H3K4me1 recruits DNA repair proteins in plants.

DNA repair proteins can be recruited by their histone reader domains to specific epigenomic features, with consequences on intragenomic mutation rate variation. Here, we investigated H3K4me1-associated hypomutation in plants. We first examined two proteins which, in plants, contain Tudor histone reader domains: PRECOCIOUS DISSOCIATION OF SISTERS 5 (PDS5C), involved in homology-directed repair, and MUTS HOMOLOG 6 (MSH6), a mismatch repair protein. The MSH6 Tudor domain of Arabidopsis (Arabidopsis thaliana) binds to H3K4me1 as previously demonstrated for PDS5C, which localizes to H3K4me1-rich gene bodies and essential genes. Mutations revealed by ultradeep sequencing of wild-type and msh6 knockout lines in Arabidopsis show that functional MSH6 is critical for the reduced rate of single base substitution mutations in gene bodies and H3K4me1-rich regions. We explored the breadth of these mechanisms among plants by examining a large rice (Oryza sativa) mutation dataset. H3K4me1-associated hypomutation is conserved in rice as are the H3K4me1 binding residues of MSH6 and PDS5C Tudor domains. Recruitment of DNA repair proteins by H3K4me1 in plants reveals convergent, but distinct, epigenome-recruited DNA repair mechanisms from those well described in humans. The emergent model of H3K4me1-recruited repair in plants is consistent with evolutionary theory regarding mutation modifier systems and offers mechanistic insight into intragenomic mutation rate variation in plants.

Optineurin provides a mitophagy contact site for TBK1 activation.

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.

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

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.

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

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.

Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization.

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.

cIAP1-based degraders induce degradation via branched ubiquitin architectures.

Targeted protein degradation through chemical hijacking of E3 ubiquitin ligases is an emerging concept in precision medicine. The ubiquitin code is a critical determinant of the fate of substrates. Although two E3s, CRL2VHL and CRL4CRBN, frequently assemble with proteolysis-targeting chimeras (PROTACs) to attach lysine-48 (K48)-linked ubiquitin chains, the diversity of the ubiquitin code used for chemically induced degradation is largely unknown. Here we show that the efficacy of cIAP1-targeting degraders depends on the K63-specific E2 enzyme UBE2N. UBE2N promotes degradation of cIAP1 induced by cIAP1 ligands and subsequent cancer cell apoptosis. Mechanistically, UBE2N-catalyzed K63-linked ubiquitin chains facilitate assembly of highly complex K48/K63 and K11/K48 branched ubiquitin chains, thereby recruiting p97/VCP, UCH37 and the proteasome. Degradation of neo-substrates directed by cIAP1-recruiting PROTACs also depends on UBE2N. These results reveal an unexpected role for K63-linked ubiquitin chains and UBE2N in degrader-induced proteasomal degradation and demonstrate the diversity of the ubiquitin code used for chemical hijacking.

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

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.

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

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.

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

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 (K D < 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 (IC 50 = 46 pM, 0.62 ng/ml), and the Alpha (IC 50 = 77 pM, 1.0 ng/ml), Beta (IC 50 = 0.54 nM, 7.2 ng/ml), Gamma (IC 50 = 0.55 nM, 7.4 ng/ml), and Delta (IC 50 = 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.

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

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. Graphic abstract: Construction of a highly diverse mRNA library.

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

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 for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.

cDNA TRAP display for rapid and stable in vitro selection of antibody-like proteins.

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.

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

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.

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

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.

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

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.

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

We report an Fmoc-compatible and external-thiol-free method of peptide C-terminus thioesterification with cysteinylprolyl imide. The newly synthesized structure, i.e., cysteinylprolyl-thiazolidinone, provided high conversion and sequence-independent fast kinetics (90 min) in the diketopiperazine thioester formation under relatively mild conditions: pH 6.0, 37 °C. Employing this thioesterification method, we synthesized histone H3.2 bearing K56 acetylation.

Toolbox for chemically synthesized histone proteins.

Histone post-translational modifications play significant roles in gene regulation processes. Among many approaches, chemical protein synthesis has been a successful and promising method for the preparation of homogeneous products of site-specifically modified histones for elucidation of their biological significance. In this short review, we describe the recent advances in synthetic toolbox for histone proteins such as thioester precursors, chemical ubiquitination, and one-pot peptide ligation.

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

We report selective removals of N-terminal and internal Cys protecting groups using different palladium complexes to facilitate the efficient chemical protein synthesis. Utilizing the orthogonal deprotection pairs, we accomplished chemical synthesis of histone H3 containing trimethylated Lys through the combination of Pd(0)-mediated Alloc deprotection for one-pot multiple peptide ligation and Pd(II)Cl2-mediated Acm deprotection to recover native Cys residues after desulfurization.

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

Native chemical ligation (NCL) between the C-terminal peptide thioester and the N-terminal cysteinyl-peptide revolutionized the field of chemical protein synthesis. The difficulty of direct synthesis of the peptide thioester in the Fmoc method has prompted the development of crypto-thioesters that can be efficiently converted into thioesters. Cysteinylprolyl ester (CPE), which is an N-S acyl shift-driven crypto-thioester that relies on an intramolecular O-N acyl shift to displace the amide-thioester equilibrium, enabled trans-thioesterification and subsequent NCL in one pot. However, the utility of CPE is limited because of the moderate thioesterification rates and the synthetic complexity introduced by the ester group. Herein, we develop a new crypto-thioester, cysteinylprolyl imide (CPI), which replaces the alcohol leaving group of CPE with other leaving groups such as benzimidazolidinone, oxazolidinone, and pyrrolidinone. CPI peptides were efficiently synthesized by using standard Fmoc solid-phase peptide synthesis (SPPS) and subsequent on-resin imide formation. Screening of the several imide structures indicated that methyloxazolidinone-t-leucine (MeOxd-Tle) showed faster conversion into thioester and higher stability against hydrolysis under NCL conditions. Finally, by using CPMeOxd-Tle peptides, we demonstrated the chemical synthesis of affibody via N-to-C sequential, three-segment ligation and histone H2A.Z via convergent four-segment ligation. This facile and straightforward method is expected to be broadly applicable to chemical protein synthesis.