Cell-Permeable Ubiquitin and Histone Tools for Studying Post-translational Modifications.

Protein post-translational modifications (PTMs) regulate nearly all biological processes in eukaryotic cells, and synthetic PTM protein tools are widely used to detect the activity of the related enzymes and identify the interacting proteins in cell lysates. Recently, the study of these enzymes and the interacting proteome has been accomplished in live cells using cell-permeable PTM protein tools. In this concept, we will introduce cell penetrating techniques, the syntheses of cell-permeable PTM protein tools, and offer some future perspective.

Chemical synthesis of on demand-activated SUMO-based probe by a photocaged glycine-assisted strategy.

The transiently-activated SUMO probes are conducive to understand the dynamic control of SENPs activity. Here, we developed a photocaged glycine-assisted strategy for the construction of on demand-activated SUMO-ABPs. The light-sensitive groups installed at G92 and G64 backbone of SUMO-2 can temporarily block probes activity and hamper aspartimide formation, respectively, which enabled the efficient synthesis of inert SUMO-2 propargylamide (PA). The probe could be activated to capture SENPs upon photo-irradiation not only in vitro but also in intact cells, providing opportunities to further perform intracellular time-resolved proteome-wide profiling of SUMO-related enzymes.

The Ubiquitin Enigma: Progress in the Detection and Chemical Synthesis of Branched Ubiquitin Chains.

Ubiquitin chains with distinct topologies play essential roles in eukaryotic cells. Recently, it was discovered that multiple ubiquitin units can be ligated to more than one lysine residue in the same ubiquitin to form diverse branched ubiquitin chains. Although there is increasing evidence implicating these branched chains in a plethora of biological functions, few mechanistic details have been elucidated. This concept article introduces the function, detection and chemical synthesis of branched ubiquitin chains; and offers some future perspective for this exciting new field.

Chemical Synthesis of K34-Ubiquitylated H2B for Nucleosome Reconstitution and Single-Particle Cryo-Electron Microscopy Structural Analysis.

Post-translational modifications (e.g., ubiquitylation) of histones play important roles in dynamic regulation of chromatin. Histone ubiquitylation has been speculated to directly influence the structure and dynamics of nucleosomes. However, structural information for ubiquitylated nucleosomes is still lacking. Here we report an alternative strategy for total chemical synthesis of homogenous histone H2B-K34-ubiquitylation (H2B-K34Ub) by using acid-cleavable auxiliary-mediated ligation of peptide hydrazides for site-specific ubiquitylation. Synthetic H2B-K34Ub was efficiently incorporated into nucleosomes and further used for single-particle cryo-electron microscopy (cryo-EM) imaging. The cryo-EM structure of the nucleosome containing H2B-K34Ub suggests that two flexible ubiquitin domains protrude between the DNA chains of the nucleosomes. The DNA chains around the H2B-K34 sites shift and provide more space for ubiquitin to protrude. These analyses indicated local and slight structural influences on the nucleosome with ubiquitylation at the H2B-K34 site.

A statistical view of protein chemical synthesis using NCL and extended methodologies.

Native chemical ligation and extended methodologies are the most popular chemoselective reactions for protein chemical synthesis. Their combination with desulfurization techniques can give access to small or challenging proteins that are exploited in a large variety of research areas. In this report, we have conducted a statistical review of their use for protein chemical synthesis in order to provide a flavor of the recent trends and identify the most popular chemical tools used by protein chemists. To this end, a protein chemical synthesis (PCS) database (http://pcs-db.fr) was created by collecting a set of relevant data from more than 450 publications covering the period 1994-2017. A preliminary account of what this database tells us is presented in this report.

Synthesis of relaxin-2 and insulin-like peptide 5 enabled by novel tethering and traceless chemical excision.

This report presents an entirely chemical, general strategy for the synthesis of relaxin-2 and insulin-like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A-chain and B-chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization-mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine-mediated disulfide bond formation and enzyme-assisted proteolysis to generate biologically active two-chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin-like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin-like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two-chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Practical Chemical Synthesis of Atypical Ubiquitin Chains by Using an Isopeptide-Linked Ub Isomer.

Chemical ubiquitination is an effective approach for accessing structurally defined, atypical ubiquitin (Ub) chains that are difficult to prepare by other techniques. Herein, we describe a strategy that uses a readily accessible premade isopeptide-linked 76-mer (isoUb), which has an N-terminal Cys and a C-terminal hydrazide, as the key building block to assemble atypical Ub chains in a modular fashion. This method avoids the use of auxiliary-modified Lys and instead employs the canonical and therefore more robust Cys-based native chemical ligation technique. The efficiency and capacity of this isoUb-based strategy is exemplified by the cost-effective synthesis of several linkage- and length-defined atypical Ub chains, including K27-linked tetra-Ub and K11/K48-branched tri-, tetra-, penta-, and hexa-Ubs.

Blocking of the PD-1/PD-L1 Interaction by a D-Peptide Antagonist for Cancer Immunotherapy.

Blockade of the protein-protein interaction between the transmembrane protein programmed cell death protein 1 (PD-1) and its ligand PD-L1 has emerged as a promising immunotherapy for treating cancers. Using the technology of mirror-image phage display, we developed the first hydrolysis-resistant D-peptide antagonists to target the PD-1/PD-L1 pathway. The optimized compound (D) PPA-1 could bind PD-L1 at an affinity of 0.51 µM in vitro. A blockade assay at the cellular level and tumor-bearing mice experiments indicated that (D) PPA-1 could also effectively disrupt the PD-1/PD-L1 interaction in vivo. Thus D-peptide antagonists may provide novel low-molecular-weight drug candidates for cancer immunotherapy.

An efficient one-pot four-segment condensation method for protein chemical synthesis.

Successive peptide ligation using a one-pot method can improve the efficiency of protein chemical synthesis. Although one-pot three-segment ligation has enjoyed widespread application, a robust method for one-pot four-segment ligation had to date remained undeveloped. Herein we report a new one-pot multisegment peptide ligation method that can be used to condense up to four segments with operational simplicity and high efficiency. Its practicality is demonstrated by the one-pot four-segment synthesis of a plant protein, crambin, and a human chemokine, hCCL21.

Probing effects of site-specific aspartic acid isomerization on structure and stability of GB1 through chemical protein synthesis.

Chemical modifications of long-lived proteins, such as isomerization and epimerization, have been evoked as prime triggers for protein-damage related diseases. Deamidation of Asn residues, which results in formation of a mixture of l- and d-Asp and isoAsp via an intermediate aspartyl succinimide, can result in the disruption of cellular proteostasis and toxic protein depositions. In contrast to extensive data on the biological prevalence and functional implications of aspartyl succinimide formation, much less is known about the impact of the resulting altered backbone composition on properties of individual proteins at a molecular level. Here, we report the total chemical synthesis, biophysical characterization, and NMR structural analysis of a series of variants of the B1 domain of protein G from Streptococcal bacteria (GB1) in which all possible Asp isomers as well as an aspartyl succinimide were individually incorporated at a defined position in a solvent-exposed loop. Subtle local structural effects were observed; however, these were accompanied by notable differences in thermodynamic folded stability. Surprisingly, the noncanonical backbone connectivity of d-isoAsp led to a variant that exhibited enhanced stability relative to the natural protein.

Atg8-PE protein-based in vitro biochemical approaches to autophagy studies.

Macroautophagy/autophagy is an evolutionarily conserved intracellular degradation pathway that maintains cellular homeostasis. Over the past two decades, a series of scientific breakthroughs have helped explain autophagy-related molecular mechanisms and physiological functions. This tremendous progress continues to depend largely on powerful research methods, specifically, various autophagy marker Atg8-PE protein-based methods for studying membrane dynamics and monitoring autophagic activity. Recently, several biochemical approaches have been successfully developed to produce the lipidated protein Atg8-PE or its mimics in vitro, including enzyme-mediated reconstitution systems, chemically defined reconstitution systems, cell-free lipidation systems and protein chemical synthesis. These approaches have contributed important insights into the mechanisms underlying Atg8-mediated membrane dynamics and protein-protein interactions, creating a new perspective in autophagy studies. In this review, we comprehensively summarize Atg8-PE protein-based in vitro biochemical approaches and recent advances to facilitate a better understanding of autophagy mechanisms. In addition, we highlight the advantages and disadvantages of various Atg8-PE protein-based approaches to provide general guidance for their use in studying autophagy.Abbreviations: ATG: autophagy related; ATP: adenosine triphosphate; COPII: coat protein complex II; DGS-NTA: 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt); DPPE: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; DSPE: 1,2-distearoyl-sn-glycero-3-phosphoethanolamine; E. coli: Escherichia coli; EPL: expressed protein ligation; ERGIC: ER-Golgi intermediate compartment; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; GUVs: giant unilamellar vesicles; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MBP: maltose binding protein; MEFs: mouse embryonic fibroblasts; MESNa: 2-mercaptoethanesulfonic acid sodium salt; NCL: native chemical ligation; NTA: nitrilotriacetic acid; PE: phosphatidylethanolamine; PS: phosphatidylserine; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SPPS: solid-phase peptide synthesis; TEV: tobacco etch virus; WT: wild-type.

A new method of N to C sequential ligation using thioacid capture ligation and native chemical ligation.

Sequential peptide ligation strategy becomes more and more important in large protein or long peptides chemical synthesis due to the limited peptide/protein size obtained by solid phase synthesis of individual peptides or even one-step peptide ligation. Herein, we developed an alternative method which could perform the sequential peptide ligation of several segments from N to C direction based on the combined use of thioacid capture ligation and native chemical ligation. The sweet protein monellin was produced through this strategy on a scale of multi-milligrams.

Total Chemical Synthesis of Modified Histones.

In the post-genome era, epigenetics has received increasing attentions in recent years. The post-translational modifications (PTMs) of four core histones play central roles in epigenetic regulation of eukaryotic genome by either directly altering the biophysical properties of nucleosomes or by recruiting other effector proteins. In order to study the biological functions and structural mechanisms of these histone PTMs, an obligatory step is to prepare a sufficient amount of homogeneously modified histones. This task cannot be fully accomplished either by recombinant technology or enzymatic modification. In this context, synthetic chemists have developed novel protein synthetic tools and state-of-the-art chemical ligation strategies for the preparation of homologous modified histones. In this review, we summarize the recent advances in the preparation of modified histones, focusing on the total chemical synthesis strategies. The importance and potential of synthetic chemistry for the study of histone code will be also discussed.

Total chemical synthesis of a thermostable enzyme capable of polymerase chain reaction.

Polymerase chain reaction (PCR) has been a defining tool in modern biology. Towards realizing mirror-image PCR, we have designed and chemically synthesized a mutant version of the 352-residue thermostable Sulfolobus solfataricus P2 DNA polymerase IV with l-amino acids and tested its PCR activity biochemically. To the best of our knowledge, this enzyme is the largest chemically synthesized protein reported to date. We show that with optimization of PCR conditions, the fully synthetic polymerase is capable of amplifying template sequences of up to 1.5 kb. The establishment of this synthetic route for chemically synthesizing DNA polymerase IV is a stepping stone towards building a d-enzyme system for mirror-image PCR, which may open up an avenue for the creation of many mirror-image molecular tools such as mirror-image systematic evolution of ligands by exponential enrichment.