Faculty Profiles - HAYASHI Gosuke

One-pot ligation of multiple peptide segments via N-terminal thiazolidine deprotection chemistry

Optineurin provides a mitophagy contact site for TBK1 activation Open Access

Yamano, K; Sawada, M; Kikuchi, R; Nagataki, K; Kojima, W; Endo, R; Kinefuchi, H; Sugihara, A; Fujino, T; Watanabe, A; Tanaka, K; Hayashi, G; Murakami, H; Matsuda, N

EMBO JOURNAL Vol. 43 ( 5 ) page： 754 - 779 2024.3

Language：English Publishing type：Research paper (scientific journal) Publisher：EMBO Journal

Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.

DOI： 10.1038/s44318-024-00036-1

Nakamura, G; Nakatsu, K; Hayashi, G

PEPTIDE AND PEPTIDE MIMICKING TOOLS Vol. 698 page： 169 - 194 2024

Language：English Publishing type：Research paper (scientific journal) Publisher：Methods in Enzymology

Peptide ligation chemistries have revolutionized the synthesis of proteins with site-specific modifications or proteomimetics through assembly of multiple peptide segments. In order to prepare polypeptide chains consisting of 100–150 amino acid residues or larger generally assembled from three or more peptide segments, iterative purification process that decreases the product yield is usually demanded. Accordingly, methodologies for one-pot peptide ligation that omit the purification steps of intermediate peptide segments have been vigorously developed so far to improve the efficiency of chemical protein synthesis. In this chapter, we first outline the concept and recent advances of one-pot peptide ligation strategies. Then, the practical guideline for the preparation of peptide segments for one-pot peptide ligation is described with an emphasis on diketopiperazine thioester synthesis. Finally, we disclose the explicit protocols for one-pot four segment ligation via repetitive deprotection of N-terminal thiazolidine by a 2-aminobenzamide type aldehyde scavenger.

DOI： 10.1016/bs.mie.2024.04.020

Large-scale analysis of mRNA sequences localized near the start and amber codons and their impact on the diversity of mRNA display libraries Open Access

Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II Open Access

Oishi, T; Hatazawa, S; Kujirai, T; Kato, J; Kobayashi, Y; Ogasawara, M; Akatsu, M; Ehara, H; Sekine, SI; Hayashi, G; Takizawa, Y; Kurumizaka, H

NUCLEIC ACIDS RESEARCH Vol. 51 ( 19 ) page： 10364 - 10374 2023.10

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

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

DOI： 10.1093/nar/gkad754

Umemoto, S; Kondo, T; Fujino, T; Hayashi, G; Murakami, H

NUCLEIC ACIDS RESEARCH Vol. 51 ( 14 ) page： 7465 - 7479 2023.8

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

Extremely diverse libraries are essential for effectively selecting functional peptides or proteins, and mRNA display technology is a powerful tool for generating such libraries with over 1012-1013 diversity. Particularly, the protein-puromycin linker (PuL)/mRNA complex formation yield is determining for preparing the libraries. However, how mRNA sequences affect the complex formation yield remains unclear. To study the effects of N-terminal and C-terminal coding sequences on the complex formation yield, puromycin-attached mRNAs containing three random codons after the start codon (32768 sequences) or seven random bases next to the amber codon (6480 sequences) were translated. Enrichment scores were calculated by dividing the appearance rate of every sequence in protein-PuL/mRNA complexes by that in total mRNAs. The wide range of enrichment scores (0.09-2.10 for N-terminal and 0.30-4.23 for C-terminal coding sequences) indicated that the N-terminal and C-terminal coding sequences strongly affected the complex formation yield. Using C-terminal GGC-CGA-UAG-U sequences, which resulted in the highest enrichment scores, we constructed highly diverse libraries of monobodies and macrocyclic peptides. The present study provides insights into how mRNA sequences affect the protein/mRNA complex formation yield and will accelerate the identification of functional peptides and proteins involved in various biological processes and having therapeutic applications.

DOI： 10.1093/nar/gkad555

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization Open Access

Suzuki, S; Nakajima, Y; Kamo, N; Osakabe, A; Okamoto, A; Hayashi, G; Murakami, H

MOLECULES Vol. 28 ( 9 ) 2023.4

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

Thiol catalysts are essential in native chemical ligation (NCL) to increase the reaction efficiency. In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.

DOI： 10.3390/molecules28093655

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger.

Hata K, Kobayashi N, Sugimura K, Qin W, Haxholli D, Chiba Y, Yoshimi S, Hayashi G, Onoda H, Ikegami T, Mulholland CB, Nishiyama A, Nakanishi M, Leonhardt H, Konuma T, Arita K

Nucleic acids research Vol. 50 ( 21 ) page： 12527 - 12542 2022.11

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Hata K., Kobayashi N., Sugimura K., Qin W., Haxholli D., Chiba Y., Yoshimi S., Hayashi G., Onoda H., Ikegami T., Mulholland C.B., Nishiyama A., Nakanishi M., Leonhardt H., Konuma T., Arita K.

Nucleic acids research Vol. 50 ( 21 ) page： 12527 - 12542 2022.11

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

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.

DOI： 10.1093/nar/gkac1082

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Hata Keiichi, Kobayashi Naohiro, Sugimura Keita, Qin Weihua, Haxholli Deis, Chiba Yoshie, Yoshimi Sae, Hayashi Gosuke, Onoda Hiroki, Ikegami Takahisa, Mulholland Christopher B., Nishiyama Atsuya, Nakanishi Makoto, Leonhardt Heinrich, Konuma Tsuyoshi, Arita Kyohei

NUCLEIC ACIDS RESEARCH Vol. 50 ( 21 ) page： 12527 - 12542 2022.11

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

NUCLEIC ACIDS RESEARCH 2022.11

Repetitive Thiazolidine Deprotection Using a Thioester-Compatible Aldehyde Scavenger for One-Pot Multiple Peptide Ligation. International journal

Koki Nakatsu, Akimitsu Okamoto, Gosuke Hayashi, Hiroshi Murakami

Angewandte Chemie (International ed. in English) Vol. 61 ( 39 ) page： e202206240 2022.9

Language：English Publishing type：Research paper (scientific journal)

Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N',N'-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

DOI： 10.1002/anie.202206240

One-pot peptide ligation mediated by 2-aminobenzamide-type aldehyde scavengers

Nakatsu, K; Okamoto, A; Murakami, H; Hayashi, G

JOURNAL OF PEPTIDE SCIENCE Vol. 28 2022.8

Peptide selenoester synthesis via diketopiperazine formation

Hayashi, G; Hashimoto, M; Nakatsu, K; Murakami, H

JOURNAL OF PEPTIDE SCIENCE Vol. 28 2022.8

Monobodies with potent neutralizing activity against SARS-CoV-2 Delta and other variants of concern Open Access

Kondo, T; Matsuoka, K; Umemoto, S; Fujino, T; Hayashi, G; Iwatani, Y; Murakami, H

LIFE SCIENCE ALLIANCE Vol. 5 ( 6 ) 2022.6

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

Neutralizing antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are useful for patients’ treatment of the coronavirus disease 2019 (COVID-19). We report here affinity maturation of monobodies against the SARS-CoV-2 spike protein and their neutralizing activity against SARS-CoV-2 B.1.1 (Pango v.3.1.14) as well as four variants of concern. We selected matured monobodies from libraries with multi-site saturation mutagenesis on the recognition loops through in vitro selection. One clone, the C4-AM2 monobody, showed extremely high affinity (KD < 0.01 nM) against the receptor-binding domain of the SARS-CoV-2 B.1.1, even in monomer form. Furthermore, the C4-AM2 monobody efficiently neutralized the SARS-CoV-2 B.1.1 (IC50 = 46 pM, 0.62 ng/ml), and the Alpha (IC50 = 77 pM, 1.0 ng/ml), Beta (IC50 = 0.54 nM, 7.2 ng/ml), Gamma (IC50 = 0.55 nM, 7.4 ng/ml), and Delta (IC50 = 0.59 nM, 8.0 ng/ml) variants. The obtained monobodies would be useful as neutralizing proteins against current and potentially hazardous future SARS-CoV-2 variants.

DOI： 10.26508/lsa.202101322

Construction of a Highly Diverse mRNA Library for in vitro Selection of Monobodies Open Access

Kondo, T; Eguchi, M; Tsuzuki, N; Murata, N; Fujino, T; Hayashi, G; Murakami, H

BIO-PROTOCOL Vol. 11 ( 16 ) page： e4125 2021.8

Language：English Publishing type：Research paper (scientific journal) Publisher：Bio-protocol

Recently, we developed transcription/translation coupled with the association of puromycin linker (TRAP) display as a quick in vitro selection method to obtain antibody-like proteins. For the in vitro selection, it is important to prepare mRNA libraries among which the diversity is high. Here, we describe a method for the preparation of monobody mRNA libraries with greater than 1013 theoretical diversity. First, we synthesized two long single-stranded DNAs that corresponded to fragments of monobody DNA, with random codons in the BC and FG loops. These oligonucleotides were ligated by T4 DNA ligase with the support of guide oligonucleotides containing 3′ ends that were protected by a modification. After amplifying the product DNAs by PCR, one end of each DNA fragment was digested with the type II restriction enzyme BsaI, and the resulting DNA fragments were ligated using T4 DNA ligase. After amplification of the DNA product, mRNAs were synthesized by T7 RNA polymerase. This method is simple and could be used for the preparation of mRNA libraries for various antibody-like proteins.

DOI： 10.21769/BioProtoc.4125

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

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

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 in vitro 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

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

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

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

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

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.

DOI： 10.5059/yukigoseikyokaishi.78.130

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

CiNii Research

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

Hybridization-sensitive Fluorescent Oligonucleotide Probe Conjugated with Cell-penetrating Peptides for Enhanced Cellular Uptake

Hayashi Gosuke, Tamai Makoto, Okamoto Akimitsu

CHEMISTRY LETTERS Vol. 46 ( 12 ) page： 1803-1806 - 1806 2017.12

Language：English Publishing type：Research paper (scientific journal) Publisher：The Chemical Society of Japan

Intracellular delivery of oligonucleotides is essential not only for antisense- and RNAi-therapeutics, but also for live-cell RNA imaging with fluorescent nucleic acid probes. Herein, we developed oligonucleotide–peptide conjugates synthesized via Cu-catalyzed alkyne-azide cycloaddition between an exciton-controlled hybridization-sensitive oligonucleotide (ECHO) probe and linear or cyclic cell-penetrating peptides (CPPs). When lipofected to living HeLa cells, the CPP-conjugated ECHO probe showed enhanced cellular uptake efficiency compared with that of the unconjugated ECHO probe.

DOI： 10.1246/cl.170813

Regulation of the Stability of the Histone H2A-H2B Dimer by H2A Tyr57 Phosphorylation.

Sueoka T, Hayashi G, Okamoto A

Biochemistry Vol. 56 ( 36 ) page： 4767-4772 - 4772 2017.9

Chemical synthesis of dual labeled proteins via differently protected alkynes enables intramolecular FRET analysis.

Hayashi G, Kamo N, Okamoto A

Chemical communications (Cambridge, England) Vol. 53 ( 43 ) page： 5918-5921 - 5921 2017.5

Chemically-activatable alkyne-tagged probe for imaging microdomains in lipid bilayer membranes.

Yamaguchi S, Matsushita T, Izuta S, Katada S, Ura M, Ikeda T, Hayashi G, Suzuki Y, Kobayashi K, Tokunaga K, Ozeki Y, Okamoto A

Scientific reports Vol. 7 page： 41007 2017.1

Base-Resolution Analysis of 5-Hydroxymethylcytosine by One-Pot Bisulfite-Free Chemical Conversion with Peroxotungstate.

Hayashi G, Koyama K, Shiota H, Kamio A, Umeda T, Nagae G, Aburatani H, Okamoto A

Journal of the American Chemical Society Vol. 138 ( 43 ) page： 14178-14181 - 14181 2016.11

In vitro and in cell analysis of chemically synthesized histone H2A with multiple modifications.

Hayashi G, Sueoka T, Okamoto A

Chemical communications (Cambridge, England) Vol. 52 ( 28 ) page： 4999-5002 - 5002 2016.4

Diazirine Photocrosslinking Recruits Activated FTO Demethylase Complexes for Specific N-6-methyladenosine Recognition Reviewed

Hyun Seok Jeong, Gosuke Hayashi, Akimitsu Okamoto

ACS CHEMICAL BIOLOGY Vol. 10 ( 6 ) page： 1450 - 1455 2015.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER CHEMICAL SOC

N-6-methyladenosine (m(6)A) is a prevalent modification of RNAs. m(6)A exists in mRNA and plays an important role in RNA biological pathways and in RNA epigenetic regulation, We applied diazirine photocrosslinking to the event of m(6)A recognition mediated by the fat mass and obesity associated (FTO) demethylase. A highly photoreactive diazirine adjacent to m(6)A on the RNA successfully recruited activated FTO complexes with an m(6)A preference. The process of recognition of m(6)A via FTO using diazirine photocrosslinking was controlled by the alpha-ketoglutarate (alpha-KG) cosubstrate and the Peal). cofactor, which are involved in m(6)A oxidative demethylation. In addition, FTO bound to ssRNAs prior to the m(6)A recognition process. Diazirine photocrosslinking contributes to increasing the chances of capturing activated FTO complexes with specific m(6)A recognition and provides new insights into the dynamic FTO oxidative demethylation process.

DOI： 10.1021/cb5010096

Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers.

2-Oxazoline formation for selective chemical labeling of 5-hydroxylysine.

Hayashi G, Sakamoto R, Okamoto A

Chemistry, an Asian journal Vol. 10 ( 5 ) page： 1138-41 - 41 2015.5

Hybridization-sensitive fluorescent oligonucleotide probe conjugated with a bulky module for compartment-specific mRNA monitoring in a living cell.

Hayashi G, Yanase M, Takeda K, Sakakibara D, Sakamoto R, Wang DO, Okamoto A

Bioconjugate chemistry Vol. 26 ( 3 ) page： 412-7 - 7 2015.3

Yamamoto K, Chikaoka Y, Hayashi G, Sakamoto R, Yamamoto R, Sugiyama A, Kodama T, Okamoto A, Kawamura T

Mass spectrometry (Tokyo, Japan) Vol. 4 ( 1 ) page： A0039 - A0039 2015

Language：English Publishing type：Research paper (scientific journal) Publisher：The Mass Spectrometry Society of Japan

Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach.Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments.This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides.

DOI： 10.5702/massspectrometry.A0039

An orthogonal ribosome-tRNA pair via engineering of the peptidyl transferase center.

Terasaka N, Hayashi G, Katoh T, Suga H

Nature chemical biology Vol. 10 ( 7 ) page： 555-7 - 557 2014.7

Development of photoswitchable RNA aptamer-ligand complexes.

Hayashi G, Nakatani K

Methods in molecular biology (Clifton, N.J.) Vol. 1111 page： 29-40 - 40 2014

Probe design for the effective fluorescence imaging of intracellular RNA.

Hayashi G, Okamoto A

Chemical record (New York, N.Y.) Vol. 13 ( 2 ) page： 209-17 - 17 2013.4

A nucleic acid probe labeled with desmethyl thiazole orange: a new type of hybridization-sensitive fluorescent oligonucleotide for live-cell RNA imaging.

Okamoto A, Sugizaki K, Yuki M, Yanagisawa H, Ikeda S, Sueoka T, Hayashi G, Wang DO

Organic & biomolecular chemistry Vol. 11 ( 2 ) page： 362-71 - 71 2013.1

Activation of prokaryotic translation by antisense oligonucleotides binding to coding region of mRNA.

Hayashi G, Hong C, Hagihara M, Nakatani K

Biochemical and biophysical research communications Vol. 429 ( 1-2 ) page： 105-10 - 10 2012.12

The RNA origin of transfer RNA aminoacylation and beyond.

Suga H, Hayashi G, Terasaka N

Philosophical transactions of the Royal Society of London. Series B, Biological sciences Vol. 366 ( 1580 ) page： 2959-64 - 64 2011.10

[Ribosomal synthesis of nonstandard cyclic peptides and its application to drug discovery].

Hayashi G, Ohshiro Y, Suga H

Seikagaku. The Journal of Japanese Biochemical Society Vol. 82 ( 6 ) page： 505-14 2010.6

[Ribosomal synthesis of nonstandard cyclic peptides and its application to drug discovery].

Hayashi G, Ohshiro Y, Suga H

Seikagaku. The Journal of Japanese Biochemical Society Vol. 82 ( 6 ) page： 505 - 14 2010.6

Ribosome evolution for two artificial amino acids in E. coli. International journal

Hayashi G, Goto Y, Suga H

Chemistry & biology Vol. 17 ( 4 ) page： 320-1 - 1 2010.4

RNA aptamers that reversibly bind photoresponsive azobenzene-containing peptides.

Hayashi G, Hagihara M, Nakatani K

Chemistry (Weinheim an der Bergstrasse, Germany) Vol. 15 ( 2 ) page： 424-32 - 32 2009

Genotyping by allele-specific L-DNA-tagged PCR.

Hayashi G, Hagihara M, Nakatani K

Journal of biotechnology Vol. 135 ( 2 ) page： 157-60 - 60 2008.6

RNA aptamers that reversibly bind to photoresponsive peptide.

Hayashi G, Hagihara M, Nakatani K

Nucleic acids symposium series (2004) ( 52 ) page： 703-4 - 4 2008

Photoregulation of a peptide-RNA interaction on a gold surface.

Hayashi G, Hagihara M, Dohno C, Nakatani K

Journal of the American Chemical Society Vol. 129 ( 28 ) page： 8678-9 - 9 2007.7

Detection of L-DNA-tagged PCR products by surface plasmon resonance imaging.

Hayashi G, Hagihara M, Kobori A, Nakatani K

Chembiochem : a European journal of chemical biology Vol. 8 ( 2 ) page： 169-71 - 71 2007.1

Reversible regulation of binding between a photoresponsive peptide and its RNA aptamer.

Hayashi G, Hagihara M, Dohno C, Nakatani K

Nucleic acids symposium series (2004) ( 51 ) page： 93-4 - 4 2007

N,N'-Bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine stabilized a single pyrimidine bulge in duplex DNA.

Suda H, Kobori A, Zhang J, Hayashi G, Nakatani K

Bioorganic & medicinal chemistry Vol. 13 ( 14 ) page： 4507-12 - 12 2005.7

Small-molecule ligand induces nucleotide flipping in (CAG)n trinucleotide repeats.

Nakatani K, Hagihara S, Goto Y, Kobori A, Hagihara M, Hayashi G, Kyo M, Nomura M, Mishima M, Kojima C

Nature chemical biology Vol. 1 ( 1 ) page： 39-43 - 43 2005.6

Application of L-DNA as a molecular tag.

Hayashi G, Hagihara M, Nakatani K

Nucleic acids symposium series (2004) ( 49 ) page： 261-2 - 2 2005

Solution structure of a small-molecular ligand complexed with CAG trinucleotide repeat DNA.

Nakatani K, Hagihara S, Goto Y, Kobori A, Hagihara M, Hayashi G, Kyo M, Nomura M, Mishima M, Kojima C

Nucleic acids symposium series (2004) ( 49 ) page： 49-50 - 50 2005

Detection of guanine-adenine mismatches by surface plasmon resonance sensor carrying naphthyridine-azaquinolone hybrid on the surface. International journal

Hagihara S, Kumasawa H, Goto Y, Hayashi G, Kobori A, Saito I, Nakatani K

Nucleic acids research Vol. 32 ( 1 ) page： 278-86 - 86 2004

Language：English Publishing type：Research paper (scientific journal)

We have discovered a new molecule naphthyridine-azaquinolone hybrid (Npt-Azq) that strongly stabilized the guanine-adenine (G-A) mismatch in duplex DNA. In the presence of Npt-Azq, the melting temperature (T(m)) of 5'-d(CTA ACG GAA TG)-3'/3'-d(GAT TGA CTT AC)-5' containing a single G-A mismatch increased by 15.4 degrees C, whereas fully matched duplex increased its T(m) only by 2.2 degrees C. Npt-Azq was immobilized on the sensor surface for the surface plasmon resonance (SPR) assay to examine SPR detection of duplexes containing a G-A mismatch. Distinct SPR signals were observed when 27mer DNA containing a G-A mismatch was analyzed by the Npt-Azq immobilized sensor surfaces, whereas the signal of the fully matched duplex was approximately 6-fold weaker in intensity. The SPR signals for the G-A mismatch were proportional to the concentration of DNA in a range up to 1 microM, confirming that the SPR signal is in fact due to the binding of the G-A mismatch to Npt-Azq immobilized on the surface. Examination of all 16 G-A mismatches regarding the flanking sequence revealed that the sensor surface reported here is applicable to eight flanking sequences, covering 50% of all possible G-A mismatches.

DOI： 10.1093/nar/gkh171