Ubiquitin-interacting Motifs Confer Full Catalytic Activity,but Not Ubiquitin Chain Substrate Specificity,to Deubiquitinating Enzyme USP37

Ubiquitin-specific proteases (USPs) consist of a family of deubiquitinating enzymes with more than 50 members in humans. Three of them, including USP37, contain ubiquitin-interacting motifs (UIMs), an ∼20-amino acid α-helical stretch that binds to ubiquitin. However, the roles of the UIMs in these USP enzymes remain unknown. USP37 has three UIMs, designated here as UIMs 1, 2, and 3 from the N-terminal side, between the Cys and His boxes comprising the catalytic core. Here, we examined the role of the UIMs in USP37 using its mutants that harbor mutations in the UIMs. The nuclear localization of USP37 was not affected by the UIM mutations. However, mutations in UIM2 or UIM3, but not UIM1, resulted in a significant decrease in USP37 binding to ubiquitinated proteins in the cell. In vitro, a region of USP37 harboring the three UIMs also bound to both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains in a UIM2- and UIM3-dependent manner. The level of USP37 ubiquitination was also reduced by mutations in UIM2 or UIM3, suggesting their role in ubiquitination of USP37 itself. Finally, mutants lacking functional UIM2 or UIM3 exhibited a reduced isopeptidase activity toward ubiquitinated proteins in the cell and both Lys⁴⁸-linked and Lys⁶³-linked ubiquitin chains. These results suggested that the UIMs in USP37 contribute to the full enzymatic activity, but not ubiquitin chain substrate specificity, of USP37 possibly by holding the ubiquitin chain substrate in the proximity of the catalytic core.     

Structural transformation of the tandem ubiquitin-interacting motifs in ataxin-3 and their cooperative interactions with ubiquitin chains

The ubiquitin-interacting motif (UIM) is a short peptide with dual function of binding ubiquitin (Ub) and promoting ubiquitination. We elucidated the structures and dynamics of the tandem UIMs of ataxin-3 (AT3-UIM12) both in free and Ub-bound forms. The solution structure of free AT3-UIM12 consists of two α-helices and a flexible linker, whereas that of the Ub-bound form is much more compact with hydrophobic contacts between the two helices. NMR dynamics indicates that the flexible linker becomes rigid when AT3-UIM12 binds with Ub. Isothermal titration calorimetry and NMR titration demonstrate that AT3-UIM12 binds diUb with two distinct affinities, and the linker plays a critical role in association of the two helices in diUb binding. These results provide an implication that the tandem UIM12 interacts with Ub or diUb in a cooperative manner through an allosteric effect and dynamics change of the linker region, which might be related to its recognitions with various Ub chains and ubiquitinated substrates.     

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.     

The Ubiquitin-associated (UBA) 1 Domain of Schizosaccharomyces pombe Rhp23 Is Essential for the Recognition of Ubiquitin-proteasome System Substrates Both in Vitro and in Vivo

The ubiquitin-proteasome system is essential for maintaining a functional cell. Not only does it remove incorrectly folded proteins, it also regulates protein levels to ensure their appropriate spatial and temporal distribution. Proteins marked for degradation by the addition of Lys⁴⁸-linked ubiquitin (Ub) chains are recognized by shuttle factors and transported to the 26 S proteasome. One of these shuttle factors, Schizosaccharomyces pombe Rhp23, has an unusual domain architecture. It comprises an N-terminal ubiquitin-like domain that can recognize the proteasome followed by two ubiquitin-associated (UBA) domains, termed UBA1 and UBA2, which can bind Ub. This architecture is conserved up to humans, suggesting that both domains are important for Rhp23 function. Such an extent of conservation raises the question as to why, in contrast to all other shuttle proteins, does Rhp23 require two UBA domains? We performed in vitro Ub binding assays using domain swap chimeric proteins and mutated domains in isolation as well as in the context of the full-length protein to reveal that the Ub binding properties of the UBA domains are context-dependent. In vivo, the internal Rhp23 UBA1 domain provides sufficient Ub recognition for the protein to function without UBA2.     

Mapping the interactions between Lys48 and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a

Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as nonproteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide the first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin, which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.     

Evidence for cooperative and domain-specific binding of the signal transducing adaptor molecule 2 (STAM2) to Lys63-linked diubiquitin

As the upstream component of the ESCRT (endosomal sorting complexes required for transport) machinery, the ESCRT-0 complex is responsible for directing ubiquitinated membrane proteins to the multivesicular body pathway. ESCRT-0 is formed by two subunits known as Hrs (hepatocyte growth factor-regulated substrate) and STAM (signal transducing adaptor molecule), both of which harbor multiple ubiquitin-binding domains (UBDs). In particular, STAM2 possesses two UBDs, the VHS (Vps27/Hrs/Stam) and UIM (ubiquitin interacting motif) domains, connected by a 20-amino acid flexible linker. In the present study, we report the interactions of the UIM domain and VHS-UIM construct of STAM2 with monoubiquitin (Ub), Lys(48)- and Lys(63)-linked diubiquitins. Our results demonstrate that the UIM domain alone binds monoubiquitin, Lys(48)- and Lys(63)-linked diubiquitins with the same affinity and in the same binding mode. Interestingly, binding of VHS-UIM to Lys(63)-linked diubiquitin is not only avid, but also cooperative. We also show that the distal domain of Lys(63)-linked diubiquitin stabilizes the helical structure of the UIM domain and that the corresponding complex adopts a specific structural organization responsible for its greater affinity. In contrast, binding of VHS-UIM to Lys(48)-linked diubiquitin and monoubiquitin is not cooperative and does not show any avidity. These results may explain the better sorting efficiency of some cargoes polyubiquitinated with Lys(63)-linked chains over monoubiquitinated cargoes or those tagged with Lys(48)-linked chains.     

Structural and Biochemical Basis for Higher-Order Assembly between A20-Binding Inhibitor of NF-κB 1 (ABIN1) and M1-Linked Ubiquitins

Polyubiquitination is important in controlling NF-κB signaling. Excessive NF-κB activity has been linked to inflammatory disorders and autoimmune diseases, while ABIN1 could attenuate NF-κB activation to maintain immune homeostasis by utilizing UBAN to recognize linear (M1)-linked polyubiquitinated NF-κB activation mediators, including NEMO, IRAK1 and RIP1. PolyUb-mediated UBAN recruitment remains undetermined, since the recognition studies focused mostly on di-ubiquitin (diUb). Here we report three crystal structures of human ABIN1 UBAN (hABIN1^UBAN) in complex with M1-linked diUb, triUb, and tetraUb, respectively. Notably, the hABIN1^UBAN:diUb structure reveals that a diUb randomly binds one of the Ub-binding sites of the hABIN1^UBAN dimer and leaves the other site vacant. Together with the ITC and gel-filtration analyses, we found that M1-triUb and M1-tetraUb adopt two unique conformations, instead of an elongated one, and they preferentially use the N-terminal two-Ub unit to bind the primary Ub-binding site of a hABIN1^UBAN dimer and the C-terminal two-Ub unit to bind the secondary Ub-binding site of another hABIN1^UBAN dimer. Especially, our results suggest that two ABIN1^UBAN dimers cooperatively bind two UBAN-binding units of a tetraUb or vice versa. Since the UBAN family members share a conserved diUb-binding mode, our results suggest that M1-polyUb modification allows multiple copies of the two-tandem Ub unit to simultaneously coordinate multiple and/or different binding partners to increase their local concentrations and to facilitate the formation of a large signaling complex. Our study provides a structural-functional glimpse of M1-polyUb as a multiple-molecule binding platform to exert its intrinsic structural plasticity in mediating cellular signaling.     

Molecular Basis for Phosphorylation-dependent SUMO Recognition by the DNA Repair Protein RAP80

Recognition and repair of double-stranded DNA breaks (DSB) involves the targeted recruitment of BRCA tumor suppressors to damage foci through binding of both ubiquitin (Ub) and the Ub-like modifier SUMO. RAP80 is a component of the BRCA1 A complex, and plays a key role in the recruitment process through the binding of Lys⁶³-linked poly-Ub chains by tandem Ub interacting motifs (UIM). RAP80 also contains a SUMO interacting motif (SIM) just upstream of the tandem UIMs that has been shown to specifically bind the SUMO-2 isoform. The RAP80 tandem UIMs and SIM function collectively for optimal recruitment of BRCA1 to DSBs, although the molecular basis of this process is not well understood. Using NMR spectroscopy, we demonstrate that the RAP80 SIM binds SUMO-2, and that both specificity and affinity are enhanced through phosphorylation of the canonical CK2 site within the SIM. The affinity increase results from an enhancement of electrostatic interactions between the phosphoserines of RAP80 and the SIM recognition module within SUMO-2. The NMR structure of the SUMO-2·phospho-RAP80 complex reveals that the molecular basis for SUMO-2 specificity is due to isoform-specific sequence differences in electrostatic SIM recognition modules.     

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.     

Synthesis of Free and Proliferating Cell Nuclear Antigen-bound Polyubiquitin Chains by the RING E3 Ubiquitin Ligase Rad5

In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys⁶³-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13∼Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys⁶³ of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.DNA is susceptible to chemical alteration by many endogenous and exogenous agents. To counter this threat and maintain genome integrity, eukaryotic cells employ three main strategies: DNA repair pathways that directly reverse DNA damage, cell cycle checkpoints that allow time to repair the damage prior to replication, and DNA damage tolerance (DDT),² which is a method of bypassing DNA damage lesions during the DNA replication phase of the cell cycle.Proliferating cell nuclear antigen (PCNA) is a key regulatory protein in DNA replication and repair (1). At the replication fork, DNA is encircled by PCNA, a homotrimeric protein that promotes processive movement of the replicative DNA polymerase. Upon DNA damage and subsequent stalling of the replicative polymerase, Ub modifications of PCNA signal DDT, which allows a cell to bypass the lesion and proceed past this potential block in replication (2–4).In the DDT pathway, as in other Ub-dependent pathways, Ub is conjugated to a substrate by the actions of three enzymes, an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (5). The E1 enzyme initiates the pathway in a two-step reaction that utilizes ATP hydrolysis to activate the C terminus of Ub, culminating in the formation of an E1∼Ub thiolester. Subsequent transthiolation to the active site cysteine of the E2 generates an E2∼Ub thiolester. An E3 ligase then brings a substrate into close proximity to the E2∼Ub intermediate, thereby catalyzing the formation of an isopeptide bond between the amino group of a substrate lysine and the C-terminal glycine of Ub. Polyubiquitination occurs when this substrate is another Ub, either free or as part of a Ub-protein conjugate.The DDT pathway is characterized by distinct ubiquitination events on PCNA that occur in two stages (3, 4, 6). The first of these is monoubiquitination of lysine 164 on one or more of the PCNA subunits by the E2-E3 complex of Rad6-Rad18 in Saccharomyces cerevisiae (3, 4, 7). monoUb-PCNA can serve either as a signal for error-prone bypass of the DNA lesion by recruiting translesion polymerases or as a substrate for subsequent polyubiquitination by the E2 heterodimer Ubc13-Mms2 and the E3 ligase Rad5 (3, 4, 8, 9). The polyUb chain extended from the initial Ub moiety on monoUb-PCNA is linked specifically through Ub Lys⁶³ residues. This Lys⁶³-linked chain is thought to enable a template switch mechanism that allows for error-free bypass of the DNA lesion, in part by utilizing the single-strand DNA-dependent helicase activity of Rad5 (3, 4, 10, 11). Both PCNA ubiquitination events promote bypass of the DNA lesion rather than direct removal or repair of the lesion.We have been interested in the mechanism by which the yeast (Ubc13-Mms2)-Rad5 complex catalyzes the formation of Lys⁶³-linked polyUb on PCNA. Previous studies have shown that heterodimerization of the Ubc13-Mms2 E2 is essential for Lys⁶³-specific Ub-Ub conjugation in vitro and in vivo (12–15). Ubc13 is a canonical E2 enzyme with an active site cysteine that receives activated Ub by transthiolation from the E1∼Ub complex (12, 13). This Ub is referred to as the “donor Ub.” Mms2 is a Ub E2 variant protein that lacks the active site cysteine (12, 15); rather, Mms2 binds to a second Ub, the “acceptor Ub,” and positions it to facilitate nucleophilic attack on the Ubc13∼Ub thiolester bond by the ϵ-amine of Lys⁶³ (15, 16). The positioning of the acceptor Ub by Mms2 controls the specificity of polyUb assembly such that only Lys⁶³-linked chains can be formed (16).Ubc13-Mms2 can synthesize Lys⁶³-linked chains in vitro in the absence of a PCNA substrate or an E3 ligase (12, 13). However, unlike the synthesis of free Lys⁶³-linked polyUb chains by Ubc13-Mms2, little is known about the polyubiquitination of PCNA or the role of the Rad5 E3 ligase in these reactions. Rad5 can bind PCNA and Rad18, and it contains a catalytic RING domain that characterizes the largest class of E3 ligases (17–21). There is evidence that RING E3s like Rad5 may play a more active role in ubiquitination than simply bringing the substrate into close proximity with the E2∼Ub. Several RING E3s have been shown to stimulate the synthesis of unanchored polyUb chains or autoubiquitination of their cognate E2s in the absence of substrates (22–24). This stimulation may be related to the ability of RING E3s to enhance reactivity of the E2∼Ub thiolester bond through allosteric effects (25, 26).Using purified recombinant forms of Ubc13, Mms2, and Rad5, we have explored the assembly of free Lys⁶³-linked polyUb chains as well as the extension of a polyUb chain on a synthetic analog of monoUb-PCNA. We show that Rad5 facilitates ubiquitination in part by increasing the reactivity of the Ubc13∼Ub thiolester bond. With monoUb-PCNA substrates, Rad5 also stimulated polyubiquitination through direct interactions with PCNA and recruitment of Ub-charged Ubc13-Mms2. Surprisingly, Rad5 recognition of monoUb-PCNA appeared to depend on interactions only with the PCNA moiety of the conjugate, which suggests that substrate selectivity in vivo is likely to depend on additional factors.     

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.     

Dynamic behaviour of ubiquitin receptor S5a in free and complex with K48-linked diubiquitin

S5a is a critical component of proteasome and carries ubiquitin recognition function. Previous nuclear magnetic resonance (NMR) experiments have shown that K48-linked diubiquitin binds to S5a through a major and a minor conformational species. Molecular dynamics simulations have been performed on S5a and S5a:K48-linked diubiquitin complex extracted from both species to investigate the essential dynamic behaviour of the receptor S5a in free and complex with the diubiquitin. It shows that structures of S5a as well as S5a:diubiquitin complex are very mobile during the simulations, which enables the receptor to undergo a conformational interconversion from the minor to major species or vice versa, though finally the receptor alone tends to adopt a tight packed structure. The binding of diubiquitin to S5a reduces the structural mobility of the receptor, however, it is still able to cover the different conformations within each species of the complex. Despite the high mobility of the structures, the binding of ubiquitin interacting with motif 2 (UIM2) is always stronger than the UIM1 to the ubiquitin subunit. Accordingly, the current dynamic study provides a vivid view how the receptor in free and complex with diubiquitin sampled the multiple conformations as well as their exchanges revealed in two NMR structures.     

De-ubiquitylation is the most critical step in the ubiquitin-mediated homeostatic control of the NF-κB/IKK basal activity

The role of ubiquitylation in signal-induced activation of nuclear factor -κB (NF-κB) has been well established, while its involvement in maintaining NF-κB basal activity is less certain. Recent evidences demonstrate that in unstimulated cells, NF-κB homeostasis is actually the result of opposing forces: pro-activating activity of the IκB Kinase (IKK) and inhibitory activity of the Inhibitor of -κB (IκB) proteins. It is well known that endogenous de-ubiquitylating mechanisms are less effective on Ub motifs containing UbG76A. Here, we show that overexpression of a ubiquitin (Ub) G76A mutant leads to persistent activation of the IKK/NF-κB pathway in the absence of extra-cellular stimuli. In contrast, no effects on NF-κB activation were observed upon expression of UbK48R and UbK63R mutants, which are known to impair elongation of Lys⁴⁸- and Lys⁶³-linked poly-ubiquitin chains, respectively. Overall, these findings indicate that under basal conditions, the rate of de-ubiquitylation, rather than that of substrate ubiquitylation, is critical for the maintenance of appropriate levels of IKK/NF-κB activity.     

Contribution of Lysine 11-linked Ubiquitination to MIR2-mediated Major Histocompatibility Complex Class I Internalization

The polyubiquitin chain is generated by the sequential addition of ubiquitin moieties to target molecules, a reaction between specific lysine residues that is catalyzed by E3 ubiquitin ligase. The Lys⁴⁸-linked and Lys⁶³-linked polyubiquitin chains are well established inducers of proteasome-dependent degradation and signal transduction, respectively. The concept has recently emerged that polyubiquitin chain-mediated regulation is even more complex because various types of atypical polyubiquitin chains have been discovered in vivo. Here, we demonstrate that a novel complex ubiquitin chain functions as an internalization signal for major histocompatibility complex class I (MHC I) membrane proteins in vivo. Using a tetracycline-inducible expression system and quantitative mass spectrometry, we show that the polyubiquitin chain generated by the viral E3 ubiquitin ligase of Kaposi sarcoma-associated herpesvirus, MIR2, is a Lys¹¹ and Lys⁶³ mixed-linkage chain. This novel ubiquitin chain can function as an internalization signal for MHC I through its association with epsin1, an adaptor molecule containing ubiquitin-interacting motifs.     

Modulation of K11-linkage formation by variable loop residues within UbcH5A

Ubiquitination refers to the covalent addition of ubiquitin (Ub) to substrate proteins or other Ub molecules via the sequential action of three enzymes (E1, E2, and E3). Recent advances in mass spectrometry proteomics have made it possible to identify and quantify Ub linkages in biochemical and cellular systems. We used these tools to probe the mechanisms controlling linkage specificity for UbcH5A. UbcH5A is a promiscuous E2 enzyme with an innate preference for forming polyubiquitin chains through lysine 11 (K11), lysine 48 (K48), and lysine 63 (K63) of Ub. We present the crystal structure of a noncovalent complex between Ub and UbcH5A. This structure reveals an interaction between the Ub surface flanking K11 and residues adjacent to the E2 catalytic cysteine and suggests a possible role for this surface in formation of K11 linkages. Structure-guided mutagenesis, in vitro ubiquitination and quantitative mass spectrometry have been used to characterize the ability of residues in the vicinity of the E2 active site to direct synthesis of K11- and K63-linked polyubiquitin. Mutation of critical residues in the interface modulated the linkage specificity of UbcH5A, resulting in generation of more K63-linked chains at the expense of K11-linkage synthesis. This study provides direct evidence that the linkage specificity of E2 enzymes may be altered through active-site mutagenesis.     

Lysine 63-linked Polyubiquitination of TAK1 at Lysine 158 Is Required for Tumor Necrosis Factor ��- and Interleukin-1��-induced IKK/NF-��B and JNK/AP-1 Activation

Transforming growth factor-β-activated kinase 1 (TAK1) plays an essential role in the tumor necrosis factor α (TNFα)- and interleukin-1β (IL-1β)-induced IκB kinase (IKK)/nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK)/activator protein 1 (AP-1) activation. Here we report that TNFα and IL-1β induce Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue within the kinase domain. Tumor necrosis factor receptor-associated factors 2 and 6 (TRAF2 and -6) act as the ubiquitin E3 ligases to mediate Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue in vivo and in vitro. Lys⁶³-linked TAK1 polyubiquitination at the Lys¹⁵⁸ residue is required for TAK1-mediated IKK complex recruitment. Reconstitution of TAK1-deficient mouse embryo fibroblast cells with TAK1 wild type or a TAK1 mutant containing a K158R mutation revealed the importance of this site in TNFα and IL-1β-mediated IKK/NF-κB and JNK/AP-1 activation as well as IL-6 gene expression. Our findings demonstrate that Lys⁶³-linked polyubiquitination of TAK1 at Lys¹⁵⁸ is essential for its own kinase activation and its ability to mediate its downstream signal transduction pathways in response to TNFα and IL-1β stimulation.     

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.     

Quantitative affinity interaction of ubiquitinated and non-ubiquitinated proteins with proteasome subunit Rpn10

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (K _d = 2.4·10–6 M) and pyruvate kinase (K _d = 2.8·10^–5 M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10^–8 and 3.2·10^–9 M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.