34 results on '"Endo, Y."'
Search Results
2. RNA aptamers that bind to and inhibit the ribosome-inactivating protein, pepocin.
- Author
-
Hirao, I, Madin, K, Endo, Y, Yokoyama, S, and Ellington, A D
- Abstract
Pepocin, isolated from Cucurbita pepo, is a ribosome-inactivating protein (RIP). RIPs site-specifically recognize and depurinate an adenosine at position 4324 in rat 28 S rRNA, rendering the ribosome incapable of interacting with essential elongation factors. Aptamers that target pepocin were isolated from a degenerate RNA pool by in vitro selection. A conserved hairpin motif, quite different from the sequence of the toxin-substrate domain in rat 28 S rRNA, was identified in the aptamer sequences. The aptamers selectively bind to pepocin with dissociation constants between 20 and 30 nM and inhibit the N-glycosidase activity of pepocin on rat liver 28 S rRNA. Competitive binding experiments using aptamer variants suggest that the conserved hairpin region in the anti-pepocin aptamer binds near the catalytic site on pepocin and prevents the interaction of pepocin and 28 S rRNA. Anti-RIP aptamers have potential use in diagnostic systems for the detection of pepocin or could be used as therapy to prevent the action of pepocin in mammalian cells.
- Published
- 2000
3. Ribonuclease activity of rat liver perchloric acid-soluble protein, a potent inhibitor of protein synthesis.
- Author
-
Morishita, R, Kawagoshi, A, Sawasaki, T, Madin, K, Ogasawara, T, Oka, T, and Endo, Y
- Abstract
Rat liver perchloric acid-soluble protein (L-PSP) is a potent inhibitor of cell-free protein synthesis; however, its mechanism of action is not known. Here we show that the protein is a unique ribonuclease and that this activity is responsible for the inhibition of translation. The addition of perchloric acid-soluble protein to a rabbit reticulocyte cell-free system at a concentration of 6.2 microM led to an almost complete inhibition of protein synthesis. The kinetics are unlike those of hemin-controlled inhibitor, a protein that acts at the initiation step. The inhibition appears to be due to an endoribonucleolytic activity of perchloric acid-soluble protein because L-PSP directly affects mRNA template activity and induces disaggregation of the reticulocyte polysomes into 80 S ribosomes, even in the presence of cycloheximide. These effects were observed with authentic as well as recombinant L-PSP. Analysis by thin-layer chromatography of [alpha-32P]UTP-labeled mRNA incubated with the protein showed production of the ribonucleoside 3'-monophosphates Ap, Gp, Up, and Cp, providing direct evidence that the protein is an endoribonuclease. When either 5'- or 3'-32P-labeled 5 S rRNA was the substrate, L-PSP cleaved phosphodiester bonds only in the single-stranded regions of the molecule.
- Published
- 1999
4. Brain-derived neurotrophic factor induces rapid and transient release of glutamate through the non-exocytotic pathway from cortical neurons.
- Author
-
Takei, N, Numakawa, T, Kozaki, S, Sakai, N, Endo, Y, Takahashi, M, and Hatanaka, H
- Abstract
There is increasing interest in the involvement of neurotrophins in neural transmission and plasticity. Thus, we investigated the effects of brain-derived neurotrophic factor (BDNF) on glutamate release from cortical neurons. Treatment of cultured cortical neurons with BDNF induced rapid and transient release of glutamate. This effect was suggested to be mediated by TrkB activation because K252a inhibited the release of glutamate and BDNF phosphorylated TrkB within 30 s. BDNF-induced glutamate release was observed even when using Ca2+-free assay buffer but was inhibited by BAPTA-AM, a cell-permeable Ca2+ chelator. Therefore, BDNF-induced glutamate release was independent of extracelluar Ca2+ but dependent on intracellular Ca2+. Because normal neurotransmitter release is exocytotic, the involvement of the exocytotic pathway in BDNF-induced glutamate release was examined. As botulinum toxin is known to cleave exocytosis-associated proteins, thereby inhibiting exocytosis, it was applied to neurons prior to the release assay. Although botulinum toxin B cleaved VAMP2 and inhibited Ca2+-triggered glutamate release, it did not inhibit the BDNF-induced release of glutamate. These results strongly suggested that BDNF induces rapid and transient release of glutamate from cortical neurons through a non-exocytotic pathway.
- Published
- 1998
5. Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines.
- Author
-
Tanaka, N, Kaneko, K, Asao, H, Kasai, H, Endo, Y, Fujita, T, Takeshita, T, and Sugamura, K
- Abstract
STAM containing an SH3 (Src homology 3) domain and an immunoreceptor tyrosine-based activation motif was previously revealed to be implicated in signaling pathways immediately downstream of Jak2 and Jak3 tyrosine kinases associated with cytokine receptors. We molecularly cloned a novel molecule interacting with the SH3 domain of STAM, which was named AMSH (associated molecule with the SH3 domain of STAM). AMSH contains a putative bipartite nuclear localization signal and a homologous region of a c-Jun activation domain-binding protein 1 (JAB1) subdomain in addition to a binding site for the SH3 domain of STAM. AMSH mutant deleted of the C-terminal half conferred dominant negative effects on signaling for DNA synthesis and c-myc induction mediated by interleukin 2 and granulocyte macrophage-colony-stimulating factor. These results suggest that AMSH plays a critical role in the cytokine-mediated intracellular signal transduction downstream of the Jak2/Jak3.STAM complex.
- Published
- 1999
6. GPI1 stabilizes an enzyme essential in the first step of glycosylphosphatidylinositol biosynthesis.
- Author
-
Hong, Y, Ohishi, K, Watanabe, R, Endo, Y, Maeda, Y, and Kinoshita, T
- Abstract
Attachment of glycosylphosphatidylinositol (GPI) is essential for the surface expression of many proteins. Biosynthesis of glycosylphosphatidylinositol is initiated by the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol. In mammalian cells, this reaction is mediated by a complex of PIG-A, PIG-H, PIG-C, and GPI1. This complexity may be relevant for regulation and for usage of a particular phosphatidylinositol. However, the functions of the respective components have been unclear. Here we cloned the mouse GPI1 gene and disrupted it in F9 embryonal carcinoma cells. Disruption of the GPI1 gene caused a severe but not complete defect in the generation of glycosylphosphatidylinositol-anchored proteins, indicating some residual biosynthetic activity. A complex of PIG-A, PIG-H, and PIG-C decreased to a nearly undetectable level, whereas a complex of PIG-A and PIG-H was easily detected. A lack of GPI1 also caused partial decreases of PIG-C and PIG-H. Therefore, GPI1 stabilizes the enzyme by tying up PIG-C with a complex of PIG-A and PIG-H.
- Published
- 1999
7. Characterization of a novel rat brain glycosylphosphatidylinositol-anchored protein (Kilon), a member of the IgLON cell adhesion molecule family.
- Author
-
Funatsu, N, Miyata, S, Kumanogoh, H, Shigeta, M, Hamada, K, Endo, Y, Sokawa, Y, and Maekawa, S
- Abstract
In the central nervous system, many cell adhesion molecules are known to participate in the establishment and remodeling of the neural circuit. Some of the cell adhesion molecules are known to be anchored to the membrane by the glycosylphosphatidylinositol (GPI) inserted to their C termini, and many GPI-anchored proteins are known to be localized in a Triton-insoluble membrane fraction of low density or so-called "raft." In this study, we surveyed the GPI-anchored proteins in the Triton-insoluble low density fraction from 2-week-old rat brain by solubilization with phosphatidylinositol-specific phospholipase C. By Western blotting and partial peptide sequencing after the deglycosylation with peptide N-glycosidase F, the presence of Thy-1, F3/contactin, and T-cadherin was shown. In addition, one of the major proteins, having an apparent molecular mass of 36 kDa after the peptide N-glycosidase F digestion, was found to be a novel protein. The result of cDNA cloning showed that the protein is an immunoglobulin superfamily member with three C2 domains and has six putative glycosylation sites. Since this protein shows high sequence similarity to IgLON family members including LAMP, OBCAM, neurotrimin, CEPU-1, AvGP50, and GP55, we termed this protein Kilon (a kindred of IgLON). Kilon-specific monoclonal antibodies were produced, and Western blotting analysis showed that expression of Kilon is restricted to brain, and Kilon has an apparent molecular mass of 46 kDa in SDS-polyacrylamide gel electrophoresis in its expressed form. In brain, the expression of Kilon is already detected in E16 stage, and its level gradually increases during development. Kilon immunostaining was observed in the cerebral cortex and hippocampus, in which the strongly stained puncta were observed on dendrites and soma of pyramidal neurons.
- Published
- 1999
8. The RNA N-glycosidase activity of ricin A-chain. The characteristics of the enzymatic activity of ricin A-chain with ribosomes and with rRNA.
- Author
-
Endo, Y and Tsurugi, K
- Abstract
Ricin A-chain cleaves the N-glycosidic bond at A-4324 in 28 S rRNA when intact rat ribosomes are the substrate. Cleavage occurs at a concentration of the toxin of 1 X 10(-10) M, and specificity for this single residue is retained when the concentration is as high as 3 X 10(-7) M. The apparent Michaelis constant (Km) for the reaction is 2.6 microM, and the turnover number (Kcat) is 1777 min-1. The same N-glycosidic bond is cleaved by ricin A-chain in naked 28 S rRNA, but at a greatly reduced rate. The Km value for this reaction is 5.8 microM. The results suggest that the A-chain has a similar affinity for 28 S rRNA in ribosomes and in the absence of ribosomal proteins. Ricin A-chain has no effect on 23 S rRNA in Escherichia coli ribosomes, however, the N-glycosidic bond at A-2600 in naked 23 S rRNA is cleaved by the toxin; this corresponds to the ricin site in eukaryotic 28 S rRNA. Since the Km value (3.3 microM) for the reaction with E. coli 23 S rRNA approximates that obtained with rat liver ribosomes, it is possible that E. coli ribosomal protein(s) protect this site against ricin attack in intact ribosomes. Ricin A-chain also acted on naked 16 S rRNA cleaving the N-glycosidic bond of adenine at position 1014. The results suggest that ricin A-chain recognizes a specific structure in rRNA, perhaps a loop and stem having the sequence GAGA in the loop.
- Published
- 1988
- Full Text
- View/download PDF
9. Exon/intron organization, chromosome localization, alternative splicing, and transcription units of the human apolipoprotein E receptor 2 gene.
- Author
-
Kim, D H, Magoori, K, Inoue, T R, Mao, C C, Kim, H J, Suzuki, H, Fujita, T, Endo, Y, Saeki, S, and Yamamoto, T T
- Abstract
Apolipoprotein E receptor 2 is a recently identified receptor that resembles low and very low density lipoprotein receptors. Isolation and characterization of genomic clones encoding human apolipoprotein E receptor 2 revealed that the gene spans approximately 60 kilobases and contains 19 exons. The positions of the exon/intron boundaries of the gene are almost identical to those of low and very low density lipoprotein receptors. Fluorescent in situ hybridization of human chromosomes revealed that the gene is located on chromosome 1p34. Isolation of a cDNA encoding a variant receptor and reverse transcription-polymerase chain reaction indicate the presence of multiple variants with different numbers of cysteine-rich repeats in the binding domain of the receptor. We also found a variant receptor lacking a 59-amino acid insertion in the cytoplasmic domain. The transcription start site was mapped to the position 236 base pairs upstream of the AUG translation initiator codon by primer extension analysis. Sequence inspection of the 5'-flanking region revealed potential DNA elements: AP-2, GC factor, PEA3, and Sp1. The minimal promoter region and a region required for nerve growth factor inducibility in PC12 cells were also determined.
- Published
- 1997
10. The ribonuclease activity of the cytotoxin alpha-sarcin. The characteristics of the enzymatic activity of alpha-sarcin with ribosomes and ribonucleic acids as substrates.
- Author
-
Endo, Y, Huber, P W, and Wool, I G
- Abstract
The ribonuclease activity of the cytotoxic protein alpha-sarcin has been characterized. When rat liver ribosomes or 60 S ribosomal subunits were the substrates, alpha-sarcin cleaved a single oligonucleotide of about 488 residues, the alpha-fragment, from the 3' end of 28 S rRNA. In contrast, 40 S ribosomal subunits were not affected by alpha-sarcin. The alpha-fragment was cleaved from 28 S rRNA in 80 S ribosomes when the concentration of alpha-sarcin was 3 x 10(-8) M and the toxin retained its specificity even when the concentration was 3 x 10(-5) M. The turnover number (kcat) for the reaction of alpha-sarcin with ribosomes was 55 min-1, establishing that the toxin acts catalytically. When total rRNA or 28 S rRNA was the substrate, alpha-sarcin caused extensive progressive digestion of the nucleic acids; however, no formation of the alpha-fragment occurred. The extent of the digestion of 28 S rRNA was related to the concentration of alpha-sarcin, but the amount of the toxin required was somewhat greater than that needed with ribosomes. Digestion of homopolynucleotides with alpha-sarcin indicated that the protein is specific for purines. When [32P]5 S rRNA was the substrate, alpha-sarcin cleaved on the 3' side of purines in both single- and double-stranded regions of the molecule. The results suggest that the unusual specificity of alpha-sarcin, in that it cleaves only one of more than 7000 phosphodiester bonds in the ribosome, is a property both of the cytotoxin and of the ribosome.
- Published
- 1983
- Full Text
- View/download PDF
11. Hrs is associated with STAM, a signal-transducing adaptor molecule. Its suppressive effect on cytokine-induced cell growth.
- Author
-
Asao, H, Sasaki, Y, Arita, T, Tanaka, N, Endo, K, Kasai, H, Takeshita, T, Endo, Y, Fujita, T, and Sugamura, K
- Abstract
We previously reported a new type of signal-transducing adaptor molecule, STAM, which was shown to be involved in cytokine-mediated intracellular signal transduction. In this study, we molecularly cloned a 110-kDa phosphotyrosine protein inducible by stimulation with interleukin 2 (IL-2). The 110-kDa molecule was found to be a human counterpart of mouse Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and to be associated with STAM. Tyrosine phosphorylation of Hrs is induced rapidly after stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor as well as hepatocyte growth factor. The mutual association sites of Hrs and STAM include highly conserved coiled-coil sequences, suggesting that their association is mediated by the coiled-coil structures. Exogenous introduction of the wild-type Hrs significantly suppressed DNA synthesis upon stimulation with IL-2 and granulocyte-macrophage colony-stimulating factor, while the Hrs mutant deleted of the STAM-binding site lost such suppressive ability. These results suggest that Hrs counteracts the STAM function which is critical for cell growth signaling mediated by the cytokines.
- Published
- 1997
12. A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin.
- Author
-
Matsushita, M, Endo, Y, Taira, S, Sato, Y, Fujita, T, Ichikawa, N, Nakata, M, and Mizuochi, T
- Abstract
Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens. We report here a novel Ca(2+)-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence. When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins. P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains. The latter contained two potential Ca(2+)-binding sites that may be involved in carbohydrate binding. The overall sequence of P35 was highly homologous to porcine ficolins alpha and beta. Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver. P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region. P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides. Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.
- Published
- 1996
13. The cytotoxins alpha-sarcin and ricin retain their specificity when tested on a synthetic oligoribonucleotide (35-mer) that mimics a region of 28 S ribosomal ribonucleic acid.
- Author
-
Endo, Y, Chan, Y L, Lin, A, Tsurugi, K, and Wool, I G
- Abstract
An oligoribonucleotide (35-mer) that mimics the alpha-sarcin and the ricin region of eukaryotic 28 S rRNA was transcribed in vitro from a synthetic template with T7 RNA polymerase and was used to test whether the specificity of the hydrolysis by the toxins was retained. alpha-Sarcin, at a low concentration, cleaved a single phosphodiester bond on the 3' side of a guanosine residue in the synthetic oligomer that corresponds to G-4325 in 28 S rRNA, the site of action of the toxin in intact ribosomes. At a high concentration of alpha-sarcin, the substrate (35-mer) was hydrolyzed after each of its purines. alpha-Sarcin was without an effect on a synthetic RNA (20-mer) that reproduces the near universal sequence of nucleotides in the loop, but lacks the stem, of the toxin's domain. Thus, the specificity of the attack of alpha-sarcin on a precise region of 28 S rRNA appears to be contingent on the sequence of the nucleotides and the structure of the domain. Ricin depurinated a nucleotide in the synthetic oligomer (35-mer), and in the presence of aniline the phosphoribose backbone was cleaved at a position that conforms to A-4324 in 28 S rRNA, the site of action of the toxin in vivo.
- Published
- 1988
- Full Text
- View/download PDF
14. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins.
- Author
-
Endo, Y., Mitsui, K., Motizuki, M., and Tsurugi, K.
- Abstract
Ricin is a potent cytotoxic protein derived from the higher plant Ricinus communis that inactivates eukaryotic ribosomes. In this paper we have studied the mechanism of action of ricin A-chain on rat liver ribosomes in vitro. Our findings indicate that the toxin inactivates the ribosomes by modifying both or either of two nucleoside residues, G4323 and A4324, in 28 S rRNA. These nucleotides are located close to the alpha-sarcin cleavage site and become resistant to all ribonucleases tested. The examination of the lability of phosphodiester bonds of these nucleotides to both mild alkaline digestion and aniline treatment at acidic pH suggests that the base of A4324 is removed by the toxin. This unique activity of ricin A-chain was also observed when naked 28 S rRNA is used as a substrate, indicating that the toxin directly acts on the RNA. Similar activity on 28 S rRNA is also exhibited by abrin and modeccin, ricin-related toxins, suggesting a general mechanistic pathway for ribosome inactivation by lectin toxins.
- Published
- 1987
- Full Text
- View/download PDF
15. Location of 5.8 S rRNA contact sites in 28 S rRNA and the effect of alpha-sarcin on the association of 5.8 S rRNA with 28 S rRNA.
- Author
-
Walker, T A, Endo, Y, Wheat, W H, Wool, I G, and Pace, N R
- Abstract
We have constructed phage M13mp7 clones each containing the coding strand from one of three restriction fragments which collectively span the mouse 28 S rRNA gene with the exception of the 3'-terminal approximately 500 base pairs. When hybridized to 28 S rRNA, only the fragment containing the 5'-terminal 1400 nucleotides of the gene inhibited the annealing of 5.8 S rRNA to the 28 S rRNA. The same results were obtained when either the 5'- or 3'-terminal fragment of 5.8 S rRNA was used in lieu of intact 5.8 S rRNA, each of which had been shown to contain an independent 28 S rRNA contact site. However, alpha-sarcin, a cytotoxin that inhibits protein synthesis by hydrolyzing a phosphodiester bond near the 3' end of 28 S rRNA, produces a 3'-terminal 488-nucleotide fragment which exhibits a marginal capacity to anneal to 5.8 S rRNA. These results indicate that 5.8 S rRNA interacts predominantly with a structural domain near the 5' end of 28 S rRNA. This conclusion is consistent with base-pairing interactions between 5.8 S rRNA and 28 S rRNA based on the proposed secondary structures for Escherichia coli 23 S and yeast 26 S rRNAs. However, alpha-sarcin treatment of ribosomes affects the stability of the binding of 5.8 S rRNA to the 28 S rRNA, even though the toxin hydrolyzes a phosphodiester bond several thousand nucleotides from the proposed contact regions. Finally, mouse 5.8 S rRNA was shown to lack two internal nucleotides reported to be present in rat 5.8 S rRNA.
- Published
- 1983
- Full Text
- View/download PDF
16. The site of action of alpha-sarcin on eukaryotic ribosomes. The sequence at the alpha-sarcin cleavage site in 28 S ribosomal ribonucleic acid.
- Author
-
Endo, Y and Wool, I G
- Abstract
alpha-Sarcin is a cytotoxic protein that inhibits eukaryotic elongation factor 1 catalyzed binding of aminoacyl-tRNA to eukaryotic ribosomes as a result of cleaving a fragment from the large RNA in the 60 S ribosomal subunit. Rat liver polysomes are sensitive to alpha-sarcin in vitro only if the treatment is carried out in the presence of EDTA. The oligonucleotide produced by alpha-sarcin action on rat liver ribosomes is derived from the 3' end of 28 S rRNA and has a molecular weight of approximately 161,000 (about 488 bases). The sequence at the site of cleavage (the 5' end of the oligonucleotide produced by alpha-sarcin action on rat liver ribosomes) was AGGAAC for both rat liver 28 S rRNA and yeast 25 S rRNA. Based on the size of the alpha-fragment derived from Escherichia coli ribosomes (about 243 nucleotides), it is likely that a similar sequence, AGGACC, occurs at the site of cleavage of prokaryotic 23 S rRNA. It is also likely that the sequence on the 5' side of the alpha-sarcin cleavage site is UAGUACGAG. The sequence UAGUACGAGAGGAAC must be important because it is conserved and because hydrolysis of a single phosphodiester bond there inactivates the ribosome. We suggest that this region of the large nucleic acid in the 60 S subparticle is involved in binding aminoacyl-tRNA to the ribosome, first, because it is the eukaryotic elongation factor 1 catalyzed reaction that is inhibited by alpha-sarcin, and second, because peptidyl-tRNA seems to protect polysomes against the toxin. The function of EDTA appears to be to remove peptidyl-tRNA since puromycin also sensitizes polysomes to alpha-sarcin.
- Published
- 1982
- Full Text
- View/download PDF
17. Structural studies of the sugar chains of human parotid alpha-amylase.
- Author
-
Yamashita, K., primary, Tachibana, Y., additional, Nakayama, T., additional, Kitamura, M., additional, Endo, Y., additional, and Kobata, A., additional
- Published
- 1980
- Full Text
- View/download PDF
18. RNA N-glycosidase activity of ricin A-chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes.
- Author
-
Endo, Y, primary and Tsurugi, K, additional
- Published
- 1987
- Full Text
- View/download PDF
19. The sequence of the nucleotides at the alpha-sarcin cleavage site in rat 28 S ribosomal ribonucleic acid.
- Author
-
Chan, Y L, primary, Endo, Y, additional, and Wool, I G, additional
- Published
- 1983
- Full Text
- View/download PDF
20. Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells.
- Author
-
Koizumi M, Kama Y, Hirano KI, Endo Y, Tanaka T, Hozumi K, and Hosokawa H
- Subjects
- Chromatography, Liquid, Tandem Mass Spectrometry, Hematopoietic Stem Cells metabolism, Cell Differentiation, LIM Domain Proteins genetics, LIM Domain Proteins metabolism, Lymphoid Progenitor Cells physiology, Transcription Factors genetics, Transcription Factors metabolism
- Abstract
Hematopoietic stem and progenitor cells can differentiate into all types of blood cells. Regulatory mechanisms underlying pluripotency in progenitors, such as the ability of lymphoid progenitor cells to differentiate into T-lineage, remain unclear. We have previously reported that LIM domain only 2 (Lmo2), a bridging factor in large transcriptional complexes, is essential to retain the ability of lymphoid progenitors to differentiate into T-lineage. However, biochemical characterization of Lmo2 protein complexes in physiological hematopoietic progenitors remains obscure. Here, we identified approximately 600 Lmo2-interacting molecules in a lymphoid progenitor cell line by two-step affinity purification with LC-MS/MS analysis. Zinc finger and BTB domain containing 1 (Zbtb1) and CBFA2/RUNX1 partner transcriptional corepressor 3 (Cbfa2t3) were found to be the functionally important binding partners of Lmo2. We determined CRISPR/Cas9-mediated acute disruption of Zbtb1 or Cbfa2t3 in the lymphoid progenitor or bone marrow-derived primary hematopoietic progenitor cells causes significant defects in the initiation of T-cell development when Notch signaling is activated. Our transcriptome analysis of Zbtb1- or Cbfa2t3-deficient lymphoid progenitors revealed that Tcf7 was a common target for both factors. Additionally, ChIP-seq analysis showed that Lmo2, Zbtb1, and Cbfa2t3 cobind to the Tcf7 upstream enhancer region, which is occupied by the Notch intracellular domain/RBPJ transcriptional complex after Notch stimulation, in lymphoid progenitors. Moreover, transduction with Tcf7 restored the defect in the T-lineage potential of Zbtb1-deficient lymphoid progenitors. Thus, in lymphoid progenitors, the Lmo2/Zbtb1/Cbfa2t3 complex directly binds to the Tcf7 locus and maintains responsiveness to the Notch-mediated inductive signaling to facilitate T-lineage differentiation., 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
- Full Text
- View/download PDF
21. HYBID (alias KIAA1199/CEMIP) and hyaluronan synthase coordinately regulate hyaluronan metabolism in histamine-stimulated skin fibroblasts.
- Author
-
Yoshida H, Aoki M, Komiya A, Endo Y, Kawabata K, Nakamura T, Sakai S, Sayo T, Okada Y, and Takahashi Y
- Subjects
- Cell Line, Fibroblasts drug effects, Gene Expression Regulation drug effects, Humans, Hyaluronan Synthases genetics, Hyaluronoglucosaminidase genetics, Mast Cells drug effects, Mast Cells metabolism, Molecular Weight, Phosphatidylinositol 3-Kinases metabolism, Protein Kinase C-delta metabolism, Proto-Oncogene Proteins c-akt metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, Receptors, Histamine metabolism, Signal Transduction drug effects, Skin Aging drug effects, Time Factors, Fibroblasts metabolism, Histamine pharmacology, Hyaluronan Synthases metabolism, Hyaluronic Acid metabolism, Hyaluronoglucosaminidase metabolism, Skin cytology
- Abstract
The immune-regulatory compound histamine is involved in the metabolism of the essential skin component hyaluronan (HA). We previously reported that histamine up-regulates the expression of HYBID (hyaluronan-binding protein involved in hyaluronan depolymerization, also called CEMIP or KIAA1199), which plays a key role in HA degradation. However, no information is available about histamine's effects on HA synthase (HAS) expression, the molecular sizes of HA species produced, and histamine receptors and their signaling pathways in skin fibroblasts. Moreover, histamine's effects on photoaged skin remain elusive. Here, we show that histamine increases HA degradation by up-regulating HYBID and down-regulating HAS2 in human skin fibroblasts in a dose- and time-dependent manner and thereby decreases the total amounts and sizes of newly produced HA. Histamine H1 blocker abrogated the histamine effects on HYBID up-regulation, HAS2 suppression, and HA degradation. Histamine H1 agonist exhibited effects on HA levels, composition, and breakdown similar to those of histamine. Of note, blockade of protein kinase Cδ or PI3K-Akt signaling abolished histamine-mediated HYBID stimulation and HAS2 suppression, respectively. Immunohistochemical experiments revealed a significant ∼2-fold increase in tryptase-positive mast cells in photoaged skin, where HYBID and HAS2 expression levels were increased and decreased, respectively, compared with photoprotected skin. These results indicate that histamine controls HA metabolism by up-regulating HYBID and down-regulating HAS2 via distinct signaling pathways downstream of histamine receptor H1. They further suggest that histamine may contribute to photoaged skin damage by skewing HA metabolism toward degradation., Competing Interests: The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Yoshida et al.)
- Published
- 2020
- Full Text
- View/download PDF
22. Benzolactam-related compounds promote apoptosis of HIV-infected human cells via protein kinase C-induced HIV latency reversal.
- Author
-
Matsuda K, Kobayakawa T, Tsuchiya K, Hattori SI, Nomura W, Gatanaga H, Yoshimura K, Oka S, Endo Y, Tamamura H, Mitsuya H, and Maeda K
- Subjects
- CD4-Positive T-Lymphocytes pathology, CD4-Positive T-Lymphocytes virology, Caspase 3 biosynthesis, Caspase 3 genetics, Cell Cycle Proteins, Female, Gene Expression Regulation, Enzymologic drug effects, HIV Infections genetics, HIV Infections pathology, Humans, Male, Nuclear Proteins antagonists & inhibitors, Nuclear Proteins genetics, Nuclear Proteins metabolism, Protein Kinase C genetics, Transcription Factors antagonists & inhibitors, Transcription Factors genetics, Transcription Factors metabolism, Apoptosis drug effects, Benzodiazepinones pharmacology, CD4-Positive T-Lymphocytes metabolism, HIV Infections metabolism, HIV-1 physiology, Protein Kinase C metabolism, Virus Latency drug effects
- Abstract
Latency-reversing agents (LRAs) are considered a potential strategy for curing cells of HIV-1 infection. Certain protein kinase C (PKC) activators have been previously reported to be LRAs because they can reverse HIV latency. In the present study, we examined the activities of a panel of benzolactam derivatives against cells latently infected with HIV. Using determination of p24 antigen in cell supernatants or altered intracellular GFP expression to measure HIV reactivation from latently infected cells along with a cytotoxicity assay, we found that some of the compounds exhibited latency-reversing activity, which was followed by enhanced release of HIV particles from the cells. One derivative, BL-V8-310, displayed activity in ACH-2 and J-Lat cells latently infected with HIV at a concentration of 10 nm or higher, which was superior to the activity of another highly active PKC activator, prostratin. These results were confirmed with peripheral blood cells from HIV-infected patients. We also found that these drugs up-regulate the expression of caspase 3 and enhance apoptosis specifically in latently HIV-infected cells. Moreover, combining BL-V8-310 with a bromodomain-containing 4 (BRD4) inhibitor, JQ1, not only enhanced HIV latency-reversing activity, but also reduced the effect on cytotoxic cytokine secretion from CD4
+ T-cells induced by BL-V8-310 alone. Our results suggest that BL-V8-310 and its related benzolactam derivatives are potential LRA lead compounds that are effective in reversing HIV latency and reducing viral reservoirs in HIV-positive individuals with few adverse effects.- Published
- 2019
- Full Text
- View/download PDF
23. Methylation of Gata3 protein at Arg-261 regulates transactivation of the Il5 gene in T helper 2 cells.
- Author
-
Hosokawa H, Kato M, Tohyama H, Tamaki Y, Endo Y, Kimura MY, Tumes DJ, Motohashi S, Matsumoto M, Nakayama KI, Tanaka T, and Nakayama T
- Subjects
- Amino Acid Sequence, Animals, Arginine metabolism, Blotting, Western, Cell Differentiation, Cell Proliferation, Cells, Cultured, Chaperonin 60 antagonists & inhibitors, Chaperonin 60 genetics, Chaperonin 60 metabolism, Chromatin Assembly and Disassembly, Chromatin Immunoprecipitation, Cytokines genetics, Cytokines metabolism, Flow Cytometry, Fluorescent Antibody Technique, GATA3 Transcription Factor metabolism, Immunoprecipitation, Interleukin-5 metabolism, Mice, Mice, Inbred C57BL, Mitochondrial Proteins antagonists & inhibitors, Mitochondrial Proteins genetics, Mitochondrial Proteins metabolism, Molecular Sequence Data, Promoter Regions, Genetic genetics, RNA, Messenger genetics, RNA, Small Interfering genetics, Real-Time Polymerase Chain Reaction, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Th1 Cells immunology, Th1 Cells metabolism, Th2 Cells immunology, Arginine genetics, DNA Methylation, GATA3 Transcription Factor genetics, Interleukin-5 genetics, Th2 Cells metabolism, Transcriptional Activation
- Abstract
Gata3 acts as a master regulator for T helper 2 (Th2) cell differentiation by inducing chromatin remodeling of the Th2 cytokine loci, accelerating Th2 cell proliferation, and repressing Th1 cell differentiation. Gata3 also directly transactivates the interleukin-5 (Il5) gene via additional mechanisms that have not been fully elucidated. We herein identified a mechanism whereby the methylation of Gata3 at Arg-261 regulates the transcriptional activation of the Il5 gene in Th2 cells. Although the methylation-mimicking Gata3 mutant retained the ability to induce IL-4 and repress IFNγ production, the IL-5 production was selectively impaired. We also demonstrated that heat shock protein (Hsp) 60 strongly associates with the methylation-mimicking Gata3 mutant and negatively regulates elongation of the Il5 transcript by RNA polymerase II. Thus, arginine methylation appears to play a pivotal role in the organization of Gata3 complexes and the target gene specificity of Gata3., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
24. Mitochondria and the lectin pathway of complement.
- Author
-
Brinkmann CR, Jensen L, Dagnæs-Hansen F, Holm IE, Endo Y, Fujita T, Thiel S, Jensenius JC, and Degn SE
- Subjects
- Animals, Blood Proteins metabolism, Complement C3 metabolism, Complement C4 metabolism, Humans, Interleukin-6 blood, Lectins genetics, Lectins metabolism, Lipopolysaccharides pharmacology, Lung immunology, Lung pathology, Mannose-Binding Lectin metabolism, Mannose-Binding Protein-Associated Serine Proteases physiology, Mice, Mice, Inbred C57BL, Mice, Knockout, Mitochondria, Liver metabolism, Protein Binding, Rats, Receptors, Pattern Recognition metabolism, Ficolins, Complement Pathway, Mannose-Binding Lectin, Mitochondria, Liver immunology
- Abstract
Mitochondria, the powerhouses of our cells, are remnants of a eubacterial endosymbiont. Notwithstanding the evolutionary time that has passed since the initial endosymbiotic event, mitochondria have retained many hallmarks of their eubacterial origin. Recent studies have indicated that during perturbations of normal homeostasis, such as following acute trauma leading to massive necrosis and release of mitochondria, the immune system might mistake symbiont for enemy and initiate an inappropriate immune response. The innate immune system is the first line of defense against invading microbial pathogens, and as such is the primary suspect in the recognition of mitochondria-derived danger-associated molecular patterns and initiation of an aberrant response. Conversely, innate immune mechanisms are also central to noninflammatory clearance of innocuous agents. Here we investigated the role of a central humoral component of innate immunity, the lectin pathway of complement, in recognition of mitochondria in vitro and in vivo. We found that the soluble pattern recognition molecules, mannan-binding lectin (MBL), L-ficolin, and M-ficolin, were able to recognize mitochondria. Furthermore, MBL in complex with MBL-associated serine protease 2 (MASP-2) was able to activate the lectin pathway and deposit C4 onto mitochondria, suggesting that these molecules are involved either in homeostatic clearance of mitochondria or in induction of untoward inflammatory reactions. We found that following mitochondrial challenge, C3 was consumed in vivo in the absence of overt inflammation, indicating a potential role of complement in noninflammatory clearance of mitochondria. Thus, we report here the first indication of involvement of the lectin pathway in mitochondrial immune handling.
- Published
- 2013
- Full Text
- View/download PDF
25. Structural basis for specific recognition of Rpt1p, an ATPase subunit of 26 S proteasome, by proteasome-dedicated chaperone Hsm3p.
- Author
-
Takagi K, Kim S, Yukii H, Ueno M, Morishita R, Endo Y, Kato K, Tanaka K, Saeki Y, and Mizushima T
- Subjects
- Adenosine Triphosphatases genetics, Amino Acid Substitution, Binding Sites, Crystallography, X-Ray, Models, Molecular, Molecular Chaperones genetics, Mutagenesis, Site-Directed, Proteasome Endopeptidase Complex genetics, Protein Binding, Protein Interaction Domains and Motifs, Protein Multimerization, Protein Structure, Quaternary, Protein Structure, Secondary, Saccharomyces cerevisiae Proteins genetics, Surface Properties, Adenosine Triphosphatases chemistry, Molecular Chaperones chemistry, Proteasome Endopeptidase Complex chemistry, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins chemistry
- Abstract
The 26 S proteasome is a 2.5-MDa molecular machine that degrades ubiquitinated proteins in eukaryotic cells. It consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of 6 ATPase (Rpt) and 13 non-ATPase (Rpn) subunits. Multiple proteasome-dedicated chaperones facilitate the assembly of the proteasome, but little is known about the detailed mechanisms. Hsm3, a 19 S RP dedicated chaperone, transiently binds to the C-terminal domain of the Rpt1 subunit and forms a tetrameric complex, Hsm3-Rpt1-Rpt2-Rpn1, during maturation of the ATPase ring of 19 S RP. To elucidate the structural basis of Hsm3 function, we determined the crystal structures of Hsm3 and its complex with the C-terminal domain of the Rpt1 subunit (Rpt1C). Hsm3 has a C-shaped structure that consists of 11 HEAT repeats. The structure of the Hsm3-Rpt1C complex revealed that the interacting surface between Hsm3 and Rpt1 is a hydrophobic core and a complementary charged surface. Mutations in the Hsm3-Rpt1 surface resulted in the assembly defect of the 26 S proteasome. Furthermore, a structural model of the Hsm3-Rpt ring complex and an in vitro binding assay suggest that Hsm3 can bind Rpt2 in addition to Rpt1. Collectively, our results provide the structural basis of the molecular functions of Hsm3 for the RP assembly.
- Published
- 2012
- Full Text
- View/download PDF
26. Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase.
- Author
-
Shimada H, Hirai K, Simamura E, Hatta T, Iwakiri H, Mizuki K, Hatta T, Sawasaki T, Matsunaga S, Endo Y, and Shimizu S
- Subjects
- Animals, Cell-Free System, HeLa Cells, Humans, Hydrogen Peroxide pharmacology, Male, Microscopy, Electron methods, Mitochondria metabolism, NADH, NADPH Oxidoreductases metabolism, Rats, Rats, Wistar, Reactive Oxygen Species, Voltage-Dependent Anion Channel 1 metabolism, Herbicides toxicity, NAD metabolism, NADH, NADPH Oxidoreductases physiology, Paraquat toxicity, Voltage-Dependent Anion Channel 1 physiology
- Abstract
Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning are unreliable, and numerous deaths have been attributed inappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O(2)(*)) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O(2)(*) production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches.
- Published
- 2009
- Full Text
- View/download PDF
27. GLI1 is a direct transcriptional target of EWS-FLI1 oncoprotein.
- Author
-
Beauchamp E, Bulut G, Abaan O, Chen K, Merchant A, Matsui W, Endo Y, Rubin JS, Toretsky J, and Uren A
- Subjects
- Animals, Cell Line, Cell Proliferation, Chlorocebus aethiops, Gene Expression Regulation, Neoplastic, Humans, Oncogene Proteins, Fusion genetics, Promoter Regions, Genetic genetics, Protein Binding, Proto-Oncogene Protein c-fli-1 genetics, RNA, Small Interfering genetics, RNA-Binding Protein EWS, Transcription Factors genetics, Transcription, Genetic genetics, Zinc Finger Protein GLI1, Oncogene Proteins, Fusion metabolism, Proto-Oncogene Protein c-fli-1 metabolism, Transcription Factors metabolism
- Abstract
Ewing sarcoma family of tumors (ESFT) is an undifferentiated neoplasm of the bone and soft tissue. ESFT is characterized by a specific chromosomal translocation occurring between chromosome 22 and (in most cases) chromosome 11, which generates an aberrant transcription factor, EWS-FLI1. The function of EWS-FLI1 is essential for the maintenance of ESFT cell survival and tumorigenesis. The Hedgehog pathway is activated in several cancers. Oncogenic potential of the Hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence suggests that EWS-FLI1 increases expression of GLI1 by an unknown mechanism. Our data from chromatin immunoprecipitation and promoter reporter studies indicated GLI1 as a direct transcriptional target of EWS-FLI1. Expression of EWS-FLI1 in non-ESFT cells increased GLI1 expression and GLI-dependent transcription. We also detected high levels of GLI1 protein in ESFT cell lines. Pharmacological inhibition of GLI1 protein function decreased proliferation and soft agar colony formation of ESFT cells. Our results establish GLI1 as a direct transcriptional target of EWS-FLI1 and suggest a potential role for GLI1 in ESFT tumorigenesis.
- Published
- 2009
- Full Text
- View/download PDF
28. Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes.
- Author
-
Watanabe K, Nureki O, Fukai S, Endo Y, and Hori H
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Arginine genetics, Base Sequence, Binding Sites, Catalysis, Catalytic Domain, Chromatography, Affinity, Chromatography, Gel, Conserved Sequence, Electrophoretic Mobility Shift Assay, Lysine genetics, Models, Molecular, Mutagenesis, Site-Directed, Mutation, Protein Structure, Tertiary, RNA, Transfer genetics, Sequence Homology, Amino Acid, tRNA Methyltransferases genetics, tRNA Methyltransferases metabolism, Arginine metabolism, Lysine metabolism, RNA, Transfer metabolism, S-Adenosylmethionine metabolism, tRNA Methyltransferases chemistry
- Abstract
Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.
- Published
- 2006
- Full Text
- View/download PDF
29. Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.
- Author
-
Watanabe K, Nureki O, Fukai S, Ishii R, Okamoto H, Yokoyama S, Endo Y, and Hori H
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Asparagine chemistry, Binding Sites, Catalysis, Catalytic Domain, Chromatography, Chromatography, Affinity, Chromatography, Gel, Circular Dichroism, Cysteine chemistry, Kinetics, Models, Biological, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Protein Structure, Tertiary, RNA, Transfer chemistry, Sequence Homology, Amino Acid, Thermus enzymology, Time Factors, tRNA Methyltransferases physiology, tRNA Methyltransferases chemistry
- Abstract
Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apoand AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl-l-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl-l-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.
- Published
- 2005
- Full Text
- View/download PDF
30. Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2.
- Author
-
Endo Y, Wolf V, Muraiso K, Kamijo K, Soon L, Uren A, Barshishat-Küpper M, and Rubin JS
- Subjects
- Adaptor Proteins, Signal Transducing, Animals, CHO Cells, Cricetinae, Cricetulus, Culture Media, Conditioned pharmacology, Dishevelled Proteins, Enzyme Activation drug effects, Mice, Phosphoproteins, Proteins metabolism, Signal Transduction, Wnt Proteins, Wnt3 Protein, Wnt3A Protein, Cell Shape drug effects, Chemotaxis drug effects, Proteins pharmacology, rhoA GTP-Binding Protein metabolism
- Abstract
Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.
- Published
- 2005
- Full Text
- View/download PDF
31. Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus.
- Author
-
Okamoto H, Watanabe K, Ikeuchi Y, Suzuki T, Endo Y, and Hori H
- Subjects
- Amino Acid Sequence, Base Sequence, Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Escherichia coli metabolism, Escherichia coli Proteins genetics, Genome, Bacterial, Guanine chemistry, Kinetics, Mass Spectrometry, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Open Reading Frames, Protein Conformation, Protein Structure, Tertiary, RNA, Transfer chemistry, Sequence Homology, Amino Acid, tRNA Methyltransferases genetics, tRNA Methyltransferases metabolism, Bacteria enzymology, RNA, Transfer metabolism
- Abstract
Transfer RNA (m7G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m7G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNAPhe and E. coli tRNAThr. It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.
- Published
- 2004
- Full Text
- View/download PDF
32. Aquifex aeolicus tRNA (Gm18) methyltransferase has unique substrate specificity. TRNA recognition mechanism of the enzyme.
- Author
-
Hori H, Kubota S, Watanabe K, Kim JM, Ogasawara T, Sawasaki T, and Endo Y
- Subjects
- Amino Acid Sequence, Bacterial Proteins metabolism, Eubacterium genetics, Genome, Bacterial, Molecular Sequence Data, Substrate Specificity, Eubacterium enzymology, RNA, Transfer metabolism, tRNA Methyltransferases metabolism
- Abstract
Transfer RNA (guanosine-2')-methyltransferase (Gm-methylase) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine to 2'-OH of G18 in the D-loop of tRNA. Based on their mode of tRNA recognition, Gm-methylases can be divided into the following two types: type I having broad specificity toward the substrate tRNA, and type II that methylates only limited tRNA species. Protein synthesized by in vitro cell-free translation revealed that Gm-methylase encoded in the Aquifex aeolicus genome is a novel type II enzyme. Experiments with chimeric tRNAs and mini- and micro-helix RNAs showed that the recognition region of this enzyme is included within the D-arm structure of tRNALeu and that a bulge is essentially required. Variants of tRNALeu, tRNASer, and tRNAPhe revealed that a combination of certain base pairs in the D-stem is strongly recognized by the enzyme, that 4 bp in the D-stem enhance methyl acceptance activity, and that the Py16Py17G18G19 sequence is important for efficient methyl transfer. The methyl acceptance activities of all the A. aeolicus tRNA genes, which can be classified into 14 categories on the basis of their D-arm structure, were tested. The results clearly showed that the substrate recognition mechanism elucidated by the variant experiments was applicable to their native substrates.
- Published
- 2003
- Full Text
- View/download PDF
33. Ribosomal proteins at the stalk region modulate functional rRNA structures in the GTPase center.
- Author
-
Uchiumi T, Honma S, Endo Y, and Hachimori A
- Subjects
- Animals, Endoribonucleases chemistry, Nucleic Acid Conformation, Peptide Elongation Factor 2 metabolism, Rats, Ribosomes metabolism, Ricin chemistry, Fungal Proteins, GTP Phosphohydrolases chemistry, RNA, Ribosomal chemistry, Ribosomal Proteins chemistry
- Abstract
Replacement of the L10.L7/L12 protein complex and L11 in Escherichia coli ribosomes with the respective rat counterparts P0.P1/P2 and eukaryotic L12 causes conversion of ribosomal specificity for elongation factors from prokaryotic elongation factor (EF)-Tu/EF-G to eukaryotic EF (eEF)-1alpha/eEF-2. Here we have investigated the effects of protein replacement on the structure and function of two rRNA domains around positions 1070 and 2660 (sarcin/ricin loop) of 23 S rRNA. Protein replacement at the 1070 region in E. coli 50 S subunits was demonstrated by chemical probing analysis. Binding of rat proteins to the 1070 region caused increased accessibility of the 2660 and 1070 regions to ligands for eukaryotic ribosomes: the ribotoxin pepocin for the 2660 region (E. coli numbering), anti-28 S autoantibody for the 1070 region, and eEF-2 for both regions. Moreover, binding of the E. coli L10.L7/L12 complex and L11 to the 1070 region was shown to be responsible for E. coli ribosomal accessibility to another ribotoxin, gypsophilin. Ribosomal proteins at the 1070 region appear to modulate the structures and functions of the 2660 and 1070 RNA regions in slightly different modes in prokaryotes and eukaryotes.
- Published
- 2002
- Full Text
- View/download PDF
34. Cloning and characterization of novel ficolins from the solitary ascidian, Halocynthia roretzi.
- Author
-
Kenjo A, Takahashi M, Matsushita M, Endo Y, Nakata M, Mizuochi T, and Fujita T
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Blotting, Northern, Carrier Proteins chemistry, Carrier Proteins metabolism, Cloning, Molecular, DNA Primers, Glycoproteins metabolism, Humans, Molecular Sequence Data, Phylogeny, Protein Binding, Sequence Homology, Amino Acid, Urochordata, Ficolins, Carrier Proteins genetics, Lectins
- Abstract
Ficolins are animal lectins with collagen-like and fibrinogen-like domains. They are involved in the first line of host defense against pathogens. Human ficolin/P35 as well as mannose-binding lectin (MBL) activates the complement lectin pathway in association with MBL-associated serine proteases. To elucidate the origin and evolution of ficolins, we separated approximately 40 kDa (p40) and approximately 50 kDa (p50) N-acetylglucosamine-binding lectins from hemolymph plasma of the solitary ascidian. Binding assays revealed that p40 recognizes N-acetyl groups in association with a pyranose ring and that p50 recognizes N-acetylglucosamine alone. Based on the amino acid sequences of the proteins, we isolated two clones each of p40 and p50 from the ascidian hepatopancreas cDNA and determined the entire coding sequences of these clones. Because all of the clones contained both collagen-like and fibrinogen-like domains, we concluded that these were homologs of the mammalian ficolin family and designated ascidian ficolins (AsFCNs). The fibrinogen-like domain of the AsFCNs shows 45.4-52.4% amino acid sequence identity with the mammalian ficolin family. A phylogenetic tree of the fibrinogen-like sequences shows that all the fibrinogen-like domains may have evolved from a common ancestor that branched off an authentic fibrinogen. These results suggest that AsFCNs play an important role with respect to ascidian hemolymph lectin activity and the correlation of different functions with binding specificity.
- Published
- 2001
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.