Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK12Oub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK12Oub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

Long-chain scorpion toxin AaH-II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage-gated sodium ion channel Na(v)1.7 responsible for pain sensation. Efficient chemical synthesis of AaH-II and its derivatives is beneficial to the study of the function and mechanism of Na(v)1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid-phase peptide synthesis, hydrazide-based two-segment native chemical ligation, and hydrazide-based three-segment native chemical ligation for the synthesis of AaH-II. The hydrazide-based two-segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH-II to study the biological functions and modulation of Na(v)1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.Deubiquitinases (DUBs) targeting Met1-linked ubiquitin chains have important functions in mammals but are barely studied in plants. Here, the authors identify rice OTUB1 as a Met1-targeting DUB, characterize the structural determinants of this activity, and show that these features are conserved in green plants.

Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked β-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation. © 2022 American Chemical Society.

The beta(2)-adrenergic receptor (beta(2)AR) is a G-proteincoupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. beta(2)AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length beta(2)AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length beta(2)AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length beta(2)ARs with distinct modification patterns, including a full-length beta(2)AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length beta(2)AR receptors, we found that different phosphorylation patterns mediate different interactions with beta-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between beta(2)AR and beta-arrestin1. Access to full-length beta(2)AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of beta(2)AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

The replacement of disulfide bridges with metabolically stable isosteres is a promising strategy to improve the stability of disulfide-rich polypeptides towards reducing agents and isomerases. A diaminodiacid-based strategy is one of the most effective methods to construct disulfide bond mimics, but modified diaminodiacids have not been developed till now. Inspired by the fact that alkylation of disulfide bonds can regulate the activity of polypeptides, herein, we report the first example of thioether bridged diaminodiacids incorporating Cys C-beta dimethyl modification, obtained by penicillamine (Pen)-based thiol alkylation. The utility of these new diaminodiacids was demonstrated by the synthesis of disulfide surrogates of oxytocin containing a short-span disulfide bond and of KIIIA with large-span disulfide bonds. This new type of synthetic bridge further extends the diaminodiacid toolbox to facilitate the study of the structure-activity relationship of disulfide-rich peptides.

A mirror-image protein-based information barcoding and storage technology wherein D-amino acids are used to encode information into mirror-image proteins that are chemically synthesized is described. These mirror-image proteins were then fused into various materials from which information-encoded objects were produced. Subsequently, the mirror-image proteins were extracted from the objects using biotin-streptavidin resin-mediated specific enrichment and cleaved using an Ni(II)-mediated selective peptide cleavage. Protein sequencing was accomplished using liquid chromatography/tandem mass spectrometry (LC-MS/MS) and then transcoded into the recorded information. We demonstrated the use of this technology to encode Chinese words into mirror-image proteins, which were then fused onto a poly(ethylene terephthalate) (PET) film and retrieved and decoded by LC-MS/MS sequencing. Compared to information barcoding and storage technologies using natural biopolymers, the mirrorimage biopolymers used in our technology may be more stable and durable. (c) 2021 Science China Press. Published by Elsevier B.V. and Science China Press. All rights reserved.

Main observation and conclusion The synthesis of an Asp lactam derivative of A-183, a selective inhibitor of Factor 7a with good anticoagulant and antithrombotic activity, is described. Our synthesis depends on the use of a removable backbone modification (RBM) strategy to prevent aspartimide formation, which thwarted all attempts to synthesize this target using direct solid-phase peptide synthesis. Validation of the RBM strategy in the synthesis of a second Asp lactam derivative was also accomplished. The RBM strategy is therefore proposed as a general method for the synthesis of Asp lactam cyclic peptides.

Lactam cyclic peptides are a class of interesting and pharmaceutically active molecules, but their previous syntheses have required the use of heavy metals and/or forcing conditions. Here, we describe the efficient application of the previously reported tert-butyl disulfide-protected amino acids and their use in the efficient, solid-phase synthesis of a series of lactam cyclic peptides under mild, metal-free conditions.