Takeshi Tenno

Nagoya University, Nagoya, Aichi, Japan

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Publications (29)132.22 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Hef is an archaeal protein that probably functions in stalled replication fork repair mainly. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high-speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and ten candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.
    The Journal of biological chemistry. 06/2014;
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    ABSTRACT: open access at http://www.mdpi.com/1420-3049/18/8/9567/pdf In silico approaches have become indispensable for drug discovery as well as drug repositioning and adverse effect prediction. We have developed the eF-seek program to predict protein-ligand interactions based on the surface structure of proteins using a clique search algorithm. We have also developed a special protein structure prediction pipeline and accumulated predicted 3D models in the Structural Atlas of the Human Genome (SAHG) database. Using this database, genome-wide prediction of non-peptide ligands for proteins in the human genome was performed, and a subset of predicted interactions including 14 PDZ domains was then confirmed by NMR titration. Surprisingly, diclofenac, a non-steroidal anti-inflammatory drug, was found to be a non-peptide PDZ domain ligand, which bound to 5 of 15 tested PDZ domains. The critical residues for the PDZ-diclofenac interaction were also determined. Pharmacological implications of the accidental PDZ-diclofenac interaction are further discussed.
    Molecules 01/2013; 18(8):9567-81. · 2.43 Impact Factor
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    ABSTRACT: Intrinsically disordered proteins (IDPs) do not adopt stable three-dimensional structures in physiological conditions, yet these proteins play crucial roles in biological phenomena. In most cases, intrinsic disorder manifests itself in segments or domains of an IDP, called intrinsically disordered regions (IDRs), but fully disordered IDPs also exist. Although IDRs can be detected as missing residues in protein structures determined by X-ray crystallography, no protocol has been developed to identify IDRs from structures obtained by Nuclear Magnetic Resonance (NMR). Here, we propose a computational method to assign IDRs based on NMR structures. We compared missing residues of X-ray structures with residue-wise deviations of NMR structures for identical proteins, and derived a threshold deviation that gives the best correlation of ordered and disordered regions of both structures. The obtained threshold of 3.2 Å was applied to proteins whose structures were only determined by NMR, and the resulting IDRs were analyzed and compared to those of X-ray structures with no NMR counterpart in terms of sequence length, IDR fraction, protein function, cellular location, and amino acid composition, all of which suggest distinct characteristics. The structural knowledge of IDPs is still inadequate compared with that of structured proteins. Our method can collect and utilize IDRs from structures determined by NMR, potentially enhancing the understanding of IDPs.
    Journal of Structural Biology 11/2012; · 3.36 Impact Factor
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    ABSTRACT: Katanin p60 (p60-katanin) is a microtubule (MT)-severing enzyme and its activity is regulated by the p80 subunit (adaptor-p80). p60-katanin consists of an N-terminal domain, followed by a single ATPase associated with various cellular activities (AAA) domain. We have previously shown that the N-terminal domain serves as the binding site for MT, the substrate of p60-katanin. In this study, we show that the same domain shares another interface with the C-terminal domain of adaptor-p80. We further show that Ca(2+) ions inhibit the MT-severing activity of p60-katanin, whereas the MT-binding activity is preserved in the presence of Ca(2+). In detail, the basal ATPase activity of p60-katanin is stimulated twofold by both MTs and the C-terminal domain of adaptor-p80, whereas Ca(2+) reduces elevated ATPase activity to the basal level. We identify the Ca(2+) -binding site at the end of helix 2 of the N-terminal domain, which is different from the MT-binding interface. On the basis of these observations, we propose a speculative model in which spatial rearrangement of the N-terminal domain relative to the C-terminal AAA domain may be important for productive ATP hydrolysis towards MT-severing. Our model can explain how Ca(2+) regulates both severing and ATP hydrolysis activity, because the Ca(2+) -binding site on the N-terminal domain moves close to the AAA domain during MT severing.
    FEBS Journal 02/2012; 279(7):1339-52. · 4.25 Impact Factor
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    ABSTRACT: p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.
    Journal of Biological Chemistry 09/2011; 286(36):31864-31874. · 4.65 Impact Factor
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    ABSTRACT: p62/SQSTM1/A170 is a multimodular protein that is found in ubiquitin-positive inclusions associated with neurodegenerative diseases. Recent findings indicate that p62 mediates the interaction between ubiquitinated proteins and autophagosomes, leading these proteins to be degraded via the autophagy-lysosomal pathway. This ubiquitin-mediated selective autophagy is thought to begin with recognition of the ubiquitinated proteins by the C-terminal ubiquitin-associated (UBA) domain of p62. We present here the crystal structure of the UBA domain of mouse p62 and the solution structure of its ubiquitin-bound form. The p62 UBA domain adopts a novel dimeric structure in crystals, which is distinctive from those of other UBA domains. NMR analyses reveal that in solution the domain exists in equilibrium between the dimer and monomer forms, and binding ubiquitin shifts the equilibrium toward the monomer to form a 1:1 complex between the UBA domain and ubiquitin. The dimer-to-monomer transition is associated with a structural change of the very C-terminal end of the p62 UBA domain, although the UBA fold itself is essentially maintained. Our data illustrate that dimerization and ubiquitin binding of the p62 UBA domain are incompatible with each other. These observations reveal an autoinhibitory mechanism in the p62 UBA domain and suggest that autoinhibition plays a role in the function of p62.
    Journal of Biological Chemistry 06/2011; 286(36):31864-74. · 4.65 Impact Factor
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    ABSTRACT: The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2(UD)). NVL2(UD) contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of β-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (∼300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2(UD) potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2(UD) has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis.
    Journal of Biological Chemistry 06/2011; 286(24):21732-41. · 4.65 Impact Factor
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    ABSTRACT: Reversible interactions between cytosolic proteins and membrane lipids such as phosphoinositides play important roles in membrane morphogenesis driven by actin polymerization. In this paper, we identify a novel lipid-binding module, which we call the SYLF domain (after the SH3YL1, Ysc84p/Lsb4p, Lsb3p, and plant FYVE proteins that contain it), that is highly conserved from bacteria to mammals. SH3YL1 (SH3 domain containing Ysc84-like 1) strongly bound to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P(3)) and several D5-phosphorylated phosphoinositides through its SYLF domain and was localized to circular dorsal ruffles induced by platelet-derived growth factor stimulation. Interestingly, SHIP2 (the PI(3,4,5)P(3) 5-phosphatase, src-homology 2-containing inositol-5-phosphatase 2) was identified as a binding partner of SH3YL1, and knockdown of these proteins significantly suppressed dorsal ruffle formation. Phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), which is mainly synthesized from PI(3,4,5)P(3) by the action of SHIP2, was enriched in dorsal ruffles, and PI(3,4)P(2) synthesis strongly correlated with formation of the circular membrane structure. These results provide new insight into the molecular mechanism of dorsal ruffle formation and its regulation by phosphoinositide metabolism.
    The Journal of Cell Biology 05/2011; 193(5):901-16. · 10.82 Impact Factor
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    ABSTRACT: Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe(6), Tyr(10), Leu(13), and Met(17) found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water-micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.
    Biochimica et Biophysica Acta 03/2011; 1814(5):724-30. · 4.66 Impact Factor
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    ABSTRACT: Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8 A resolution, respectively. The tetraubiquitin crystals belonged to space group C222(1), with unit-cell parameters a = 58.795, b = 76.966, c = 135.145 A. The hexaubiquitin crystals belonged to space group P2(1), with unit-cell parameters a = 51.248, b = 102.668, c = 51.161 A. Structural analysis by molecular replacement is in progress.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2010; 66(Pt 7):834-7. · 0.55 Impact Factor
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    ABSTRACT: DEER (double electron-electron resonance) enables the observation of long-range dipole interactions (1.5-8 nm) between electron spin centers and has become a unique method for structural analysis of site-directed spin-labeled (SDSL) proteins. The method was applied to proteins inside eukaryotic cells, Xenopus laevis oocytes. DEER measurements of the oocytes, into which SDSL-ubiquitin derivates were injected, gave rise to interpretable signals and allowed us to perform in situ analyses of the interspin distances of the proteins.
    Journal of the American Chemical Society 06/2010; 132(24):8228-9. · 10.68 Impact Factor
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    ABSTRACT: Katanin p60 (kp60), a microtubule-severing enzyme, plays a key role in cytoskeletal reorganization during various cellular events in an ATP-dependent manner. We show that a single domain isolated from the N terminus of mouse katanin p60 (kp60-NTD) binds to tubulin. The solution structure of kp60-NTD was determined by NMR. Although their sequence similarities were as low as 20%, the structure of kp60-NTD revealed a striking similarity to those of the microtubule interacting and trafficking (MIT) domains, which adopt anti-parallel three-stranded helix bundle. In particular, the arrangement of helices 2 and 3 is well conserved between kp60-NTD and the MIT domain from Vps4, which is a homologous protein that promotes disassembly of the endosomal sorting complexes required for transport III membrane skeleton complex. Mutation studies revealed that the positively charged surface formed by helices 2 and 3 binds tubulin. This binding mode resembles the interaction between the MIT domain of Vps4 and Vps2/CHMP1a, a component of endosomal sorting complexes required for transport III. Our results show that both the molecular architecture and the binding modes are conserved between two AAA-ATPases, kp60 and Vps4. A common mechanism is evolutionarily conserved between two distinct cellular events, one that drives microtubule severing and the other involving membrane skeletal reorganization.
    Journal of Biological Chemistry 03/2010; 285(22):16822-9. · 4.65 Impact Factor
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    ABSTRACT: In-cell NMR is an isotope-aided multi-dimensional NMR technique that enables observations of conformations and functions of proteins in living cells at the atomic level. This method has been successfully applied to proteins overexpressed in bacteria, providing information on protein-ligand interactions and conformations. However, the application of in-cell NMR to eukaryotic cells has been limited to Xenopus laevis oocytes. Wider application of the technique is hampered by inefficient delivery of isotope-labelled proteins into eukaryote somatic cells. Here we describe a method to obtain high-resolution two-dimensional (2D) heteronuclear NMR spectra of proteins inside living human cells. Proteins were delivered to the cytosol by the pyrenebutyrate-mediated action of cell-penetrating peptides linked covalently to the proteins. The proteins were subsequently released from cell-penetrating peptides by endogenous enzymatic activity or by autonomous reductive cleavage. The heteronuclear 2D spectra of three different proteins inside human cells demonstrate the broad application of this technique to studying interactions and protein processing. The in-cell NMR spectra of FKBP12 (also known as FKBP1A) show the formation of specific complexes between the protein and extracellularly administered immunosuppressants, demonstrating the utility of this technique in drug screening programs. Moreover, in-cell NMR spectroscopy demonstrates that ubiquitin has much higher hydrogen exchange rates in the intracellular environment, possibly due to multiple interactions with endogenous proteins.
    Nature 04/2009; 458(7234):106-9. · 38.60 Impact Factor
  • Seikagaku. The Journal of Japanese Biochemical Society 11/2008; 80(10):948-58. · 0.04 Impact Factor
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    ABSTRACT: Insulin-like growth factor binding proteins (IGFBPs) have various IGF-independent cellular activities, including receptor-independent cellular uptake followed by transcriptional regulation, although mechanisms of cellular entry remain unclear. Herein, we focused on their receptor-independent cellular entry mechanism in terms of protein transduction domain (PTD) activity, which is an emerging technique useful for clinical applications. The peptides of 18 amino acid residues derived from IGFBP-3 and IGFBP-5, which involve heparin-binding regions, mediated cellular delivery of an exogenous protein into NIH3T3 and HeLa cells. Relative protein delivery activities of IGFBP-3/5-derived peptides were approximately 20–150% compared to that of the HIV-Tat peptide, a potent PTD. Heparin inhibited the uptake of the fusion proteins with IGFBP-3 and IGFBP-5, indicating that the delivery pathway is heparin-dependent endocytosis, similar to that of HIV-Tat. The delivery of GST fused to HIV-Tat was competed by either IGFBP-3 or IGFBP-5-derived synthetic peptides. Therefore, the entry pathways of the three PTDs are shared. Our data has shown a new approach for designing protein delivery systems using IGFBP-3/5 derived peptides based on the molecular mechanisms of IGF-independent activities of IGFBPs.
    Experimental Cell Research 08/2008; · 3.56 Impact Factor
  • Takeshi Tenno, Hidekazu Hiroaki
    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 04/2008; 53(3):256-64.
  • Analytical Biochemistry 01/2008; 371(2):247-9. · 2.58 Impact Factor
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    ABSTRACT: Cellular protein delivery is an emerging technique, by which exogenous recombinant proteins are delivered into mammalian cells across the membrane. We have developed an E. coli expression vector suited for protein cellular delivery experiments. The plasmid is designed to generate a C-terminal fusion with the 12 amino acid HIV-Tat peptide as a protein transduction domain (PTD), whereas the protein N-terminus is fused to an 17-residue peptide lanthanide-binding tag (LBT). LBT is used for both purification by affinity chromatography and fluorescent detection with Tb(3+) as a coordinating metal. We have employed the TA-cloning site between the two tags, LBT and PTD, according to the PRESAT-vector methodology [N. Goda, T. Tenno, H. Takasu, H. Hiroaki, M. Shirakawa, The PRESAT-vector: asymmetric T-vector for high-throughput screening of soluble protein domains for structural proteomics, Protein Sci. 13 (2004) 652-658], which facilitates unidirectional cloning of any PCR-amplified DNA fragments corresponding to the protein of interest. A simple three-step protocol consisting of affinity purification of LBT/PTD dual-tagged proteins has also been developed, in which the proteins are purified by heparin-, then immobilized Ni(2+)-, and then heparin-affinity chromatography, in this order. The purified protein is ready for protein delivery experiment, and the delivered protein is visible by fluorescent microscopy. Our LBT/PTD dual-tagged PRESAT-vector provides a powerful research tool for exploring cellular functions of proteins in the post-genomic era.
    Biochimica et Biophysica Acta 03/2007; 1773(2):141-6. · 4.66 Impact Factor

Publication Stats

452 Citations
132.22 Total Impact Points

Institutions

  • 2013
    • Nagoya University
      • Graduate School of Information Science
      Nagoya, Aichi, Japan
  • 2006–2012
    • Kyoto University
      • Department of Molecular Engineering
      Kyoto, Kyoto-fu, Japan
  • 2008–2011
    • Kobe University
      • • Graduate School of Medicine
      • • Department of Biochemistry and Molecular Biology
      • • Division of Structural Biology
      Kōbe, Hyōgo, Japan
  • 2004–2008
    • Yokohama City University
      Yokohama, Kanagawa, Japan
    • Ehime University
      Matuyama, Ehime, Japan