An E1-Catalyzed Chemoenzymatic Strategy to Isopeptide-N-Ethylated Deubiquitylase-Resistant Ubiquitin Probes.

Triazole-based deubiquitylase (DUB)-resistant ubiquitin (Ub) probes have recently emerged as effective tools for the discovery of Ub chain-specific interactors in proteomic studies, but their structural diversity is limited. A new family of DUB-resistant Ub probes is reported based on isopeptide-N-ethylated dimeric or polymeric Ub chains, which can be efficiently prepared by a one-pot, ubiquitin-activating enzyme (E1)-catalyzed condensation reaction of recombinant Ub precursors to give various homotypic and even branched Ub probes at multi-milligram scale. Proteomic studies using label-free quantitative (LFQ) MS indicated that the isopeptide-N-ethylated Ub probes may complement the triazole-based probes in the study of Ub interactome. Our study highlights the utility of modern protein synthetic chemistry to develop structurally and new families of tool molecules needed for proteomic studies.

One-Step Chemical Synthesis of Native Met1-Linked Poly-Ubiquitin Chains.

The enzyme-mediated construction of poly-ubiquitin (Ub) chains on target proteins leads to a variety of cellular responses and is involved in processes ranging from protein degradation to cell cycle control and immune responses. This complex post-translational modification system is under intense investigation, but generation of specific Ub chains and tools made thereof is not always trivial. We discovered that native methionine-1-linked polymeric ubiquitin chains can be constructed in a single chemical reaction. We validate correct folding and regioselective attachment of such chains using linkage specific proteases and further demonstrate that these poly-Ub chains can be converted into thioesters by the activating E1-enzyme. Subsequent ligation reactions using these in situ prepared thioesters leads to poly-ubiquitinated peptides.

Synthesis and purification of linkage-specific polyubiquitin chains of distinct length for structural studies.

Polyubiquitylation is one of the most versatile post-translational modifications involved in the regulation of numerous intracellular signaling processes. An assembly procedure that is simple, robust, and efficient to synthesize and purify linkage-specific polyubiquitin chains of defined length at a preparative scale is required in biophysical and structural studies. Here, we have optimized known enzymatic procedures in the form of a protocol to obtain multi-milligrams of Lys48-and Lys63-linked polyubiquitin chain types with more than 99% purity. Mass spectrometry (ESI/MS) analysis of K48- and K63-linked diubiquitin confirmed that the enzymes used in the preparation generated homogeneous linkages with no promiscuity.

Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis.

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

The linkage specificity determination of Ube2g2-gp78 mediated polyubiquitination.

Polyubiquitin chain linkage specificity or topology is essential for its role in diverse cellular processes. Previous studies pay more attentions to the linkage specificity of the first ubiquitin moieties, whereas, little is known about the editing mechanism of linkage specificity in longer polyubiquitin chains. gp78 and its cognate E2-Ube2g2 catalyze lysine48 (K48)-linked polyubiquitin chains to promote the degradation of targeted proteins. Here, we show that the linkage specificity of the entire polyubiquitin chain is determined by the conjugation manner of the first ubiquitin molecule but not the following ones. Further study discovered that the gp78 CUE domain works as a proofreading machine during the growth of K48-linked polyubiquitin chains to ensure the linkage specificity. Together, our studies uncover a novel mechanism underlying the linkage specificity determination of longer polyubiquitin chains.

Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin.

The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with (15)N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.

The ubiquitin-conjugating enzyme, UbcM2, is restricted to monoubiquitylation by a two-fold mechanism that involves backside residues of E2 and Lys48 of ubiquitin.

Proteins can be modified on lysines (K) with a single ubiquitin (Ub) or with polymers of Ub (polyUb). These different configurations and their respective topologies are primary factors for determining whether substrates are targeted to the proteasome for degradation or directed to nonproteolytic outcomes. We report here on the intrinsic ubiquitylation properties of UbcM2 (UBE2E3/UbcH9), a conserved Ub-conjugating enzyme linked to cell proliferation, development, and the cellular antioxidant defense system. Using a fully recombinant ubiquitylation assay, we show that UbcM2 is severely limited in its ability to synthesize polyUb chains with wild-type Ub. Restriction to monoubiquitylation is governed by multiple residues on the backside of the enzyme, far removed from its active site, and by lysine 48 of Ub. UbcM2 with mutated backside residues can synthesize K63-linked polyUb chains and to a lesser extent K6- and K48-linked chains. Additionally, we identified a single residue on the backside of the enzyme that promotes monoubiquitylation. Together, these findings reveal that a combination of noncatalytic residues within the Ubc catalytic core domain of UbcM2 as well as a lysine(s) within Ub can relegate a Ub-conjugating enzyme to monoubiquitylate its cognate targets despite having the latent capacity to construct polyUb chains. The two-fold mechanism for restricting activity to monoubiquitylation provides added insurance that UbcM2 will not build polyUb chains on its substrates, even under conditions of high local Ub concentrations.

Chemical strategies to understand the language of ubiquitin signaling.

Ubiquitin (Ub) is a small 76 amino acid long protein that is highly conserved in all eukaryotes studied to date. In humans, more than 600 ligases are involved in the reversible modification of specific lysine side-chain amines in substrate proteins by conjugation with the C-terminal carboxylate of Ub. Initially monoubiquitylated proteins can undergo repetitive ubiquitylation starting from one of seven lysine residues or the α-amine in the first Ub to generate a variety of polyUb chains with different topologies and functions. The most well known role for protein ubiquitylation is in targeting substrates for proteolytic destruction by 26S proteasomes. However, a growing body of evidence indicates that both mono- and polyubiquitylation play proteasome-independent roles in modulating the structure, function, and localization of protein substrates. Understanding the complexity of Ub-mediated functions in our cells is a major challenge for modern biology. In addition to well-established in vivo genetic methods, biochemical and biophysical investigations of ubiquitylated proteins in vitro can shed light on the direct mechanistic roles for Ub in different contexts. Such studies have traditionally been limited by the ability to obtain sufficient quantities of homogenously ubiquitylated proteins with precisely defined linkages. This review focuses on recent advances in both synthetic and recombinant protein-based methods that have yielded access to homogenously site-specifically ubiquitylated proteins. Mechanistic studies of the roles for protein ubiquitylation and of the enzymes involved in protein deubiquitylation that are enabled by these chemical tools are highlighted.

Synthetic polyubiquitinated α-Synuclein reveals important insights into the roles of the ubiquitin chain in regulating its pathophysiology.

Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow site-specific introduction of ubiquitin (Ub) chains to a specific substrate. Here, we describe chemical and semisynthetic strategies for site-specific incorporation of K48-linked di- or tetra-Ub chains onto the side chain of Lys12 of α-Synuclein (α-Syn). These advances provided unique opportunities to elucidate the role of ubiquitination and Ub chain length in regulating α-Syn stability, aggregation, phosphorylation, and clearance. In addition, we investigated the cross-talk between phosphorylation and ubiquitination, the two most common α-Syn pathological modifications identified within Lewy bodies and Parkinson disease. Our results suggest that α-Syn functions under complex regulatory mechanisms involving cross-talk among different posttranslational modifications.

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies.

Polymeric chains of a small protein ubiquitin are involved in regulation of nearly all vital processes in eukaryotic cells. Elucidating the signaling properties of polyubiquitin requires the ability to make these chains in vitro. In recent years, chemical and chemical-biology tools have been developed that produce fully natural isopeptide-linked polyUb chains with no need for linkage-specific ubiquitin-conjugating enzymes. These methods produced unbranched chains (in which no more than one lysine per ubiquitin is conjugated to another ubiquitin). Here we report a nonenzymatic method for the assembly of fully natural isopeptide-linked branched polyubiquitin chains. This method is based on the use of mutually orthogonal removable protecting groups (e.g., Boc- and Alloc-) on lysines combined with an Ag-catalyzed condensation reaction between a C-terminal thioester on one ubiquitin and a specific ε-amine on another ubiquitin, and involves genetic incorporation of more than one Lys(Boc) at the desired linkage positions in the ubiquitin sequence. We demonstrate our method by making a fully natural branched tri-ubiquitin containing isopeptide linkages via Lys11 and Lys33, and a (15)N-enriched proximal ubiquitin, which enabled monomer-specific structural and dynamical studies by NMR. Furthermore, we assayed disassembly of branched and unbranched tri-ubiquitins as well as control di-ubiquitins by the yeast proteasome-associated deubiquitinase Ubp6. Our results show that Ubp6 can recognize and disassemble a branched polyubiquitin, wherein cleavage preferences for individual linkages are retained. Our spectroscopic and functional data suggest that, at least for the chains studied here, the isopeptide linkages are effectively independent of each other. Together with our method for nonenzymatic assembly of unbranched polyubiquitin, these developments now provide tools for making fully natural polyubiquitin chains of essentially any type of linkage and length.

Synthesis and analysis of K11-linked ubiquitin chains.

Much has been learned about protein ubiquitination by studying the structural, biochemical, and biophysical properties of ubiquitin chains in vitro. However, these analyses were limited to K48-, K63-linked, and linear ubiquitin chains. Only recently, enzymatic and chemical assembly systems for the remaining chain types have been developed. Here, we describe a method to synthesise K11-linked ubiquitin chains in vitro by exploiting the intrinsic K11-specificity of the E2 enzyme UBE2S.

Synthesis and analysis of linear ubiquitin chains.

Previously, polyubiquitin chains have been believed to be generated through isopeptide linkages between C-terminal of carboxyl group of ubiquitin and ε-amino group of one of the seven lysine residues in another ubiquitin. In 2006, a new type of polyubiquitin chain was identified in which the C-terminal carboxyl group of one ubiquitin is conjugated to α-amino group of the N-terminal methionine of another ubiquitin. The new type of polyubiquitin was named as the linear polyubiquitin chain. Linear polyubiquitin chains were shown to be involved in NF-κB activation. Here, we describe methods to synthesize linear polyubiquitin chains in vitro and to detect linear chains in vivo.

Formation of ubiquitin dimers via azide-alkyne click reaction.

The conjugation of poly-ubiquitin chains is a widespread post-translational modification of proteins that plays a role in many different cellular processes. Notably, the biological function of the attached ubiquitin chain depends on which lysine residue is used for chain formation. Here, we report a method for the modular synthesis of site-specifically linked ubiquitin dimers, which is based on click reaction between two artificial amino acids. In this way, it is possible to synthesize all seven naturally occurring ubiquitin connectivities, thus giving access to all ubiquitin dimers. Furthermore, this method can be generally applied to link ubiquitin to any substrate protein or even to link any two proteins site specifically.

Synthesis of atypical diubiquitin chains.

Post-translational modification of proteins with ubiquitin (Ub) and Ub chains controls numerous biochemical events. Although it has been proven that all Ub-Ub linkages are formed in cells, studies have been limited for a long time to K48 and K63 chains as these can be generated biochemically. Access to the remaining (atypical) Ub-Ub chain types has been hampered by a lack of specific E2 enzymes. In this chapter we present a solution to this problem by using a native chemical ligation approach to obtain all other (i.e. K6, K11, K27, K29 and K33) diubiquitin chains.

Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.

Crystal structure and solution NMR studies of Lys48-linked tetraubiquitin at neutral pH.

Ubiquitin modification of proteins is used as a signal in many cellular processes. Lysine side-chains can be modified by a single ubiquitin or by a polyubiquitin chain, which is defined by an isopeptide bond between the C terminus of one ubiquitin and a specific lysine in a neighboring ubiquitin. Polyubiquitin conformations that result from different lysine linkages presumably differentiate their roles and ability to bind specific targets and enzymes. However, conflicting results have been obtained regarding the precise conformation of Lys48-linked tetraubiquitin. We report the crystal structure of Lys48-linked tetraubiquitin at near-neutral pH. The two tetraubiquitin complexes in the asymmetric unit show the complete connectivity of the chain and the molecular details of the interactions. This tetraubiquitin conformation is consistent with our NMR data as well as with previous studies of diubiquitin and tetraubiquitin in solution at neutral pH. The structure provides a basis for understanding Lys48-linked polyubiquitin recognition under physiological conditions.

Controlled synthesis of polyubiquitin chains.

Many intracellular signaling processes depend on the modification of proteins with polymers of the conserved 76-residue protein ubiquitin. The ubiquitin units in such polyubiquitin chains are connected by isopeptide bonds between a specific lysine residue of one ubiquitin and the carboxyl group of G76 of the next ubiquitin. Chains linked through K48-G76 and K63-G76 bonds are the best characterized, signaling proteasome degradation and nonproteolytic outcomes, respectively. The molecular determinants of polyubiquitin chain recognition are under active investigation; both the chemical structure and the length of the chain can influence signaling outcomes. In this article, we describe the protein reagents necessary to produce K48- and K63-linked polyubiquitin chains and the use of these materials to produce milligram quantities of specific-length chains for biochemical and biophysical studies. The method involves reactions catalyzed by linkage-specific conjugating factors, in which proximally and distally blocked monoubiquitins (or chains) are joined to produce a particular chain product in high yield. Individual chains are then deblocked and joined in another round of reaction. Successive rounds of deblocking and synthesis give rise to a chain of the desired length.