Structure-function studies of bacterial mitoNEET system

• Project/Area Number: 26670215
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Bacteriology (including mycology)
• Research Institution: Nippon Medical School
• Principal Investigator: Iwasaki Toshio 日本医科大学, 医学部, 講師 (40277497)
• Co-Investigator(Renkei-kenkyūsha): KUSANO TERUO 日本医科大学, 医学部, 助教 (30434129) ; KUMASAKA TAKASHI 高輝度光科学研究センター, タンパク質結晶解析推進室, 室長代理 (30291066) ; IWASAKI HIDEO 早稲田大学, 理工学術院, 教授 (00324393)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,510,000 (Direct Cost: ¥2,700,000、Indirect Cost: ¥810,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: mitoNEET / 鉄硫黄クラスター / 構造生物学 / オミックス解析 / 構造機能 / 鉄硫黄タンパク質 / 好熱菌 / プロテオーム / 構造生物 / 機能生理 / 構造、生理 / システム生理学 / 細菌

Outline of Final Research Achievements

MitoNEET is a novel outer-mitochondrial membrane iron-sulfur protein, recently identified as a potential mitochondrial target that binds pioglitazone, an insulin sensitizer for the treatment of type II diabetes. Recently we constructed a null-deletion mutant strain of Thermus thermophilus lacking TthNEET (the thermophile homolog of mitoNEET), and found a “prokaryotic glucose intolerance” for this delta-TthNEET null strain. In this work, we characterized TthNEET mutant proteins and strains with altered [2Fe-2S] cluster redox potentials by X-ray crystallographic and physiological analyses to better understand the structure-function relationships of this class of proteins, and explored the protein interaction network of TthNEET by monitoring the whole-cell global changes of the Thermus protein components, using pulldown assays and two-dimensional gel electrophoresis.

Research Products

• [Int'l Joint Research] Univ. of Illinois at Urbana-Champaign(米国)
• [Journal Article] Escherichia coli auxotroph host strains for amino acid-selective isotope labeling of recombinant proteins (2015)
  • Author(s): Lin, M. T., Fukazawa, R., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Iwasaki, T., and Gennis, R. B.
  • Journal Title: Methods Enzymol. (Isotope Labeling of Biomolecules - Labeling Methods) Volume: 565 Pages: 45-66
  • DOI: 10.1016/bs.mie.2015.05.012
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Ligand mutagenesis of TthNEET, a thermophile homolog of mitoNEET. (2014)
  • Author(s): Iwasaki, T., Hagiuda, E., Fukazawa, R., Hayashi-Iwasaki, Y., Oshima, T., Hasegawa, K., and Kumasaka, T.
  • Journal Title: J. Biol. Inorg. Chem. Volume: 19 (Suppl 2)
• [Journal Article] A set of Escherichia coli amino acid auxotrophic expression host strains for selectively labelled metalloenzyme research. (2014)
  • Author(s): Fukazawa, R., Lin, M. T., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Baldansuren, A., Dikanov, S. A., Gennis, R. B., and Iwasaki, T.
  • Journal Title: J. Biol. Inorg. Chem. Volume: 19 (Suppl 2)
• [Presentation] Exploring the through-bond outer-sphere interactions in biological [2Fe-2S] proteins by two-dimensional 13Cbeta(cysteine) pulsed EPR (2016)
  • Author(s): Taguchi, A. T., Dikanov, S. A., and Iwasaki, T.
  • Organizer: Gordon Conference Metals in Biology 2016
  • Place of Presentation: Ventura, CA, USA
  • Year and Date: 2016-01-24
  • Int'l Joint Research
• [Presentation] Mapping the electron spin distribution in [2Fe-2S] proteins by 13C(beta) cysteine labeling: implications in electron transport pathways (2015)
  • Author(s): Taguchi, A. T., Miyajima-Nakano, Y., Fukazawa, R., Baldansuren, A., Samoilova, R. I., Hasegawa, K., Kumasaka, T., Dikanov, S. A., and Iwasaki, T.
  • Organizer: 17th International Conference on Bioinorganic Chemistry
  • Place of Presentation: Beijing, China
  • Year and Date: 2015-07-20
  • Int'l Joint Research
• [Presentation] X線結晶構造解析と分光学的手法の接点-鉄硫黄蛋白質を例に- (2014)
  • Author(s): 岩崎 俊雄
  • Organizer: 第87回日本生化学会大会「フォーラム: 蛋白質化学に立脚した“超域”生命化学への期待」
  • Place of Presentation: 京都
  • Year and Date: 2014-10-16
  • Invited
• [Presentation] Ligand mutagenesis of TthNEET, a thermophile homolog of mitoNEET. (2014)
  • Author(s): Iwasaki, T., Hagiuda, E., Fukazawa, R., Hayashi-Iwasaki, Y., Oshima, T., Hasegawa, K., and Kumasaka, T.
  • Organizer: 12th European Biological Inorganic Chemistry Conference (EuroBIC 12)
  • Place of Presentation: Zurich, Switzerland
  • Year and Date: 2014-08-24 – 2014-08-28
• [Presentation] A set of Escherichia coli amino acid auxotrophic expression host strains for selectively labelled metalloenzyme research. (2014)
  • Author(s): Fukazawa, R., Lin, M. T., Miyajima-Nakano, Y., Matsushita, S., Choi, S. K., Baldansuren, A., Dikanov, S. A., Gennis, R. B., and Iwasaki, T.
  • Organizer: 12th European Biological Inorganic Chemistry Conference (EuroBIC 12)
  • Place of Presentation: Zurich, Switzerland
  • Year and Date: 2014-08-24 – 2014-08-28
• [Remarks] 研究グループ ホームページ
  • URL: http://www.nms.ac.jp/fesworld/top.html