The E2 Ubiquitin-conjugating Enzymes Direct Polyubiquitination to Preferred Lysines

The ubiquitin-proteasome pathway plays a crucial role in many cellular processes by degrading substrates tagged by polyubiquitin chains, linked mostly through lysine 48 of ubiquitin. Although polymerization of ubiquitin via its six other lysine residues exists in vivo as part of various physiological pathways, the molecular mechanisms that determine the type of polyubiquitin chains remained largely unknown. We undertook a systematic, in vitro, approach to evaluate the role of E2 enzymes in determining the topology of polyubiquitin. Because this study was performed in the absence of an E3 enzyme, our data indicate that the E2 enzymes are capable of directing the ubiquitination process to distinct subsets of ubiquitin lysines, depending on the specific E2 utilized. Moreover, our findings are in complete agreement with prior analyses of lysine preference assigned to certain E2s in the context of E3 (in vitro and in vivo). Finally, our findings support the rising notion that the functional unit of E2 is a dimer. To our knowledge, this is the first systematic indication for the involvement of E2 enzymes in specifying polyubiquitin chain assembly.     

A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved alpha/beta-fold core domain of approximately 150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c approximately Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.     

USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity.     

Direct catalysis of lysine 48-linked polyubiquitin chains by the ubiquitin-activating enzyme

Within the ubiquitin degradation pathway, the canonical signal is a lysine 48-linked polyubiquitin chain that is assembled upon an internal lysine residue of a substrate protein. Once constructed, this ubiquitin chain becomes the principle signal for recognition and target degradation by the 26S proteasome. The mechanism by which polyubiquitin chains are assembled on a substrate protein, however, has yet to be clearly defined. In an in vitro model system, purified E2-ubiquitin thiolester was unable to catalyze the formation of polyubiquitin chains in the absence of the ubiquitin-activating enzyme E1. Mutagenesis of key residues within the E1 active site revealed that its conserved catalytic cysteine residue is essential for the formation of these chains. Moreover, inactivation of the E2 active site had no effect on the ability of E1 to catalyze ubiquitin chain formation. These findings strongly suggest E1 is responsible for not only the activation of ubiquitin but also for the direct catalytic extension of a lysine 48-linked polyubiquitin chain.     

Mass spectrometric analysis of tumor necrosis factor receptor-associated factor 1 ubiquitination mediated by cellular inhibitor of apoptosis 2

Signaling complexes formed on tumor necrosis factor receptor 2 (TNF-R2) contain adaptor proteins TNF-R-associated factors (TRAFs) 1 and 2, and cellular inhibitors of apoptosis (cIAPs) 1 and 2 which function as regulators of programmed cell death. TRAF2, cIAP1 and cIAP2 all have RING finger domains known to possess E3 ubiquitin ligase activity, implying that ubiquitination may play an important role in the TNF signaling pathway. In this report, we have shown that cIAP2 specifically mediated ubiquitination and proteasome-dependent degradation of TRAF1. To identify the sites for cIAP2-mediated ubiquitination of TRAF1, we used high pressure liquid chromatography coupled with tandem mass spectrometry. Lys185 and Lys193 of TRAF1 were found to be modified with ubiquitin chains. Mutation of Lys185 and Lys193 to Arg almost completely blocked cIAP2-mediated ubiquitination of TRAF1, indicating that they are the major, if not the only, sites of TRAF1 ubiquitination. Our data suggest that cIAP2 may regulate the turnover of TRAF1 by adding polyubiquitin chains on Lys185 or Lys193 following its recruitment to TNF-R signaling complexes.     

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes.     

TRIM44 interacts with and stabilizes terf, a TRIM ubiquitin E3 ligase

Terf/TRIM17 is a member of the TRIM family of proteins, which is characterized by the RING finger, B-box, and coiled-coil domains. In the present study, we found that terf interacts with TRIM44. Terf underwent ubiquitination in vitro in the presence of the E2 enzyme UbcH6; this suggests that terf exhibits E3 ubiquitin ligase activity. It was also found that terf was conjugated with polyubiquitin chains and stabilized by the proteasome inhibitor in mammalian cells; this suggested that terf rendered itself susceptible to proteasomal degradation through polyubiquitination. We also found that TRIM44 inhibited ubiquitination of terf, and thus stabilized the protein. The N-terminal region of TRIM44 contains a zinc-finger domain found in ubiquitin hydrolases (ZF UBP) and ubiquitin specific proteases (USPs). Thus, we proposed that TRIM44 may function as a new class of the “USP-like-TRIM” which regulates the activity of associated TRIM proteins.     

Ubiquitin-Specific Protease 5 Is Required for the Efficient Repair of DNA Double-Strand Breaks

During the DNA damage response (DDR), ubiquitination plays an important role in the recruitment and regulation of repair proteins. However, little is known about elimination of the ubiquitination signal after repair is completed. Here we show that the ubiquitin-specific protease 5 (USP5), a deubiquitinating enzyme, is involved in the elimination of the ubiquitin signal from damaged sites and is required for efficient DNA double-strand break (DSB) repair. Depletion of USP5 sensitizes cells to DNA damaging agents, produces DSBs, causes delayed disappearance of γH2AX foci after Bleocin treatment, and influences DSB repair efficiency in the homologous recombination pathway but not in the non-homologous end joining pathway. USP5 co-localizes to DSBs induced by laser micro-irradiation in a RAD18-dependent manner. Importantly, polyubiquitin chains at sites of DNA damage remained for longer periods in USP5-depleted cells. Our results show that disassembly of polyubiquitin chains by USP5 at sites of damage is important for efficient DSB repair.     

The human T-cell leukemia virus type 1 Tax oncoprotein requires the ubiquitin-conjugating enzyme Ubc13 for NF-kappaB activation

Ubiquitination of the human T-cell leukemia virus 1 Tax oncoprotein provides an important regulatory mechanism that promotes the Tax-mediated activation of NF-κB. However, the type of polyubiquitin chain linkages and the host factors that are required for Tax ubiquitination have not been identified. Here, we demonstrate that Tax polyubiquitin chains are composed predominantly of lysine 63-linked chains. Furthermore, the ubiquitination of Tax is critically dependent on the E2 ubiquitin-conjugating enzyme Ubc13. Tax interacts with Ubc13, and small interfering RNA-mediated knockdown of Ubc13 expression abrogates Tax ubiquitination and the activation of NF-κB. Mouse fibroblasts lacking Ubc13 exhibit impaired Tax activation of NF-κB despite normal tumor necrosis factor- and interleukin-1-mediated NF-κB activation. Finally, the interaction of Tax with NEMO is disrupted in the absence of Tax ubiquitination and Ubc13 expression, suggesting that Tax ubiquitination is critical for NEMO binding. Collectively, our results reveal that Ubc13 is essential for Tax ubiquitination, its interaction with NEMO, and Tax-mediated NF-κB activation.     

A HECT domain E3 enzyme assembles novel polyubiquitin chains

Although polyubiquitin chains linked through Lys(29) of ubiquitin have been implicated in the targeting of certain substrates to proteasomes, the signaling properties of these chains are poorly understood. We previously described a ubiquitin-protein isopeptide ligase (E3) from erythroid cells that assembles polyubiquitin chains through either Lys(29) or Lys(48) of ubiquitin (Mastrandrea, L. D., You, J., Niles, E. G., and Pickart, C. M. (1999) J. Biol. Chem. 274, 27299-27306). Here we describe the purification of this E3 based on its affinity for a linear fusion of ubiquitin to the ubiquitin-conjugating enzyme UbcH5A. Among five major polypeptides in the affinity column eluate, the activity of interest was assigned to the product of a previously cloned human cDNA known as KIAA10 (Nomura, N., Miyajima, N., Sazuka, T., Tanaka, A., Kawarabayasi, Y., Sato, S., Nagase, T., Seki, N., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 27-35). The KIAA10 protein is a member of the HECT (homologous to E6-AP carboxyl terminus) domain family of E3s. These E3s share a conserved C-terminal (HECT) domain that functions in the catalysis of ubiquitination, while their divergent N-terminal domains function in cognate substrate binding (Huibregtse, J. M., Scheffner, M., Beaudenon, S., and Howley, P. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2563-2567). Recombinant KIAA10 catalyzed the assembly of both Lys(29)- and Lys(48)-linked polyubiquitin chains. Surprisingly, the C-terminal 428 residues of KIAA10 were both necessary and sufficient for this activity, suggesting that the ability to assemble polyubiquitin chains may be a general property of HECT domains. The N-terminal domain of KIAA10 interacted in vitro with purified 26 S proteasomes and with the isolated S2/Rpn1 subunit of the proteasome's 19 S regulatory complex, suggesting that the N-terminal domains of HECT E3s may function in proteasome binding as well as substrate binding.     

Control of AMPK-related kinases by USP9X and atypical Lys(29)/Lys(33)-linked polyubiquitin chains

AMPK (AMP-activated protein kinase)-related kinases regulate cell polarity as well as proliferation and are activated by the LKB1-tumour suppressor kinase. In the present study we demonstrate that the AMPK-related kinases, NUAK1 (AMPK-related kinase 5) and MARK4 (microtubule-affinity-regulating kinase 4), are polyubiquitinated in vivo and interact with the deubiquitinating enzyme USP9X (ubiquitin specific protease-9). Knockdown of USP9X increased polyubiquitination of NUAK1 and MARK4, whereas overexpression of USP9X inhibited ubiquitination. USP9X, catalysed the removal of polyubiquitin chains from wild-type NUAK1, but not from a non-USP9X-binding mutant. Topological analysis revealed that ubiquitin monomers attached to NUAK1 and MARK4 are linked by Lys(29) and/or Lys(33) rather than the more common Lys(48)/Lys(63). We find that AMPK and other AMPK-related kinases are also polyubiquitinated in cells. We identified non-USP9X-binding mutants of NUAK1 and MARK4 and find that these are hyper-ubiquitinated and not phosphorylated at their T-loop residue targeted by LKB1 when expressed in cells, suggesting that polyubiquitination may inhibit these enzymes. The results of the present study demonstrate that NUAK1 and MARK4 are substrates of USP9X and provide the first evidence that AMPK family kinases are regulated by unusual Lys(29)/Lys(33)-linked polyubiquitin chains.     

E2/E3-mediated assembly of lysine 29-linked polyubiquitin chains.

Polyubiquitin (Ub) chains linked through Lys-48-Gly-76 isopeptide bonds represent the principal signal by which substrates of the Ub-dependent protein degradation pathway are targeted to the 26 S proteasome, but the mechanism(s) whereby these chains are assembled on substrate proteins is poorly understood. Nor have assembly mechanisms or definitive functions been assigned to polyubiquitin chains linked through several other lysine residues of ubiquitin. We show that rabbit reticulocyte lysate harbors enzymatic components that catalyze the assembly of unanchored Lys-29-linked polyubiquitin chains. This reaction can be reconstituted using the ubiquitin-conjugating enzyme (E2) known as UbcH5A, a 120-kDa protein(s) that behaves as a ubiquitin-protein ligase (E3), and ubiquitin-activating enzyme (E1). The same partially purified E3 preparation also catalyzes the assembly of unanchored chains linked through Lys-48. Kinetic studies revealed a K(m) of approximately 9 microM for the acceptor ubiquitin in the synthesis of diubiquitin; this value is similar to the concentration of free ubiquitin in most cells. Similar kinetic behavior was observed for conjugation to Lys-48 versus Lys-29 and for conjugation to tetraubiquitin versus monoubiquitin. The properties of these enzymes suggest that there may be distinct pathways for ubiquitin-ubiquitin ligation versus substrate-ubiquitin ligation in vivo.     

Linear polyubiquitination: a new regulator of NF‐κB activation

The ubiquitin‐conjugation system regulates a vast range of biological phenomena by affecting protein function mostly through polyubiquitin conjugation. The type of polyubiquitin chain that is generated seems to determine how conjugated proteins are regulated, as they are recognized specifically by proteins that contain chain‐specific ubiquitin‐binding motifs. An enzyme complex that catalyses the formation of newly described linear polyubiquitin chains—known as linear ubiquitin chain‐assembly complex (LUBAC)—has recently been characterized, as has a particular ubiquitin‐binding domain that specifically recognizes linear chains. Both have been shown to have crucial roles in the canonical nuclear factor‐κB (NF‐κB)‐activation pathway. The ubiquitin system is intimately involved in regulating the NF‐κB pathway, and the regulatory roles of K63‐linked chains have been studied extensively. However, the role of linear chains in this process is only now emerging. This article discusses the possible mechanisms underlying linear polyubiquitin‐mediated activation of NF‐κB, and the different roles that K63‐linked and linear chains have in NF‐κB activation. Future directions for linear polyubiquitin research are also discussed.     

Localization to the proteasome is sufficient for degradation

The majority of unstable proteins in eukaryotic cells are targeted for degradation through the ubiquitin-proteasome pathway. Substrates for degradation are recognized by the E1, E2, and E3 ubiquitin conjugation machinery and tagged with polyubiquitin chains, which are thought to promote the proteolytic process through their binding with the proteasome. We describe a method to bypass the ubiquitination step artificially both in vivo and in a purified in vitro system. Seven proteasome subunits were tagged with Fpr1, and fusion reporter constructs were created with the Fpr1-rapamycin binding domain of Tor1. Reporter proteins were localized to the proteasome by the addition of rapamycin, a drug that heterodimerizes Fpr1 and Tor1. Degradation of reporter proteins was observed with proteasomes that had either Rpn10 or Pre10 subunits tagged with Fpr1. Our experiments resolved a simple but central problem concerning the design of the ubiquitin-proteasome pathway. We conclude that localization to the proteasome is sufficient for degradation and, therefore, any added functions polyubiquitin chains possess beyond tethering substrates to the proteasome are not strictly necessary for proteolysis.     

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.     

Recruitment of Ubiquitin within an E2 Chain Elongation Complex

The ubiquitin (Ub) proteolysis pathway uses an E1, E2, and E3 enzyme cascade to label substrate proteins with ubiquitin and target them for degradation. The mechanisms of ubiquitin chain formation remain unclear and include a sequential addition model, in which polyubiquitin chains are built unit by unit on the substrate, or a preassembly model, in which polyubiquitin chains are preformed on the E2 or E3 enzyme and then transferred in one step to the substrate. The E2 conjugating enzyme UBE2K has a 150-residue catalytic core domain and a C-terminal ubiquitin-associated (UBA) domain. Polyubiquitin chains anchored to the catalytic cysteine and free in solution are formed by UBE2K supporting a preassembly model. To study how UBE2K might assemble polyubiquitin chains, we synthesized UBE2K-Ub and UBE2K-Ub₂ covalent complexes and analyzed E2 interactions with the covalently attached Ub and Ub₂ moieties using NMR spectroscopy. The UBE2K-Ub complex exists in multiple conformations, including the catalytically competent closed state independent of the UBA domain. In contrast, the UBE2K-Ub₂ complex takes on a more extended conformation directed by interactions between the classic I44 hydrophobic face of the distal Ub and the conserved MGF hydrophobic patch of the UBA domain. Our results indicate there are distinct differences between the UBE2K-Ub and UBE2K-Ub₂ complexes and show how the UBA domain can alter the position of a polyubiquitin chain attached to the UBE2K active site. These observations provide structural insights into the unique Ub chain-building capacity for UBE2K.     

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys(48)-linked chains target proteins for proteasomal degradation, and Lys(63)-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)-Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13-Uev1a supports the formation of free Lys(63)-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.     

Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.