Ubiquitin chain trimming recycles the substrate binding sites of the 26 S proteasome and promotes degradation of lysine 48-linked polyubiquitin conjugates

The 26 S proteasome possesses two distinct deubiquitinating activities. The ubiquitin (Ub) chain amputation activity removes the entire polyUb chain from the substrates. The Ub chain trimming activity progressively cleaves a polyUb chain from the distal end. The Ub chain amputation activity mediates degradation-coupled deubiquitination. The Ub chain trimming activity can play a supportive or an inhibitory role in degradation, likely depending on features of the substrates. How Ub chain trimming assists degradation is not clear. We find that inhibition of the chain trimming activity of the 26 S proteasome with Ub aldehyde significantly inhibits degradation of Ub₄ (Lys-48)-UbcH10 and causes accumulation of free Ub₄ (generated from chain amputation) that can be retained on the proteasome. Also, a non-trimmable Lys-48-mimic Ub₄ efficiently targets UbcH10 to the 26 S proteasome, but it cannot support efficient degradation of UbcH10 compared with regular Lys-48 Ub₄. These results indicate that polyUb chain trimming promotes proteasomal degradation of Lys-48-linked substrates. Mechanistically, we propose that Ub chain trimming cleaves the proteasome-bound Lys-48-linked polyUb chains, which vacates the Ub binding sites of the 26 S proteasome, thus allowing continuous substrate loading.     

In vivo disassembly of free polyubiquitin chains by yeast Ubp14 modulates rates of protein degradation by the proteasome.

Degradation of many eukaryotic proteins requires their prior ligation to polyubiquitin chains, which target substrates to the 26S proteasome, an abundant cellular protease. We describe a yeast deubiquitinating enzyme, Ubp14, that specifically disassembles unanchored ('free') ubiquitin chains in vitro, a specificity shared by mammalian isopeptidase T. Correspondingly, deletion of the UBP14 gene from yeast cells results in a striking accumulation of free ubiquitin chains, which correlates with defects in ubiquitin-dependent proteolysis. Increasing the steady-state levels of ubiquitin chains in wild-type cells (by expressing a derivative of ubiquitin with an altered C-terminus) inhibits protein degradation to a degree comparable with that observed in ubp14delta cells. Inhibition of degradation is also seen when an active site mutant of Ubp14 is overproduced in vivo. Surprisingly, overproduction of wild-type Ubp14 can inhibit degradation of some proteins as well. Finally, Ubp14 and human isopeptidase T are shown to be functional homologs by complementation analysis. We propose that Ubp14 and isopeptidase T facilitate proteolysis in vivo by preventing unanchored ubiquitin chains from competitively inhibiting polyubiquitin-substrate binding to the 26S proteasome.     

Nonhydrolyzable diubiquitin analogues are inhibitors of ubiquitin conjugation and deconjugation

A series of nonhydrolyzable ubiquitin dimer analogues has been synthesized and evaluated as inhibitors of ubiquitin-dependent processes. Dimer analogues were synthesized by cross-linking ubiquitin containing a terminal cysteine (G76C) to ubiquitin containing cysteine at position 11 ((76-11)Ub(2)), 29 ((76-29)Ub(2)), 48 ((76-48)Ub(2)), or 63 ((76-63)Ub(2)). A head-to-head dimer of cysteine G76C ((76-76)Ub(2)) served as a control. These analogues are mimics of the different chain linkages observed in natural polyubiquitin chains. All analogues showed weak inhibition toward the catalytic domain of UCH-L3 and a UBP pseudogene. In the absence of ubiquitin, isopeptidase T was inhibited only by the dimer linked through residue 29. In the presence of 0.5 microM ubiquitin, isopeptidase T was inhibited by several of the dimer analogues, with the (76-29)Ub(2) dimer exhibiting a K(i) of 1.8 nM. However, USP14, the human homologue of yeast Ubp6, was not inhibited at the concentrations tested. Some analogues of ubiquitin dimer also acted as selective inhibitors of conjugation and deconjugation of ubiquitin catalyzed by reticulocyte fraction II. (76-76)Ub(2) and (76-11)Ub(2) did not inhibit the conjugation of ubiquitin, while (76-29)Ub(2), (76-48)Ub(2), and (76-63)Ub(2) were potent inhibitors of conjugation. This specificity is consistent with the known ability of cells to form K29-, K48-, and K63-linked polyubiquitin chains. While (76-11)Ub(2), (76-29)Ub(2), and (76-63)Ub(2) inhibited release of ubiquitin from a pool of total conjugates, (76-48)Ub(2) and (76-76)Ub(2) showed no significant inhibition. Isopeptidase T was shown to specifically disassemble two conjugates (assumed to be di- and triubiquitin with masses of 26 and 17 kDa) formed in the reticulocyte lysate system. This activity was inhibited differentially by all dimer analogues. The inhibitor selectivity for deconjugation of the 26 and 17 kDa conjugates was similar to that observed for isopeptidase T. The observations suggest that these two conjugated proteins of the reticulocyte lysate are specific substrates for isopeptidase T in lysates.     

Structural and biochemical studies of the open state of Lys48-linked diubiquitin

Ubiquitin (Ub) is a small protein highly conserved among eukaryotes and involved in practically all aspects of eukaryotic cell biology. Polymeric chains assembled from covalently-linked Ub monomers function as molecular signals in the regulation of a host of cellular processes. Our previous studies have shown that the predominant state of Lys48-linked di- and tetra-Ub chains at near-physiological conditions is a closed conformation, in which the Ub-Ub interface is formed by the hydrophobic surface residues of the adjacent Ub units. Because these very residues are involved in (poly)Ub interactions with the majority of Ub-binding proteins, their sequestration at the Ub-Ub interface renders the closed conformation of polyUb binding incompetent. Thus the existence of open conformation(s) and the interdomain motions opening and closing the Ub-Ub interface is critical for the recognition of Lys48-linked polyUb by its receptors. Knowledge of the conformational properties of a polyUb signal is essential for our understanding of its specific recognition by various Ub-receptors. Despite their functional importance, open states of Lys48-linked chains are poorly characterized. Here we report a crystal structure of the open state of Lys48-linked di-Ub. Moreover, using NMR, we examined interactions of the open state of this chain (at pH4.5) with a Lys48-linkage-selective receptor, the UBA2 domain of a shuttle protein hHR23a. Our results show that di-Ub binds UBA2 in the same mode and with comparable affinity as the closed state. Our data suggest a mechanism for polyUb signal recognition, whereby Ub-binding proteins select specific conformations out of the available ensemble of polyUb chain conformations. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

The complexity of recognition of ubiquitinated substrates by the 26S proteasome

The Ubiquitin Proteasome System (UPS) was discovered in two steps. Initially, APF-1 (ATP-dependent proteolytic Factor 1) later identified as ubiquitin (Ub), a hitherto known protein of unknown function, was found to covalently modify proteins. This modification led to degradation of the tagged protein by – at that time – an unknown protease. This was followed later by the identification of the 26S proteasome complex which is composed of a previously identified Multi Catalytic Protease (MCP) and an additional regulatory complex, as the protease that degrades Ub-tagged proteins. While Ub conjugation and proteasomal degradation are viewed as a continued process responsible for most of the regulated proteolysis in the cell, the two processes have also independent roles. In parallel and in the years that followed, the hallmark signal that links the substrate to the proteasome was identified as an internal Lys48-based polyUb chain. However, since these initial findings were described, our understanding of both ends of the process (i.e. Ub-conjugation to proteins, and their recognition and degradation), have advanced significantly. This enabled us to start bridging the ends of this continuous process which suffered until lately from limited structural data regarding the 26S proteasomal architecture and the structure and diversity of the Ub chains. These missing pieces are of great importance because the link between ubiquitination and proteasomal processing is subject to numerous regulatory steps and are found to function improperly in several pathologies. Recently, the molecular architecture of the 26S proteasome was resolved in great detail, enabling us to address mechanistic questions regarding the various molecular events that polyubiquitinated (polyUb) substrates undergo during binding and processing by the 26S proteasome. In addition, advancement in analytical and synthetic methods enables us to better understand the structure and diversity of the degradation signal. The review summarizes these recent findings and addresses the extrapolated meanings in light of previous reports. Finally, it addresses some of the still remaining questions to be solved in order to obtain a continuous mechanistic view of the events that a substrate undergoes from its initial ubiquitination to proteasomal degradation. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.     

S5a promotes protein degradation by blocking synthesis of nondegradable forked ubiquitin chains

Ubiquitin (Ub)–protein conjugates formed by purified ring‐finger or U‐box E3s with the E2, UbcH5, resist degradation and disassembly by 26S proteasomes. These chains contain multiple types of Ub forks in which two Ub's are linked to adjacent lysines on the proximal Ub. We tested whether cells contain factors that prevent formation of nondegradable conjugates and whether the forked chains prevent proteasomal degradation. S5a is a ubiquitin interacting motif (UIM) protein present in the cytosol and in the 26S proteasome. Addition of S5a or a GST‐fusion of S5a's UIM domains to a ubiquitination reaction containing 26S proteasomes, UbcH5, an E3 (MuRF1 or CHIP), and a protein substrate, dramatically stimulated its degradation, provided S5a was present during ubiquitination. Mass spectrometry showed that S5a and GST–UIM prevented the formation of Ub forks without affecting synthesis of standard isopeptide linkages. The forked Ub chains bind poorly to 26S proteasomes unlike those synthesized with S5a present or linked to Lys63 or Lys48 chains. Thus, S5a (and presumably certain other UIM proteins) function with certain E3/E2 pairs to ensure synthesis of efficiently degraded non‐forked Ub conjugates.     

Mammalian Ddi2 is a shuttling factor containing a retroviral protease domain that influences binding of ubiquitylated proteins and proteasomal degradation

Although several proteasome subunits have been shown to bind ubiquitin (Ub) chains, many ubiquitylated substrates also associate with 26S proteasomes via “shuttling factors.” Unlike the well-studied yeast shuttling factors Rad23 and Dsk2, vertebrate homologs Ddi2 and Ddi1 lack a Ub-associated domain; therefore, it is unclear how they bind Ub. Here, we show that deletion of Ddi2 leads to the accumulation of Ub conjugates with K11/K48 branched chains. We found using affinity copurifications that Ddi2 binds Ub conjugates through its Ub-like domain, which is also required for Ddi2 binding to proteasomes. Furthermore, in cell extracts, adding Ub conjugates increased the amount of Ddi2 associated with proteasomes, and adding Ddi2 increased the binding of Ub conjugates to purified proteasomes. In addition, Ddi2 also contains a retroviral protease domain with undefined cellular roles. We show that blocking the endoprotease activity of Ddi2 either genetically or with the HIV protease inhibitor nelfinavir increased its binding to Ub conjugates but decreased its binding to proteasomes and reduced subsequent protein degradation by proteasomes leading to further accumulation of Ub conjugates. Finally, nelfinavir treatment required Ddi2 to induce the unfolded protein response. Thus, Ddi2 appears to function as a shuttling factor in endoplasmic reticulum–associated protein degradation and delivers K11/K48-ubiquitylated proteins to the proteasome. We conclude that the protease activity of Ddi2 influences this shuttling factor activity, promotes protein turnover, and helps prevent endoplasmic reticulum stress, which may explain nelfinavir’s ability to enhance cell killing by proteasome inhibitors.     

A pervasive role of ubiquitin conjugation in activation and termination of IkappaB kinase pathways

The nuclear factor (NF)-kappaB pathway is a paradigm for gene expression control by ubiquitin-mediated protein degradation. In stimulated cells, phosphorylation by the IkappaB kinase (IKK) complex primes NF-kappaB-inhibiting IkappaB molecules for lysine (Lys)-48-linked polyubiquitination and subsequent destruction by the 26S proteasome. However, recent studies indicate that the ubiquitin (Ub) system controls NF-kappaB pathways at many levels. Ub ligases are activated by different upstream signalling pathways, and they function as central regulators of IKK and c-Jun amino-terminal kinase activation. The assembly of Lys 63 polyUb chains provides docking surfaces for the recruitment of IKK-activating complexes, a reaction that is counteracted by deubiquitinating enzymes. Furthermore, Ub conjugation targets upstream signalling mediators as well as nuclear NF-kappaB for post-inductive degradation to limit the duration of signalling.     

Deubiquitination by proteasome is coordinated with substrate translocation for proteolysis in vivo

The 26S proteasome mediates degradation of protein substrates labeled with polyUb chains. After recognition by the 19S proteasome regulatory complex, polyUb chains are disassembled and substrates are processed in the 20S core of proteasome. However, the exact relationship of degradation-associated deubiquitination to substrate processing remains unclear. Here, using Ub-based tagging strategies, we provided evidence that removable polyUb chains serve as the signal for proteolytic processing of ubiquitinated substrates. We showed that inhibition of the proteasome by proteasome inhibitor MG132 results in trapping of the substrate in the proteasome. Such a trapping allows proteasomal cleavage of attached non-removable Ub mutant (UbV75,76), which is otherwise a "difficult" deubiquitination substrate. Characterization of deubiquitination and degradation intermediates, generated due to incomplete proteolytic inhibition, revealed changes in proteolytic cleavage sites, within the Gal4-VP16 model substrate, suggesting that the copy number of attached UbV75,76 affects substrate processing. Conversion of lysine48 to arginine48 in UbV75,76 did not have significant effect on in vivo polyubiquitination of multiple Ub-fused substrates, but considerably reduced proteolytic intermediates. Taken together, the results support a model in which deubiquitination process is a crucial event for proteolysis of ubiquitinated substrates and such an event is coordinated with substrate translocation.     

A new method of purification of proteasome substrates reveals polyubiquitination of 20 S proteasome subunits

The 26 S proteasome is implicated in the control of many major biological functions but a reliable method for the identification of its major substrates, i.e. polyubiquitin (Ub) conjugates, is still lacking. Based on the steps present in cells, i.e. recognition and deubiquitination, we developed an affinity matrix-based purification of polyUb conjugates suitable for any biological sample. Ub-conjugates were first purified from proteasome inhibitor-treated C2C12 cells using the Ub binding domains of the S5a proteasome subunit bound to an affinity matrix and then deubiquitinated by the catalytic domain of the USP2 enzyme. This two step purification of proteasome substrates involving both protein-protein interactions and enzyme-mediated release allowed highly specific isolation of polyUb 26 S proteasome substrates, which were then resolved on two-dimensional gels post-deubiquitination. To establish our method, we focused on a gel area where spots were best resolved. Surprisingly, spot analysis by mass spectrometry identified alpha2, alpha6, alpha7, beta2, beta3, beta4, and beta5 20 S proteasome subunits as potential substrates. Western blots using an anti-beta3 proteasome subunit antibody confirmed that high molecular weight forms of beta3 were present, particularly in proteasome inhibitor-treated cells. Sucrose gradients of cell lysates suggested that the proteasome was first disassembled before subunits were polyubiquitinated. Altogether, we provide a technique that enables large scale identification of 26 S proteasome substrates that should contribute to a better understanding of this proteolytic machinery in any living cell and/or organ/tissue. Furthermore, the data suggest that proteasome homeostasis involves an autoregulatory mechanism.     

Characterization of ubiquitin and ubiquitin-like-protein isopeptidase activities

Conjugation or deconjugation of ubiquitin (Ub) or ubiquitin-like proteins (UBLs) to or from cellular proteins is a multifaceted and universal means of regulating cellular physiology, controlling the lifetime, localization, and activity of many critical proteins. Deconjugation of Ub or UBL from proteins is performed by a class of proteases called isopeptidases. Herein is described a readily quantifiable novel isopeptidase assay platform consisting of Ub or UBL fused to the reporter enzyme phospholipase A(2) (PLA(2)). Isopeptidase activity releases PLA(2), which cleaves its substrate, generating a signal that is linear with deubiquitylase (DUB) concentration and is able to discriminate DUB, deSUMOylase, deNEDDylase, and deISGylase activities. The power and sensitivity of the UBL-PLA(2) assay are demonstrated by its ability to differentiate the contrasting deISGylase and DUB activities of two coronavirus proteases: severe acute respiratory syndrome papain-like protease (SARS-CoV PLpro) and NL63 CoV papain-like protease 2 (PLP2). Furthermore, direct comparisons with the current Ub-7-amino-4-methylcoumarin (Ub-AMC) assay demonstrated that the Ub-PLA(2) assay is an effective tool for characterizing modulators of isopeptidase activity. This observation was expanded by profiling the inhibitory activity of the nonselective isopeptidase inhibitor NSC 632839 against DUBs and deSUMOylases. Taken together, these studies illustrate the utility of the reporter-based approach to measuring isopeptidase activity.     

Conformational dynamics of wild-type Lys-48-linked diubiquitin in solution

Proteasomal degradation is mediated through modification of target proteins by Lys-48-linked polyubiquitin (polyUb) chain, which interacts with several binding partners in this pathway through hydrophobic surfaces on individual Ub units. However, the previously reported crystal structures of Lys-48-linked diUb exhibit a closed conformation with sequestered hydrophobic surfaces. NMR studies on mutated Lys-48-linked diUb indicated a pH-dependent conformational equilibrium between closed and open states with the predominance of the former under neutral conditions (90% at pH 6.8). To address the question of how Ub-binding proteins can efficiently access the sequestered hydrophobic surfaces of Ub chains, we revisited the conformational dynamics of Lys-48-linked diUb in solution using wild-type diUb and cyclic forms of diUb in which the Ub units are connected through two Lys-48-mediated isopeptide bonds. Our newly determined crystal structure of wild-type diUb showed an open conformation, whereas NMR analyses of cyclic Lys-48-linked diUb in solution revealed that its structure resembled the closed conformation observed in previous crystal structures. Comparison of a chemical shift of wild-type diUb with that of monomeric Ub and cyclic diUb, which mimic the open and closed states, respectively, with regard to the exposure of hydrophobic surfaces to the solvent indicates that wild-type Lys-48-linked diUb in solution predominantly exhibits the open conformation (75% at pH 7.0), which becomes more populated upon lowering pH. The intrinsic properties of Lys-48-linked Ub chains to adopt the open conformation may be advantageous for interacting with Ub-binding proteins.     

Molecular Recognition of M1-Linked Ubiquitin Chains by Native and Phosphorylated UBAN Domains

Although the Ub-binding domain in ABIN proteins and NEMO (UBAN) is highly conserved, UBAN-containing proteins exhibit different Ub-binding properties, resulting in their diverse biological roles. Post-translational modifications further control UBAN domain specificity for poly-Ub chains. However, precisely, how the UBAN domain structurally confers such functional diversity remains poorly understood. Here we report crystal structures of ABIN-1 alone and in complex with one or two M1-linked di-Ub chains. ABIN-1 UBAN forms a homo-dimer that provides two symmetrical Ub-binding sites on either side of the coiled-coil structure. Moreover, crystal structures of ABIN1 UBAN in complex with di-Ub chains reveal a concentration-dependency of UBAN/di-Ub binding stoichiometry. Analysis of UBAN/M1-linked di-Ub binding characteristics indicates that phosphorylated S473 in OPTN and its corresponding phospho-mimetic residue in ABIN-1 (E484) are essential for high affinity interactions with M1-linked Ub chains. Also, a phospho-mimetic mutation of A303 in NEMO, corresponding to S473 of OPTN, increases binding affinity for M1-linked Ub chains. These findings are in line with the diverse physiological roles of UBAN domains, as phosphorylation of OPTN UBAN is required to enhance its binding to Ub during mitophagy.     

Polyubiquitin binding and cross-reactivity in the USP domain deubiquitinase USP21

Modification of proteins by ubiquitin (Ub) and Ub-like (Ubl) modifiers regulates a variety of cellular functions. The ability of Ub to form chains of eight structurally and functionally distinct types adds further complexity to the system. Ub-specific proteases (USPs) hydrolyse polyUb chains, and some have been suggested to be cross-reactive with Ubl modifiers, such as neural precursor cell expressed, developmentally downregulated 8 (NEDD8) and interferon-stimulated gene 15 (ISG15). Here, we report that USP21 cleaves Ub polymers, and with reduced activity also targets ISG15, but is inactive against NEDD8. A crystal structure of USP21 in complex with linear diUb aldehyde shows how USP21 interacts with polyUb through a previously unidentified second Ub- and ISG15-binding surface on the USP domain core. We also rationalize the inability of USP21 to target NEDD8 and identify differences that allow USPs to distinguish between structurally related modifications.     

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.     

The structures of ubiquitin conjugates of yeast Iso-2-cytochrome c.

Ubiquitin (Ub) conjugates to Saccharomyces cerevisiae iso-2-cytochrome c were formed in vitro in a rabbit reticulocyte extract (Fraction II). In the presence of ubiquitin-aldehyde, used to inhibit ubiquitin-protein isopeptidases in Fraction II, mono-, di-, and triubiquitinated cytochrome c conjugates accumulated in a 1.2:1.0:0.2 molar ratio. CNBr digestions showed that, in all three conjugates, Ub attachment was within the first 73 amino acids of the cytochrome c. For the two most abundant conjugates, this region was further narrowed to the first 30 residues by peptide mapping with Staphylococcus aureus V8 protease. N-terminal protein sequencing identified Lys-13 as the major ubiquitination site in each conjugate. For di- and triubiquitinated iso-2-cytochrome c, this suggested that Ub2 and Ub3 multiubiquitin chains extend from Lys-13. This conclusion was supported by a variation of protein sequencing in which polypeptides recovered after Edman degradation were analyzed to determine at which cycle(s) radiolabeled Ub or Ubn was cleaved from the conjugate. Because of the sensitivity afforded by the use of 125I-Ub in this "stutter-step" sequencing method, minor ubiquitination at Lys-8 also was detected. Thus, Ub2-iso-2-cytochrome c conjugates contain mostly Ub2 at Lys-13 with a small fraction of conjugates having single Ubs on 2 residues, Lys-8 and Lys-13. Similarly, Ub3-iso-2-cytochrome c predominantly has a Ub3 chain on Lys-13, although minor species with combinations of Ub1 and Ub2 distributed on Lys-8 and Lys-13 also may be present. This specificity is discussed in the context of iso-2-cytochrome c structure.     

A single MIU motif of MINDY‐1 recognizes K48‐linked polyubiquitin chains

The eight different types of ubiquitin (Ub) chains that can be formed play important roles in diverse cellular processes. Linkage‐selective recognition of Ub chains by Ub‐binding domain (UBD)‐containing proteins is central to coupling different Ub signals to specific cellular responses. The motif interacting with ubiquitin (MIU) is a small UBD that has been characterized for its binding to monoUb. The recently discovered deubiquitinase MINDY‐1/FAM63A contains a tandem MIU repeat (tMIU) that is highly selective at binding to K48‐linked polyUb. We here identify that this linkage‐selective binding is mediated by a single MIU motif (MIU2) in MINDY‐1. The crystal structure of MIU2 in complex with K48‐linked polyubiquitin chains reveals that MIU2 on its own binds to all three Ub moieties in an open conformation that can only be accommodated by K48‐linked triUb. The weak Ub binder MIU1 increases overall affinity of the tMIU for polyUb chains without affecting its linkage selectivity. Our analyses reveal new concepts for linkage selectivity and polyUb recognition by UBDs.     

Cyclization of polyubiquitin by the E2-25K ubiquitin conjugating enzyme

For most substrates of ubiquitin (Ub)-dependent degradation, recognition by the proteasome is mediated by a covalently attached signal assembled from multiple ubiquitins linked to each other via the C terminus of one Ub and the epsilon-amine of Lys(48) of another Ub. Among Ub-conjugating enzymes, E2-25K is unique in its ability to synthesize in vitro unanchored Lys(48)-linked poly-Ub chains from mono- or poly-Ub, E1, and ATP; thus, E2-25K has distinct binding sites for donor and acceptor (poly)Ub. During studies of chain assembly by E2-25K, we observed that Lys(48)-linked tri-Ub was efficiently converted to a new species that upon SDS-polyacrylamide gel electrophoresis migrated between linear di-Ub and tri-Ub. Analysis of this product by mass spectrometry and tryptic digestion showed that it was a cyclic form of tri-Ub. Cyclization of tri-Ub requires E1, E2-25K, ATP, and that the linear substrate has a free Gly(76) C terminus on the proximal end Ub and a Lys(48) side chain available on the distal end Ub. E2-25K similarly can catalyze the cyclization of longer poly-Ub chains, including tetra- and penta-Ub. Although cyclic tri-Ub resists hydrolysis by the PA700 or isopeptidase T deubiquitinating enzymes, it can be disassembled to Ub monomers by isopeptidase(s) in a red blood cell extract. Thus, if cyclic poly-Ub forms in vivo, it will not accumulate as a dead-end product.