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α-Conotoxins are well-known probes for the characterization of the various subtypes of nicotinic acetylcholine receptors (nAChRs). Identifying new α-conotoxins with different pharmacological profiles can provide further insights into the physiological or pathological roles of the numerous nAChR isoforms found at the neuromuscular junction, the central and peripheral nervous systems, and other cells such as immune cells. This study focuses on the synthesis and characterization of two novel α-conotoxins obtained from two species endemic to the Marquesas Islands, namely Conus gauguini and Conus adamsonii. Both species prey on fish, and their venom is considered a rich source of bioactive peptides that can target a wide range of pharmacological receptors in vertebrates. Here, we demonstrate the versatile use of a one-pot disulfide bond synthesis to achieve the α-conotoxin fold [Cys 1-3; 2-4] for GaIA and AdIA, using the 2-nitrobenzyl (NBzl) protecting group of cysteines for effective regioselective oxidation. The potency and selectivity of GaIA and AdIA against rat nicotinic acetylcholine receptors were investigated electrophysiologically and revealed potent inhibitory activities. GaIA was most active at the muscle nAChR (IC50 = 38 nM), whereas AdIA was most potent at the neuronal α6/3 β2β3 subtype (IC50 = 177 nM). Overall, this study contributes to a better understanding of the structure–activity relationships of α-conotoxins, which may help in the design of more selective tools.

A rapid and efficient cyclization of unprotected N-propargylated peptides using the Au(I) organometallic complex is reported. The method relies on the activation of the propargyl functionality using gold(I) to produce a new linkage with the N-terminus amine at the cyclization site. The presented method features a fast reaction rate (within 20 min), mild conditions, chemoselectivity, wide sequence scope, and high yields (up to 87%). The strategy was successfully tested on a wide variety of 30 unprotected peptides having various sequences and lengths, thus providing access to structurally distinct cyclic peptides. The practical usefulness of this method was demonstrated in producing peptides that bind efficiently to Lys48-linked di- and tetra-ubiquitin chains. The new cyclic peptide modulators exhibited high permeability to living cells and promoted apoptosis via binding with the endogenous Lys48-linked ubiquitin chains., A rapid and efficient cyclization of unprotected N-propargylated peptides using (JohnPhos)Au(ACN)SbF6 complex is reported and features a fast reaction rate (within 20 min) and chemoselectivity.

Deciphering the protein posttranslational modification (PTM) code is one of the greatest biochemical challenges of our time. Phosphorylation and ubiquitylation are key PTMs that dictate protein function, recognition, sub-cellular localization, stability, turnover and fate. Hence, failures in their regulation leads to various disease. Chemical protein synthesis allows preparation of ubiquitinated and phosphorylated proteins to study their biochemical properties in great detail. However, monitoring these modifications in intact cells or in cell extracts mostly depends on antibodies, which often have off-target binding. Here, we report that the most widely used antibody for ubiquitin (Ub) phosphorylated at serine 65 (pUb) has significant off-targets that appear during mitosis. These off-targets are connected to polo-like kinase 1 (PLK1) mediated phosphorylation of cell cycle-related proteins and the anaphase promoting complex subunit 1 (APC1).

In this study, the live-cell delivery of structurally different synthetic diubiquitin chains was examined. We found that the combination of structural variations of the Ub chains (intrinsic factors); nature of CPP and CPP-protein linkage (extrinsic factors) influence their delivery.

Development of modulators targeting specific interactions of ubiquitin‐based conjugates with their partners is a formidable task since it requires a suitable screening assay and homogeneous ubiquitin conjugates. We developed a novel high‐throughput strategy for screening ligands for Lys48‐linked tetraubiquitin chain in a relatively simple, fast, and affordable manner. This approach combined with a state‐of‐the‐art toolbox of chemical protein synthesis and a specially optimized Cys deprotection protocol enabled us to design highly potent, Lys48‐linked tetraubiquitin chain selective “next generation” dimeric peptide modulators. The dimeric peptide exhibited cancer cell permeability and induced cell death with higher efficiency compared to its monocyclic analogue. These features make our dimeric peptide a promising candidate for further studies using in vivo models. Our assay can be adopted for other various ubiquitin chains in their free or anchored forms as well as conjugates for Ub‐like modifiers., A fluorescence‐based competitive assay was developed, which enabled the high‐throughput screening of various cyclic peptides targeting the Lys48‐linked tetraubiquitin chain. This assay combined with chemical tools enabled the design of a potent dimeric cyclic peptide, which induced cell death with higher efficiency compared to its monocyclic analogue.

Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We develop an approach based on the combination of a small molecule, ultraviolet-light, and palladium for chemo- and regio-selective activation of cysteine, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepare bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery., Synthesis of peptides and proteins containing multiple disulfide bonds is challenging, limiting the elucidation of their biological functions. Here, the authors report a general synthetic strategy for fast formation of two and three disulfide bonds in peptides and proteins, and apply it to prepare several therapeutically important peptides.

Targeting the cell nucleus remains a challenge for drug delivery. Here we present a universal platform for smart design of nano-particles (NPs) decoration that allows recruitment of multiple dynein motors to drive their active motion towards the nucleus. The uniqueness of our approach is based on using: (i) a spacer polymer, commonly Biotin-Polyethylene-glycol-thiol (B-PEG-SH), whose grafting density and molecular weight can be tuned thereby allowing NP transport optimization, and (ii) protein binding peptides, like cell penetrating, NLS, or cancer targeting, peptides. Universal chemistry is employed to link peptides to the PEG free-end. To manifest our platform, we use a SV40T large antigen-originating NLS peptide. Our modular design allows tuning the number of recruited motors, and to replace the NLS by a variety of other localization signal molecules. Our control of the NP decoration scheme, and the modularity of our platform, carries great advantage for nano-carrier design for drug delivery applications.

Modifying cyclic cell-penetrating deca-arginine (cR10) peptides with 4-(4-dimethylaminophenylazo)benzoic acid (DABCYL) improves the uptake efficiency of synthetic ubiquitin (Ub) cargoes into living cells. To probe the role of the DABCYL moiety, we performed time-lapse microscopy and fluorescence lifetime imaging microscopy (FLIM) of fluorescent DABCYL-R10 to evaluate the impact on cell entry by the formation of nucleation zones. Furthermore, we performed a structure-uptake relationship study with 13 DABCYL derivatives coupled to CPP to examine their effect on the cell-uptake efficiency when conjugated to mono-Ub through disulfide linkages. Our results show that through structure variations of the DABCYL moiety alone we could reach, at nanomolar concentration, an additional threefold increase in the cytosolic delivery of Ub, which will enable studies on various intracellular processes related to Ub signaling.

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.

In fundamental research and drug discovery, there is still a need for effective and straightforward chemical approaches for generating cyclic peptides. The divergent synthesis of cyclic peptides remains a challenge, in particular when cyclization is carried out in the presence of unprotected side chains and a nonpeptidic component within the cycle is needed. Herein, we describe a novel and efficient strategy based on Au(I)-mediated cyclization of unprotected peptides through rapid (30−60 min) amine addition on a propargyl group to generate an imine linkage. Mechanistic insights reveal that the reaction proceeds via regioselective Markovnikov’s addition of the amine on the Au(I)- activated propargyl. This strategy was successfully applied to prepare efficiently (56−94%) over 35 diverse cyclic peptides having different sequences and lengths. We have also achieved stereoselective reduction of cyclic imines employing chiral ligands. The practicality of our method was extended for the synthesis of cyclic peptides that bind Lys48-linked di-ubiquitin chains with high affinity, leading to apoptosis of cancer cells.

Chemists have been interested in the N-alkylation of a peptide bond because such a modification alters the conformation of the amide bond, interferes with hydrogen bond formation, and changes other properties of the peptide (e.g., solubility). This modification also opens the door for attaching functional groups for various applications. Nonetheless, the irreversibility of some of these modifications and the harsh conditions required for their removal currently limits the wide utility of this approach. Herein, we report applying a propargyl group for peptide bond modification at diverse junctions, which can be removed under mild and aqueous conditions via treatment with gold(I). Considering the straightforward conditions for both the installation and removal of this group, the propargyl group provides access to the benefits of backbone N-alkylation, while preserving the ability for on-demand depropargylation and full recovery of the native amide bond. This reversible modification was found to improve solid-phase peptide synthesis as demonstrated in the chemical synthesis of NEDD8 protein, without the use of special dipeptide analogues. Also, the reported approach was found to be useful in decaging a broad range of propargyl-based protecting groups used in chemical protein synthesis. Remarkably, reversing the order of the two residues in the propargylation site resulted in rapid amide bond cleavage, which extends the applicability of this approach beyond a removable backbone modification to a cleavable linker. The easy attach/detach of this functionality was also examined in loading and releasing of biotinylated peptides from streptavidin beads.

Ubiquitin (Ub) and its related small Ub like modifier (SUMO) are among the most influential protein post-translational modifications in eukaryotes. Unfortunately, visualizing these modifications in live cells is a challenging task. Chemical protein synthesis offers great opportunities in studying and further understanding Ub and SUMO biology. Nevertheless, the low cell permeability of proteins limits these studies mainly for in vitro applications. Here, we introduce a multiplexed protein cell delivery approach, termed MBL (multiplexed bead loading), for simultaneous loading of up to four differentially labeled proteins with organic fluorophores. We applied MBL to visualize ubiquitination and SUMOylation events in live and untransfected cells without fluorescent protein tags or perturbation to their endogenous levels. Our study reveals unprecedented involvements of Ub and SUMO2 in lysosomes depending on conjugation states. We envision that this approach will improve our understanding of dynamic cellular processes such as formation and disassembly of membraneless organelles.

Nuclear factor κB (NF-κB) is an important transcriptional regulator that is involved in numerous cellular processes, including cell proliferation, immune response, cell survival, and malignant transformation. It relies on the ubiquitin–proteasome system (UPS) for several of the steps in the concerted cascade of its activation. Previously, we showed that the ubiquitin (Ub) ligase KPC1 is involved in ubiquitination and limited proteasomal processing of the NF-κB1 p105 precursor to generate the p50 active subunit of the “canonical” heterodimeric transcription factor p50–p65. Overexpression of KPC1 with the generation of an excessive amount of p50 was shown to suppress tumors, an effect which is due to multiple mechanisms. Among them are suppression of expression of programmed cell death-ligand 1 (PD-L1), overexpression of a broad array of tumor suppressors, and secretion of cytokines which results in recruitment of suppressive immune cells into the tumor. Here, we show that the site of KPC1 to which p105 binds is exceptionally short and is made up of the seven amino acids WILVRLW. Attachment of this short stretch to a small residual part (∼20%) of the ligase that also contains the essential Really Interesting New Gene (RING)-finger domain was sufficient to bind p105, conjugate to it Ub, and suppress tumor growth in an animal model. Fusion of the seven amino acids to a Von Hippel–Lindau protein (pVHL)-binding ligand (which serves as a “universal” ligase for many proteolysis-targeting chimeras; PROTACs) resulted in a compound that stimulated conjugation of Ub to p105 in a cell-free system and its processing to p50 in cells and restricted cell growth.

The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins., The 20S particle is part of the 26S proteasome, but also exists as a free complex. Here, the authors outline signature activities of the 20S and combine chemical, structural, functional and proteomic assays to show that the 20S can degrade ubiquitin tags along with conjugated substrates.

The removal of ubiquitin (Ub) from a modified protein or Ub chain is a process that occurs regularly by the ubiquitin-proteasome system. This process is known to be mediated by various deubiquitinating enzymes (DUBs) in order to control the protein's half-life and its expression levels among many other signaling processes. Since the function of DUBs is also involved in numerous human diseases, such as cancer, there is an obvious need for an effective diagnostic probe that can monitor the activity of these enzymes. We have developed the first chemiluminescence probe for detection of DUBs activity. The probe was prepared by conjugation of the chemically synthesized C-terminally activated Ub(1-75) with a Gly-enolether precursor. Subsequent oxidation, under aqueous conditions, of the enolether conjuagate with singlet-oxygen furnished the dioxetane probe Ub-CL. This synthesis provides the first example of a dioxetane-luminophore protein conjugate. The probe's ability to detect deubiquitinating activity was successfully validated with three different DUBs. In order to demonstrate the advantage of our new probe, comparison measurements for detection of DUB UCH-L3 activity were performed between the chemiluminescent probe Ub-CL and the well-known Ub-AMC probe. The obtained data showed significantly higher S/N, for probe Ub-CL (>93-fold) in comparison to that observed for Ub-AMC (1.5-fold). We anticipate that the successful design and synthesis of the turn-ON protein-dioxetane conjugate probe, demonstrated in this work, will provide the insight and motivation for preparation of other relevant protein-dioxetane conjugates.

Protein post-translational modifications are involved in essentially all aspects of cellular signaling. Their dynamic nature and the difficulties in installing them using enzymatic approaches limits their direct study in human cells. Reported herein is the first synthesis, delivery and cellular study of a stable phosphoubiquitin probe. Our results compare Parkin’s substrate preference during mitophagy via direct visualization of a phosphorylated ubiquitin probe in the cellular environment

Chemical protein synthesis and biorthogonal modification chemistries allow production of unique proteins for a range of biological studies. Bond-forming reactions for site-selective protein labeling are commonly used in these endeavors. Selective bond-cleavage reactions, however, are much less explored and still pose a great challenge. In addition, most of studies with modified proteins prepared by either total synthesis or semisynthesis have been applied mainly for in vitro experiments with very limited extension to live cells. Reported here is an approach for studying uniquely modified proteins containing a traceless cell delivery unit and palladium-based cleavable element for chemical activation, and monitoring the effect of these proteins in live cells. This approach is demonstrated for the synthesis of a caged ubiquitin-aldehyde, which was decaged for the inhibition of deubiquitinases in live cells.

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.

One of the enigmas in the ubiquitin (Ub) field is the requirement for a poly-Ub chain as a proteasomal targeting signal. The canonical chain appears to be longer than the distance between the two Ub-binding proteasomal receptors. Furthermore, genetic manipulation has shown that one receptor subunit is sufficient, which suggests that a single Ub can serve as a degradation signal. To shed light on this mystery, we chemically synthesized tetra-Ub, di-Ub (K48-based), and mono-Ub adducts of HA-α-globin, where the distal or proximal Ub moieties were tagged differentially with either Myc or Flag. When incubated in a crude cell extract, the distal Ub moiety in the tetra-Ub adduct was mostly removed by deubiquitinating enzymes (DUBs) and reconjugated to other substrates in the extract. In contrast, the proximal moiety was most likely degraded with the substrate. The efficacy of degradation was proportionate to the chain length; while tetra-Ub globin was an efficient substrate, with mono-Ub globin, we observed rapid removal of the Ub moiety with almost no degradation of the free globin. Taken together, these findings suggest that the proximal moieties are necessary for securing the association of the substrate with the proteasome along the proteolytic process, whereas the distal moieties are important in protecting the proximal moieties from premature deubiquitination. Interestingly, when the same experiment was carried out using purified 26S proteasome, mono- and tetra-Ub globin were similarly degraded, highlighting the roles of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive. Recently, we introduced a strategy for one-pot disulfide bonds formation, tackling most of the previous limitations. Nevertheless, the effect of the order of oxidation remains an underexplored issue. Here we report on the synthetic flexibility and the full rainbow of oxidation orders of three disulfide bonds in targets that lack the knot motif. In contrast, our study reveals an essential order of disulfide bond formation in the EETI-II knotted miniprotein. This synthetic strategy was straightforwardly applied for the synthesis of novel analogues of the plectasin antimicrobial peptide with enhanced activities against methicillin-resistantn Staphylococcus n aureus (MRSA) , a notorious human pathogen. .

The attachment of ubiquitin and ubiquitin chains are critical post-translational modifications. Diseases, like cancer, often involve dysregulation of this ubiquitin system. Here, we report the discovery of small, highly non-proteinogenic cyclic peptides which can tightly bind and modulate ubiquitin chains with particular Lys48-linkages. Our cyclic peptides block the action of deubiquitinases and the proteasome, induce apoptosis in vitro, and attenuate tumor growth in vivo. We conclude that modulating ubiquitin chains is a promising avenue for future anti-cancer therapeutics.

The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels−Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g. knotted mini proteins). Here we show a de novo general synthetic strategy for the ultrafast, high-yielding formation of two and three disulfide bonds in peptides and proteins. We developed an approach based on the combination of a small molecule, UV-light, and palladium for chemo- and regio-selective activation of Cys, which enables the one-pot formation of multiple disulfide bonds in various peptides and proteins. We prepared bioactive targets of high therapeutic potential, including conotoxin, RANTES, EETI-II, and plectasin peptides and the linaclotide drug. We anticipate that this strategy will be a game-changer in preparing millions of inaccessible targets for drug discovery.

Peptides and proteins can be either synthesized using solid-phase peptide synthesis (SPPS) or by applying a combination of SPPS and ligation approaches to address fundamental questions related to human health and disease, among others. The demand for their production either by chemical or biological methods continues to raise significant interests from the synthetic community. In this context, transition metals such as Pd, Ag, Hg, Tl, Au, Zn, Ni, and Cu have also contributed to the field of peptide and protein synthesis such as in peptide conjugation, extending native chemical ligation (NCL), and for regioselective disulfide bonds formation. In this review, we highlight, summarize, and evaluate the use of various transition metals in the chemical synthesis of peptides and proteins with emphasis on recent developments in this exciting research area.

The maleimide group is a widely used reagent for bio-conjugation of peptides, proteins and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various maleimide derivatives in aqueous conditions. Maleimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segments ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a maleimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

Interferon-stimulated gene 15 (ISG15) is a member of the ubiquitin-like modifiers (ULM) family, which adopts a β- grasp fold domain(s) similar to ubiquitin (Ub) with only minor sequence homology. ISG15 consists of two Ub-like domains and aids the immune system in neutralizing infections by numerous pathogens and plays an important role in defending cells against many viruses including influenza A. Recently, Ub was found to be a substrate for ISG15, which can be ISGylated on Lys29 and Lys48, while the former is more dominant. The discovery of such hybrid ISG15-Ub chains brought forward various fundamental questions regarding the nature and effect of this conjugation. To further investigate the role of hybrid ISG15-Ub chains, the pure homogeneous material of these chains is needed in workable quantities. By applying advanced chemical strategies for protein synthesis, we report the total chemical synthesis of a 231-residue ISG15-Lys29-Ub hybrid chain. During the synthesis we encountered insoluble peptide fragments, and therefore we developed a new reversible Acm based solubilizing tag to efficiently tackle this hurdle. This new Acm tag was compared with the known Arg based Acm solubilizing tag and was found to be more reliable in terms of incorporation and efficiency as demonstrated in the synthesis of the native ISG15-Ub hybrid chain.

Messenger RNA display of peptides containing non-proteinogenic amino acids, referred to as RaPID system, has become one of the leading methods to express libraries consisting of more than trillion-members of macrocyclic peptides, which allows for discovering de novo bioactive ligands. Ideal macrocyclic peptides should have dissociation constants (KD ) as low as single-digit values in the nanomolar range towards a specific target of interest. Here, a twofold strategy to discover optimized macrocyclic peptides within this affinity regime is described. First, benzyl thioether cyclized peptide libraries were explored to identify tight binding hits. To obtain more insights into critical sequence information, sequence alignment was applied to guide rational mutagenesis for the improvement of their binding affinity. Using this twofold strategy, benzyl thioether macrocyclic peptide binders against Lys48-linked ubiquitin dimer (K48-Ub2) were successfully obtained that display KD values in the range 0.3-1.2 nm, which indicate binding two orders of magnitude stronger than those of macrocyclic peptides recently reported. Most importantly, this macrocyclic peptide also showed an improved cellular inhibition of the K48-Ub2 recognition by deubiquitinating enzymes and the 26S proteasome, resulting in the promotion of apoptosis in cancer cells.

p19INK4d plays an important role in the regulation of the cell cycle by inhibiting the function of cyclin-dependent kinases 4/6 that is responsible for the phosphorylation and deactivation of the retinoblastoma protein (pRb) tumour suppressor. Recently, it was reported that phosphorylation of p19INK4d at Ser76 and Ser66 causes structural changes, which lead to its ubiquitination and degradation. Yet the exact contribution of each phosphorylation site remains unclear. To shed light on the role of these sites, we developed the chemical synthesis of unmodified, mono- and doubly phosphorylated p19INK4d using state of the art methods for chemical protein synthesis. The synthesized proteins were characterized by circular dichroism and biochemical methods to examine the effect of phosphorylation on the thermal stability and ubiquitination, respectively. Our results provide clear determination of p19INK4d stability upon phosphorylation at different sites and reveal that phosphorylation of both Ser residues might be necessary for promoting ubiquitination of p19INK4d.

Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.

We report a general and novel semisynthetic strategy for the preparation of ubiquitinated protein-activity-based probes on the basis of sequential dehydroalanine formation on expressed proteins. We applied this approach to construct a physiologically and therapeutically relevant ubiquitinated α-globin probe, which was used for the enrichment and proteomic identification of α-globin-modulating deubiquitinases. We found USP15 as a potential deubiquitinase for the modulation of α-globin, an excess of which aggravates β-thalassemia symptoms. This development opens new opportunities for activity-based-probe design to shed light on the important aspects underlying ubiquitination and deubiquitination in health and disease.

An efficient native chemical ligation approach at Asp and Glu sites is reported applying a hydrazide precursor, as a peptide thioester, and allyl protection at the side chain of Asp and Glu. The allyl protection was efficiently removed, after the ligation step, using the water-soluble palladium complex [Pd(allyl)Cl]2 and glutathione within a few minutes under fully aqueous conditions.

Ester-linked ubiquitinated proteins have been reported by several groups to be involved in ubiquitin signalling. However, due to the lack of the suitable tools to homogeneously produce such conjugates, their exact physiological roles and biochemical behavior remain enigmatic. Here, we report for the first time on the development of a novel synthetic strategy based on total chemical synthesis of proteins to construct ubiquitinated proteins, where ubiquitin is linked to the substrate via an ester bond. In this study, we prepared ester- and isopeptide-linked ubiquitinated α-globin and examined their relative behaviors with various deubiquitinases. We found that deubiquitinases are able to cleave the ester linkage with different efficiency relative to the isopeptide-linked substrate. These results may indicate that ester-linked ubiquitinated proteins are natural substrates for deubiquitinases.