Synthesis of ubiquitinated proteins for biochemical and functional analysis
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Synthesis of ubiquitinated proteins for biochemical and functional analysis Julia Kriegesmann and Ashraf Brik * Ubiquitination plays a crucial role in controlling various biological processes such as translation, DNA repair and immune response. Protein degradation for example, is one of the main processes which is controlled by the ubiquitin system and has significant implications on human health. In order to investigate these processes and the roles played by different ubiquitination patterns on biological systems, homogeneously ubiquitinated proteins are needed. Notably, these conjugates that are made enzymatically in cells cannot be easily obtained in large amounts and high homogeneity by employing such strategies. Therefore, chemical and semisynthetic approaches have emerged to prepare different ubiquitinated proteins. In this review, we will present the key synthetic strategies and their applications for the preparation of various ubiquitinated proteins. Furthermore, the use of these precious conjugates in different biochemical and functional studies will be highlighted. Introduction Ubiquitination Ubiquitination is one of the most important posttranslational modications (PTMs), which inuences a wide range of cellular processes, such as protein degradation by the proteasome, DNA damage response and intracellular trafficking. 1,2 Ubiquitination is catalyzed by three enzymes known as the E1 ubiquitin (Ub) activating enzymes, E2 Ub conjugating enzymes and E3 Ub protein ligases (Fig. 1). In a rst step, Ub, a highly conserved protein of 76 amino acids, is activated by the E1 enzyme in an ATP-dependent manner to form a thioester intermediate, which is then transferred to the E2 enzyme. Aerwards, Ub is transferred to the substrate protein by E3 ligases. 3,4 Notably, there are only two E1 enzymes, 30–40 E2 enzymes and several hundred E3 ligases. In this process, the C-terminus carboxy group of Ub is attached to the 3-amine of a Lys residue or the N-terminal amine 5 and to a lesser extent to the side chain of Ser/Thr/Cys 6 of the substrate protein. Ub can be attached as a single moiety or as a polymeric chain in which several Ub moieties are linked internally through isopeptide bonds. Based on the linkage type, the formed Ub chains adopt different conformations. This leads to a great variety of signals within cells, since all the seven lysine Julia Kriegesmann received her MSc in Chemical Biology from the TU Dortmund, Germany in 2016 and her PhD in Chemistry from the University of Vienna, Austria, in 2021 under the supervision of Prof. Dr Christian F. W. Becker. Julia is currently a postdoctoral researcher in the group of Prof. Ashraf Brik at the Technion –Israel Institute of Technology, where she is working on the chemical ubiquitination of proteins. Ashraf Brik is a professor at the Schulich Faculty of Chemistry, Technion –Israel Institute of Technology, holding the Jordan and Irene Tark Academic Chair. He received his BSc in Chemistry from the Ben-Gurion University of the Negev and MSc in Chemistry from the Technion. He obtained his PhD in Chemistry in 2001 from the Technion. From 2002 to 2006, he was a Research Associate at the Scripps Research Institute. In 2007, he joined the Department of Chemistry in the Ben-Gurion University as an assistant professor and was promoted to full professor in 2012. In 2015, Prof. Brik moved to Technion. Schulich Faculty of Chemistry, Technion –Israel Institute of Technology, Haifa, Israel. E-mail: [email protected] Cite this: Chem. Sci.,2023,14, 10025 Received 17th July 2023 Accepted 27th August 2023 DOI: 10.1039/d3sc03664b rsc.li/chemical-science © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2023,14,10025–10040 | 10025 Chemical Science REVIEW Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue
residues within Ub (K6, K11, K27, K29, K33, K48, K63) and Met1 can be involved in ubiquitination (Fig. 2). 7 If the same residue is modied during elongation, the Ub chains are called homotypic, whereas different linkages lead to mixed chains. If a single Ub is modied with multiple Ubs, this leads to branched chains. 1,5 Notably, all possible linkages have been detected in cells. 8,9 Ubiquitination is a reversible PTM, in which a family of enzymes known as deubiquitinases (DUBs) removes Ub or Ub chains, which stops or edits the respective signal in cells. 10,11 As ubiquitination and deubiquitination play important roles in many cellular signaling pathways that are relevant for human health and disease, understanding the great details of this signal is extremely important for basic research and for the development of novel therapeutics for various diseases, such as cancer, among several others. Chemical synthesis of proteins To study the role and mechanism of PTMs such as ubiquitination in great details, the modied protein must be prepared in homogeneous form and workable quantities. The generation of dened modied conjugates by enzymatic methods is challenging, especially in the context of ubiquitination, as most of the E3 ligases are promiscuous and ubiquitinate target proteins either on several lysine residues or lead to mono-as well as polyubiquitination. In the last two decades, several synthetic and semisynthetic strategies have been developed to generate homogeneously modied proteins containing either the native bond or unnatural linkages between the respective modication and the substrate. These methods rely on protein expression, chemical synthesis or semisynthesis (Fig. 3) and have been reviewed elsewhere. 12–18 Genetic code expansion has been used to express proteins containing for example an azide and alkyne functionality in order to link the Ub and the protein substrate using Cucatalyzed azide–alkyne cycloaddition (CuAAC). 19 Ligation methods such as Native Chemical Ligation (NCL) have been proven to be a very suitable tool for chemically synthesizing proteins, by linking peptide segments to form the polypeptide chains. In NCL, oen a peptide containing a C-terminal thioester moiety is ligated with another segment bearing an Nterminal cysteine residue to form the native bond at the ligation site. 20 Chemical synthesis of ubiquitinated proteins While there are several excellent reviews about the chemistry and biology of ubiquitin signaling, 21–24 in this review we will focus only on the different strategies for the preparation of ubiquitinated proteins, containing native or unnatural linkages. We will also emphasize the studies that have been performed with these conjugates to shed light on interesting biochemical, structural and functional aspects of the Ub system. Synthesis of monoubiquitinated proteins containing a native isopeptide linkage and their biological implications Histones. Genetic information in eukaryotic cells is tightly packaged in a nucleoprotein complex called chromatin. The repeating unit of chromatin is the nucleosome, which consists of DNA wrapped around an octamer of the four core histones (H2A, H2B, H3 and H4). Additionally, the linker histone H1 binds to the nucleosome in a dynamic manner to form higherorder chromatin structures. 25 Different PTMs of histones such as acetylation, methylation, phosphorylation and ubiquitination have been described, Fig. 1 The process of ubiquitination is catalysed by the three enzymes E1, E2 and E3. Ubiquitination starts by activation of Ub by E1, followed by transfer to E2 and finally to the substrate protein by an E3 ligase. A subset of E3 ligases accepts Ub on an active site thiol before transferring it to substrates, while most of the E3 ligases only position the substrate lysine close to the E2–Ub conjugate without involving thioester formation with Ub. Fig. 2 Ubiquitination of a substrate protein can either be monoor polyubiquitination. Based on the linkages within the Ub chain, polyubiquitination can lead to homotypic or heterotypic chains, which can either be mixed or branched. xand ystand for the position of the lysine residue that is linked to the next Ub moiety. Fig. 3 Schematic presentation of the different strategies for the preparation of ubiquitinated proteins based on protein expression, chemical and semisynthetic strategies. 10026 |Chem. Sci.,2023,14,10025–10040 © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
which can in general either disrupt contacts between nucleosomes or recruit non-histone proteins. This regulates different processes such as transcription, DNA repair and replication. 26 Crosstalk between these PTMs either on the same histone or within different histones provides an additional layer of regulation and specicity as the activity of the enzyme for the second PTM is oen controlled by the rst modication. 27 Ubiquitination of histones produces the most representative functional interplays with other histone PTMs as it provides steric bulk and interacting surfaces. 28 Ubiquitinationdependent histone crosstalk can be classied into three types: in the rst case, histone ubiquitination promotes the installation or removal of the second modication by increasing the binding affinity of an enzyme for the nucleosome. In the second case, the Ub on the histone directly interacts with the enzymes to restrict the active conformations, but without increasing the binding affinities of enzymes for the nucleosome. In the last case, ubiquitination leads to structural rearrangements of an enzyme to ease auto-inhibition or activating activity of the catalytic subunit. 29 Decoding the role of histone PTMs is important to understand fundamental processes of epigenetic regulation in health and diseases. It is known that monoubiquitination at K120 in human histone H2B (H2BK120-Ub) plays important roles in transcriptional elongation and trans-tail histone H3 methylation. 30 The proteins hSet1, a member of the MLL protein family, and hDot1L have been shown to methylate the H3 residues K4 and K79, respectively, in a H2BK120-Ub-dependent manner. 31,32 Chromosomal translocation of the MLL protein, which results in its fusion with protein partners such as AF-10, have been shown to mistarget hDot1L activity to a subset of hox genes. This leads to hypermethylation and overexpression of these genes and causes acute myeloid or acute lymphoblastic leukemias. 33 Understanding how H2B-Ub stimulates trans-tail hDot1L activity would not only enhance our understanding of the role of this modication in epigenetic control mechanisms but could also lead to new therapeutic strategies to target leukemias mediated by hDot1L activity. 34,35 In 2008, the Muir group aimed to shed light on the role of H2BK120-Ub in H3 K79 methylation. 32 The group developed a strategy for the synthesis of homogeneously ubiquitinated H2B based on expressed protein ligation (EPL) of three different fragments (Fig. 4A). EPL is used to link recombinant and synthetic polypeptides by an amide bond, one containing a Cterminal thioester and the other one an N-terminal cysteine. As no native cysteine is present in H2B and Ub, two traceless ligation strategies were needed to prepare native H2BK120-Ub. For the rst ligation, the group used a photolytically removable thiol-bearing ligation auxiliary linked to the K120 side chain of H2B. To exclude double ubiquitination during ligation with the recombinant Ub thioester, the cysteine of this H2B fragment was protected with the photolabile S-(o-nitrobenzyl) group. Aer removal of the auxiliary and the cysteine protecting group, the ubiquitinated H2B fragment was ligated to the recombinant thioester H2B fragment. A nal desulfurization step gave the native H2BK120-Ub. The H2BK120-Ub was incorporated into histone octamers with recombinant H2A, H3 and H4 proteins and these were used to reconstitute mononucleosomes. With these nucleosomes, the effect of H2BK120-Ub on Dot1 methyltransferase activity was investigated by a 3 H-SAM methyltransferase assay. Methyltransferase activity was only detected in mononucleosomes containing H2BK120-Ub and not in unmodied nucleosomes. This can be explained by the proximity of H3 K79 and H2B K120, which builds the structural basis for this crosstalk as was revealed by cryo-electron microscopy (EM) studies. 36,37 Docking studies have shown that the catalytic domain of hDot1L is located adjacent to the H2B ubiquitination. 38 As H2B ubiquitination is correlated with increased levels of diand trimethylation of H3 K79 in humans 39 and yeast, 40 respectively, the group aimed to determine the degree of methylation occurring in their assays. In unmodied nucleosomes some monomethylation and no diand trimethylation was observed, whereas mononucleosomes containing H2BK120-Ub showed monoand dimethylation. No trimethylation was observed, which is in accordance with analysis of H3 K79 methylation in human cell lines. 41 Recently, Liu and co-workers compared Dot1L activities stimulated by histone H2B ubiquitinated at K34 and K120 (H2BK34-Ub and H2BK120-Ub) nucleosomes. The group prepared the natively ubiquitinated histone variants by chemical protein synthesis and auxiliary-mediated site-specic ubiquitination (Fig. 4B). 29 The acid-labile auxiliary was linked to the lysine side chain (K34 or K120) of the H2B fragment containing a C-terminal hydrazide. Aer ligation to the Ub Fig. 4 Two different auxiliary-based ligation strategies for the preparation of ubiquitinated H2B. (A) The Muir group used an H2B fragment with a photolytically removable auxiliary containing a nitrobenzylprotected cysteine that was linked to the Ub-thioester. After cysteine deprotection, the second H2B thioester fragment was ligated. (B) The Liu group used an H2B fragment with an acid-labile auxiliary and a Cterminal hydrazide. After ligation to the Ub-thioester, the hydrazide was activated and ligated with the second H2B cysteine fragment. © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2023,14,10025–10040 | 10027 Review Chemical Science Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
thioester and auxiliary removal, the hydrazide was oxidized and converted to a thioester, which allowed the ligation with the second H2B cysteine fragment. Desulfurization gave the native ubiquitinated H2B variants. These proteins were reconstituted into octamers with recombinantly expressed unmodied H2A, H3 and H4 and the methyltransferase activities of Dot1L were compared. Ubiquitinated H2B variants stimulated the catalytic activity of Dot1L on H3 K79 methylation. The activity on H2BK34-Ub and H2BK120-Ub nucleosomes was fourand seven-fold higher than on unmodied nucleosomes, respectively. Next, the group used cryo-EM to determine the structure of Dot1L bound to the H2BK34-Ub nucleosome and to elucidate the molecular details of Dot1L stimulation by H2BK34-Ub. Notably, K79 of H3 was mutated to norleucine, which was known to increase the binding affinity between the methyltransferase and a nucleosome and traps the enzyme in an active state in a SAM-dependent manner. 42 These structural investigations in combination with site-directed mutagenesis of Dot1L, maleimide footprinting and Ub displacement revealed the biochemical and structural basis for the crosstalk between histone H2B K34 ubiquitination and methylation of H3 K79 by Dot1L. This study revealed that H2BK34-Ub restricts the orientation of Dot1L, without any direct Ub-Dot1L interaction. It induces a nucleosome distortion, which orients Dot1L on the disk face of the nucleosome and positions its catalytic pocket to face H3 K79, leading to stimulation of Dot1L. The stimulation of Dot1L by H2BK34-Ub by reshaping the nucleosome core structure to accommodate the activity of histone-modifying enzymes, represents a new mode of trans-histone crosstalk, which may also account for other histone crosstalks. 29 Notably, the Brik group previously prepared histone H2B monoubiquitinated at K34 by convergent chemical synthesis. 43 This method also allowed the synthesis of doubly modied H2B with Ub and N-acetylglucosamine. 44 The glycosylation of H2B on S112 (H2BS112-GlcNAc) was found to promote its ubiquitination on K120 (ref. 45) and the preparation of H2BS112GlcNAc-K120-Ub will allow to understand the mechanism behind this. Interestingly, the group compared the total chemical synthesis of four H2B variants (monoubiquitinated at K34 or K120, glycosylation at S112 and doubly modied H2B at S112 and K120) from four fragments by convergent and one-pot approaches. This can be used as a guideline when selecting the most efficient approach for the preparation of complex protein targets. In the context of PTM crosstalks, experiments with oligonucleosomes reconstituted from homo sapiens histones and containing uniformly ubiquitinated H2A at K119 (H2AK119Ub), puried from mammalian cells, have shown that H2A K119 ubiquitination inhibits the activities of several H3 K36specic methyltransferases, leading to negative regulation of H3 K36 methylation levels. 46 However, it remained unclear if the inhibition was caused by the Ub present in the same or the neighbouring nucleosome in folded oligonucleosomes. Therefore, the Rhodes and Liu groups prepared monoubiquitinated histone H2A at K119 (H2AK119-Ub) by genetic incorporation of azidonorleucine in combination with auxiliaryassisted NCL and compared the enzymatic activity of NSD2 and SETD2 (methyltransferases for H3 K36-specic diand trimethylation, respectively) on unmodied and ubiquitinated nucleosome core particles. 47 The levels of diand trimethylation were signicantly decreased on ubiquitinated nucleosome core particles (NCPs). In order to conrm that this was indeed caused by Ub, the group removed Ub from H2AK119-Ub by USP21 treatment, which restored the levels of diand tri-methylation. Furthermore, the group conrmed the inhibitory role of H2A ubiquitination on H3 K36 methylation by the specic methyltransferases in mononucleosomes reconstituted from Xenopus laevis histones, which explains the observation that H2A ubiquitination and H3 K36 methylation rarely coexist in vivo. 46 Although mostly the crosstalk of PTMs within different histones was investigated, an example for the preparation of one histone containing two PTMs was also reported. The Li group developed the total chemical synthesis of H3 containing K56 acetylation and K122 ubiquitination (H3K56-Ac/K122-Ub). 48 Combining standard hydrazide-based NCL with auxiliarymediated ligation for site-specic ubiquitination led to the desired product on the tens of milligrams scale. The critical and rate-limiting step in nucleosome assembly is believed to be the deposition of (H3–H4) 2 tetramers, 49 which has been speculated to be facilitated by the site-specic acetylation and ubiquitination of histone H3. With this doubly modied H3 variant the group aimed to investigate this hypothesis, but during reconstitution of the three tetramers (H3, H3K56-Ac and H3K56-Ac/K122-Ub) with unmodied H4, the experiment with the ubiquitinated variant failed. It was suggested that chaperones are required to place H3K56-Ac/K122-Ub in an appropriate conformation to prevent nonspecic interactions in the formation of (H3–H4) 2 tetramers. Another important aspect in the context of histones is histone deubiquitination, which is involved in DNA damage repair, gene activation inhibition and chromosome condensation. 50 Aberration on deubiquitination is highly associated with human diseases such as cancer, but also with aging and infertility. 51 Therefore, studying DUB-mediated deubiquitination is of current interest in medicine. 52 While most of the histone-associated DUBs act on a variety of substrates, 53 also some site-selective histone-associated DUBs have been identied. USP51 was found to be able to cleave H2A with ubiquitination at K13 and K15 (H2AK13-Ub and H2AK15Ub), but not K119 (H2AK119-Ub) in vivo. 54 In order to study the mechanism and selectivity of USP51, the Liu group prepared H2A ubiquitinated at K13, K15 or K119 by their auxiliary-based ligation strategy. 55 In contrast to the in vivo results, USP51 did not favour H2A ubiquitinated at K13 and K15 against ubiquitination at K119 in vitro. Interestingly, H2AK119-Ub was cleaved more quickly than H2AK13/15-Ub. These results suggest that other factors such as other histonemodications or competitive reader proteins that bind to H2AK119-Ub might inuence the selectivity and activity of USP51. H2B is deubiquitinated by the Spt-Ada-Gcn5 acetyltransferase (SAGA) coactivator, which contains a subcomplex consisting 10028 |Chem. Sci.,2023,14,10025–10040 © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 30 August 2023. 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of the four proteins Ubp8, Sgf11, Sus1 and Sgf37, also known as the SAGA DUB module. 56–58 Deubiquitination of H2B was found to be inhibited by phosphorylation of Y57 in histone H2A. 59 To study this interaction, the Wolberger and Brik groups prepared phosphorylated histone H2A (H2AY57-P) by total chemical synthesis and ubiquitinated histone H2B by semisynthesis. 60 The histones H2BK120-Ub, H3, H4 and the unmodied H2A or H2AY57-P were reconstituted into octamers and the ability of the yeast SAGA DUB module to deubiquitinate H2B was compared for both cases. In nucleosomes containing H2AY57P, the deubiquitination showed a 30-fold reduction compared to the nucleosomes containing unmodied H2A. This conrms the inhibitory effect of H2AY57-P on H2BK120-Ub deubiquitination. This is in accordance with investigations of the crystal structure of the SAGA DUB module bound to ubiquitinated nucleosomes, 61 which will be described in the section related to ubiquitinated proteins containing unnatural linkages. a-Synuclein. a-Synuclein (a-Syn) is a protein that plays a critical role in the pathogenesis of Parkinson's disease (PD) and other neurodegenerative diseases. A characteristic of the pathology of PD is the loss of dopaminergic neurons and the formation of intracellular inclusions, called Lewy bodies (LB). 62 It has been shown that different PTMs such as phosphorylation and ubiquitination are associated with PD pathology. 63 Therefore, understanding the role of these modications in the regulation of a-Syn pathophysiology is an important step towards identifying novel therapeutic targets for the treatment of PD. Most a-Syn species found in LBs are monoor diubiquitinated at multiple lysine residues. 64 But as the directed sitespecic ubiquitination of lysine residue(s) within a-Syn has not been possible for a long time, it was difficult to investigate the effect of ubiquitination at specic lysine residues. In order to address this problem, the Brik and Lashuel groups devised a synthetic strategy to prepare homogeneously monoubiquitinated forms of a-Syn by using the d-mercaptolysine strategy (Fig. 5). 65 The latter was installed to prompt transthioesterication with a Ub thioester followed by an S–N acyl transfer step to form the modied isopeptide linkage between the Ub and lysine of the substrate. As the sequence of a-Syn does not contain Cys residues, A19 was chosen as ligation site and the desulfurization of the dmercaptolysine aer ligation led to the native monoubiquitinated protein. In more details, the fragment a-Syn 19–140 containing an N-terminal Cys was expressed in E. coli and the aSyn 1–18 thioester containing a d-mercaptolysine protected with Acm (acetamidomethyl) at position K6 was prepared using Boc SPPS. These two fragments were linked by NCL under denaturing conditions. Aer purication, the Acm protecting group was removed and the free d-mercaptolysine containing polypeptide was ligated with the Ub thioester, which was followed by desulfurization to give the desired monoubiquitinated a-Syn in the puried form as conrmed by different methods. In order to determine the effect of ubiquitination on a-Syn bril formation, the groups compared the bril formation of monoubiquitinated a-Syn at K6 and the wildtype (wt) protein in a thioavin T (ThT) assay and with transmission electron microscopy (TEM). The a-Syn wt was found to form extensive brillar structures, whereas monoubiquitination at K6 inhibited bril formation, suggesting that ubiquitination could occur aer brilization. The data obtained during this study contradict the results of other studies that indicated enhancement of a-Syn aggregation by ubiquitination in vitro and in cell cultures. 66 Notably, in these studies heterogenous mixtures of unmodied a-Syn and a-Syn ubiquitinated at different lysine residues have been used. This further highlights the importance of the preparation of homogeneously ubiquitinated proteins. Furthermore, it allows to investigate the crosstalk between different PTMs. In this case, the group aimed to explore the effect of ubiquitination at K6 on a-Syn phosphorylation at S87 and S129 by three different kinases, which was found not to signicantly inuence the extent of a-Syn phosphorylation. a-Globin. Although most proteins are linked to Ub or polyUb via lysine residues, there is growing evidence about the existence and physiological relevance of the so called “non-canonical”ubiquitination. 6 Proteins lacking lysine or having the lysine residue mutated to arginine can still be ubiquitinated and targeted for the proteasomal or ER-associated degradation. 67 The Brik group aimed to prepare esterand isopeptidelinked ubiquitinated a-globin (modied at T127 and K127, respectively) in order to compare their behaviour with various DUBs. 68 Only a few examples for the incorporation of an ester unit into proteins have been reported. 69–71 The Brik group reported the rst total chemical synthesis of a ubiquitinated protein, which contains an ester-linked Ub unit (Fig. 6). In detail, the group divided HA-a-globin in four fragments that were prepared by SPPS. The fragment containing the ester linkage at position T127 (Ub 46–76 –a-globin 120–150 ) was also prepared via standard SPPS up to position 128. Aerwards, the group had to overcome the problem of cyclization at two Fig. 5 Synthesis strategy for the preparation of ubiquitinated a-Syn using d-mercaptolysine. The N-terminal fragment of a-Syn containing Acm-protected d-mercaptolysine at position K6 with C-terminus thioester was first linked to the recombinantly expressed C-terminal fragment containing a cysteine. After Acm removal, the Ub thioester was ligated followed by desulfurization to give the desired ubiquitinated a-Syn. © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2023,14,10025–10040 | 10029 Review Chemical Science Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
different synthesis steps. Aer coupling of Fmoc-Thr-OH, the next amino acid was coupled to prevent an intramolecular attack of the Thr free amine on the ester bond. The ester bond was formed by coupling allyloxycarbonyl (Alloc) protected glycine to the free Thr hydroxyl group. Aer nishing the synthesis of the backbone peptide, the Alloc protecting group of the branched glycine was removed and the following Arg–Gly was coupled as a dipeptide to prevent intramolecular diketopiperazine formation. The remaining peptide was synthesized using standard SPPS. Ligations of the four fragments followed by desulfurization gave the nal HA-a-globin-Ub analogue. With both HA-a-globin-Ub variants in hand, the group compared the recognition and stability of the esterand isopeptide linkage in DUBs cleavage assays. Therefore, the variants were incubated with different puried DUBs and the cleavage efficiency was evaluated. A detailed comparison of the cleavage efficiency by USP2 showed that the isopeptide linked HA-aglobin-Ub undergoes faster cleavage than the ester-linked variant. Aer a short incubation time, over 70% of the isopeptide variant underwent cleavage, while it was only around 30% for the ester-linked variant. Analysis with USP15 showed comparable differences in the cleavage of the two variants, but it was faster for both compared to USP2. Importantly, these ndings show, for the rst time, that DUBs can cleave an ester bond between Ub and another protein. Other proteins. In addition to modied histones, a-synuclein and a-globin, other natively monoubiquitinated proteins were prepared. For example, the Offer group aimed to investigate the effect of ubiquitination of NEMO (NF-kB essential modulator) using the monoubiquitinated form of this protein (NEMOK302-Ub) prepared by chemical synthesis. 72 Linear ubiquitination of NEMO promotes activation of a kinase complex which is important for intracellular immune signalling. The only known ligase complex to synthesize linear Ub is the linear Ub chain assembly complex (LUBAC), a hetero complex consisting of three proteins. It has been unclear if the catalytic core is sufficient for linear ubiquitination or if the entire LUBAC complex is required. The group provided support that Ub elongation does not require additional structural elements outside the catalytic core and suggested that the linear ubiquitination of NEMO occurs in two stages. First, Ub is attached to NEMO by the LUBAC complex and second, another Ub is linked to the M1 of the Ub on NEMO, leading to chain elongation, which only requires the catalytic core activity. Okamoto and co-workers developed a one-pot ligation strategy, which enabled the introduction of several PTMs into the HP1aprotein, including ubiquitination, phosphorylation, citrullination and acetylation. 73 In the context of HP1aubiquitination, they focused on the K154 ubiquitination site which has been proposed to promote the degradation of HP1a through the autophagy pathway to enable efficient DNA repair. As K154 is located at the chromo shadow domain, which is responsible for the self-dimerization of HP1a, the group suggested that steric hindrance caused by the Ub disturbs the dimerization. Synthesis of polyubiquitinated proteins containing a native isopeptide linkage and their biological implications In addition to monoubiquitinated proteins, polyubiquitinated proteins containing a natural linkage between the substrate protein and the Ub chain have been synthesized, however to a lesser extent. For the rst time, the synthesis of diubiquitinated histone H2A (H2AKX-di-Ub) was recently established by the Liu group 74 to investigate its interaction with p53-binding protein 1 (53BP1), which is a critical regulator of cellular response to DNA double strand breaks (DSBs). 53BP1 binds to an NCP containing dimethylated H4 at K20 and ubiquitinated H2A. 53BP1 was considered as a specic reader of H2AK15-Ub, 75 but as the Ub ligase RNF168 ubiquitinates H2A at K15 and K13 without selectivity and introduces polyubiquitination, this raised the question whether 53BP1 in addition to these events, is also a reader of H2AK13-Ub or diubiquitinated H2A. To answer this question, the Liu group synthesized ubiquitinated H2A through convergent ligation combined with desulfurization. 74 The four synthetic H2A variants (monoand di-ubiquitinated at K13 or K15) were incorporated into histone octamers and nucleosomes with recombinant H2B, H3 and chemically synthesized histone H4 dimethylated at K20 (H4K20-di-Me). In order to investigate the interaction between 53BP1 and the modied NCPs, the group performed pull-down experiments Fig. 6 Synthesis strategy for the preparation of ubiquitinated a-globin containing an ester bond. The a-globin 121–150 fragment was synthesized by SPPS and introduction of an Alloc-protected glycine onto a tyrosine side chain led to the desired ester bond between Ub and aglobin. 10030 |Chem. Sci.,2023,14,10025–10040 © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. 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with GST-53BP1 fusion proteins. 75 53BP1 selectively bound to NCPs containing H2AK15-Ub, but not H2AK13-Ub and these interactions were only detected between 53BP1 and H4K20-diMe-containing NCPs, consistent with previous studies. 75,76 Furthermore, 53BP1 was found to bind to H2AK13-di-Ub and H2AK15-di-Ub in the presence of H4K20-di-Me. The group proposed a binding model in which the 53BP1 and NCPs containing diubiquitinated H2A interact via the hydrophobic patch, H4K20-di-Me and the nucleosome acidic patch. a-Synuclein. The Brik group prepared ubiquitinated a-Syn variants by site-specically incorporating K48-linked dior tetraUb onto the side chain of K12 of a-Syn via a native isopeptide bond. 77 This allowed to elucidate the role of ubiquitination in regulating a-Syn stability, aggregation, phosphorylation and clearance. Furthermore, the group was able to investigate the crosstalk between phosphorylation and ubiquitination, which are the two most common a-Syn pathological modications in Lewy bodies and Parkinson disease. For the preparation of a-Syn, the a-Syn 30–140 fragment, containing an N-terminal Cys, was recombinantly expressed in E. coli, while the a-Syn 1–29 thioester peptide containing a d-mercaptolysine at K12 was chemically synthesized. Ligation of these fragments gave a-Syn bearing the d-mercaptolysine. This allowed the introduction of the tetra-Ub chain via two sequential ligation steps of di-Ub-thioester (Fig. 7). Desulfurization followed by purication via gel-eluted liquid fraction entrapment electrophoresis led to the desired tetraubiquitinated a-Syn 1–140 in high homogeneity. In order to investigate the effect of ubiquitination on the stability of a-Syn and its degradation, the mono-, diand tetraubiquitinated a-Syn variants were incubated in crude cell extract. The results clearly showed that diand tetraubiquitinated a-Syn are more resistant to DUBs than monoubiquitinated a-Syn and are degraded by the proteasome. In a next step, the aggregation of wt and tetraubiquitinated a-Syn were compared to nd out about the effect of Ub chain length on a-Syn bril formation. While monoubiquitination was found to stabilize monomeric a-Syn, tetraubiquitination led to the formation of nonbrillar aggregates. These ndings suggest that ubiquitination of a-Syn inhibits bril formation and probably occurs aer bril formation. This possibly suggests that ubiquitination by the longer chains is an active cellular response to disassociate these aggregates and promote clearance of a-Syn brils by degradation by the proteasome. The researchers have also studied the interplay of ubiquitination and phosphorylation and showed that the effect of phosphorylation at Y125 on a-Syn aggregation is dependent on the length of the poly-Ub chain. Monoubiquitinated a-Syn was found not to aggregate upon phosphorylation at Y125, while tetraubiquitinated a-Syn exhibited higher aggregation propensity. It has been hypothesized that the combination of tetraubiquitination and phosphorylation at Y125 induces conformational changes that lead to the formation of an aggregation-prone structure. Notably, when a-Syn is either ubiquitinated or phosphorylated, this does not inuence its aggregation. Cyclin B1. The Ub proteasome system (UPS) is the major proteolytic pathway for the removal of proteins. 4,78 The target proteins are selected by covalent Ub-tagging (typically with K48linked Ub chains), followed by proteolysis within the 26S proteasome in an ATP-dependent manner. 79 The 26S proteasome holoenzyme consists of a 19S regulatory particle, which recognizes the Ub signal and unfolds the target protein, as well as a 20S core particle, which hydrolyzes the unfolded polypeptide into short peptides. The proteasome is usually found as a mixture of 30S, 26S and 20S complexes with common catalytic sites, which makes it difficult to dissect their specic roles. Changes in the cellular ratio between the 20S and the 26S proteasomes may be part of an adaptive response to meet cellular needs. 80 Misfolded or inherently disordered proteins are oen ubiquitinated in cells, but it is unclear whether they are degraded by 20S or 26S proteasomes. Cyclin B1 is a known substrate for ubiquitin-dependent 26S proteasome degradation, but potentially a substrate for both 20S and 26S proteasomes. It has a disordered N-terminal region, which contains 15 lysine residues that can be modi- ed by Ub. 81 The chemical synthesis of homogenously ubiquitinated proteins allows a comparison of 20S and 26S proteasomes with regard to their substrate selection and peptide-product generation. Therefore, the Brik and Glickman groups prepared mono-, diand tetraubiquitinated cyclin B1 by using d-mercaptolysine assisted ubiqutination. 82 The Ub units were linked via K48 and Fig. 7 Synthetic strategy for the preparation of tetraubiquitinated aSyn based on the use of d-mercaptolysine. The ligation of two di-Ub fragments to the a-Syn, followed by desulfurization gave the natively tetraubiquitinated protein. © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2023,14,10025–10040 | 10031 Review Chemical Science Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
attached to K64 of cyclin B1. To facilitate the tracking of specic Ub units in the chain, the proximal Ub in all chains was tagged with Myc peptide and the distal Ub with Flag. In vitro, unmodied cyclin B1 was proteolyzed faster by puried 20S proteasomes than by 26S proteasomes. In contrast, tetra-Ub-cyclin B1 was proteolyzed faster by 26S proteasomes. The rate of cyclin B1 degradation by the 20S proteasome was proportionate to the number of Ub units attached to the same substrate. For the 26S proteasome, the inverse behavior was observed. Interestingly, proteolysis by the 26S proteasome led to Ub recycling whereas in the 20S proteasome the Ub was proteolyzed along with its attached target protein. While the 26S proteasome showed a distinct cleavage pattern and generated longer peptides, the 20S proteasome showed greater exibility to access potential cleavage sites and generated longer peptides. Ub units facilitated the degradation of a tagged substrate by the 26S proteasome by binding to Ub receptors. This was not the case for the 20S proteasome as it lacks Ub receptors. a-Globin. The Brik and Ciechanover groups aimed to shed light on the degradation of tetra-Ub linked to HA-a-globin and examine the fate of the Ub moieties in the degradation process. 83,84 Therefore, distal or proximal Ub moieties were tagged differently with either Myc or Flag peptides in order to study the fate of each independently. Two constructs were synthesized, in which the rst contained a Myc-tag at the Nterminus of the proximal Ub and a Flag-tag at the N-terminus of the distal Ub, while the second construct was prepared with reversed tagging. The samples were incubated in rabbit fraction II, a crude cell extract containing all the UPS components required for conjugation and degradation, including E1, most of the E2s and E3s, the proteasome and a broad array of DUBs. As also observed during the studies with cyclin B1, the degradation efficiency was proportionate to the Ub chain length. The distal Ub moiety was removed by DUBs and reconjugated to other substrates in the extract. In contrast to this, the proximal Ub moiety was degraded with the substrate. For mono-Ub HA-a-globin, the Ub moiety was removed rapidly, leading to nearly no degradation of the substrate, while the tetra-Ub was found to be an effective degradation signal (Fig. 8). These ndings suggest that proximal Ub moieties are necessary for securing the association of the substrate with the proteasome during the proteolytic process, whereas the distal Ub moieties are important for protecting the proximal moieties from premature deubiquitination. Furthermore, these studies highlight the importance of the entire repertoire of cellular DUBs in regulating the degradation of proteasomal substrates, as the monoand tetra-Ub HAa-globin were similarly degraded when using puried 26S proteasome for the experiments. Synthesis of ubiquitinated proteins with unnatural linkages and their biological implications In addition to the synthesis of native Ub conjugates, various strategies for the preparation of Ub conjugates containing unnatural linkages have been developed. Monoubiquitinated proteins Histone H2B. As described previously, ubiquitination of K120 in histone H2B plays an important role for the methylation of histone H3. Examination of the nucleosome structure revealed that several lysine residues in the C-terminal helix of H2B are solvent exposed. H2B K125 in humans 85 as well as H2B K111 in yeast 86 (analogous to K108 in mammals) have been reported to undergo ubiquitination in vivo, but their possible biological roles and effect on hDot1L are unknown. The Muir group aimed to investigate the crosstalk between ubiquitination and methylation. They wanted to study if the ability of H2B ubiquitination to stimulate H3 K79 methylation by hDot1L is strictly dependent on the Ub attachment site. Therefore, they envisioned the incorporation of a suitable sulfur-containing linker as a replacement of the isopeptide linkage and chose to use a disulde-directed strategy for site specic ubiquitination of histones. 87 This method is well suited especially in the context of histones, as there is only one cysteine residue in the four core mammalian histones (C110 in H3) and the mutation of this residue to alanine has no major effect on nucleosome function. 88 Furthermore, Ub does not contain a cysteine residue. For this semisynthetic strategy (Fig. 9), the fusion construct Ub-GyrA was expressed in E. coli and aer purication was incubated overnight with cysteamine to yield the free Ub containing a C-terminal aminoethanethiol linker. The H2B K120C mutant was expressed in E. coli and the cysteine was activated with 2,2 ′ -dithiobis(5-nitropyridine) (DTNP). Incubation of the activated H2B K120C mutant with two-fold molar excess of Ub at pH 6.9 for 1 h gave the desired disulde linked monoubiquitinated H2B (H2B-Ub(ss)). The resulting linkage between Ub and H2B was around 2.4 Å longer than the isopeptide linkage and due to the disulde bond, no reducing agents could be used in biochemical assays. Therefore, the group examined if Fig. 8 Investigation of ubiquitinated a-globin variants. For the monoubiquitinated a-globin, the Ub gets cleaved by DUBs, making degradation by the proteasome impossible. In contrast, for the tetraubiquitinated a-globin, the distal Ub gets cleaved by DUBs, but the remaining Ub chain is still a sufficient degradation signal. The free Ub moieties are reconjugated to other protein substrates. 10032 |Chem. Sci.,2023,14,10025–10040 © 2023 The Author(s). Published by the Royal Society of Chemistry Chemical Science Review Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
these changes have any effect on biochemical activity, especially on hDot1L stimulation. Therefore, they prepared octamers consisting of the core histones H2A and H4, the H3 C110A mutant and either H2B, native H2B-Ub or H2B-Ub(ss). The H3 C110A mutant was used to preclude disulde exchange with the H2B-Ub(ss) during octamer formation. Notably, mononucleosomes reconstituted with H2B ubiquitinated at the positions 108 and 116 migrated faster than the other ubiquitinated mononucleosomes during native gel electrophoresis, indicating that Ub inuences the structure and/or surface charge in a position-dependent manner. Structure– activity relationship studies narrowed down the region of the Ub surface required to stimulate hDot1L activity. Furthermore, it was revealed that this stimulatory effect is not strictly dependent on the Ub attachment point. Chromatin compaction can be regulated by histone PTMs as described in the previous section. 89 Acetylation of histone H4 (H4-Ac) at position K16 for example leads to ber decompaction 90 whereas its trimethylation at position K20 results in increased folding. 91 It has been suggested that H2B ubiquitination increases nucleosome and DNA access to downstream factors by locally open the chromatin structure. 92 But as no detailed analysis of the speciceffect of H2B ubiquitination on chromatin structure has been reported, the Muir group aimed to chemically synthesize H2B-Ub and investigate the conformation and accessibility of ubiquitinated and unmodi- ed chromatin bers in solution. The H2B-Ub was prepared by the disulde-directed methodology for the site-specic ubiquitination at K120. 93 The group devised a method based on homo-FRET between nucleosomes that directly reports on internucleosomal distance changes in equilibrium. This allowed them to show that divalent cation-induced chromatin ber compaction involves conformationally heterogenous intermediates. Interestingly, H4-Ac and H2B-Ub showed different effects. H4-Ac affects compaction throughout the folding transition by H4 tail binding to the H2A acidic patch. 94 This leads to a reduction of closely interacting nucleosomes and prevents full ber folding because of counteracting electrostatic repulsion. In contrast to this, H2B-Ub interferes with later stages of compaction. While transient interactions between nucleosomes are not impaired, upon further compaction the regular ber packing is impaired, which may lead to ber instability and local unfolding. It was hypothesized that specic interactions between Ub and the nucleosomal surface might be required to prevent escape of Ub from the interface between nucleosomes during compaction, as the similar-sized protein Hub1 could not substitute for Ub in impairing chromatin folding. In addition, ubiquitination of H2A at the opposite site of the nucleosomal surface does not hinder ber compaction. 95 Therefore, it remains unclear how exactly Ub can inuence chromatin ber compaction. In order to gain insights into the mechanism of chromatin decompaction, the Muir group used a hydrogen–deuterium exchange strategy coupled with NMR spectroscopy to map the parts of Ub responsible for structural effects on chromatin. 96 The group prepared ubiquitinated H2B at position K120C histone via the disulde strategy. 87 Previous studies have suggested that features on the Ub surface are responsible for decompaction. 93 The current study conrmed this hypothesis by revealing that the amino acids E16 and E18, within the acidic patch on Ub, are essential for decompaction as they mediate electrostatic interactions with the basic histone proteins. Interestingly, Ub-nucleosome interactions seem to be Mg 2+ dependent. The addition of Mg 2+ brings the surfaces of neighboring nucleosomes closer that are too far apart for electrostatic interactions in the absence of Mg 2+ . Besides Ub-nucleosome interactions, also Ub–Ub contacts occur in the chromatin environment, and they are important for the solubilization of chromatin polymers by preventing the establishment of a close interface between nucleosomes. 96 Monoubiquitinated H2B at K120 in human (and K123 in yeast) also plays multiple roles in transcription activation 97 and has been shown to be deubiquitinated by the SAGA DUB module. 57 The crystal structure of the DUB module bound to ubiquitinated nucleosomes was determined by the Wolberger and Brik groups. 61 Two DUB module heterotetramers are bound to a Xenopus laevis NCP containing two copies of H2B which is ubiquitinated at K120 via a non-hydrolyzable dichloroacetone linkage. The crystal structure shows the interaction of an arginine cluster on the Sgf11 zinc nger domain with the acidic patches in H2A and H2B. Furthermore, the Ubp8 catalytic domain provides additional contacts with H2B as well as with the conjugated Ub. Fig. 9 Semisynthetic strategy for the preparation of monoubiquitinated histone H2B containing a disulfide linkage. The histone H2B and the Ub were expressed in E. coli. Modification of the Ub with a thiol and activation of the H2B cysteine with DTNP allowed the formation of a disulfide bond. © 2023 The Author(s). Published by the Royal Society of Chemistry Chem. Sci.,2023,14,10025–10040 | 10033 Review Chemical Science Open Access Article. Published on 30 August 2023. Downloaded on 11/6/2025 7:50:32 AM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online
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