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 <i>in vitro</i> 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