Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting.
ubiquitination; ubiquitin acceptor; (APC/C); KEN; degron; Aurora A
Protein ubiquitination is an essential posttranslational modification regulating neurodevelopment, synaptic plasticity, learning and memory, and its dysregulation contributes to the pathogenesis of neurological diseases. Here we report a systematic analysis of ubiquitinated proteome (ubiquitome) in rat brain using a newly developed monoclonal antibody that recognizes the diglycine tag on lysine residues in trypsinized peptides (K-GG peptides). Initial antibody specificity analysis showed that the antibody can distinguish K-GG peptides from linear GG peptides or pseudo K-GG peptides derived from iodoacetamide. To evaluate the false discovery rate of K-GG peptide matches during database search, we introduced a null experiment using bacterial lysate that contains no such peptides. The brain ubiquitome was then analyzed by this antibody enrichment with or without strong cation exchange (SCX) prefractionation. During SCX chromatography, although the vast majority of K-GG peptides were detected in the fractions containing at least three positive charged peptides, specific K-GG peptides with two positive charges (e.g. protein N-terminal acetylated and C-terminal non-K/R peptides) were also identified in early fractions. The reliability of C-terminal K-GG peptides was also extensively investigated. Finally, we collected a dataset of 1786 K-GG sites on 2064 peptides in 921 proteins and estimated their abundance by spectral counting. The study reveals a wide range of ubiquitination events on key components in presynaptic region (e.g. Bassoon, NSF, SNAP25, synapsin, synaptotagmin, and syntaxin) and postsynaptic density (e.g. PSD-95, GKAP, CaMKII, as well as receptors for NMDA, AMPA, GABA, serotonin, and acetylcholine). We also determined ubiquitination sites on amyloid precursor protein and alpha synuclein that are thought to be causative agents in Alzhermer’s and Parkinson’s disorders, respectively. As K-GG peptides can also be produced from Nedd8 or ISG15 modified proteins, we quantified these proteins in the brain and found that their levels are less than 2% of ubiquitin. Together, this study demonstrates that a large number of neuronal proteins are modified by ubiquitination, and provides a feasible method for profiling the ubiquitome in the brain.
ubiquitin; synapse; antibody; proteomics; mass spectrometry
The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3
ligase enzymes to sequentially transfer the small modifier protein ubiquitin to
a substrate protein. During the last step of this cascade different types of E3
ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or
form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a
substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique
group of E3 ubiquitin ligases that includes the Human Homologue of
Drosophila Ariadne (HHARI). These E3
ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses
facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate.
We now present the solution structure of the HHARI RING2 domain, the key portion
of this E3 ligase required for the RING/HECT hybrid mechanism. The structure
shows the domain possesses two Zn2+-binding sites and a single
exposed cysteine used for ubiquitin catalysis. A structural comparison of the
RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the
cysteine and histidine residues in the catalytic site. Further, a tandem pair of
aromatic residues exists near the C-terminus of the HHARI RING2 domain that is
conserved in other RBR E3 ligases. One of these aromatic residues is remotely
located from the catalytic site that is reminiscent of the location found in
HECT E3 enzymes where it is used for ubiquitin catalysis. These observations
provide an initial structural rationale for the RING/HECT hybrid mechanism for
ubiquitination used by the RBR E3 ligases.
Ubiquitination is a widely-studied regulatory modification involved in protein degradation, DNA damage repair and the immune response. Conjugation of ubiquitin to a substrate lysine occurs in an enzymatic cascade involving an E1 ubiquitin activating enzyme, and E2 ubiquitin conjugating enzyme, and an E3 ubiquitin ligase. Assays for ubiquitin conjugation include electrophoretic mobility shift assays and detection of epitope-tagged or radiolabeled ubiquitin, which are difficult to quantitate accurately and are not amenable to high throughput screening. We have developed a colorimetric assay that quantifies ubiquitin conjugation by monitoring pyrophosphate released in the first enzymatic step in ubiquitin transfer, the ATP-dependent charging of the E1 enzyme. The assay is rapid, does not rely on radioactive labeling, and requires only a spectrophotometer for detection of pyrophosphate formation. We show that pyrophosphate production by E1 is dependent on ubiquitin transfer and describe how to optimize assay conditions to measure E1, E2, or E3 activity. The kinetics of polyubiquitin chain formation by Ubc13-Mms2 measured by this assay are similar to those determined by gel assays, indicating that the data produced by this method are comparable to methods that measure ubiquitin transfer directly. This assay is adaptable to high-throughput screening of ubiquitin and ubiquitin-like conjugating enzymes.
Ubiquitin Conjugation; Ubiquitin-like proteins; Ubc13-Mms2; ubiquitin-activating enzyme
Ubiquitination is crucial for many cellular processes such as protein degradation, DNA repair, transcription regulation and cell signaling. Ubiquitin attachment takes place via a sequential enzymatic cascade involving ubiquitin-activation (by E1 enzymes), ubiquitin-conjugation (by E2 enzymes), and ubiquitin substrate-tagging (by E3 enzymes). E3 ligases mediate ubiquitin transfer from E2s to substrates and as such confer substrate specificity. Although E3s can interact and function with numerous E2s, it is still unclear how they choose which E2 to use. Identifying all E2 partners of an E3 is essential for inferring the principles guiding E2 selection by an E3. Here we model the interactions of E3 and E2 proteins in a large, proteome-scale strategy based on interface structural motifs, which allows elucidation of 1) which E3s interact with which E2s in the human ubiquitination pathway; and 2) how they interact with each other. Interface analysis of E2-E3 complexes reveals that loop L1 of E2s is critical for binding; the residue in the sixth position in loop L1 is widely utilized as an interface hot spot and appears indispensible for E2 interactions. Other loop L1 residues also confer specificity on the E2-E3 interactions: HECT E3s are in contact with the residue in the second position in loop L1 of E2s; but this is not the case for the RING finger type E3s. Our modeled E2-E3 complexes illuminate how slight sequence variations in E2 residues may contribute to specificity in E3 binding. These findings may be important for discovering drug candidates targeting E3s, which have been implicated in many diseases.
ubiquitination; E2; E3; proteomics; protein-protein interactions; protein-protein interfaces; degradation; proteome-scale structural maps
Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate.
The ubiquitination–proteasome and degradation system is an essential process that regulates protein homeostasis. This system is involved in the regulation of cell proliferation, differentiation and survival, and dysregulations in this system lead to pathologies including cancers. The ubiquitination system is an enzymatic cascade that mediates the marking of target proteins by an ubiquitin label and thereby directs their degradation through the proteasome pathway. The ubiquitination of proteins occurs through a three-step process involving ubiquitin activation by the E1 enzyme, allowing for the transfer to a ubiquitin-conjugated enzyme E2 and to the targeted protein via ubiquitin-protein ligases (E3), the most abundant group of enzymes involved in ubiquitination. Significant advances have been made in our understanding of the role of E3 ubiquitin ligases in the control of bone turnover and tumorigenesis. These ligases are implicated in the regulation of bone cells through the degradation of receptor tyrosine kinases, signaling molecules and transcription factors. Initial studies showed that the E3 ubiquitin ligase c-Cbl, a multi-domain scaffold protein, regulates bone resorption by interacting with several molecules in osteoclasts. Further studies showed that c-Cbl controls the ubiquitination of signaling molecules in osteoblasts and in turn regulates osteoblast proliferation, differentiation and survival. Recent data indicate that c-Cbl expression is decreased in primary bone tumors, resulting in excessive receptor tyrosine kinase signaling. Consistently, c-Cbl ectopic expression reduces bone tumorigenesis by promoting tyrosine kinase receptor degradation. Here, we review the mechanisms of action of E3 ubiquitin ligases in the regulation of normal and pathologic bone formation, and we discuss how targeting the interactions of c-Cbl with some substrates may be a potential therapeutic strategy to promote osteogenesis and to reduce tumorigenesis.
ubiquitin ligases; proteasome; receptor tyrosine kinases; bone tumors; Cbl proteins; ubiquitination
E3 ubiquitin ligases are a large family of proteins that are engaged in the regulation of the turnover and activity of many target proteins. Together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates for targeted degradation through the 26S proteasome, as well as for nonproteolytic regulation of their functions or subcellular localizations. E3 ubiquitin ligases, therefore, play an essential role in the regulation of many biologic processes. Increasing amounts of evidence strongly suggest that the abnormal regulation of some E3 ligases is involved in cancer development. Furthermore, some E3 ubiquitin ligases are frequently overexpressed in human cancers, which correlates well with increased chemoresistance and poor clinic prognosis. In this review, E3 ubiquitin ligases (such as murine double minute 2, inhibitor of apoptosis protein, and Skp1-Cullin-F-box protein) will be evaluated as potential cancer drug targets and prognostic biomarkers. Extensive study in this field would lead to a better understanding of the molecular mechanism by which E3 ligases regulate cellular processes and of how their deregulations contribute to carcinogenesis. This would eventually lead to the development of a novel class of anticancer drugs targeting specific E3 ubiquitin ligases, as well as the development of sensitive biomarkers for cancer treatment, diagnosis, and prognosis.
Apoptosis; biomarkers; cancer targets; E3 ubiquitin ligases; protein degradation
A ubiquitin ligase (E3) functions at the crossroad between ubiquitin activation and the attachment of ubiquitin to protein substrates. During this process, the E3 interacts with both a substrate and a ubiquitin-conjugating enzyme (E2). Although a major goal when investigating an E3 is to identify its substrates, recent evidence indicates that the E2 dictates the type of ubiquitin modification that will occur on the substrate. There are ~ 30 E2s identified in the human genome, many of which remain to be characterized. We found that the RING E3 BRCA1/BARD1 can interact with 10 different E2s. The ability of BRCA1 to interact with multiple E2s is likely to be a common feature among other RING and U-box E3s. We and others have also found that certain E2s show a preference for attaching either the first ubiquitin to a substrate lysine or ubiquitin to itself (chain building), suggesting that E2s may play a role in dictating product formation. Therefore, when investigating the functions of an E3 it is advisable to identify all E2s that interact with the E3 so that these can be used in E3-dependent substrate-ubiquitination assays. We describe a method used to identify all the E2s that interact with BRCA1. Defining the set of E2s that interact with other RING and U-box E3s will open the door for predictive models and lead to a better understand of substrate ubiquitination.
BRCA1; NMR; protein-protein interactions; RING domain; UbcH5; Ubc13; ubiquitin ligase; ubiquitination; ubiquitin-conjugating enzyme; yeast two-hybrid
Ubiquitination is an important post-translational modification involved in diverse biological processes. Therefore, genomewide representation of the ubiquitination system for a species is important.
SCUD is a web-based database for the ubiquitination system in Saccharomyces cerevisiae (Baker's yeast). We first searched for all the known enzymes involved in the ubiquitination process in yeast, including E1, E2, E3, and deubiquitination enzymes. Then, ubiquitinated substrates were collected by literature search. Especially, E3 and deubiquitination enzymes are classified into classes and subclasses by their shared domains and unique functions. As a result, 42 different E3 enzymes were grouped into corresponding classes and subclasses, and 940 ubiquitinated substrates including mutant substrates were identified. All the enzyme and substrate information are interconnected by hyperlinks, which makes it easy to view the enzyme-specific ubiquitination information.
This database aims to represent a comprehensive yeast ubiquitination system, and is easily expandable with the further experimental data. We expect that this database will be useful for the research on the ubiquitination systems of other higher organisms.
SCUD is accessible at
Ubiquitin and UBL (ubiquitin-like) modifiers are small proteins that covalently modify other proteins to alter their properties or behaviours. Ubiquitin modification (ubiquitylation) targets many substrates, often leading to their proteasomal degradation. NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is the UBL most closely related to ubiquitin, and its best-studied role is the activation of CRLs (cullin-RING ubiquitin ligases) by its conjugation to a conserved C-terminal lysine residue on cullin proteins. The attachment of UBLs requires three UBL-specific enzymes, termed E1, E2 and E3, which are usually well insulated from parallel UBL pathways. In the present study, we report a new mode of NEDD8 conjugation (NEDDylation) whereby the UBL NEDD8 is linked to proteins by ubiquitin enzymes in vivo. We found that this atypical NEDDylation is independent of classical NEDD8 enzymes, conserved from yeast to mammals, and triggered by an increase in the NEDD8 to ubiquitin ratio. In cells, NEDD8 overexpression leads to this type of NEDDylation by increasing the concentration of NEDD8, whereas proteasome inhibition has the same effect by depleting free ubiquitin. We show that bortezomib, a proteasome inhibitor used in cancer therapy, triggers atypical NEDDylation in tissue culture, which suggests that a similar process may occur in patients receiving this treatment.
bortezomib; MG132; MLN4924; neural-precursor-cell-expressed developmentally down-regulated 8 (NEDD8)-activating enzyme (NAE); proteasome; ubiquitinactivating enzyme; BCA3, breast cancer-associated gene 3; CHO, Chinese-hamster ovary; CRL, cullin-RING ubiquitin ligase; EGFR, epidermal growth factor receptor; FBS, fetal bovine serum; HA, haemagglutinin; HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate; NAE, NEDD8-activating enzyme; NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; siRNA, small interfering RNA; SUMO, small ubiquitin-like modifier; TCA, trichloroacetic acid; UBL, ubiquitin-like; WT, wild-type
Ubiquitin-protein ligases (E3s) are often in the precarious position of ubiquitinating themselves, mediating their own destruction. The intrinsically disordered E3 San1 prevents its own autoubiquitination and degradation by minimizing Lys residues and hydrophobic stretches in its disordered regions.
Ubiquitin-protein ligases (E3s) that ubiquitinate substrates for proteasomal degradation are often in the position of ubiquitinating themselves due to interactions with a charged ubiquitin-conjugating enzyme (E2). This can mediate the E3’s proteasomal degradation. Many E3s have evolved means to avoid autoubiquitination, including protection by partner or substrate binding, preventative modifications, and deubiquitinating enzyme reversal of ubiquitination. Here we describe another adaptation for E3 self-protection discovered while exploring San1, which ubiquitinates misfolded nuclear proteins in yeast for proteasomal degradation. San1 is highly disordered in its substrate-binding regions N- and C-terminal to its RING domain. In cis autoubiquitination could occur if these flexible regions come in proximity to the E2. San1 prevents this by containing no lysines in its disordered regions; thus the canonical residue used for ubiquitin attachment has been selectively eliminated. San1’s target substrates have lost their native structures and expose hydrophobicity. To avoid in trans autoubiquitination, San1 possesses little concentrated hydrophobicity in its disordered regions, and thus the that feature San1 recognizes in misfolded substrates has also been selectively eliminated. Overall the presence of key residues in San1 have been evolutionarily minimized to avoid self-destruction either in cis or in trans. Our work expands the ways in which E3s protect themselves from autoubiquitination.
Post-translational modification of proteins by ubiquitin (Ub) regulates a host of cellular processes including protein quality control, DNA repair, endocytosis and cellular signaling. In the ubiquitination cascade, a thioester-linked conjugate between the Ub C-terminus and the active site cysteine of a ubiquitin-conjugating enzyme (E2) is formed. The E2~Ub conjugate interacts with a ubiquitin ligase (E3) to transfer Ub to a lysine residue on a target protein. The flexibly-linked E2~Ub conjugates have been shown to form a range of structures in solution. In addition, select E2~Ub conjugates oligomerize through a noncovalent “backside” interaction between Ub and E2 components of different conjugates. Additional studies are needed to bridge the gap between the dynamic monomeric conjugates, E2~Ub oligomers and the mechanisms of ubiquitination. We present a new 2.35 Å crystal structure of an oligomeric UbcH5c~Ub conjugate. The conjugate forms a staggered linear oligomer that differs substantially from the “infinite spiral” helical arrangement of the sole previously reported structure of an oligomeric conjugate. Our structure also differs in intra-conjugate conformation from other structurally characterized conjugates. Despite these differences, we find that the backside interaction mode is conserved in different conjugate oligomers and is independent of intra-conjugate relative E2/Ub orientations. We delineate a common intra-conjugate E2-binding surface on Ub. In addition, we demonstrate that an E3 ligase CHIP (carboxyl terminus of Hsp70 interacting protein) interacts directly with UbcH5c~Ub oligomers, not only with conjugate monomers. These results provide insights into the conformational diversity of E2~Ub conjugates and conjugate oligomers, and into their compatibility and interactions with E3 ligases, which have important consequences for the ubiquitination process.
RNA interference experiments in Caenorhabditis elegans suggest functional overlap in many ubiquitin-conjugating enzymes (UBCs). Phylogenetic analysis of C. elegans, Drosophila, and human genes implies that the numbers of UBCs increases with developmental complexity.
The eukaryotic ubiquitin-conjugation system sets the turnover rate of many proteins and includes activating enzymes (E1s), conjugating enzymes (UBCs/E2s), and ubiquitin-protein ligases (E3s), which are responsible for activation, covalent attachment and substrate recognition, respectively. There are also ubiquitin-like proteins with distinct functions, which require their own E1s and E2s for attachment. We describe the results of RNA interference (RNAi) experiments on the E1s, UBC/E2s and ubiquitin-like proteins in Caenorhabditis elegans. We also present a phylogenetic analysis of UBCs.
The C. elegans genome encodes 20 UBCs and three ubiquitin E2 variant proteins. RNAi shows that only four UBCs are essential for embryogenesis: LET-70 (UBC-2), a functional homolog of yeast Ubc4/5p, UBC-9, an ortholog of yeast Ubc9p, which transfers the ubiquitin-like modifier SUMO, UBC-12, an ortholog of yeast Ubc12p, which transfers the ubiquitin-like modifier Rub1/Nedd8, and UBC-14, an ortholog of Drosophila Courtless. RNAi of ubc-20, an ortholog of yeast UBC1, results in a low frequency of arrested larval development. A phylogenetic analysis of C. elegans, Drosophila and human UBCs shows that this protein family can be divided into 18 groups, 13 of which include members from all three species. The activating enzymes and the ubiquitin-like proteins NED-8 and SUMO are required for embryogenesis.
The number of UBC genes appears to increase with developmental complexity, and our results suggest functional overlap in many of these enzymes. The ubiquitin-like proteins NED-8 and SUMO and their corresponding activating enzymes are required for embryogenesis.
Conjugation of ubiquitin-like protein Nedd8 to cullin (i.e. neddylation) is essential for the function of cullin-RING ubiquitin ligases (CRLs). Here we show that neddylation stimulates recruitment of ubiquitin-conjugating enzyme (E2) esterified with ubiquitin (E2~Ub), helps bridge the ~50 Å gap between E2 and substrate bound to SCF to enable their reaction, and facilitates formation of amide bonds in E2's active site. Together, these effects potently stimulate transfer of ubiquitin to substrate. We propose that the initiator ubiquitin spans the gap, and the impact of neddylation on transfer of subsequent ubiquitins by the E2 Cdc34 arises from improved E2 recruitment and enhanced amide bond formation in the E2 active site. The combined effects of neddylation greatly enhance the probability that a substrate molecule acquires ≥4 ubiquitins in a single encounter with a CRL. The surprisingly diverse effects of Nedd8 conjugation underscore the complexity of CRL regulation and suggest that modification of other ubiquitin ligases with ubiquitin or ubiquitin-like proteins may likewise have major functional consequences.
Tagging a small molecule ubiquitin to a protein substrate, or protein ubiquitination, plays an important role in the immune responses. This process is catalyzed by a cascade of enzymatic reactions, with the E3 ubiquitin ligases being the critical enzymes that determine the specificity of substrate recognition. The E3 ligase Itch was identified from a mutant mouse which displays skin scratching and abnormal immune disorders. In the past few years, much progress has been made in our understanding of Itch-promoted protein ubiquitination, modulation of its ligase activity by upstream kinases, and the kinase-ligase interaction in T cell differentiation and tolerance induction.
Itch; ubiquitin; E3 ligase; T helper type 2; Jun proteins; anergy; asthma
Ubiquitination involves the attachment of ubiquitin to lysine residues on substrate proteins or itself, which can result in protein monoubiquitination or polyubiquitination. Ubiquitin attachment to different lysine residues can generate diverse substrate-ubiquitin structures, targeting proteins to different fates. The mechanisms of lysine selection are not well understood. Ubiquitination by the largest group of E3 ligases, the RING-family E3 s, is catalyzed through co-operation between the non-catalytic ubiquitin-ligase (E3) and the ubiquitin-conjugating enzyme (E2), where the RING E3 binds the substrate and the E2 catalyzes ubiquitin transfer. Previous studies suggest that ubiquitination sites are selected by E3-mediated positioning of the lysine toward the E2 active site. Ultimately, at a catalytic level, ubiquitination of lysine residues within the substrate or ubiquitin occurs by nucleophilic attack of the lysine residue on the thioester bond linking the E2 catalytic cysteine to ubiquitin. One of the best studied RING E3/E2 complexes is the Skp1/Cul1/F box protein complex, SCFCdc4, and its cognate E2, Cdc34, which target the CDK inhibitor Sic1 for K48-linked polyubiquitination, leading to its proteasomal degradation. Our recent studies of this model system demonstrated that residues surrounding Sic1 lysines or lysine 48 in ubiquitin are critical for ubiquitination. This sequence-dependence is linked to evolutionarily conserved key residues in the catalytic region of Cdc34 and can determine if Sic1 is mono- or poly-ubiquitinated. Our studies indicate that amino acid determinants in the Cdc34 catalytic region and their compatibility to those surrounding acceptor lysine residues play important roles in lysine selection. This may represent a general mechanism in directing the mode of ubiquitination in E2 s.
Posttranslational modification of a protein by ubiquitin usually results in rapid degradation of the ubiquitinated protein by the proteasome. The transfer of ubiquitin to substrate is a multistep process. Cdc4p is a component of a ubiquitin ligase that tethers the ubiquitin-conjugating enzyme Cdc34p to its substrates. Among the domains of Cdc4p that are crucial for function are the F-box, which links Cdc4p to Cdc53p through Skp1p, and the WD-40 repeats, which are required for binding the substrate for Cdc34p. In addition to Cdc4p, other F-box proteins, including Grr1p and Met30p, may similarly act together with Cdc53p and Skp1p to function as ubiquitin ligase complexes. Because the relative abundance of these complexes, known collectively as SCFs, is important for cell viability, we have sought evidence of mechanisms that modulate F-box protein regulation. Here we demonstrate that the abundance of Cdc4p is subject to control by a peptide segment that we term the R-motif (for “reduced abundance”). Furthermore, we show that binding of Skp1p to the F-box of Cdc4p inhibits R-motif-dependent degradation of Cdc4p. These results suggest a general model for control of SCF activities.
Protein ubiquitination is catalyzed by ubiquitin-conjugating enzymes (E2s) in collaboration with ubiquitin-protein ligases (E3s). This process depends on nucleophilic attack by a substrate lysine on a thioester bond linking the C-terminus of ubiquitin to a cysteine in the E2 active site. Different E2 family members display specificity for lysines in distinct contexts. We addressed the mechanistic basis for this lysine selectivity in Ubc1, an E2 that catalyzes the ubiquitination of lysine 48 (K48) in ubiquitin, leading to the formation of K48-linked polyubiquitin chains. We identified a cluster of polar residues near the Ubc1 active site, as well as a residue in ubiquitin itself, that are required for catalysis of K48-specific ubiquitin ligation but not for general activity toward other lysines. Our results suggest that the active site of Ubc1, as well as the surface of ubiquitin, contain specificity determinants that channel specific lysines to the central residues involved directly in catalysis.
Ubiquitin signaling plays an essential role in controlling cellular processes in eukaryotes, and the impairment of ubiquitin regulation contributes to the pathogenesis of a wide range of human diseases. During the last decade, mass spectrometry–based proteomics has emerged as an indispensable approach for identifying ubiquitinated proteins, ubiquitin modification sites, the structure of complex ubiquitin chains, as well as the interactome of ubiquitin enzymes. In particular, implementation of quantitative strategies allows the detection of dynamic changes in the ubiquitinated proteome, enhancing the ability to differentiate between function-relevant protein targets and false positives arising from biological and experimental variations. The profiling of total cell lysate and ubiquitinated proteome in the same sets of samples has become a powerful tool, revealing a subset of substrates that are modulated by specific physiological and pathological conditions, such as gene mutations in ubiquitin signaling. This strategy is equally useful for dissecting the pathways of ubiquitin-like proteins.
ubiquitin; proteasome; E3; DUB; mass spectrometry; proteomics; SILAC
Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for Ub transfer to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2~Ub conjugate. Therefore, full characterization of the structure and dynamics of E2~Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2~Ub conjugates of two human enzymes, UbcH5c~Ub and Ubc13~Ub, in solution as determined by NMR and SAXS. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: UbcH5c~Ub populates an array of extended conformations and the population of Ubc13~Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces Helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2~Ub species and thus provide insight into the diverse E2~Ub protein interactome, particularly regarding interaction with Ub ligases.
ubiquitin; ubiquitin conjugating enzyme; ubiquitination; UbcH5; Ubc13; NMR; spin label; SAXS
The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred – E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions.
Ubiquitin is a small polypeptide that is conjugated to proteins and commonly serves as a degradation signal. The attachment of ubiquitin (Ub) to a substrate proceeds through a multi-enzyme cascade involving an activating enzyme (E1), a conjugating enzyme (E2), and a protein ligase (E3). We previously demonstrated that a murine E2, UbcM2, is imported into nuclei by the transport receptor importin-11. We now show that the import mechanism for UbcM2 and two other human class III E2s (UbcH6 and UBE2E2) uniquely requires the covalent attachment of Ub to the active site cysteine of these enzymes. This coupling of E2 activation and transport arises from the selective interaction of importin-11 with the Ub-loaded forms of these enzymes. Together, these findings reveal that Ub charging can function as a nuclear import trigger, and identify a novel link between E2 regulation and karyopherin-mediated transport.
The pathway by which ubiquitin chains are generated on substrate via a cascade of enzymes consisting of an E1, E2 and E3 remains unclear. Multiple distinct models involving chain assembly on E2 or substrate have been proposed. However, the speed and complexity of the reaction have precluded direct experimental tests to distinguish between potential pathways. Here we introduce new theoretical and experimental methodologies to address both limitations. A quantitative framework based on product distribution predicts that the really interesting new gene (RING) E3s SCFCdc4 and SCFβ-TrCP work with the E2 Cdc34 to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrate that substrate polyubiquitylation proceeds sequentially. Our results present an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminate the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.