研究者業績

町田 幸大

マチダ コウダイ  (Kodai Machida)

基本情報

所属
兵庫県立大学 大学院 工学研究科 准教授
学位
博士(再生医科学)(2009年3月 鳥取大学)

研究者番号
20553093
J-GLOBAL ID
202001018700160800
researchmap会員ID
R000009667

論文

 29
  • Kodai Machida, Rin Tanaka, Seraya Miki, Shotaro Noseda, Mayumi Yuasa-Sunagawa, Hiroaki Imataka
    BioTechniques 76(4) 161-168 2024年4月  査読有り
    Programmed-1 ribosomal frameshifting (-1 PRF) is a translational mechanism adopted by some viruses, including SARS-CoV-2. To find a compound that can inhibit -1 PRF in SARS-CoV-2, we set up a high-throughput screening system using a HeLa cell extract-derived cell-free protein synthesis (CFPS) system. A total of 32,000 compounds were individually incubated with the CFPS system programmed with a -1 PRF-EGFP template. Several compounds were observed to decrease the -1 PRF-driven fluorescence, and one of them had some suppressive effect on -1 PRF of a SARS-CoV-2 genome sequence in transfected cells. Thus the CFPS system can be used as a tool for a high-throughput screening of chemicals.
  • Hayato Ito, Kodai Machida, Mayuka Hasumi, Morio Ueyama, Yoshitaka Nagai, Hiroaki Imataka, Hideki Taguchi
    Scientific reports 13(1) 22826-22826 2023年12月20日  査読有り
    Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.
  • Yosuke Ito, Yuhei Chadani, Tatsuya Niwa, Ayako Yamakawa, Kodai Machida, Hiroaki Imataka, Hideki Taguchi
    Nature communications 13(1) 7451-7451 2022年12月2日  査読有り
    Robust translation elongation of any given amino acid sequence is required to shape proteomes. Nevertheless, nascent peptides occasionally destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains can stochastically induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, using budding yeast and a human factor-based reconstituted translation system, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in their N-terminal regions alter canonical ribosome dynamics, stochastically aborting translation. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we find many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk of failure. Indeed, a bioinformatics analysis reveals that the N-terminal regions of ORFs lack D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.
  • Kodai Machida, Shoma Miyawaki, Kuru Kanzawa, Taiki Hakushi, Tomonori Nakai, Hiroaki Imataka
    ACS synthetic biology 10(11) 3158-3166 2021年11月19日  査読有り筆頭著者責任著者
    In vitro reconstitution of whole cellular events is one of the important goals in synthetic biology. Using a cell-free protein synthesis (CFPS) system reconstituted with human translation factors and chaperones, we reproduced the biogenesis of β-actin, synthesis, folding, and polymerization in a test tube. This system enabled us to define which step of the β-actin biogenesis was defective in genetic mutations related to diseases. Hence, the CFPS system reconstituted with human factors may be a useful tool for analyzing proteostasis in eukaryotes.
  • Chihiro Hirayama, Kodai Machida, Kentaro Noi, Tadayoshi Murakawa, Masaki Okumura, Teru Ogura, Hiroaki Imataka, Kenji Inaba
    iScience 24(4) 102296-102296 2021年4月23日  査読有り
    The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.

MISC

 1

共同研究・競争的資金等の研究課題

 6