Formation Of Polyubiquitin Chains Research Articles

Nuclear localization of Chfr is crucial for its checkpoint function.

Chfr, a checkpoint with FHA and RING finger domains, plays an important role in cell cycle progression and tumor suppression. Chfr possesses the E3 ubiquitin ligase activity and stimulates the formation of polyubiquitin chains by Ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins, including Plk1 and Aurora A. While Chfr is a nuclear protein that functions within the cell nucleus, how Chfr is localized in the nucleus has not been clearly demonstrated. Here, we show that nuclear localization of Chfr is mediated by nuclear localization signal (NLS) sequences. To reveal the signal sequences responsible for nuclear localization, a short lysine-rich stretch (KKK) at amino acid residues 257-259 was replaced with alanine, which completely abolished nuclear localization. Moreover, we show that nuclear localization of Chfr is essential for its checkpoint function but not for its stability. Thus, our results suggest that NLS-mediated nuclear localization of Chfr leads to its accumulation within the nucleus, which may be important in the regulation of Chfr activation and Chfr-mediated cellular processes, including cell cycle progression and tumor suppression.

Cloning and Characterization of DULP, a Novel Ubiquitin-Like Molecule from Human Dendritic Cells

We identified a novel ubiquitin-like molecule DULP from human dendritic cells. DULP contains a domain that shares 26% identity and 34% similarity with ubiquitin, and it possesses the corresponding Ile-44 hydrophobic patch used by mono- or poly-ubiquitin to interact with a ubiquitin-interaction motif (UIM) or ubiquitin-associated domain (UBA). Lysine residue corresponding to 6 of ubiquitin, which is involved in the formation of a multi-ubiquitin chain that can bind proteasomal subunit Rpn10/S5a, is also conserved in its ubiquitin-homology domain. However, DULP does not possess the highly conserved C-terminus Gly-Gly required for ubiquitin conjugation or the Lys-48 required for the formation of polyubiquitin chain to target substrates for degradation, suggesting it might be a novel ubiquitin-domain protein (UDP). DULP was found widely expressed in many cells and the ubiquitin-homology domain was not cleaved. We also confirmed that DULP expression was enriched in the nucleus and much weaker in the cytosol. Besides, we found that overexpression of DULP in 293T cells induced apoptosis, which might not be associated with the mitochondrial or proteasome pathway, with the specific mechanism remaining unclear. Further investigations are needed to identify the precise biological functions of DULP.

Crystallographic structure of ubiquitin in complex with cadmium ions

BackgroundUbiquitination plays a critical role in regulating many cellular processes, from DNA repair and gene transcription to cell cycle and apoptosis. It is catalyzed by a specific enzymatic cascade ultimately leading to the conjugation of ubiquitin to lysine residues of the target protein that can be the ubiquitin molecule itself and to the formation of poly-ubiquitin chains.FindingsWe present the crystal structure at 3.0 Å resolution of bovine ubiquitin crystallized in presence of cadmium ions. Two molecules of ubiquitin are present in the asymmetric unit. Interestingly this non-covalent dimeric arrangement brings Lys-6 and Lys-63 of each crystallographically-independent monomer in close contact with the C-terminal ends of the other monomer. Residues Leu-8, Ile-44 and Val-70 that form a hydrophobic patch at the surface of the Ub monomer are trapped at the dimer interface.ConclusionsThe structural basis for signalling by poly-Ub chains relies on a visualization of conformations of alternatively linked poly-Ub chains. This arrangement of ubiquitin could illustrate how linkages involving Lys-6 or Lys-63 of ubiquitin are produced in the cell. It also details how ubiquitin molecules can specifically chelate cadmium ions.

Mechanistic Insight into Site-Restricted Monoubiquitination of FANCD2 by Ube2t, FANCL, and FANCI

A key step in the Fanconi anemia (FA) tumor suppressor pathway is the site-specific monoubiquitination of the FANCD2 protein. Genetic studies indicate that this crucial modification requires eight known FA gene products and the E2-conjugating enzyme Ube2t. Here, we minimally reconstitute this monoubiquitination reaction with Ube2t and the FANCL protein, revealing that monoubiquitination is stimulated by a conserved RWD-like domain in FANCL. Furthermore, addition of the FANCI protein enhances monoubiquitination and also restricts it to the in vivo substrate lysine residue on FANCD2. This work therefore establishes a system that provides mechanistic insight into the functions of FANCL and FANCI in the catalysis of FANCD2 monoubiquitination.

Regulation of the stability and transcriptional activity of NFATc4 by ubiquitination

Nuclear factor of activated T cells (NFATc4) has been implicated as a critical regulator of the cardiac development and hypertrophy. However, the mechanisms for regulating NFATc4 stability and transactivation remain unclear. We showed that NFATc4 protein was predominantly ubiquitinated through the formation of Lysine 48-linked polyubiquitin chains, and this modification decreased NFATc4 protein levels and its transcriptional activity. Furthermore, activation of GSK3β markedly enhanced NFATc4 ubiquitination and decreased its transactivation, whereas inhibition of GSK3β had opposite effects. Importantly, ubiquitination and phosphorylation induced by GSK3β repressed NFATc4-dependent cardiac-specific gene expression. These results demonstrate that the ubiquitin–proteasome system plays an important role in regulating NFATc4 stability and transactivation. Structured summary MINT- 6798349: NFATc4 (uniprotkb: Q14934) physically interacts (MI: 0218) with Ubiquitin (uniprotkb: P62988) by anti bait coimmunoprecipitation (MI: 0006) MINT- 6798334: NFATc4 (uniprotkb: Q14934) physically interacts (MI: 0218) with Ubiquitin (uniprotkb: P62988) by anti tag coimmunoprecipitation (MI: 0007) MINT- 6798321: Ubiquitin (uniprotkb: P62988) physically interacts (MI: 0218) with NFATc4 (uniprotkb: Q14934) by pull down (MI: 0096)

Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

Pellino 3b Negatively Regulates Interleukin-1-induced TAK1-dependent NFκB Activation

HMMS2 serves a redundant role in human PCNA polyubiquitination

BackgroundIn yeast, DNA damage leads to the mono and polyubiquitination of the sliding clamp PCNA. Monoubiquitination of PCNA is controlled by RAD18 (E3 ligase) and RAD6 (E2 conjugating enzyme), while the extension of the monoubiquitinated PCNA into a polyubiquitinated substrate is governed by RAD5, and the heterodimer of UBC13/MMS2. Each modification directs a different branch of the DNA damage tolerance pathway (DDT). While PCNA monoubiquitination leads to error-prone bypass via TLS, biochemical studies have identified MMS2 along with its heteromeric partner UBC13 to govern the error-free repair of DNA lesions by catalyzing the formation of lysine 63-linked polyubiquitin chains (K63-polyUb). Recently, it was shown that PCNA polyubiquitination is conserved in human cells and that this modification is dependent on RAD18, UBC13 and SHPRH. However, the role of hMMS2 in this process was not specifically addressed.ResultsIn this report we show that mammalian cells in which MMS2 was reduced by siRNA-mediated knockdown maintains PCNA polyubiquitination while a knockdown of RAD18 or UBC13 abrogates PCNA ubiquitination. Moreover, the additional knockdown of a UEV1A (MMS2 homolog) does not deplete PCNA polyubiquitination. Finally, mouse embryonic stem cells null for MMS2 with or without the additional depletion of mUEV1A continue to polyubiquitinated PCNA with normal kinetics.ConclusionOur results point to a high level of redundancy in the DDT pathway and suggest the existence of another hMMS2 variant (hMMSv) or complex that can compensate for its loss.

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.

Pirh2 interacts with and ubiquitylates signal recognition particle receptor β subunit

Pirh2 is a RING finger type ubiquitin ligase which ubiquitylates various proteins including p53, p27(Kip1), HDAC1, and epsilon-COP. In this study, we identified signal recognition particle receptor beta subunit (SRbeta), an integral membrane protein of the endoplasmic reticulum (ER), as a novel Pirh2-interacting protein by yeast two-hybrid screening. We confirmed that Pirh2 interacted with SRbeta in mammalian cells. An immunofluorescent staining revealed that Pirh2 colocalized with SRbeta in the ER. Pirh2 poly-ubiquitylated SRbeta in an intact RING finger domain-dependent manner in vivo and in vitro. Unexpectedly, different from other Pirh2 substrates, neither overexpression of Pirh2 nor depletion of cellular Pirh2 affected SRbeta protein stability. Pirh2 preferentially utilized lysine residues 6 and 29 of the ubiquitin to mediate the formation of polyubiquitin chains on SRbeta. These results suggest that Pirh2 may regulate SRbeta function by mediating poly-ubiquitylation of SRbeta without affecting the stability of SRbeta protein per se.

Mechanism of extracellular ubiquitination in the mammalian epididymis

Posttranslational modification by ubiquitination marks defective or outlived intracellular proteins for proteolytic degradation by the 26S proteasome. The ATP-dependent, covalent ligation and formation of polyubiquitin chains on substrate proteins requires the presence and activity of a set of ubiquitin activating and conjugating enzymes. While protein ubiquitination typically occurs in the cell cytosol or nucleus, defective mammalian spermatozoa become ubiquitinated on their surface during post-testicular sperm maturation in the epididymis, suggesting an active molecular mechanism for sperm quality control. Consequently, we hypothesized that the bioactive constituents of ubiquitin-proteasome pathway were secreted in the mammalian epididymal fluid (EF) and capable of ubiquitinating extrinsic substrates. Western blotting indeed detected the presence of the ubiquitin-activating enzyme E1 and presumed E1-ubiquitin thiol-ester intermediates, ubiquitin-carrier enzyme E2 and presumed E2-ubiquitin thiol-ester intermediates and the ubiquitin C-terminal hydrolase PGP 9.5/UCHL1 in the isolated bovine EF. Thiol-ester assays utilizing recombinant ubiquitin-activating and ubiquitin-conjugating enzymes, biotinylated substrates, and isolated bovine EF confirmed the activity of the ubiquitin activating and conjugating enzymes within EF. Ubiquitinated proteins were found to be enriched in the defective bull sperm fraction and appropriate proteasomal deubiquitinating and proteolytic activities were measured in the isolated EF by specific fluorescent substrates. The apocrine secretion of cytosolic proteins was visualized in transgenic mice and rats expressing the enhanced green fluorescent protein (eGFP) under the direction of ubiquitin-C promoter. Accumulation of eGFP, ubiquitin and proteasomes was detected in the apical blebs, the apocrine secretion sites of the caput epididymal epithelia of both the rat and mouse epididymal epithelium, although region-specific differences exist. Secretion of eGFP and proteasomes continued during the prolonged culture of the isolated rat epididymal epithelial cells in vitro. This study provides evidence that the activity of the ubiquitin system is not limited to the intracellular environment, contributing to a greater understanding of the sperm maturation process during epididymal passage.

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.

Polyubiquitylation of PARP‐1 through ubiquitin K48 is modulated by activated DNA, NAD+, and dipeptides

Poly(ADP-ribose) polymerase-1 (PARP-1) is the most abundant and the best-studied isoform of a family of enzymes that catalyze the polymerization of ADP-ribose from NAD(+) onto target proteins. PARP-1 is well known to involve in DNA repair, genomic stability maintenance, transcription regulation, apoptosis, and necrosis. Polyubiquitylation targets proteins towards degradation and regulates cell cycle progression, transcription, and apoptosis. Here we report polyubiquitylation of PARP-1 in mouse fibroblasts in the presence of proteasome inhibitor and in full-length recombinant PARP-1 in vitro under standard ubiquitylation assay conditions by immunoprecipitation and immunoblotting. Mutation of ubiquitin K48R but not ubiquitin K63R abolishes polyubiquitylation of PARP-1, indicating that K48 of ubiquitin was used in the formation of polyubiquitin chain and that ubiquitylated PARP-1 is likely destined for degradation. Full-length PARP-1 was ubiquitylated most likely at the N-terminal 24 kDa domain of PARP-1 as suggested by the inhibition of ubiquitylation by activated DNA and the absence of polyubiquitin in the C-terminal 89 kDa PARP-1 derived from caspase-catalyzed cleavage. NAD(+) inhibited ubiquitylation of PARP-1, while dipeptides ArgAla and LeuAla enhanced ubiquitylation of PARP-1. ATP inhibited the synthesis of poly(ADP-ribose) by PARP-1 and affinity purified polyubiquitylated PARP-1 was active in PAR synthesis. The results suggest polyubiquitylation of PARP-1 could regulate poly(ADP-ribosyl)ation of nuclear proteins by PARP-1 and consequently apoptosis and PARP-1 regulated cellular processes through ubiquitin-dependent degradation pathways.

Triad1 Regulates Myelopoiesis through Different Ubiquitin Ligase Activities.

The modification of cellular proteins with poly-ubiquitin chains plays an essential role in hematopoiesis. Different types of ubiquitin chains may have opposite effects on the marked proteins. Chains linked through lysine 48 of ubiquitin are recognized by the proteasome resulting in progressive degradation of the ubiquitylated proteins. Ubiquitin chains linked through lysine 63 are not recognized by the proteasome. Instead, these chains can bind proteins that regulate signal transduction and gene transcription. Although it is known that ubiquitylation is essential for hematopoiesis and that alterations in ubiquitylation have been implicated in malignant hematopoiesis the ubiquitin ligases that catalyze protein ubiquitylation remain largely unknown. Triad1 is an ubiquitin ligase that inhibits the proliferation of myeloid progenitor cells through its ligase activity. Triad1 belongs to a unique class of ligases that harbor two RING finger protein domains. This domain specifically binds ubiquitin conjugating enzymes (Ubcs). Together with the Ubcs the ligases determine which type of ubiquitin chain is catalyzed. To understand how Triad1 regulates myelopoiesis we screened a panel of Ubcs for Triad1 interaction and found that Triad1 binds UbcH7 through its N-terminal RING domain and Ubc13 through its C-terminal RING domain. UbcH7 catalyzes the formation of ubiquitin chains linked through lysine 48 that are recognized by the proteasome. Importantly, ubiquitin chains catalyzed by Ubc13 are linked through lysine 63 and are not recognized by the proteasome. In agreement with these interactions, in vitro ubiquitylation assays using different ubiquitin mutants containing only one lysine residue showed that Triad1 can catalyze the formation of both types of ubiquitin chains. The relevance of these findings in myelopoiesis was studied by generating a panel of Triad1 deletion mutants that lack the coiled coil, DRIL or RING domains. Next, the growth inhibitory effect of these mutants was tested in clonogenic assays by retroviral transduction of U937 cells. Like in primary cells, wild type Triad1 inhibited U937 colony formation by over 60% compared to empty vector transduced cells. Three Triad1 mutants lacking the DRIL domain or either one or two of the coiled coil domains inhibited clonogenic growth at a comparable rate as wild type Triad1. In contrast, deletion of either the N- or C-terminal RING finger completely abrogated the inhibitory effect of Triad1 in clonogenic growth. Thus, loss of either the UbcH7 or Ubc13 binding domain of Triad1 affects its inhibitory function in myeloid cell proliferation. Recently, we reported that Triad1 binds the transcription factor Gfi1. Gfi1 plays an important role during many hematopoietic developmental stages and is essential for neutrophilic differentiation. Remarkably, Triad1 inhibited Gfi1 ubiquitylation and proteasomal degradation. The finding here that Triad1 can catalyze the formation of different ubiquitin chains might suggest that Triad1 may modify Gfi1 with ubiquitin in a functional way, rather than marking it for proteasomal degradation. Indeed, in vivo ubiquitylation experiments showed that Gfi1 can be modified with ubiquitin chains not linked through lysine 48. The relevance of this finding and the role of Triad1 in this process is currently studied. Together, these data indicate that the dual ubiquitin ligase activity that results in the formation of different poly-ubiquitin chains is crucial to the central role of Triad1 in myelopoiesis.

Coordinated Regulation of Toll-Like Receptor and NOD2 Signaling by K63-Linked Polyubiquitin Chains

K63 polyubiquitin chains spatially and temporally link innate immune signaling effectors such that cytokine release can be coordinated. Crohn's disease is a prototypical inflammatory disorder in which this process may be faulty as the major Crohn's disease-associated protein, NOD2 (nucleotide oligomerization domain 2), regulates the formation of K63-linked polyubiquitin chains on the I kappa kinase (IKK) scaffolding protein, NEMO (NF-kappaB essential modifier). In this work, we study these K63-linked ubiquitin networks to begin to understand the biochemical basis for the signaling cross talk between extracellular pathogen Toll-like receptors (TLRs) and intracellular pathogen NOD receptors. This work shows that TLR signaling requires the same ubiquitination event on NEMO to properly signal through NF-kappaB. This ubiquitination is partially accomplished through the E3 ubiquitin ligase TRAF6. TRAF6 is activated by NOD2, and this activation is lost with a major Crohn's disease-associated NOD2 allele, L1007insC. We further show that TRAF6 and NOD2/RIP2 share the same biochemical machinery (transforming growth factor beta-activated kinase 1 [TAK1]/TAB/Ubc13) to activate NF-kappaB, allowing TLR signaling and NOD2 signaling to synergistically augment cytokine release. These findings suggest a biochemical mechanism for the faulty cytokine balance seen in Crohn's disease.

Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity

Chfr, a mitotic stress checkpoint, plays an important role in cell cycle progression, tumor suppression and the processes that require the E3 ubiquitin ligase activity mediated by the RING finger domain. Chfr stimulates the formation of polyubiquitin chains by ub-conjugating enzymes, and induces the proteasome-dependent degradation of a number of cellular proteins including Plk1 and Aurora A. In this study, we identified USP7 (also known as HAUSP), which is a member of a family of proteins that cleave polyubiquitin chains and/or ubiquitin precursors, as an interacting protein with Chfr by immunoaffinity purification and mass spectrometry, and their interaction greatly increases the stability of Chfr. In fact, USP7 can remove ubiquitin moiety from the autoubiquitinated Chfr both in vivo and in vitro, which results in the accumulation of Chfr in the cell. Thus, our finding suggests that USP7-mediated deubiquitination of Chfr leads to its accumulation, which might be a key regulatory step for Chfr activation and that USP7 may play an important role in the regulation of Chfr-mediated cellular processes including cell cycle progression and tumor suppression.

Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[ a]pyrene-diol-epoxide-induced mutagenicity

Benzo[ a]pyrene exerts its mutagenic effects via induction of benzo[ a]pyrene-diol-epoxide (BPDE)–DNA adducts. Such helix-distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway. Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. In the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLη), causing increased BPDE-induced mutagenicity. To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin ( K63R- Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin ( WT- Ub). We show that PCNA is poly-ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity. In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R- Ub expressing cells, of which the majority (74%) was G → T transversion. BPDE treatment caused an enhanced recruitment of POLη to the replication machinery of the K63R- Ub expressing cells, where it co-localized with PCNA. Suppression of POLη expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis-mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability.

Regulation of the Drosophila ubiquitin ligase DIAP1 is mediated via several distinct ubiquitin system pathways

Inhibitors of apoptosis proteins (IAPs) suppress cell death by inactivating proapoptotic regulators, and therefore play important roles in controlling apoptosis in normal and malignant cells. Many IAPs are ubiquitin ligases, and their activity is mediated via ubiquitination and subsequent degradation of their targets. Here we corroborate a previous observation that DIAP1 (Drosophila IAP1) can be degraded via a two-step mechanism: (i) limited caspase-mediated cleavage and (ii) degradation of the released fragment via the ubiquitin N-end rule pathway. Yet, we demonstrate that this pathway is not the only one involved in DIAP1 degradation, and the intact protein can be degraded independent of prior caspase cleavage. Importantly, this mode of degradation does not require the RING-finger-mediated autoubiquitinating activity of DIAP1, believed to target many RING-finger E3s for self-destruction. Our preliminary data suggest that DIAP2 mediates DIAP1 degradation, suggesting a novel regulatory loop within the apoptotic pathway. Studying the role of the autoubiquitinating activity of DIAP1, we demonstrate that it does not involve formation of Lys48-based polyubiquitin chains, but probably chains linked via Lys63. Our preliminary data suggest that the autoubiquitination serves to attenuate the ligase activity of DIAP1 towards its exogenous substrates.

Mms2–Ubc13 covalently bound to ubiquitin reveals the structural basis of linkage-specific polyubiquitin chain formation

Lys63-linked polyubiquitin chains participate in nonproteolytic signaling pathways, including regulation of DNA damage tolerance and NF-kappaB activation. E2 enzymes bound to ubiquitin E2 variants (UEV) are vital in these pathways, synthesizing Lys63-linked polyubiquitin chains, but how these complexes achieve specificity for a particular lysine linkage has been unclear. We have determined the crystal structure of an Mms2-Ubc13-ubiquitin (UEV-E2-Ub) covalent intermediate with donor ubiquitin linked to the active site residue of Ubc13. In the structure, the unexpected binding of a donor ubiquitin of one Mms2-Ubc13-Ub complex to the acceptor-binding site of Mms2-Ubc13 in an adjacent complex allows us to visualize at atomic resolution the molecular determinants of acceptor-ubiquitin binding. The structure reveals the key role of Mms2 in allowing selective insertion of Lys63 into the Ubc13 active site and suggests a molecular model for polyubiquitin chain elongation.

Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin–protein ligase with monoubiquitylation capacity

Mutations in the parkin gene, encoding an E3 ubiquitin-protein ligase, are a frequent cause of autosomal recessive parkinsonism and are also involved in sporadic Parkinson's disease. Loss of Parkin function is thought to compromise the polyubiquitylation and proteasomal degradation of specific substrates, leading to their deleterious accumulation. Several studies have analyzed the effects of parkin gene mutations on the biochemical properties of the protein. However, the absence of a cell-free system for studying intrinsic Parkin activity has limited the interpretation of these studies. Here we describe the biochemical characterization of Parkin and 10 pathogenic variants carrying amino-acid substitutions throughout the sequence. Mutations in the RING fingers or the ubiquitin-like domain decreased the solubility of the protein in detergent and increased its tendency to form visible aggregates. None of the mutations studied compromised the binding of Parkin to a series of known protein partners/substrates. Moreover, only two variants with substitutions of conserved cysteine residues of the second RING finger were inactive in a purely in vitro ubiquitylation assay, demonstrating that loss of ligase activity is a minor pathogenic mechanism. Interestingly, in this in vitro assay, Parkin catalyzed the linkage of single ubiquitin molecules only, whereas the ubiquitin-protein ligases CHIP and Mdm2 promoted the formation of polyubiquitin chains. Similarly, in mammalian cells Parkin promoted the multimonoubiquitylation of its substrate p38, rather than its polyubiquitylation. Thus, Parkin may mediate polyubiquitylation or proteasome-independent monoubiquitylation depending on the protein context. The discovery of monoubiquitylated Parkin species in cells hints at a novel post-translational modification potentially involved in the regulation of Parkin function.

Arabidopsis thaliana UBC13: Implication of Error-free DNA Damage Tolerance and Lys63-linked Polyubiquitylation in Plants

Ubiquitylation is an important biochemical reaction found in all eukaryotic organisms and is involved in a wide range of cellular processes. Conventional ubiquitylation requires the formation of polyubiquitin chains linked through Lys48 of the ubiquitin, which targets specific proteins for degradation. Recently polyubiquitylation through a noncanonical Lys63 chain has been reported, and is required for error-free DNA damage tolerance (or postreplication repair) in yeast. To date, Ubc13 is the only known ubiquitin-conjugating enzyme (Ubc) capable of catalyzing the Lys63-linked polyubiquitylation reaction and this function requires interaction with the Ubc variant Mms2. No information is available on either Lys63-linked ubiquitylation or error-free damage tolerance in plants. We thus cloned and functionally characterized two Arabidopsis thaliana UBC13 genes, AtUBC13A and AtUBC13B. The two genes are highly conserved with respect to chromosomal structure and protein sequence, suggesting that they are derived from a recent gene duplication event. Both AtUbc13 proteins are able to physically interact with yeast or human Mms2, implying that plants also employ the Lys63-linked polyubiquitylation reaction. Furthermore, AtUBC13 genes are able to functionally complement the yeast ubc13 null mutant for spontaneous mutagenesis and sensitivity to DNA damaging agents, suggesting the existence of an error-free DNA damage tolerance pathway in plants. The AtUBC13 genes appear to express ubiquitously and are not induced by various conditions tested.