Faculty Profiles - HAYASHI Gosuke

Papers - HAYASHI Gosuke

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Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest

Koyama, K; Hayashi, G; Ueda, H; Ota, S; Nagae, G; Aburatani, H; Okamoto, A

ORGANIC & BIOMOLECULAR CHEMISTRY Vol. 19 ( 29 ) page： 6478 - 6486 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Organic and Biomolecular Chemistry

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method forhm5C in RNA by applying peroxotungstate-mediated chemical conversion ofhm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at thethT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of thehm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.

DOI： 10.1039/d1ob00995h

Web of Science

Scopus

PubMed

Silyl-protected propargyl glycine for multiple labeling of peptides by chemoselective silyl-deprotection Open Access

Naoki Kamo, Gosuke Hayashi, Akimitsu Okamoto

Tetrahedron Letters Vol. 73 2021.6

Language：English Publishing type：Research paper (scientific journal) Publisher：Elsevier Ltd

We synthesized Fmoc-propargyl glycine derivatives bearing different silyl protecting groups that can be readily introduced by using a standard solid-phase peptide coupling procedures. Taking advantage of the orthogonality between the different silyl protecting groups, chemoselective incorporation of functional molecules into a 19-mer peptide through click reactions was demonstrated.

DOI： 10.1016/j.tetlet.2021.153093

Web of Science

Scopus

Organoruthenium-catalyzed chemical protein synthesis to elucidate the functions of epigenetic modifications on heterochromatin factors† Open Access

Kamo, N; Kujirai, T; Kurumizaka, H; Murakami, H; Hayashi, G; Okamoto, A

CHEMICAL SCIENCE Vol. 12 ( 16 ) page： 5926 - 5937 2021.4

Language：English Publishing type：Research paper (scientific journal) Publisher：Chemical Science

The application of organometallic compounds for protein science has received attention. Recently, total chemical protein synthesis using transition metal complexes has been developed to produce various proteins bearing site-specific posttranslational modifications (PTMs). However, in general, significant amounts of metal complexes were required to achieve chemical reactions of proteins bearing a large number of nucleophilic functional groups. Moreover, syntheses of medium-size proteins (>20 kDa) were plagued by time-consuming procedures due to cumbersome purification and isolation steps, which prevented access to variously decorated proteins. Here, we report a one-pot multiple peptide ligation strategy assisted by an air-tolerant organoruthenium catalyst that showed more than 50-fold activity over previous palladium complexes, leading to rapid and quantitative deprotection on a protein with a catalytic amount (20 mol%) of the metal complex even in the presence of excess thiol moieties. Utilizing the organoruthenium catalyst, heterochromatin factors above 20 kDa, such as linker histone H1.2 and heterochromatin protein 1α (HP1α), bearing site-specific PTMs including phosphorylation, ubiquitination, citrullination, and acetylation have been synthesized. The biochemical assays using synthetic proteins revealed that the citrullination at R53 in H1.2 resulted in the reduced electrostatic interaction with DNA and the reduced binding affinity to nucleosomes. Furthermore, we identified a key phosphorylation region in HP1α to control its DNA-binding ability. The ruthenium chemistry developed here will facilitate the preparation of a variety of biologically and medically significant proteins containing PTMs and non-natural amino acids.

DOI： 10.1039/d1sc00731a

cDNA TRAP display for rapid and stable <i>in vitro</i> selection of antibody-like proteins

Kondo, T; Eguchi, M; Kito, S; Fujino, T; Hayashi, G; Murakami, H

CHEMICAL COMMUNICATIONS Vol. 57 ( 19 ) page： 2416 - 2419 2021.3

Language：English Publishing type：Research paper (scientific journal) Publisher：Chemical Communications

We developed a cDNA TRAP display for the rapid selection of antibody-like proteins in various conditions. By modifying the original puromycin linker in the TRAP display, a monobody was covalently attached to the cDNA. As a proof-of-concept, we demonstrated a rapid model selection of an anti-EGFR1 monobody in a solution containing ribonuclease.

DOI： 10.1039/d0cc07541h

Web of Science

Scopus

PubMed

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain Open Access

Hao, FF; Murphy, KJ; Kujirai, T; Kamo, N; Kato, J; Koyama, M; Okamato, A; Hayashi, G; Kurumizaka, H; Hayes, JJ

NUCLEIC ACIDS RESEARCH Vol. 48 ( 20 ) page： 11510 - 11520 2020.11

Language：English Publishing type：Research paper (scientific journal) Publisher：Nucleic Acids Research

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

DOI： 10.1093/nar/gkaa949

L-DNA-tagged fluorescence: In situ hybridization for highly sensitive imaging of RNAs in single cells

Motoyuki Ogata, Gosuke Hayashi, Anri Ichiu, Akimitsu Okamoto

Organic and Biomolecular Chemistry Vol. 18 ( 40 ) page： 8084 - 8088 2020.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Royal Society of Chemistry

We report an effective fluorescence in situ hybridization strategy, named l-DNA tagged FISH (LT-FISH), for highly sensitive RNA detection in fixed cultured cells. LT-FISH includes two-step hybridization processes with a l-d chimera oligonucleotide probe and a fluorescence-labeled PCR product tethering a l-DNA tag. The degree of fluorescence enhancement, depending on the length of PCR products, was up to 14-fold when the 606 bp product was used. Endogenous mRNA and miRNA in cancer cells were visualized by utilizing this l-DNA-mediated signal amplification technique.

DOI： 10.1039/d0ob01635g

Web of Science

Scopus

PubMed

Toolbox for chemically synthesized histone proteins. Open Access

Nakatsu K, Hayashi G, Okamoto A

Current opinion in chemical biology Vol. 58 page： 10 - 19 2020.10

Antibody-like proteins that capture and neutralize SARS-CoV-2 Open Access

Kondo, T; Iwatani, Y; Matsuoka, K; Fujino, T; Umemoto, S; Yokomaku, Y; Ishizaki, K; Kito, S; Sezaki, T; Hayashi, G; Murakami, H

SCIENCE ADVANCES Vol. 6 ( 42 ) 2020.10

Language：English Publishing type：Research paper (scientific journal) Publisher：Science Advances

To combat severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) and any unknown emerging pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore, the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration, 0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly a future, pandemic.

DOI： 10.1126/sciadv.abd3916

Fmoc-Compatible and C-terminal-Sequence-Independent Peptide Alkyl Thioester Formation Using Cysteinylprolyl Imide

Nakatsu Koki, Yanase Masafumi, Hayashi Gosuke, Okamoto Akimitsu

ORGANIC LETTERS Vol. 22 ( 12 ) page： 4670 - 4674 2020.6

Novel Strategies of Peptide Ligation for Accelerating Chemical Protein Synthesis

Hayashi Gosuke, Okamoto Akimitsu

JOURNAL OF SYNTHETIC ORGANIC CHEMISTRY JAPAN Vol. 78 ( 2 ) page： 130 - 139 2020.2

Novel Strategies of Peptide Ligation for Accelerating Chemical Protein Synthesis

Hayashi Gosuke, Okamoto Akimitsu

JOURNAL OF SYNTHETIC ORGANIC CHEMISTRY JAPAN Vol. 78 ( 2 ) page： 130-139 2020.2

タンパク質化学合成を活用した翻訳後修飾研究

林剛介

ファルマシア Vol. 56 ( 1 ) page： 46-50 - 50 2020

Language：English Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本薬学会

タンパク質化学合成法の発展により、多様な翻訳後修飾を持つタンパク質の作製が可能なってきた。本稿では、タンパク質化学合成法について解説するとともに、化学合成タンパク質を用いた翻訳後修飾研究について紹介する。特に、エピジェネティクス研究で中心的役割を果たすヒストンタンパク質の翻訳後修飾研究、また本特集のテーマであるユビキチン鎖やユビキチン化タンパク質を化学的に合成し、応用した研究例について紹介する。

DOI： 10.14894/faruawpsj.56.1_46

CiNii Research

Novel Strategies of Peptide Ligation for Accelerating Chemical Protein Synthesis

Hayashi Gosuke, Okamoto Akimitsu

Journal of Synthetic Organic Chemistry, Japan Vol. 78 ( 2 ) page： 130 - 139 2020

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：The Society of Synthetic Organic Chemistry, Japan

<p>Chemical protein synthesis (CPS) that consists of solid-phase peptide synthesis and peptide ligation generates not only naturally-occurring proteins with/without posttranslational modifications (PTMs), but also a variety of artificial proteins including unnatural amino-acids such as ᴅ-amino acids and fluorophore-labeled amino acids. This unique property offers new analytical methods for protein structure and interaction in terms of PTMs. In fact, we have chemically synthesized histone proteins with PTMs, which play an important role in regulation of gene expression in eukaryotic cells, and analyzed the effects of PTMs such as methylation, acetylation, and phosphorylation. However, current CPS is still in developing process and includes several issues to be solved such as difficult handling in hydrophobic protein synthesis and time-consuming multistep process for large protein synthesis. We have approached these issues by creating new strategies for peptide ligation. One-pot ligation of five peptide segments was demonstrated for the first time by utilizing multifunctionality of thiophenol compound in deprotection of allyloxycarbonyl group by palladium complex. New thioester precursors for native chemical ligation, which have potential to offer a novel two-way one-pot ligation, have also been developed recently. Furthermore, we have been developing a new strategy for simultaneous ligation of multiple peptides on DNA scaffold, which can connect peptides in highly diluted condition. This article also describes the background and current situation of CPS with our future perspectives.</p>

DOI： 10.5059/yukigoseikyokaishi.78.130

Scopus

CiNii Research

Chemical Synthesis of Cys-Containing Protein via Chemoselective Deprotection with Different Palladium Complexes

Kamo Naoki, Hayashi Gosuke, Okamoto Akimitsu

ORGANIC LETTERS Vol. 21 ( 20 ) page： 8378 - 8382 2019.10

Cysteinylprolyl imide (CPI) peptide: a highly reactive and easily accessible crypto-thioester for chemical protein synthesis Open Access

Yanase Masafumi, Nakatsu Koki, Cardos Charlane Joy, Konda Yoshiki, Hayashi Gosuke, Okamoto Akimitsu

CHEMICAL SCIENCE Vol. 10 ( 23 ) page： 5967 - 5975 2019.6

Reaction design for highly efficient chemical protein synthesis

Hayashi Gosuke, Okamoto Akimitsu

ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY Vol. 257 page： . 2019.3

Simultaneous and Traceless Ligation of Peptide Fragments on DNA Scaffold.

Hayashi G, Yanase M, Nakatsuka Y, Okamoto A

Biomacromolecules Vol. 20 ( 3 ) page： 1246-1253 - 1253 2019.3

Triple Function of 4-Mercaptophenylacetic Acid Promotes One-Pot Multiple Peptide Ligation.

Kamo N, Hayashi G, Okamoto A

Angewandte Chemie (International ed. in English) Vol. 57 ( 50 ) page： 16533-16537 - 16537 2018.12

Chemistry-Driven Epigenetic Investigation of Histone and DNA Modifications.

Sueoka T, Koyama K, Hayashi G, Okamoto A

Chemical record (New York, N.Y.) Vol. 18 ( 12 ) page： 1727-1744 - 1744 2018.12

Efficient peptide ligation between allyl-protected Asp and Cys followed by palladium-mediated deprotection.

Kamo N, Hayashi G, Okamoto A

Chemical communications (Cambridge, England) Vol. 54 ( 34 ) page： 4337-4340 - 4340 2018.4

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Papers - HAYASHI Gosuke