Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from Japanese society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：International academic award (Japan or overseas)  Country：Japan

Award type：Award from international society, conference, symposium, etc.  Country：Japan

Award type：International academic award (Japan or overseas)  Country：Japan

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

DOI： 10.1093/nar/gkac1082

PubMed

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

Scopus

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

DOI： 10.1093/nar/gkac1082

Web of Science

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

DOI： 10.1093/nar/gkac1082

Web of Science

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

Web of Science

Scopus

PubMed

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

Web of Science

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

Web of Science

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

Web of Science

Scopus

PubMed

CiNii Research

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

Web of Science

Scopus

PubMed

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

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

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

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

Web of Science

Scopus

PubMed

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

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

Web of Science

Scopus

PubMed

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

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

DOI： 10.1016/j.cbpa.2020.04.016

Web of Science

Scopus

PubMed

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

Web of Science

Scopus

PubMed

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

DOI： 10.1021/acs.orglett.0c01450

Web of Science

Scopus

PubMed

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

Web of Science

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

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

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

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

DOI： 10.1021/acs.orglett.9b03152

Web of Science

Scopus

PubMed

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

DOI： 10.1039/c9sc00646j

Web of Science

Scopus

PubMed

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

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

DOI： 10.1021/acs.biomac.8b01655

PubMed

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

DOI： 10.1002/anie.201809765

PubMed

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

DOI： 10.1002/tcr.201800040

PubMed

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

DOI： 10.1039/c8cc01965g

PubMed

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

<p>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.</p>

DOI： 10.1246/cl.170813

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

DOI： 10.1021/acs.biochem.7b00504

PubMed

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

DOI： 10.1039/c7cc02612a

PubMed

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

DOI： 10.1038/srep41007

PubMed

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

DOI： 10.1021/jacs.6b06428

PubMed

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

DOI： 10.1039/c5cc10555b

PubMed

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

Web of Science

PubMed

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

DOI： 10.1002/asia.201500172

PubMed

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

DOI： 10.1021/acs.bioconjchem.5b00090

PubMed

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

PubMed

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

DOI： 10.1038/nchembio.1549

PubMed

CiNii Books

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

DOI： 10.1007/978-1-62703-755-6_3

PubMed

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

DOI： 10.1002/tcr.201200026

PubMed

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

DOI： 10.1039/c2ob26707a

PubMed

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

DOI： 10.1016/j.bbrc.2012.10.072

PubMed

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

DOI： 10.1098/rstb.2011.0137

PubMed

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

Publishing type：Research paper (scientific journal)  

PubMed

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

DOI： 10.1016/j.chembiol.2010.04.005

PubMed

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

DOI： 10.1002/chem.200800936

PubMed

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

DOI： 10.1016/j.jbiotec.2008.03.011

PubMed

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

DOI： 10.1093/nass/nrn355

PubMed

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

DOI： 10.1021/ja071298x

PubMed

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

DOI： 10.1002/cbic.200600477

PubMed

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

DOI： 10.1093/nass/nrm047

PubMed

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

DOI： 10.1016/j.bmc.2005.04.035

PubMed

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

DOI： 10.1038/nchembio708

PubMed

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

DOI： 10.1093/nass/49.1.261

PubMed

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

DOI： 10.1093/nass/49.1.49

PubMed

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

PubMed

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

DOI： 10.1093/nass/48.1.129

PubMed

Language：English  

DOI： 10.1002/asia.201500172

PubMed

Language：English  

DOI： 10.1021/acs.bioconjchem.5b00090

PubMed

Language：English  

DOI： 10.5702/massspectrometry.A0039

PubMed

Language：English  

DOI： 10.1038/nchembio.1549

PubMed

Language：English  

DOI： 10.1007/978-1-62703-755-6_3

PubMed

Language：English  

DOI： 10.1002/tcr.201200026

PubMed

Language：English  

DOI： 10.1039/c2ob26707a

PubMed

Language：English  

DOI： 10.1016/j.bbrc.2012.10.072

PubMed

Language：English  

DOI： 10.1098/rstb.2011.0137

PubMed

Language：Japanese  

PubMed

Language：English  

DOI： 10.1016/j.chembiol.2010.04.005

PubMed

Language：English  

DOI： 10.1002/chem.200800936

PubMed

Language：English  

DOI： 10.1016/j.jbiotec.2008.03.011

PubMed

Language：English  

DOI： 10.1093/nass/nrn355

PubMed

Language：English  

DOI： 10.1021/ja071298x

PubMed

Language：English  

DOI： 10.1002/cbic.200600477

PubMed

Language：English  

DOI： 10.1093/nass/nrm047

PubMed

Language：English  

DOI： 10.1016/j.bmc.2005.04.035

PubMed

Language：English  

DOI： 10.1038/nchembio708

PubMed

Language：English  

DOI： 10.1093/nass/49.1.261

PubMed

Language：English  

DOI： 10.1093/nass/49.1.49

PubMed

Language：English  

DOI： 10.1093/nass/48.1.129

PubMed

Language：English  

DOI： 10.1093/nar/gkh171

PubMed