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Research

Research

We aim to develop new therapeutic and diagnostic methods for intractable diseases for which there is no effective therapeutic or prophylactic treatment, such as arteriosclerosis, cancer, Alzheimer's disease and viral infection, through elucidation of the mechanisms of the diseases by biochemical and molecular biological approaches.

Factors involved in the development of these diseases, from signaling through cell surface receptors to regulation of gene transcription, are considered to be controlled by dynamic networks consisting of multiple complexes of different proteins.

Through antigen presentation using a baculovirus expression system, we have produced monoclonal antibodies against proteins involved in the diseases and analyzed them by sensitive proteomics approaches in order to reveal dynamic control mechanisms of the protein complexes, the analysis of which has been difficult, and to develop new therapeutic methods.

Production and design of functional antibodies

Therapeutic Antibody making, design,

With the aim of generating antibodies for the therapy and diagnosis of intractable diseases such as cancer, we have produced functional antibodies including membrane proteins by using baculovirus display. We are also developing new applications of these antibodies for use in highly complex structural analysis and attempting to use computational design to produce additional functional therapeutic antibodies.

Main members:
Yasuhiro Mochizuki, Hiroko Iwanari, Osamu Arai, Yuichiro Takamatsu
Kiyoshi Nakagawa, Toshiyoshi Yamamoto, Satoru Akimoto, Kenichi Mitsui
Kazuaki Takahashi
Hideaki Fujitani1, Takefumi Yamashita1, Keiko Shinoda1

1 Biosimulation, RCAST
Related major projects:
Molecular Dynamics for Antibody Drug Development (MDADD) (FIRST Program)
Targeted Proteins Research Program (TPRP)
Development of New Functional Antibody Technologies funded by the New Energy and Industrial Technology Development Organization (NEDO) (ended in FY2010)

Publications

  1. Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin. Hayashi I, Takatori S, Urano Y, Miyake Y, Takagi J, Sakata-Yanagimoto M, Iwanari H, Osawa S, Morohashi Y, Li T, Wong PC, Chiba S, Kodama T, Hamakubo T, Tomita T, Iwatsubo T. Oncogene. 2011 Jul 4. doi: 10.1038/onc.2011.265.
  2. Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression. Kanki Y, Kohro T, Jiang S, Tsutsumi S, Mimura I, Suehiro J, Wada Y, Ohta Y, Ihara S, Iwanari H, Naito M, Hamakubo T, Aburatani H, Kodama T, Minami T. EMBO J. 2011 Jun 10;30(13):2582-95
  3. A method of generating antibodies against exogenously administered self-antigen by manipulating CD4+CD25+ regulatory T cells. Iwanari H, Nakada-Nakura Y, Kusano-Arai O, Suzuki N, Kodama T, Sakihama T, Hamakubo T. J Immunol Methods. 2011 Jun 30;369(1-2):108-14.
  4. Kunii R, Jiang S, Hasegawa G, Yamamoto T, Umezu H, Watanabe T, Tsuchida M, Hashimoto T, Hamakubo T, Kodama T, Sasai K, Naito M. The predominant expression of hepatocyte nuclear factor 4α (HNF4α) in thyroid transcription factor-1 (TTF-1)-negative pulmonary adenocarcinoma. Histopathology. 2011 Feb;58(3):467-76.
  5. Akazawa YO, Saito Y, Hamakubo T, Masuo Y, Yoshida Y, Nishio K, Shichiri M, Miyasaka T, Iwanari H, Mochizuki Y, Kodama T, Noguchi N, Niki E. Elevation of oxidized DJ-1 in the brain and erythrocytes of Parkinson disease model animals. Neurosci Lett. 2010 Oct 15;483(3):201-5.
  6. Kuwako K, Kakumoto K, Imai T, Igarashi M, Hamakubo T, Sakakibara S, Tessier-Lavigne M, Okano HJ, Okano H. Neural RNA-binding protein Musashi1 controls midline crossing of precerebellar neurons through posttranscriptional regulation of Robo3/Rig-1 expression.　Neuron. 2010 Aug 12;67(3):407-21.
  7. Seki M, Watanabe A, Enomoto S, Kawamura T, Ito H, Kodama T, Hamakubo T, Aburatani H. Human ROBO1 is cleaved by metalloproteinases and gamma-secretase and migrates to the nucleus in cancer cells FEBS Lett. 2010 Jul 2;584(13):2909-15.
  8. Suehiro JI, Hamakubo T, Kodama T, Aird WC, Minami T. Vascular endothelial growth factor activation of endothelial cells is mediated by early growth response-3. Blood. 2010 Mar 25;115(12):2520-32.
  9. Saito Y, Hamakubo T, Yoshida Y, Ogawa Y, Hara Y, Fujimura H, Imai Y, Iwanari H, Mochizuki Y, Shichiri M, Nishio K, Kinumi T, Noguchi N, Kodama T, Niki E. Preparation and application of monoclonal antibodies against oxidized DJ-1. Significant elevation of oxidized DJ-1 in erythrocytes of early-stage Parkinson disease patients. Neurosci Lett. 2009 Nov 6;465(1):1-5.
  10. Hayashi I, Takatori S, Urano Y, Iwanari H, Isoo N, Osawa S, Fukuda MA, Kodama T, Hamakubo T, Li T, Wong PC, Tomita T, Iwatsubo T. Single chain variable fragment against nicastrin inhibits the gamma-secretase activity. J Biol Chem. 2009 Oct 9;284(41):27838-47.
  11. Minami T, Yano K, Miura M, Kobayashi M, Suehiro J, Reid PC, Hamakubo T, Ryeom S, Aird WC, Kodama T. The Down syndrome critical region gene 1 short variant promoters direct vascular bed-specific gene expression during inflammation in mice. J Clin Invest. 2009 Aug;119(8):2257-70.
  12. Noritake J, Fukata Y, Iwanaga T, Hosomi N, Tsutsumi R, Matsuda N, Tani H, Iwanari H, Mochizuki Y, Kodama T, Matsuura Y, Bredt DS, Hamakubo T, Fukata M. Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95. J Cell Biol. 2009 Jul 13;186(1):147-60.
  13. Wakabayashi K, Okamura M, Tsutsumi S, Nishikawa NS, Tanaka T, Sakakibara I, Kitakami J, Ihara S, Hashimoto Y, Hamakubo T, Kodama T, Aburatani H, Sakai The peroxisome proliferator-activated receptor gamma/retinoid X receptor alpha heterodimer targets the histone modification enzyme PR-Set7/Setd8 gene and regulates adipogenesis through a positive feedback loop. J.Mol Cell Biol. 2009 Jul;29(13):3544-55.
  14. Okamura M, Kudo H, Wakabayashi K, Tanaka T, Nonaka A, Uchida A, Tsutsumi S, Sakakibara I, Naito M, Osborne TF, Hamakubo T, Ito S, Aburatani H, Yanagisawa M, Kodama T, Sakai J. COUP-TFII acts downstream of Wnt/beta-catenin signal to silence PPARgamma gene expression and repress adipogenesis. Proc Natl Acad Sci U S A. 2009 Apr 7;106(14):5819-24.
  15. Saitoh R, Ohtomo T, Yamada Y, Kamada N, Nezu J, Kimura N, Funahashi S, Furugaki K, Yoshino T, Kawase Y, Kato A, Ueda O, Jishage K, Suzuki M, Fukuda R, Arai M, Iwanari H, Takahashi K, Sakihama T, Ohizumi I, Kodama T, Tsuchiya M, Hamakubo T. J Immunol Methods. 2007 Apr 30;322(1-2):104-17.

Living cell imaging by FRET

FRET imaging, cell migration

By using fluorescence resonance energy transfer (FRET) probes, transduction of signals from external sources by membrane receptors such as G protein-coupled receptor (GPCR) is imaged in real time in living cells.
We also reveal how cells migrate by computer image analysis.

Main members:
Kazuyuki Masuda
Sigeo Ihara1, Jun-ichi Kitakami1
Tohru Kozasa2, Nobuchika Suzuki2, Nicole Hajicek2

1Dynamical Bioinformatics Lab., RCAST
2Division of Signal Transduction, RCAST
Related major projects:
MDADD (FIRST Program)
Grant-in-Aid for Scientific Research (S)

Publications

  1. Masuda K, Itoh H, Sakihama T, Akiyama C, Takahashi K, Fukuda R, Yokomizo T, Shimizu T, Kodama T, Hamakubo T. A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor. J Biol Chem. 278(27):24552-62. 2003.
  2. Motoyoshi Baba, Tohru Kozasa, Takao Hamakubo, Hiroto Kuroda, Kazuyuki Masuda, Shin Yoneya and Tatsuhiko Kodama　Soft X-ray Laser Microscopy of Lipid Rafts towards GPCR-Based Drug Discovery Using Time-Resolved FRET Spectroscopy　Pharmaceuticals 2011, 4(3), 524-550

RNA splicing mechanism

RNA processing

We have identified Wilms tumor 1-associating protein (WTAP), which is involved in cell cycling and RNA processing.
By producing specific antibodies against WTAP and conducting comprehensive analyses, including an analysis of protein complexes by proteomics approaches and an analysis of bound RNA, we have revealed the splicing mechanism and the cell cycle regulatory mechanism.

Main members:
Keiko Horiuchi
Sigeo Ihara1, Yohishiro Ohta1

1Dynamical Bioinformatics Lab., RCAST
Related major projects:
Grant-in-Aid for Scientific Research (S)

Publications

  1. Horiuchi K, Umetani M, Minami T, Okayama H, Takada S, Yamamoto M, Aburatani H, Reid PC, Housman DE, Hamakubo T, Kodama T. Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA. Proc Natl Acad Sci U S A. Nov 14;103(46):17278-83. 2006
  2. Umetani M, Mataki C, Minegishi N, Yamamoto M, Hamakubo T, Kodama T. Function of GATA transcription factors in induction of endothelial vascular cell adhesion molecule-1 by tumor necrosis factor-alpha. Arterioscler Thromb Vasc Biol. 21(6):917-22. 2001.

Proteomics of protein complexes

proteomics

We have comprehensively analyzed components of protein complexes by sensitive proteomics approaches using high-affinity antibodies and magnetic beads. We have also analyzed changes of modules of the complexes by informatics to reveal the dynamism of transcriptional regulation and complicated pathologies of diseases, the analysis of which has been difficult.

Main members:
Kenji Daigo, Takeshi Kawamura
Sigeo Ihara1, Yohishiro Ohta1

1Dynamical Bioinformatics Lab., RCAST
Related major projects:
MDADD (FIRST Program)
Grant-in-Aid for Scientific Research (S)

Publications

  1. Daigo K, Kawamura T, Ohta Y, Ohashi R, Katayose S, Tanaka T, Aburatani H, Naito M, Kodama T, Ihara S, Hamakubo T. Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors. J Biol Chem. 2011 Jan 7;286(1):674-86.
  2. Seki M, Watanabe A, Enomoto S, Kawamura T, Ito H, Kodama T, Hamakubo T, Aburatani H. Human ROBO1 is cleaved by metalloproteinases and gamma-secretase and migrates to the nucleus in cancer cells FEBS Lett. 2010 Jul 2;584(13):2909-15.
  3. Ohguchi H, Tanaka T, Uchida A, Magoori K, Kudo H, Kim I, Daigo K, Sakakibara I, Okamura M, Harigae H, Sasaki T, Osborne TF, Gonzalez FJ, Hamakubo T, Kodama T, Sakai J. Hepatocyte nuclear factor 4alpha contributes to thyroid hormone homeostasis by cooperatively regulating the type 1 iodothyronine deiodinase gene with GATA4 and Kruppel-like transcription factor 9. Mol Cell Biol. 2008 Jun;28(12):3917-31.
  4. Sumi K, Tanaka T, Uchida A, Magoori K, Urashima Y, Ohashi R, Ohguchi H, Okamura M, Kudo H, Daigo K, Maejima T, Kojima N, Sakakibara I, Jiang S, Hasegawa G, Kim I, Osborne TF, Naito M, Gonzalez FJ, Hamakubo T, Kodama T, Sakai J. Cooperative interaction between Hepatocyte Nuclear Factor 4{alpha} and GATA transcription factors Regulates ATP-binding cassette sterol transporters ABCG5 and ABCG8. Mol Cell Biol. 2007 Apr 2

Baculovirus technology

Baculovirus expression system

By using functional expression of membrane proteins on baculovirus particles, we have constructed reaction/screening systems using membrane protein enzymes, including reconstruction of odorant receptors and sugar chain synthesis, which have previously proved difficult, and identified novel functional substances.

Main members:
Toshiko Sakihama, Kazuyuki Masuda, Kazuaki Takahashi, Kenichi Mitsui
Related major projects:
MDADD (FIRST Program)
Grant-in-Aid for Scientific Research (S)

Publications

  1. Kakutani H, Takahashi A, Kondoh M, Saito Y, Yamaura T, Sakihama T, Hamakubo T, Yagi K. A novel screening system for claudin binder using baculoviral display. PLoS One. 2011 Feb 14;6(2):e16611.
  2. Uchida H, Kondoh M, Hanada T, Takahashi A, Hamakubo T, Yagi K. Mucosal vaccination using claudin-4-targeting. Biochem Pharmacol. 2010 May 15;79(10):1437-44.
  3. Saeki R, Kondoh M, Kakutani H, Tsunoda S, Mochizuki Y, Hamakubo T, Tsutsumi Y, Horiguchi Y, Yagi K. A novel tumor-targeted therapy using a claudin-4-targeting molecule. Mol Pharmacol. 2009 Oct;76(4):918-26.
  4. Sakihama T, Sato T, Iwanari H, Kitamura T, Sakaguchi S, Kodama T, Hamakubo T. A simple detection method for low-affinity membrane protein interactions by baculoviral display. PLoS ONE. 2008;3(12):e4024.
  5. Sakihama T, Masuda K, Sato T, Doi T, Kodama T, Hamakubo T. Functional reconstitution of G protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system. J Biotechnol. 2008 May 20;135(1):28-33.
  6. Saitoh R, Ohtomo T, Yamada Y, Kamada N, Nezu J, Kimura N, Funahashi S, Furugaki K, Yoshino T, Kawase Y, Kato A, Ueda O, Jishage K, Suzuki M, Fukuda R, Arai M, Iwanari H, Takahashi K, Sakihama T, Ohizumi I, Kodama T, Tsuchiya M, Hamakubo T. J Immunol Methods. 2007 Apr 30;322(1-2):104-17.
  7. Saitoh R, Ohtomo T, Ito Y, Nezu J, Kimura N, Funahashi S, Aso Y, Ohizumi I, Kodama T, Hamakubo T, Tsuchiya M. Recovery of functional peptide transporter PepT1 in budded baculovirus fraction.
  8. Ikuo Hayashi, Yasuomi Urano, Rie Fukuda, Noriko Isoo, Tatsuhiko Kodama, Takao Hamakubo Taisuke Tomita, and Takeshi Iwatsubo. Selective reconstitution and recovery of functional gamma-secretase complex on budded baculovirus particles. J Biol Chem. Sep 3;279(36):38040-6. 2004
  9. Urano Y, Yamaguchi M, Fukuda R, Masuda K, Takahashi K, Uchiyama Y, Iwanari H, Jiang SY, Naito M, Kodama T, Hamakubo T. A novel method for viral display of ER membrane proteins on budded baculovirus. Biochem Biophys Res Commun. 308(1):191-6. 2003
  10. Masuda K, Itoh H, Sakihama T, Akiyama C, Takahashi K, Fukuda R, Yokomizo T, Shimizu T, Kodama T, Hamakubo T. A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor. J Biol Chem. 278(27):24552-62. 2003.

Development of soft X-ray microscopy

Soft X-ray microscopy

We have been developing soft X-ray microscopy techniques to observe three-dimensional internal structures of cells and tissues with high (nanoscale) resolution. The observation of living tissues, such as cells, consisting of light elements, requires low-energy, or soft, X-ray microscopes. Owing to subtle differences in absorption contrast caused by minute differences in density (r), the use of 1–3 keV soft X-rays enables observation of the absorption contrast of almost all light elements. We aim to realize a technique for soft X-ray imaging of living cells and tissues that can be performed in standard laboratories.
Using a white soft X-ray microscope with a peak around 2 keV, we have observed labeled cells and living tissues. At the same time, we have conducted studies on labeling and immobilization techniques optimized for X-ray microscopy.
In addition, we have been developing a water-window (W-W) X-ray microscope to observe untreated hydrated cells under near-natural conditions.

Main members:
Motosuke Miyoshi, Keiko Horiuchi, Satoshi Murakami
Related major projects:
Innovation Promotion Program funded by NEDO (ended in FY2010)

Publications

  1. Takayoshi Koike, Atsuo Jouzuka, Tomonori Nakamura, Yoshihiro Onizuka, Motosuke Miyoshi and Hidenori Mimura. Structural and electrical properties if inflamed graphite at high temperature as a new field emitter, Proc. 24th International Vacuum Nanoelectronics Conference 2011 (IVNC2011), Jul. 2011, 35-36
  2. Motoyoshi Baba, Tohru Kozasa, Takao Hamakubo, Hiroto Kuroda, Kazuyuki Masuda, Shin Yoneya and Tatsuhiko Kodama. Soft X-ray Laser Microscopy of Lipid Rafts towards GPCR-Based Drug Discovery Using Time-Resolved FRET Spectroscopy Pharmaceuticals 2011, 4(3), 524-550
  3. Motosuke Miyoshi, Takao Hamakubo, Tatsuhiko Kodama, Masatoshi Tsuchya, Atsushi Koishikawa and Nobutada Aoki. Development of the low-energy soft x-ray CT instrument for the soft material structural analysis, J.Vac.Sci.Technol. B26(6), Nov/Dec 2008, 2356-2361