Reading the ubiquitin postal code

Polyubiquitin chains are assembled through the formation of an isopeptide bond between a lysine side-chain or terminal amino group of a proximal ubiquitin moiety and the carboxy-terminal of a distal ubiquitin moiety. Protein substrates tagged by polyubiquitin chains of different linkages undergo different fates. Many polyubiquitin chain types have been characterized so far, notably Lys11, Lys48, Lys63 and linear chains. These different types of chains are synthesized, disassembled and recognized by selective enzymes and receptors. Here I survey the structural basis for the selective binding of polyubiquitin chains of specific linkages, with an emphasis on recent advances in our understanding of polyubiquitin chain structure and functions. Recent work suggests linkage-type discrimination by members of the NF-κb signalling and DNA repair pathways and a specific role for Lys48-linked polyubiquitin chain recognition by proteasome-associated proteins.     

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.     

线性泛素化修饰研究进展

蛋白质泛素化修饰过程在调节各种细胞生物学功能的过程中发挥了非常重要的作用,如细胞周期进程、DNA损伤修复、信号转导和各种蛋白质膜定位等。泛素化修饰可分为多聚泛素化修饰和单泛素化修饰。多聚泛素化修饰系统可以通过对底物连接不同类型的多泛素化链调节蛋白质的功能。多聚泛素化修饰中已知7种泛素链连接方式均为泛素内赖氨酸连接方式。近几年发现了第8种类型的泛素链连接形式即线性泛素化,其泛素链的连接方式是由泛素甲硫氨酸的氨基基团与另一泛素甘氨酸的羧基基团相连形成泛素链标记。目前研究表明线性泛素化修饰在先天性免疫和炎症反应等多个过程中发挥着非常重要的作用。募集线性泛素链的泛素连接酶E3被称为LUBAC复合体,其组成底物以及其活性调控机制和功能所知甚少。本文综述了募集线性泛素化链的泛素连接酶、去泛素化酶、底物等活性调控机制及其在先天性免疫等多个领域中的功能,分析了后续研究方向,以期为相关研究提供参考。     

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.     

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.     

The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains

Ubiquitin chain complexity in cells is likely regulated by a diverse set of deubiquitinating enzymes (DUBs) with distinct ubiquitin chain preferences. Here we show that the polyglutamine disease protein, ataxin-3, binds and cleaves ubiquitin chains in a manner suggesting that it functions as a mixed linkage, chain-editing enzyme. Ataxin-3 cleaves ubiquitin chains through its amino-terminal Josephin domain and binds ubiquitin chains through a carboxyl-terminal cluster of ubiquitin interaction motifs neighboring the pathogenic polyglutamine tract. Ataxin-3 binds both Lys(48)- or Lys(63)-linked chains yet preferentially cleaves Lys(63) linkages. Ataxin-3 shows even greater activity toward mixed linkage polyubiquitin, cleaving Lys(63) linkages in chains that contain both Lys(48) and Lys(63) linkages. The ubiquitin interaction motifs regulate the specificity of this activity by restricting what can be cleaved by the protease domain, demonstrating that linkage specificity can be determined by elements outside the catalytic domain of a DUB. These findings establish ataxin-3 as a novel DUB that edits topologically complex chains.     

Lysine 63‐linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome

Recruitment of substrates to the 26S proteasome usually requires covalent attachment of the Lys48‐linked polyubiquitin chain. In contrast, modifications with the Lys63‐linked polyubiquitin chain and/or monomeric ubiquitin are generally thought to function in proteasome‐independent cellular processes. Nevertheless, the ubiquitin chain‐type specificity for the proteasomal targeting is still poorly understood, especially in vivo. Using mass spectrometry, we found that Rsp5, a ubiquitin‐ligase in budding yeast, catalyzes the formation of Lys63‐linked ubiquitin chains in vitro. Interestingly, the 26S proteasome degraded well the Lys63‐linked ubiquitinated substrate in vitro. To examine whether Lys63‐linked ubiquitination serves in degradation in vivo, we investigated the ubiquitination of Mga2‐p120, a substrate of Rsp5. The polyubiquitinated p120 contained relatively high levels of Lys63‐linkages, and the Lys63‐linked chains were sufficient for the proteasome‐binding and subsequent p120‐processing. In addition, Lys63‐linked chains as well as Lys48‐linked chains were detected in the 26S proteasome‐bound polyubiquitinated proteins. These results raise the possibility that Lys63‐linked ubiquitin chain also serves as a targeting signal for the 26S proteaseome in vivo.     

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.     

The role of atypical ubiquitination in cell regulation

Ubiquitination is a type of intracellular proteins post-translational modification (PTM) characterized by covalent attachment of ubiquitin molecules to target proteins. This includes monoubiquitination (attachment of one ubiquitin molecule), multiple monoubiquitination also known as multiubiquitination (attachment of several monomeric ubiquitin molecules to a target protein), and polyubiquitination (attachment of ubiquitin chains consisting of several, most frequently four ubiquitin monomers to a target protein). In the case of polyubiquitination, linear or branched polyubiquitin chains are formed. Their formation involves various lysine residues of monomeric ubiquitin. The best studied is Lys48-linked polyubiquitination, which targets proteins for proteasomal degradation. In this review we have considered examples of so-called atypical polyubiquitination, which mainly involves other lysine residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys63) and also N-terminal methionine. The considered examples convincingly demonstrate that polyubiquitination of proteins (not necessarily) targets proteins for their proteolytic degradation in proteasomes. Atypically polyubiquitinated proteins are involved in regulation of various processes including immune response, genome stability, signal transduction, etc. Alterations of ubiquitination machinery is crucial for development of serious diseases.     

Affinity makes the difference: nonselective interaction of the UBA domain of Ubiquilin-1 with monomeric ubiquitin and polyubiquitin chains

Ubiquilin/PLIC proteins belong to the family of UBL-UBA proteins implicated in the regulation of the ubiquitin-dependent proteasomal degradation of cellular proteins. A human presenilin-interacting protein, ubiquilin-1, has been suggested as potential therapeutic target for treating Huntington's disease. Ubiquilin's interactions with mono- and polyubiquitins are mediated by its UBA domain, which is one of the tightest ubiquitin binders among known ubiquitin-binding domains. Here we report the three-dimensional structure of the UBA domain of ubiquilin-1 (UQ1-UBA) free in solution and in complex with ubiquitin. UQ1-UBA forms a compact three-helix bundle structurally similar to other known UBAs, and binds to the hydrophobic patch on ubiquitin with a K_d of 20 μM. To gain structural insights into UQ1-UBA's interactions with polyubiquitin chains, we have mapped the binding interface between UQ1-UBA and Lys48- and Lys63-linked di-ubiquitins and characterized the strength of UQ1-UBA binding to these chains. Our NMR data show that UQ1-UBA interacts with the individual ubiquitin units in both chains in a mode similar to its interaction with mono-ubiquitin, although with an improved binding affinity for the chains. Our results indicate that, in contrast to UBA2 of hHR23A that has strong binding preference for Lys48-linked chains, UQ1-UBA shows little or no binding selectivity toward a particular chain linkage or between the two ubiquitin moieties in the same chain. The structural data obtained in this study provide insights into the possible structural reasons for the diversity of polyubiquitin chain recognition by UBA domains.     

Atypical ubiquitylation - the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages

Ubiquitylation is one of the most abundant and versatile post-translational modifications (PTMs) in cells. Its versatility arises from the ability of ubiquitin to form eight structurally and functionally distinct polymers, in which ubiquitin moieties are linked via one of seven Lys residues or the amino terminus. Whereas the roles of Lys48- and Lys63-linked polyubiquitin in protein degradation and cellular signalling are well characterized, the functions of the remaining six 'atypical' ubiquitin chain types (linked via Lys6, Lys11, Lys27, Lys29, Lys33 and Met1) are less well defined. Recent developments provide insights into the mechanisms of ubiquitin chain assembly, recognition and hydrolysis and allow detailed analysis of the functions of atypical ubiquitin chains. The importance of Lys11 linkages and Met1 linkages in cell cycle regulation and nuclear factor-κB activation, respectively, highlight that the different ubiquitin chain types should be considered as functionally independent PTMs.     

Preparation of distinct ubiquitin chain reagents of high purity and yield

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.     

Emerging roles for Lys11-linked polyubiquitin in cellular regulation

Polyubiquitin chains are assembled via one of seven lysine (Lys) residues or the N terminus. The cellular roles of Lys48- and Lys63-linked polyubiquitin have been extensively studied; however, the cellular functions of Lys11-linked chains are less well understood. Recent insights into Lys11-linked ubiquitin chains have revealed their important function in cell cycle control. Additionally, Lys11 linkages have been identified in the context of mixed chains in many other cellular pathways. In this review, we introduce the specific enzymes that mediate Lys11-linked chain assembly and disassembly, and discuss the diverse cellular processes in which Lys11 linkages participate. Notably, mechanistic insights have revealed how the E2 ubiquitin-conjugating enzyme UBE2S achieves its Lys11 linkage specificity, and two structures of Lys11-linked polyubiquitin highlight the dynamic nature of this compact chain type.     

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.     

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

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

Mass spectrometry insights into a tandem ubiquitin‐binding domain hybrid engineered for the selective recognition of unanchored polyubiquitin

Unanchored polyubiquitin chains are emerging as important regulators of cellular physiology with diverse roles paralleling those of substrate‐conjugated polyubiquitin. However tools able to discriminate unanchored polyubiquitin chains of different isopeptide linkages have not been reported. We describe the design of a linker‐optimized ubiquitin‐binding domain hybrid (t‐UBD) containing two UBDs, a ZnF‐UBP domain in tandem with a linkage‐selective UBA domain, which exploits avidity effects to afford selective recognition of unanchored Lys48‐linked polyubiquitin chains. Utilizing native MS to quantitatively probe binding affinities we confirm cooperative binding of the UBDs within the synthetic protein, and desired binding specificity for Lys48‐linked ubiquitin dimers. Furthermore, MS/MS analyses indicate that the t‐UBD, when applied as an affinity enrichment reagent, can be used to favor the purification of endogenous unanchored Lys48‐linked polyubiquitin chains from mammalian cell extracts. Our study indicates that strategies for the rational design and engineering of polyubiquitin chain‐selective binding in nonbiological polymers are possible, paving the way for the generation of reagents to probe unanchored polyubiquitin chains of different linkages and more broadly the ‘ubiquitome’. All MS data have been deposited in the ProteomeXchange with identifier PXD004059 ( http://proteomecentral.proteomexchange.org/dataset/PXD004059 ).     

Binding of polyubiquitin chains to ubiquitin-associated (UBA) domains of HHR23A

Ubiquitin-associated (UBA) domains are small protein domains that occur in the context of larger proteins and are likely to function as inter- and intramolecular communication elements in ubiquitin/polyubiquitin signaling. Although monoubiquitin/UBA complexes are well characterized, much less is known about UBA/polyubiquitin complexes, even though polyubiquitin chains are believed to be biologically relevant ligands of many UBA domain proteins. Here, we report the results of a quantitative study of the interaction of K48-linked polyubiquitin chains with UBA domains of the DNA repair/proteolysis protein HHR23A, using surface plasmon resonance and other approaches. We present evidence that the UBL domain of HHR23A negatively regulates polyubiquitin/UBA interactions and identify leucine 8 of ubiquitin as an important determinant of chain recognition. A striking relationship between binding affinity and chain length suggests that maximum affinity is associated with a conformational feature that is fully formed in chains of n = 4-6 and can be recognized by a single UBA domain of HHR23A. Our findings provide new insights into polyubiquitin chain recognition and set the stage for future structural investigations of UBA/polyubiquitin complexes.     

Solution structure of a CUE-ubiquitin complex reveals a conserved mode of ubiquitin binding

Monoubiquitination serves as a regulatory signal in a variety of cellular processes. Monoubiquitin signals are transmitted by binding to a small but rapidly expanding class of ubiquitin binding motifs. Several of these motifs, including the CUE domain, also promote intramolecular monoubiquitination. The solution structure of a CUE domain of the yeast Cue2 protein in complex with ubiquitin reveals intermolecular interactions involving conserved hydrophobic surfaces, including the Leu8-Ile44-Val70 patch on ubiquitin. The contact surface extends beyond this patch and encompasses Lys48, a site of polyubiquitin chain formation. This suggests an occlusion mechanism for inhibiting polyubiquitin chain formation during monoubiquitin signaling. The CUE domain shares a similar overall architecture with the UBA domain, which also contains a conserved hydrophobic patch. Comparative modeling suggests that the UBA domain interacts analogously with ubiquitin. The structure of the CUE-ubiquitin complex may thus serve as a paradigm for ubiquitin recognition and signaling by ubiquitin binding proteins.     

Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies

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

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.