Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages

It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.     

Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of Alzheimer disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys(48)), 38% (Lys(63)), 8% (Lys(29)), 2% (Lys(11)), and 0.5% or less for linear, Lys(6), Lys(27), and Lys(33) linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys(6), Lys(11), Lys(27), Lys(29), and Lys(48) mediate proteasomal degradation, whereas Lys(63) chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys(48), may also contribute to lysosomal targeting, as both Lys(63) and Lys(48) linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys(11), Lys(48), and Lys(63) but not Lys(29) linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.     

Exploring the Linkage Dependence of Polyubiquitin Conformations Using Molecular Modeling

Posttranslational modification of proteins by covalent attachment of a small protein ubiquitin (Ub) or a polymeric chain of Ub molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one Ub and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between two adjacent Ub monomers. By contrast, K63-linked chains, which act as a nonproteolytic regulatory signal, adopt an extended conformation that lacks hydrophobic interubiquitin contact. Little is known about the functional roles of the so-called “noncanonical” chains (linked via K6, K11, K27, K29, or K33, or linked head-to-tail), and no structural information on these chains is available, except for information on the crystal structure of the head-to-tail-linked diubiquitin (Ub₂). In this study, we use molecular modeling to examine whether any of the noncanonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible Ub₂ chains can be divided into two groups: chains linked via K6, K11, K27, or K48 are predicted to form a closed conformation, whereas chains linked via K29, K33, or K63, or linked head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail-linked chains. Our study also predicts structural models for Ub₂ chains linked via K6, K11, or K27. The implications of these findings for linkage-selective recognition of noncanonical polyubiquitin signals by various receptors are discussed.     

Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23.

Ubiquitin (Ub) regulates important cellular processes through covalent attachment to its substrates. The fate of a substrate depends on the number of ubiquitin moieties conjugated, as well as the lysine linkage of Ub-Ub conjugation. The major function of Ub is to regulate the in vivo half-life of its substrates. Once a multi-Ub chain is attached to a substrate, it must be shielded from deubiquitylating enzymes for the 26 S proteasome to recognize it. Molecular mechanisms of the postubiquitylation processes are poorly understood. Here, we have characterized a family of proteins that preferentially binds ubiquitylated substrates and multi-Ub chains through a motif termed the ubiquitin-associated domain (UBA). Our in vivo genetic analysis demonstrates that such interactions require specific lysine residues of Ub that are important for Ub chain formation. We show that Saccharomyces cerevisiae cells lacking two of these UBA proteins, Dsk2 and Rad23, are deficient in protein degradation mediated by the UFD pathway and that the intact UBA motif of Dsk2 is essential for its function in proteolysis. Dsk2 and Rad23 can form a complex(es), suggesting that they cooperate to recognize a subset of multi-Ub chains and deliver the Ub-tagged substrates to the proteasome. Our results suggest a molecular mechanism for differentiation of substrate fates, depending on the precise nature of the mono-Ub or multi-Ub lysine linkage, and provide a foundation to further investigate postubiquitylation events.     

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.     

Molecular determinants of polyubiquitin linkage selection by an HECT ubiquitin ligase

Ubiquitin (Ub)-protein ligases (E3s) frequently modify their substrates with multiple Ub molecules in the form of a polyubiquitin (poly-Ub) chain. Although structurally distinct poly-Ub chains (linked through different Ub lysine (Lys) residues) can confer different fates on target proteins, little is known about how E3s select the Lys residue to be used in chain synthesis. Here, we used a combination of mutagenesis, biochemistry, and mass spectrometry to map determinants of linkage choice in chain assembly catalyzed by KIAA10, an HECT (Homologous to E6AP C-Terminus) domain E3 that synthesizes K29- and K48-linked chains. Focusing on the Ub molecule that contributes the Lys residue for chain formation, we found that specific surface residues adjacent to K48 and K29 are critical for the usage of the respective Lys residues in chain synthesis. This direct mechanism of linkage choice bears similarities to the mechanism of substrate site selection in sumoylation catalyzed by Ubc9, but is distinct from the mechanism of chain linkage selection used by the Mms2/Ubc13 (Ub E2 variant (UEV)/E2) complex.     

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.     

Molecular basis of Lys-63-linked polyubiquitination inhibition by the interaction between human deubiquitinating enzyme OTUB1 and ubiquitin-conjugating enzyme UBC13

UBC13 is the only known E2 ubiquitin (Ub)-conjugating enzyme that produces Lys-63-linked Ub chain with its cofactor E2 variant UEV1a or MMS2. Lys-63-linked ubiquitination is crucial for recruitment of DNA repair and damage response molecules to sites of DNA double-strand breaks (DSBs). A deubiquitinating enzyme OTUB1 suppresses Lys-63-linked ubiquitination of chromatin surrounding DSBs by binding UBC13 to inhibit its E2 activity independently of the isopeptidase activity. OTUB1 strongly suppresses UBC13-dependent Lys-63-linked tri-Ub production, whereas it allows di-Ub production in vitro. The mechanism of this non-canonical OTUB1-mediated inhibition of ubiquitination remains to be elucidated. Furthermore, the atomic level information of the interaction between human OTUB1 and UBC13 has not been reported. Here, we determined the crystal structure of human OTUB1 in complex with human UBC13 and MMS2 at 3.15 Å resolution. The presented atomic-level interactions were confirmed by surface-plasmon resonance spectroscopy with structure-based mutagenesis. The designed OTUB1 mutants cannot inhibit Lys-63-linked Ub chain formation in vitro and histone ubiquitination and 53BP1 assembly around DSB sites in vivo. Finally, we propose a model for how capping of di-Ub by the OTUB1-UBC13-MMS2/UEV1a complex efficiently inhibits Lys-63-linked tri-Ub formation.     

The DNA repair protein rad23 is a negative regulator of multi-ubiquitin chain assembly

Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Delta was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.     

A 25-kilodalton ubiquitin carrier protein (E2) catalyzes multi-ubiquitin chain synthesis via lysine 48 of ubiquitin

Target protein multi-ubiquitination involving lysine 48 of ubiquitin (Ub) is known to occur during protein degradation in the ATP- and Ub-dependent proteolytic pathway (Chau, V., Tobias, J. W., Bachmair, A., Marriott, D., Ecker, D. J., Gonda, D. K., and Varshavsky, A. (1989) Science 243, 1576-1583). However, little is known about the enzymatic mechanism of multi-ubiquitination. We show that a purified Ub carrier protein, E2(25)K, catalyzes multi-Ub chain synthesis from purified Ub. Incubation of E2(25)K with Ub activating enzyme (E1), MgATP, and radiolabeled Ub (Mr = 8500) resulted in time dependent appearance of a "ladder" of radiolabeled Ub conjugates with molecular masses of 8.5n kDa, where n = 1, 2, 3, 4... (up to at least n = 10). The kinetics of this conjugative process were consistent with Ub2 acting as a steady-state intermediate. The putative Ub2 product of E2(25)K catalysis was purified and cleaved with a partially purified isopeptidase preparation. The sole cleavage product (Mr = 8500) had a tryptic digest identical to that of authentic Ub, confirming that the original conjugate was Ub2. Tryptic digestion of intact Ub2 gave products consistent with the existence of an isopeptide linkage between the COOH terminus of one Ub and Lys-48 of the other; this structure was confirmed by sequence analysis of the unique Ub2 tryptic fragment. Tryptic digestion of higher order Ubn adducts (n greater than or equal to 4) yielded fragments identical to those of Ub2, indicating that E2(25)K ligates successive Ub molecules primarily or exclusively via Lys-48. Although several other E2s supported synthesis of an apparent Ub2 adduct of undetermined linkage, only E2(25)K was capable of synthesizing multi-Ub chains from isolated Ub. Quantitative analysis of single turnovers showed that transfer from E2(25)K-Ub to Ub and Ub2 occurred with kappa 2 = 488 and 1170 M-1 min-1, respectively, at pH 7.3 and 37 degrees C. These results show that increasing the number of Ub molecules in a chain increases susceptibility to further ubiquitination by E2(25)K. Ub2 was a good substrate for activation by E1 and was readily transferred to E2(25)K. The labile E2(25)K-Ub2 adduct was catalytically active, and exhibited preference for Ub2 (versus Ub) as acceptor. These results suggest that E2(25)K may function as a multi-ubiquitinating enzyme in the Ub-dependent proteolytic pathway.     

Isolation of a cDNA encoding a mammalian multiubiquitinating enzyme (E225K) and overexpression of the functional enzyme in Escherichia coli.

The ubiquitin (Ub)-conjugating enzyme E2(25K) catalyzes the synthesis of multi-Ub chains in which successive Ub units are linked by an isopeptide bond involving the epsilon-amino group of Lys-48 of Ubn, and the COOH-terminal Gly residue of Ubn+1 (Chen, Z., and Pickart, C. M. (1990) J. Biol. Chem., 265, 21835-21842). We now describe the polymerase chain reaction (PCR)-based cloning of an E2(25K)-encoding cDNA from a bovine thymus library, using degenerate oligonucleotide primers based on the sequences of two E2(25K) peptides. The cDNA encodes a 200-residue protein whose sequence bears similarities of 66 and 59%, respectively, to the sequences of the Ub-conjugating enzymes encoded by the UBC1 and UBC4/UBC5 genes of the yeast Saccharomyces cerevisiae. These three yeast E2s play key roles in Ub-dependent proteolysis (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4535-4541). Comparison of the amino acid sequence of E2(25K) with other known E2 sequences strongly suggests that Cys-92, one of two E2(25K) Cys residues, forms the Ub thiol ester adduct that is an intermediate in E2-catalyzed multiubiquitination. The E2(25K)-encoding cDNA was overexpressed in Escherichia coli, and the recombinant E2(25K) protein was purified to electrophoretic homogeneity; enzymatic assays showed that its multiubiquitinating activity was quantitatively identical with that of the native protein. The availability of a cloned cDNA will allow us to assess the physiological role of E2(25K).     

Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome

Polyubiquitin is a diverse signal both in terms of chain length and linkage type. Lysine 48-linked ubiquitin is essential for marking targets for proteasomal degradation, but the significance and relative abundance of different linkages remain ambiguous. Here we dissect the relationship of two proteasome-associated polyubiquitin-binding proteins, Rpn10 and Dsk2, and demonstrate how Rpn10 filters Dsk2 interactions, maintaining proper function of the ubiquitin-proteasome system. Using quantitative mass spectrometry of ubiquitin, we found that in S. cerevisiae under normal growth conditions the majority of conjugated ubiquitin was linked via lysine 48 and lysine 63. In contrast, upon DSK2 induction, conjugates accumulated primarily in the form of lysine 48 linkages correlating with impaired proteolysis and cytotoxicity. By restricting Dsk2 access to the proteasome, extraproteasomal Rpn10 was essential for alleviating the cellular stress associated with Dsk2. This work highlights the importance of polyubiquitin shuttles such as Rpn10 and Dsk2 in controlling the ubiquitin landscape.     

A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.     

Molecular and structural insight into lysine selection on substrate and ubiquitin lysine 48 by the ubiquitin-conjugating enzyme Cdc34

The attachment of ubiquitin (Ub) to lysines on substrates or itself by ubiquitin-conjugating (E2) and ubiquitin ligase (E3) enzymes results in protein ubiquitination. Lysine selection is important for generating diverse substrate-Ub structures and targeting proteins to different fates; however, the mechanisms of lysine selection are not clearly understood. The positioning of lysine(s) toward the E2/E3 active site and residues proximal to lysines are critical in their selection. We investigated determinants of lysine specificity of the ubiquitin-conjugating enzyme Cdc34, toward substrate and Ub lysines. Evaluation of the relative importance of different residues positioned −2, −1, +1 and +2 toward ubiquitination of its substrate, Sic1, on lysine 50 showed that charged residues in the −1 and −2 positions negatively impact on ubiquitination. Modeling suggests that charged residues at these positions alter the native salt-bridge interactions in Ub and Cdc34, resulting in misplacement of Sic1 lysine 50 in the Cdc34 catalytic cleft. During polyubiquitination, Cdc34 showed a strong preference for Ub lysine 48 (K48), with lower activity towards lysine 11 (K11) and lysine 63 (K63). Mutating the −2, −1, +1 and +2 sites surrounding K11 and K63 to mimic those surrounding K48 did not improve their ubiquitination, indicating that further determinants are important for Ub K48 specificity. Modeling the ternary structure of acceptor Ub with the Cdc34~Ub complex as well as in vitro ubiquitination assays unveiled the importance of K6 and Q62 of acceptor Ub for Ub K48 polyubiquitination. These findings provide molecular and structural insight into substrate lysine and Ub K48 specificity by Cdc34.     

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.     

Enhancement of BRCA1 E3 ubiquitin ligase activity through direct interaction with the BARD1 protein

The breast and ovarian cancer-specific tumor suppressor RING finger protein BRCA1 has been identified as an E3 ubiquitin (Ub) ligase through in vitro studies, which demonstrated that its RING finger domain can autoubiquitylate and monoubiquitylate histone H2A when supplied with Ub, E1, and UBC4 (E2). Here we report that the E3 ligase activity of the N-terminal 110 amino acid residues of BRCA1, which encodes a stable domain containing the RING finger, as well as that of the full-length BRCA1, was significantly enhanced by the BARD1 protein (residues 8-142), whose RING finger domain itself lacked Ub ligase activity in vitro. The results of mutagenesis studies indicate that the enhancement of BRCA1 E3 ligase activity by BARD1 depends on direct interaction between the two proteins. Using K48A and K63A Ub mutants, we found that BARD1 stimulated the formation of both Lys(48)- and Lys(63)-linked poly-Ub chains. However, the enhancement of BRCA1 autoubiquitylation by BARD1 mostly resulted in poly-Ub chains linked through Lys(63), which could potentially activate biological pathways other than BRCA1 degradation. We also found that co-expression of BRCA1 and BARD1 in living cells increased the abundance and stability of both proteins and that this depended on their ability to heterodimerize.     

The RING finger protein RNF8 recruits UBC13 for lysine 63-based self polyubiquitylation

The heterodimeric ubiquitin conjugating enzyme (E2) UBC13-UEV mediates polyubiquitylation through lysine 63 of ubiquitin (K63), rather than lysine 48 (K48). This modification does not target proteins for proteasome-dependent degradation. Searching for potential regulators of this variant polyubiquitylation we have identified four proteins, namely RNF8, KIA00675, KF1, and ZNRF2, that interact with UBC13 through their RING finger domains. These domains can recruit, in addition to UBC13, other E2s that mediate canonical (K48) polyubiquitylation. None of these RING finger proteins were known previously to recruit UBC13. For one of these proteins, RNF8, we show its activity as a ubiquitin ligase that elongates chains through either K48 or K63 of ubiquitin, and its nuclear co-localization with UBC13. Thus, our screening reveals new potential regulators of non-canonical polyubiquitylation.     

Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.     

In vitro assembly and recognition of Lys-63 polyubiquitin chains

Polyubiquitin chains assembled through lysine 48 (Lys-48) of ubiquitin act as a signal for substrate proteolysis by 26 S proteasomes, whereas chains assembled through Lys-63 play a mechanistically undefined role in post-replicative DNA repair. We showed previously that the products of the UBC13 and MMS2 genes function in error-free post-replicative DNA repair in the yeast Saccharomyces cerevisiae and form a complex that assembles Lys-63-linked polyubiquitin chains in vitro. Here we confirm that the Mms2.Ubc13 complex functions as a high affinity heterodimer in the chain assembly reaction in vitro and report the results of a kinetic characterization of the polyubiquitin chain assembly reaction. To test whether a Lys-63-linked polyubiquitin chain can signal degradation, we conjugated Lys-63-linked tetra-ubiquitin to a model substrate of 26 S proteasomes. Although the noncanonical chain effectively signaled substrate degradation, the results of new genetic epistasis studies agree with previous genetic data in suggesting that the proteolytic activity of proteasomes is not required for error-free post-replicative repair.     

Solution conformation of Lys63-linked di-ubiquitin chain provides clues to functional diversity of polyubiquitin signaling

Diverse cellular events are regulated by post-translational modification of substrate proteins via covalent attachment of one or a chain of ubiquitin molecules. The outcome of (poly)ubiquitination depends upon the specific lysine residues involved in the formation of polyubiquitin chains. Lys48-linked chains act as a universal signal for proteasomal degradation, whereas Lys63-linked chains act as a specific signal in several non-degradative processes. Although it has been anticipated that functional diversity between alternatively linked polyubiquitin chains relies on linkage-dependent differences in chain conformation/topology, direct structural evidence in support of this model has been lacking. Here we use NMR methods to determine the structure of a Lys63-linked di-ubiquitin chain. The structure is characterized by an extended conformation, with no direct contact between the hydrophobic residues Leu8, Ile44, and Val70 on the ubiquitin units. This structure contrasts with the closed conformation observed for Lys48-linked di-ubiquitin wherein these residues form the interdomain interface (Cook, W. J., Jeffrey, L. C., Carson, M., Zhijian, C., and Pickart, C. M. (1992) J. Biol. Chem. 267, 16467-16471; Varadan, R., Walker, O., Pickart, C., and Fushman, D. (2002) J. Mol. Biol. 324, 637-647). Consistent with the open conformation of the Lys(63)-linked di-ubiquitin, our binding studies show that both ubiquitin domains in this chain can bind a ubiquitin-associated domain from HHR23A independently and in a mode similar to that for mono-ubiquitin. In contrast, Lys48-linked di-ubiquitin binds in a different, higher affinity mode that has yet to be determined. This is the first experimental evidence that alternatively linked polyubiquitin chains adopt distinct conformations.