Y., and Kitaoka, S. (1979) J. Nutr. Sci. Vitaminol. 25, 299-307 ). However, genetic evidence proving that the pathway exists has not been obtained yet. We report here the identification of a gene encoding aldonolactonase, which catalyzes a penultimate step of the biosynthesis of AsA in Euglena. By a BLAST search, we identified one candidate for the enzyme having significant sequence identity with rat gluconolactonase, a key enzyme for the production of AsA via d-glucuronate in animals. The purified recombinant aldonolactonase expressed in Escherichia coli catalyzed the reversible reaction of L-galactonate and L-galactono-1,4-lactone with zinc ion as a cofactor. The apparent K(m) values for L-galactonate and L-galactono-1,4-lactone were 1.55 +/- 0.3 and 1.67 +/- 0.39 mm, respectively. The cell growth of Euglena was arrested by silencing the expression of aldonolactonase through RNA interference and then restored to the normal state by supplementation with L-galactono-1,4-lactone. Euglena cells accumulated more AsA on supplementation with d-galacturonate than d-glucuronate. The present results indicate that aldonolactonase is significant for the biosynthesis of AsA in Euglena cells, which predominantly utilize the pathwayviad-galacturonate/L-galactonate. The identification of aldonolactonase provides the first insight into the biosynthesis of AsA via uronic acids as the intermediate in photosynthetic algae including Euglena.
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- 2008
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25. Identification of Alix-type and Non-Alix-type ALG-2-binding sites in human phospholipid scramblase 3: differential binding to an alternatively spliced isoform and amino acid-substituted mutants.
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Shibata H, Suzuki H, Kakiuchi T, Inuzuka T, Yoshida H, Mizuno T, and Maki M
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- Annexin A7 genetics, Annexin A7 metabolism, Annexins genetics, Annexins metabolism, Apoptosis Regulatory Proteins genetics, Binding Sites genetics, Calcium-Binding Proteins genetics, Carrier Proteins genetics, Carrier Proteins metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Line, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Endosomal Sorting Complexes Required for Transport, Phospholipid Transfer Proteins genetics, Protein Binding genetics, Protein Isoforms genetics, Protein Isoforms metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Transcription Factors genetics, Transcription Factors metabolism, Vesicular Transport Proteins genetics, Vesicular Transport Proteins metabolism, Alternative Splicing physiology, Amino Acid Substitution, Apoptosis Regulatory Proteins metabolism, Calcium-Binding Proteins metabolism, Phospholipid Transfer Proteins metabolism
- Abstract
ALG-2, a prototypic member of the penta-EF-hand protein family, interacts with Alix at its C-terminal Pro-rich region containing four tandem PXY repeats. Human phospholipid scramblase 3 (PLSCR3) has a similar sequence (ABS-1) in its N-terminal region. In the present study, we found that ALG-2 interacts with PLSCR3 expressed in HEK293 cells in a Ca(2+)-dependent manner by co-immunoprecipitation, pulldown with glutathione S-transferase (GST) fused ALG-2 and an overlay assay using biotin-labeled ALG-2. The GST fusion protein of an alternatively spliced isoform of ALG-2, GST-ALG-2(DeltaGF122), pulled down green fluorescent protein (GFP)-fused PLSCR3 but not GFP Alix. Deletion of a region containing ABS-1 was not sufficient to abrogate the binding. A second ALG-2-binding site (ABS-2) was essential for interaction with ALG-2(DeltaGF122). Real-time interaction analyses with a surface plasmon resonance biosensor using synthetic oligopeptides and recombinant proteins corroborated direct Ca(2+)-dependent binding of ABS-1 to ALG-2 and that of ABS-2 to ALG-2 as well as to ALG-2(DeltaGF122). The sequence of ABS-2 contains multiple prolines and two phenylalanines, among which Phe(49) was found to be critical, because its substitution with Ala or Tyr caused a loss of binding ability by pulldown assays using oligopeptide-immobilized beads. ALG-2-interacting proteins were classified into two groups based on binding ability to ALG-2(DeltaGF122): (i) isoform-non-interactive (ABS-1) types, including Alix, annexin A7, annexin A11, and TSG101 and (ii) isoform-interactive (ABS-2) types including PLSCR3, PLSCR4 and Sec31A. GST-pulldown assays using single amino acid-substituted ALG-2 mutants revealed differences in binding specificities between the two groups, suggesting structural flexibility in ALG-2-ligand complex formation.
- Published
- 2008
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26. Characterization of the interaction between recombinant human peroxin Pex3p and Pex19p: identification of TRP-104 IN Pex3p as a critical residue for the interaction.
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Sato Y, Shibata H, Nakano H, Matsuzono Y, Kashiwayama Y, Kobayashi Y, Fujiki Y, Imanaka T, and Kato H
- Subjects
- Amino Acid Substitution, Base Sequence, Binding Sites physiology, Cell Line, Escherichia coli genetics, Humans, Lipoproteins genetics, Membrane Proteins genetics, Molecular Sequence Data, Peroxins, Peroxisomes genetics, Protein Structure, Tertiary physiology, Recombinant Proteins genetics, Recombinant Proteins metabolism, Spectrometry, Fluorescence, Surface Plasmon Resonance, Tryptophan genetics, Lipoproteins metabolism, Membrane Proteins metabolism, Peroxisomes metabolism, Tryptophan metabolism
- Abstract
Proteins required for peroxisome biogenesis are termed peroxins. The peroxin Pex3p is a peroxisomal membrane protein (PMP), involved in peroxisomal membrane biogenesis. It acts as a docking receptor for another peroxin Pex19p, which is a specific carrier protein for newly synthesized PMPs. Here we have determined the physicochemical properties and binding manners of Pex3p-Pex19p interaction, in terms of the affinity, the stoichiometry, and the binding site in Pex3p. The cytosolic domain of human Pex3p was overproduced, using an Escherichia coli expression system and was highly purified by two chromatography steps. Gel filtration chromatography analyses and intrinsic tryptophan fluorescence titrations revealed that a one-to-one complex is formed between monomeric Pex3p and monomeric Pex19p. The tryptophan fluorescence spectrum of Pex3p showed a large 18-nm blue shift of the maximum emission wavelength by the binding of Pex19p. This result indicates that either one or two tryptophan residues of Pex3p (Trp-104 and Trp-224) are directly involved in binding to Pex19p. We investigated the binding activities of the wild-type and tryptophan mutants of Pex3p by pull-down assays and surface plasmon resonance analyses. As a result, the wild-type and the W104A and W104F mutants showed K(D) values of 3.4 nm, 1080 nm, and 66.2 nm, respectively. The affinity differences with mutation affected their peroxisome restoring activities in pex3 ZPG208 cells. These findings suggest that the indole ring of Trp-104 directly interacts with Pex19p to facilitate the specific peroxisomal translocation of the Pex19p-PMP complexes.
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- 2008
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27. Creation and X-ray structure analysis of the tumor necrosis factor receptor-1-selective mutant of a tumor necrosis factor-alpha antagonist.
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Shibata H, Yoshioka Y, Ohkawa A, Minowa K, Mukai Y, Abe Y, Taniai M, Nomura T, Kayamuro H, Nabeshi H, Sugita T, Imai S, Nagano K, Yoshikawa T, Fujita T, Nakagawa S, Yamamoto A, Ohta T, Hayakawa T, Mayumi T, Vandenabeele P, Aggarwal BB, Nakamura T, Yamagata Y, Tsunoda S, Kamada H, and Tsutsumi Y
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Crystallography, X-Ray, Humans, Kinetics, L Cells, Mice, Models, Molecular, Protein Conformation, Receptors, Tumor Necrosis Factor, Type I drug effects, Receptors, Tumor Necrosis Factor, Type I genetics, Reverse Transcriptase Polymerase Chain Reaction, Tumor Necrosis Factor-alpha genetics, Receptors, Tumor Necrosis Factor, Type I chemistry, Tumor Necrosis Factor-alpha antagonists & inhibitors
- Abstract
Tumor necrosis factor-alpha (TNF) induces inflammatory response predominantly through the TNF receptor-1 (TNFR1). Thus, blocking the binding of TNF to TNFR1 is an important strategy for the treatment of many inflammatory diseases, such as hepatitis and rheumatoid arthritis. In this study, we identified a TNFR1-selective antagonistic mutant TNF from a phage library displaying structural human TNF variants in which each one of the six amino acid residues at the receptor-binding site (amino acids at positions 84-89) was replaced with other amino acids. Consequently, a TNFR1-selective antagonistic mutant TNF (R1antTNF), containing mutations A84S, V85T, S86T, Y87H, Q88N, and T89Q, was isolated from the library. The R1antTNF did not activate TNFR1-mediated responses, although its affinity for the TNFR1 was almost similar to that of the human wild-type TNF (wtTNF). Additionally, the R1antTNF neutralized the TNFR1-mediated bioactivity of wtTNF without influencing its TNFR2-mediated bioactivity and inhibited hepatic injury in an experimental hepatitis model. To understand the mechanism underlying the antagonistic activity of R1antTNF, we analyzed this mutant using the surface plasmon resonance spectroscopy and x-ray crystallography. Kinetic association/dissociation parameters of the R1antTNF were higher than those of the wtTNF, indicating very fast bond dissociation. Furthermore, x-ray crystallographic analysis of R1antTNF suggested that the mutation Y87H changed the binding mode from the hydrophobic to the electrostatic interaction, which may be one of the reasons why R1antTNF behaved as an antagonist. Our studies demonstrate the feasibility of generating TNF receptor subtype-specific antagonist by extensive substitution of amino acids of the wild-type ligand protein.
- Published
- 2008
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28. Coactivation of the N-terminal transactivation of mineralocorticoid receptor by Ubc9.
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Yokota K, Shibata H, Kurihara I, Kobayashi S, Suda N, Murai-Takeda A, Saito I, Kitagawa H, Kato S, Saruta T, and Itoh H
- Subjects
- Aldosterone metabolism, Animals, COS Cells, Cell Line, Chlorocebus aethiops, Glutathione Transferase metabolism, Humans, Kidney metabolism, Mice, Protein Binding, Protein Structure, Tertiary, Two-Hybrid System Techniques, Receptors, Mineralocorticoid metabolism, Transcriptional Activation, Ubiquitin-Conjugating Enzymes chemistry, Ubiquitin-Conjugating Enzymes physiology
- Abstract
Molecular mechanisms underlying mineralocorticoid receptor (MR)-mediated gene expression are not fully understood. Various transcription factors are post-translationally modified by small ubiquitin-related modifier-1 (SUMO-1). We investigated the role of the SUMO-1-conjugating enzyme Ubc9 in MR transactivation. Yeast two-hybrid, GST-pulldown, and coimmunoprecipitation assays showed that Ubc9 interacted with N-terminal MR-(1-670). Endogenous Ubc9 is associated with stably expressing MR in 293-MR cells. Transient transfection assays in COS-1 cells showed that Ubc9 increased MR transactivation of reporter constructs containing MRE, ENaC, or MMTV promoter in a hormone-sensitive manner. Moreover, reduction of Ubc9 protein levels by small interfering RNA attenuated hormonal activation of a reporter construct as well as an endogenous target gene by MR. A sumoylation-inactive mutant Ubc9(C93S) similarly interacted with MR and potentiated aldosterone-dependent MR transactivation. An MR mutant in which four lysine residues within sumoylation motifs were mutated into arginine (K89R/K399R/K494R/K953R) failed to be sumoylated, but Ubc9 similarly enhanced transactivation by the mutant MR, indicating that sumoylation activity is dispensable for coactivation capacity of Ubc9. Coexpression of Ubc9 and steroid receptor coactivator-1 (SRC-1) synergistically enhanced MR-mediated transactivation in transient transfection assays. Indeed, chromatin immunoprecipitation assays demonstrated that endogenous MR, Ubc9, and SRC-1 were recruited to an endogenous ENaC gene promoter in a largely aldosterone-dependent manner. Coimmunoprecipitation assays showed a complex of MR, Ubc9, and SRC-1 in mammalian cells, and the endogenous proteins were colocalized in the nuclei of the mouse collecting duct cells. These findings support a physiological role of Ubc9 as a transcriptional MR coactivator, beyond the known SUMO E2-conjugating enzyme.
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- 2007
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29. Ubc9 and Protein Inhibitor of Activated STAT 1 Activate Chicken Ovalbumin Upstream Promoter-Transcription Factor I-mediated Human CYP11B2 Gene Transcription.
- Author
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Kurihara I, Shibata H, Kobayashi S, Suda N, Ikeda Y, Yokota K, Murai A, Saito I, Rainey WE, and Saruta T
- Subjects
- Animals, COUP Transcription Factor I, Cell Line, DNA-Binding Proteins metabolism, Enhancer Elements, Genetic, Humans, Promoter Regions, Genetic, Protein Inhibitors of Activated STAT, Proteins analysis, Rats, Small Ubiquitin-Related Modifier Proteins, Trans-Activators metabolism, Transcription Factors metabolism, Transcription, Genetic, Transcriptional Activation, Transfection, Two-Hybrid System Techniques, Ubiquitin-Conjugating Enzymes analysis, Zona Glomerulosa chemistry, Zona Glomerulosa cytology, Cytochrome P-450 CYP11B2 genetics, DNA-Binding Proteins physiology, Proteins metabolism, Transcription Factors physiology, Ubiquitin-Conjugating Enzymes metabolism
- Abstract
Aldosterone synthase (CYP11B2) is involved in the final steps of aldosterone biosynthesis and expressed exclusively in the adrenal zona glomerulosa cells. Using an electrophoretic mobility shift assay, we demonstrate that COUP-TFI binds to the -129/-114 element (Ad5) of human CYP11B2 promoter. Transient transfection in H295R adrenal cells demonstrated that COUP-TFI enhanced CYP11B2 reporter activity. However, the reporter construct with mutated Ad5 sequences showed reduced basal and COUP-TFI-enhanced activity, suggesting that binding of COUP-TFI to Ad5 is important for CYP11B2 transactivation. To elucidate molecular mechanisms of COUP-TFI-mediated activity, we subsequently screened for COUP-TFI-interacting proteins from a human adrenal cDNA library using a yeast two-hybrid system and identified Ubc9 and PIAS1, which have small ubiquitin-related modifier-1 (SUMO-1) conjugase and ligase activities, respectively. The coimmunoprecipitation assays confirmed that COUP-TFI forms a complex with Ubc9 and PIAS1 in mammalian cells. Immunohistochemistry showed that Ubc9 and PIAS1 are markedly expressed in rat adrenal glomerulosa cells. Coexpression of Ubc9 and PIAS1 synergistically enhanced the COUP-TFI-mediated CYP11B2 reporter activity, indicating that both proteins function as coactivators of COUP-TFI. However, sumoylation-defective mutants, Ubc9 (C93S) and PIAS1 (C351S), continued to function as coactivators of COUP-TFI, indicating that sumoylation activity are separable from coactivator ability. In addition, chromatin immunoprecipitation assays demonstrated that ectopically expressed COUP-TFI, Ubc9, and PIAS1 were recruited to an endogenous CYP11B2 promoter. Moreover, reduction of Ubc9 or PIAS1 protein levels by small interfering RNA inhibited the CYP11B2 transactivation by COUP-TFI. Our data support a physiological role of Ubc9 and PIAS1 as transcriptional coactivators in COUP-TFI-mediated CYP11B2 transcription.
- Published
- 2005
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30. Domain architecture and activity of human Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane proteins.
- Author
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Shibata H, Kashiwayama Y, Imanaka T, and Kato H
- Subjects
- ATP-Binding Cassette Transporters metabolism, Blotting, Western, Cell-Free System, Circular Dichroism, DNA, Complementary metabolism, Electrophoresis, Polyacrylamide Gel, Escherichia coli metabolism, Glutathione Transferase metabolism, Humans, Intracellular Membranes metabolism, Membrane Proteins metabolism, Myelin Proteins metabolism, Oligonucleotides chemistry, Peptides chemistry, Plasmids metabolism, Precipitin Tests, Protein Binding, Protein Biosynthesis, Protein Structure, Tertiary, Membrane Proteins chemistry, Molecular Chaperones metabolism, Peroxisomes metabolism
- Abstract
Pex19p is a peroxin involved in peroxisomal membrane biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pex19p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pex19p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex.
- Published
- 2004
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31. The ALG-2-interacting protein Alix associates with CHMP4b, a human homologue of yeast Snf7 that is involved in multivesicular body sorting.
- Author
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Katoh K, Shibata H, Suzuki H, Nara A, Ishidoh K, Kominami E, Yoshimori T, and Maki M
- Subjects
- ATPases Associated with Diverse Cellular Activities, Amino Acid Sequence, Calcium-Binding Proteins physiology, Carrier Proteins physiology, Cell Cycle Proteins, Cell Line, Cytoplasm metabolism, DNA, Complementary metabolism, Endocytosis, Endosomal Sorting Complexes Required for Transport, Endosomes metabolism, Epidermal Growth Factor metabolism, Genes, Dominant, Glutathione Transferase metabolism, HeLa Cells, Humans, Microscopy, Fluorescence, Models, Genetic, Molecular Sequence Data, Phylogeny, Plasmids metabolism, Precipitin Tests, Protein Binding, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Transfection, Two-Hybrid System Techniques, Ubiquitin metabolism, Vacuolar Proton-Translocating ATPases, Vesicular Transport Proteins, Adenosine Triphosphatases chemistry, Calcium-Binding Proteins metabolism, Carrier Proteins chemistry, Carrier Proteins metabolism, Nuclear Proteins chemistry, Repressor Proteins chemistry, Saccharomyces cerevisiae Proteins chemistry
- Abstract
Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca(2+)-binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human AlixDeltaC (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b (chromatin-modifying protein; charged multivesicular body protein), as novel binding partners of Alix. The interaction of Alix with CHMP4b was confirmed by a glutathione S-transferase pull-down assay and by co-immunoprecipitation experiments. Fluorescence microscopic analysis revealed that CHMP4b transiently expressed in HeLa cells mainly exhibited a punctate distribution in the perinuclear area and co-localized with co-expressed Alix. The distribution of CHMP4b partly overlapped the distributions of early and late endosomal marker proteins, EEA1 (early endosome antigen 1) and Lamp-1 (lysosomal membrane protein-1), respectively. Transient overexpression of CHMP4b induced the accumulation of ubiquitinated proteins as punctate patterns that were partly overlapped with the distribution of CHMP4b and inhibited the disappearance of endocytosed epidermal growth factor. In contrast, stably expressed CHMP4b in HEK293 cells was observed diffusely in the cytoplasm. Transient overexpression of AlixDeltaC in stably CHMP4b-expressing cells, however, induced formation of vesicle-like structures in which CHMP4b and AlixDeltaC were co-localized. SKD1(E235Q), a dominant negative form of the AAA type ATPase SKD1 that plays critical roles in the endocytic pathway, was co-immunoprecipitated with CHMP4b. Furthermore, CHMP4b co-localized with SKD1(E235Q) as punctate patterns in the perinuclear area, and Alix was induced to exhibit dot-like distributions overlapped with SKD1(E235Q) in HeLa cells. These results suggest that CHMP4b and Alix participate in formation of multivesicular bodies by cooperating with SKD1.
- Published
- 2003
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32. Insulin recruits GLUT4 from distinct compartments via distinct traffic pathways with differential microtubule dependence in rat adipocytes.
- Author
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Liu LB, Omata W, Kojima I, and Shibata H
- Subjects
- 3-O-Methylglucose pharmacokinetics, Aminopeptidases chemistry, Animals, Biological Transport, Cell Membrane metabolism, Cells, Cultured, Dose-Response Relationship, Drug, Endocytosis, Glucose metabolism, Glucose Transporter Type 4, Immunoblotting, Insulin metabolism, Intracellular Membranes metabolism, Microscopy, Confocal, Microscopy, Fluorescence, Monosaccharide Transport Proteins chemistry, Nocodazole pharmacology, Phosphorylation, Protein Transport, Rats, Rats, Sprague-Dawley, Subcellular Fractions, Time Factors, Trypsin pharmacology, Tubulin metabolism, Vimentin metabolism, Adipocytes metabolism, Microtubules metabolism, Monosaccharide Transport Proteins metabolism, Muscle Proteins
- Abstract
In the present study, we investigated the physiological significance of the microtubules in the subcellular localization and trafficking of GLUT4 in rat primary adipocytes. Morphological and biochemical analyses revealed a dose- and time-dependent disruption of the microtubules by treatment with nocodazole. With nearly complete disruption of the microtubules, the insulin-stimulated glucose transport activity was inhibited by 55%. This inhibition was concomitant with a comparable inhibition of GLUT4 translocation measured by the subcellular fractionation and the cell-surface GLUT4 labeling by trypsin cleavage. In addition, the time-course of insulin stimulation of the glucose transport activity was significantly delayed by microtubule disruption (t(1/2) were 7 and 2.3 min in nocodazole-treated and control cells, respectively), while the rate of GLUT4 endocytosis was little affected. The impaired insulin-stimulated glucose transport activity was not fully restored to the level in control cells by blocking GLUT4 endocytosis, suggesting that the inhibition was due to the existence of a microtubule-dependent subpopulation in the insulin-responsive GLUT4 pool. On the other hand, nocodazole partially inhibited insulin-induced translocation of the insulin-regulated aminopeptidase and the vesicle-associated membrane protein (VAMP)-2 without affecting GLUT1 and VAMP-3. In electrically permeabilized adipocytes, the insulin-stimulated glucose transport was inhibited by 40% by disruption of the microtubules whereas that stimulated with GTP gamma S was not affected. Intriguingly, the two reagents stimulated glucose transport to the comparable level by disruption of the microtubules. These data suggest that insulin recruits GLUT4 to the plasma membrane from at least two distinct intracellular compartments via distinct traffic routes with differential microtubule dependence in rat primary adipocytes.
- Published
- 2003
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33. Direct interaction of Rab4 with syntaxin 4.
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Li L, Omata W, Kojima I, and Shibata H
- Subjects
- Adipocytes drug effects, Animals, Cell Compartmentation, Cells, Cultured, Guanosine 5'-O-(3-Thiotriphosphate) pharmacology, Guanosine Diphosphate pharmacology, Guanosine Triphosphate metabolism, Insulin pharmacology, Kinetics, Membrane Proteins chemistry, Mutation, Precipitin Tests, Protein Conformation, Qa-SNARE Proteins, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins metabolism, Syntaxin 1, Thionucleotides pharmacology, rab4 GTP-Binding Proteins genetics, Adipocytes metabolism, Guanosine Diphosphate analogs & derivatives, Membrane Proteins metabolism, rab4 GTP-Binding Proteins metabolism
- Abstract
In the present study, we examined the possible interaction between Rab4 and syntaxin 4, both having been implicated in insulin-induced GLUT4 translocation. Rab4 and syntaxin 4 were coimmunoprecipitated from the lysates of electrically permeabilized rat adipocytes. The interaction between the two proteins was reduced by insulin treatment and increased by the addition of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). An in vitro binding assay revealed that the bacterially expressed Rab4 was bound to a glutathione S-transferase fusion protein containing the cytoplasmic domain of syntaxin 4 (GST-syntaxin 4-(1-273)) but not to syntaxin 1A or vesicle-associated membrane protein-2. The interaction between Rab4 and syntaxin 4 seemed to be regulated by the guanine nucleotide status of Rab4, because 1) GTPgammaS treatment of the cells significantly increased, but guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) treatment decreased the amount of Rab4 pulled down with GST-syntaxin 4-(1-273) from the cell lysates; 2) GTPgammaS loading on Rab4 caused a marked increase in the affinity of Rab4 to syntaxin 4 whereas GDPbetaS loading had little effect; and 3) a GTPase-deficient mutant of Rab4 (Rab4(Q67L)), but not a GTP-binding-defective mutant (Rab4(S22N)), was bound to GST-syntaxin 4-(1-273). Although insulin stimulated [gamma-(32)P]GTP binding to Rab4 in a time-dependent fashion, its effect on the Rab4 interaction with syntaxin 4 was apparently biphasic; an initial increase in Rab4 associated with syntaxin 4 was followed by a gradual dissociation of the GTPase from syntaxin 4. Finally, the binding of Rab4(Q67L) to GST-syntaxin 4-(1-273) was inhibited by munc-18c in a dose-dependent manner, indicating that GTP-loaded Rab4 binds to syntaxin 4 in the open conformation. These results suggest that 1) Rab4 interacts with syntaxin 4 in a direct and specific manner, and 2) the interaction is regulated by the guanine nucleotide status of Rab4 as well as by the conformational status of syntaxin 4.
- Published
- 2001
- Full Text
- View/download PDF
34. MOdified nucleosides of nuclear and nucleolar low molecular weight ribonucleic acid.
- Author
-
Reddy R, Ro-Choi TS, Henning D, Shibata H, Choi YC, and Busch H
- Subjects
- Adenine analysis, Animals, Carcinoma, Hepatocellular metabolism, Chromatography, Gel, Chromatography, Thin Layer, Cytosine analysis, Electrophoresis, Polyacrylamide Gel, Glycols analysis, Guanine analysis, Hypoxanthines analysis, Liver Neoplasms, Male, Molecular Weight, Neoplasms, Experimental metabolism, Rats, Thymine analysis, Uracil analysis, Cell Nucleolus analysis, Cell Nucleus analysis, Nucleosides analysis, RNA, Neoplasm analysis
- Published
- 1972
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