Search tips
Search criteria

Results 1-25 (1331253)

Clipboard (0)

Related Articles

1.  Exploring linkage dependence of polyubiquitin conformations using molecular modeling 
Journal of molecular biology  2009;395(4):803.
Post-translational modification of proteins by covalent attachment of a small protein ubiquitin or a polymeric chain of ubiquitin molecules (called polyubiquitin) is involved in controlling a vast variety of processes in eukaryotic cells. The question of how different polyubiquitin signals are recognized is central to understanding the specificity of various types of polyubiquitination. In polyubiquitin, the monomers are linked to each other via an isopeptide bond between the C-terminal glycine of one ubiquitin and a lysine of the other. The functional outcome of polyubiquitination depends on the particular lysine involved in the chain formation and appears to rely on linkage-dependent conformation of polyubiquitin. Thus, K48-linked chains, a universal signal for proteasomal degradation, under physiological conditions adopt a closed conformation where functionally important residues L8, I44, and V70 are sequestered at the interface between the two adjacent ubiquitin monomers. By contrast, K63-linked chains, which act as a non-proteolytic, regulatory signal, adopt an extended conformation that lacks the hydrophobic inter-ubiquitin contact. Little is known about functional roles of the so-called “non-canonical” chains, linked via K6, K11, K27, K29, K33, or head-to-tail; and no structural information on these chains is available, except for the crystal structure of the head-to-tail linked diubiquitin. In this study, we use molecular modeling to examine whether any of the non-canonical chains can adopt a closed conformation similar to that in K48-linked polyubiquitin. Our results show that the eight possible di-ubiquitin chains can be divided into two groups: K6-, K11-, K27-, and K48-linked chains are predicted to form a closed conformation, whereas chains linked via K29, K33, K63, or head-to-tail are unable to form such a contact due to steric occlusion. These predictions are validated by the known structures of K48-, K63-, and head-to-tail linked chains. Our study also predicts structural models for di-ubiquitins linked via K6, K11, 3 and K27. Implications of these findings for linkage-selective recognition of the non-canonical polyubiquitin signals by various receptors are discussed.
PMCID: PMC2813430  PMID: 19853612
polyubiquitin; non-canonical linkage; isopeptide linkage; head-to-tail; modeling
2.  Proteomic Identification and Analysis of K63-linked Ubiquitin Conjugates 
Analytical chemistry  2012;84(22):10121-10128.
Post-translational modification of proteins by covalent attachment of ubiquitin or a polyubiquitin chain is involved in myriad of processes in eukaryotic cells. The particular outcome of ubiquitination is directed by the length of the ubiquitin conjugate and its linkage composition. Among seven possible isopeptide linkage sites in ubiquitin, K48 and K63 occur most commonly and act as distinct cellular signals. Strategies are reported here for analysis of linkage sites and complexity of K63-linked polyubiquitin chains, based on rapid chemical proteolysis at aspartate residues combined with immunoprecipitation and mass spectrometry. Rapid chemical proteolysis at aspartate residues results in K63-linked peptides with truncated branches, which enable identification and characterization of stretches of consecutive K63 linkages on generally available instruments. A characteristic cleavage pattern and a characteristic fragmentation pattern allow recognition of K63 oligomers in proteolytic mixtures. Engineered K63-linked polyubiquitin chains of defined lengths were used to evaluate and demonstrate the method. In-gel microwave-supported acid hydrolysis was used to observe peptides specific to K63-linked ubiquitin dimers and trimers. Acid hydrolysis in solution, used in conjunction with linkage-specific immunoprecipitation, allowed more complex K63-linked branches to be characterized. Finally a substrate protein, UbcH5b, was conjugated to mono-ubiquitin and to polyubiquitin chains containing only K63 linkages, and the sites of conjugation and chain lengths were characterized.
PMCID: PMC3509807  PMID: 23101881
3.  Regulation of Proteolysis by Human Deubiquitinating Enzymes 
Biochimica et biophysica acta  2013;1843(1):10.1016/j.bbamcr.2013.06.027.
The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase.
PMCID: PMC3833951  PMID: 23845989
Deubiquitinating enzyme; Ubiquitin; Poly-Ubiquitin; Proteolysis; Regulation
4.  Proteomic snapshot of the EGF-induced ubiquitin network 
In this work, the authors report the first proteome-wide analysis of EGF-regulated ubiquitination, revealing surprisingly pervasive growth factor-induced ubiquitination across a broad range of cellular systems and signaling pathways.
Epidermal growth factor (EGF) triggers a novel ubiquitin (Ub)-based signaling cascade that appears to intersect both housekeeping and regulatory circuitries of cellular physiology.The EGF-regulated Ubiproteome includes scores ubiquitinating and deubiquitinating enzymes, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery.The EGF-Ubiproteome overlaps significantly with the EGF-phosphotyrosine proteome, pointing to a possible crosstalk between these two signaling mechanisms.The significant number of biological insights uncovered in our study (among which EphA2 as a novel, downstream ubiquitinated target of EGF receptor) illustrates the general relevance of such proteomic screens and calls for further analysis of the dynamics of the Ubiproteome.
Ubiquitination is a process by which one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins by E3 ligases. Deubiquitinating enzymes (DUBs) revert Ub conjugation, thus ensuring a dynamic equilibrium between pools of ubiquitinated and deubiquitinated proteins (Amerik and Hochstrasser, 2004). Traditionally, ubiquitination has been associated with protein degradation; however, it is now becoming apparent that this post-translation modification is an important signaling mechanism that can modulate the function, localization and protein/protein interaction abilities of targets (Mukhopadhyay and Riezman, 2007; Ravid and Hochstrasser, 2008).
One of the best-characterized signaling pathways involving ubiquitination is the epidermal growth factor (EGF)-induced pathway. Upon EGF stimulation, a variety of proteins are subject to Ub modification. These include the EGF receptor (EGFR), which undergoes both multiple monoubiquitination (Haglund et al, 2003) and K63-linked polyubiquitination (Huang et al, 2006), as well as components of the downstream endocytic machinery, which are modified by monoubiquitination (Polo et al, 2002; Mukhopadhyay and Riezman, 2007). Ubiquitination of the EGFR has been shown to have an impact on receptor internalization, intracellular sorting and metabolic fate (Acconcia et al, 2009). However, little is known about the wider impact of EGF-induced ubiquitination on cellular homeostasis and on the pleiotropic biological functions of the EGFR. In this paper, we attempt to address this issue by characterizing the repertoire of proteins that are ubiquitinated upon EGF stimulation, i.e., the EGF-Ubiproteome.
To achieve this, we employed two different purification procedures (endogenous—based on the purification of proteins modified by endogenous Ub from human cells; tandem affinity purification (TAP)—based on the purification of proteins modified by an ectopically expressed tagged-Ub from mouse cells) with stable isotope labeling with amino acids in cell culture-based MS to obtain both steady-state Ubiproteomes and EGF-induced Ubiproteomes. The steady-state Ubiproteomes consist of 1175 and 582 unambiguously identified proteins for the endogenous and TAP approaches, respectively, which we largely validated. Approximately 15% of the steady-state Ubiproteome was EGF-regulated at 10 min after stimulation; 176 of 1175 in the endogenous approach and 105 of 582 in the TAP approach. Both hyper- and hypoubiquitinated proteins were detected, indicating that EGFR-mediated signaling can modulate the ubiquitin network in both directions. Interestingly, many E2, E3 and DUBs were present in the EGF-Ubiproteome, suggesting that the Ub signal might be rapidly transmitted and amplified through the Ub machinery. Moreover, analysis of Ub-chain topology, performed using mass spectrometry and specific abs, suggested that the K63-linkage was the major Ub-based signal in the EGF-induced pathway.
To obtain a higher-resolution molecular picture of the EGF-regulated Ub network, we performed a network analysis on the non-redundant EGF-Ubiproteome (265 proteins). This analysis revealed that in addition to well-established liaisons with endocytosis-related pathways, the EGF-Ubiproteome intersects many circuitries of intracellular signaling involved in, e.g., DNA damage checkpoint regulation, cell-to-cell adhesion mechanisms and actin remodeling (Figure 5A).
Moreover, the EGF-Ubiproteome was enriched in hubs, proteins that can establish multiple protein/protein interaction and thereby regulate the organization of networks. These results are indicative of a crosstalk between EGFR-activated pathways and other signaling pathways through the Ub-network.
As EGF binding to its receptor also triggers a series of phosphorylation events, we examined whether there was any overlap between our EGF-Ubiproteome and published EGF-induced phosphotyrosine (pY) proteomes (Blagoev et al, 2004; Oyama et al, 2009; Hammond et al, 2010). We observed a significant overlap between ubiquitinated and pY proteins: 23% (61 of 265) of the EGF-Ubiproteome proteins were also tyrosine phosphorylated. Pathway analysis of these 61 Ub/pY-containing proteins revealed a significant enrichment in endocytic and signal-transduction pathways, while ‘hub analysis' revealed that Ub/pY-containing proteins are enriched in highly connected proteins to an even greater extent than Ub-containing proteins alone. These data point to a complex interplay between the Ub and pY networks and suggest that the flow of information from the receptor to downstream signaling molecules is driven by two complementary and interlinked enzymatic cascades: kinases/phosphatases and E3 ligases/DUBs.
Finally, we provided a proof of principle of the biological relevance of our EGF-Ubiproteome. We focused on EphA2, a receptor tyrosine kinase, which is involved in development and is often overexpressed in cancer (Pasquale, 2008). We started from the observation that EphA2 is present in the EGF-Ubiproteome and that proteins of the EGF-Ubiproteome are enriched in the Ephrin receptor signaling pathway(s). We confirmed the MS data by demonstrating that the EphA2 is ubiquitinated upon EGF stimulation. Moreover, EphA2 also undergoes tyrosine phosphorylation, indicating crosstalk between the two receptors. The EGFR kinase domain was essential for these modifications of EphA2, and a partial co-internalization with EGFR upon EGF activation was clearly detectable. Finally, we demonstrated by knockdown of EphA2 in MCF10A cells that this receptor is critically involved in EGFR biological outcomes, such as proliferation and migration (Figure 7).
Overall, our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected and highlight the ‘resource' feature of our EGF-Ubiproteome.
The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses.
PMCID: PMC3049407  PMID: 21245847
EGF; network; proteomics; signaling; ubiquitin
5.  Mapping the interactions between Lys48- and Lys63-linked di-ubiquitins and a ubiquitin-interacting motif of S5a 
Journal of molecular biology  2007;368(3):753-766.
Numerous cellular processes are regulated by (poly)ubiquitin-mediated signaling events, which involve a covalent modification of the substrate protein by a single ubiquitin or a chain of ubiquitin molecules linked via a specific lysine. Remarkably, the outcome of polyubiquitination is linkage-dependent. For example, Lys48-linked chains are the principal signal for proteasomal degradation, while Lys63-linked chains act as non-proteolytic signals. Despite significant progress in characterization of various cellular pathways involving ubiquitin, understanding of the structural details of polyubiquitin chain recognition by downstream cellular effectors is missing. Here we use NMR to study the interaction of a ubiquitin-interacting motif (UIM) of the proteasomal subunit S5a with di-ubiquitin, the simplest model for polyubiquitin chain, in order to gain insights into the mechanism of polyubiquitin recognition by the proteasome. We have mapped the binding interface and characterized the stoichiometry and the process of UIM binding to Lys48- and Lys63-linked di-ubiquitin chains. Our data provide first direct evidence that UIM binding involves a conformational transition in Lys48-linked di-ubiquitin which opens the hydrophobic interdomain interface. This allows UIM to enter the interface and bind directly to the same ubiquitin hydrophobic-patch surface as utilized in UIM:monoubiquitin complexes. The results indicate that up to two UIM molecules can bind di-ubiquitin, and the binding interface between UIM and ubiquitin units in di-ubiquitin is essentially the same for both Lys48- and Lys63-linked chains. Our data suggest possible structural models for the binding of UIM and of full-length S5a to di-ubiquitin.
PMCID: PMC1941660  PMID: 17368669
ubiquitin; polyubiquitin; ubiquitin interacting motif; S5a; chemical shift perturbation mapping; ligand binding
6.  Purification, crystallization and preliminary crystallographic studies of Lys48-linked polyubiquitin chains 
Lys48-linked tetraubiquitin, hexaubiquitin and octaubiquitin were enzymatically synthesized, purified and crystallized. X-ray diffraction data sets for tetraubiquitin and hexaubiquitin were collected at 1.6 and 1.8 Å resolution, respectively.
Post-translational modification of proteins by covalent attachment of ubiquitin regulates diverse cellular events. A Lys48-linked polyubiquitin chain is formed via an isopeptide bond between Lys48 and the C-terminal Gly76 of different ubiquitin molecules. The chain is attached to a lysine residue of a substrate protein, which leads to proteolytic degradation of the protein by the 26S proteasome. In order to reveal the chain-length-dependent higher order structures of polyubiquitin chains, Lys48-linked polyubiquitin chains were synthesized enzymatically on a large scale and the chains were separated according to chain length by cation-exchange column chromatography. Subsequently, crystallization screening was performed using the hanging-drop vapour-diffusion method, from which crystals of tetraubiquitin, hexaubiquitin and octaubiquitin chains were obtained. The crystals of the tetraubiquitin and hexaubiquitin chains diffracted to 1.6 and 1.8 Å resolution, respectively. The tetraubiquitin crystals belonged to space group C2221, with unit-cell parameters a = 58.795, b = 76.966, c = 135.145 Å. The hexaubiquitin crystals belonged to space group P21, with unit-cell parameters a = 51.248, b = 102.668, c = 51.161 Å. Structural analysis by molecular replacement is in progress.
PMCID: PMC2898474  PMID: 20606286
ubiquitin; Lys48-linked polyubiquitin
7.  Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme 
Journal of the American Chemical Society  2011;133(44):17855-17868.
Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This however has been a major impediment in the ubiquitin field. Here we present a robust, efficient, and widely accessible method for controlled iterative non-enzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the non-enzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked di-ubiquitin, and a mixed-linkage Lys33,Lys11-linked tri-ubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.
PMCID: PMC3226840  PMID: 21962295
8.  Nonenzymatic assembly of branched polyubiquitin chains for structural and biochemical studies 
Bioorganic & medicinal chemistry  2013;21(12):3421-3429.
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.
PMCID: PMC3665622  PMID: 23557636
ubiquitin; polyubiquitin; branched chain; isopeptide bond; unnatural amino acids; deubiquitination
9.  CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, accumulates at the postsynaptic density in an activity-dependent manner 
Polyubiquitin chains on proteins flag them for distinct fates depending on the type of polyubiquitin linkage. While lysine48-linked polyubiquitination directs proteins to proteosomal degradation, lysine63-linked polyubiquitination promotes different protein trafficking and is involved in autophagy. Here we show that postsynaptic density (PSD) fractions from adult rat brain contain deubiquitinase activity that targets both lysine48 and lysine63-linked polyubiquitins. Comparison of PSD fractions with parent subcellular fractions by Western immunoblotting reveals that CYLD, a deubiquitinase specific for lysine63-linked polyubiquitins, is highly enriched in the PSD fraction. Electron microscopic examination of hippocampal neurons in culture under basal conditions shows immunogold label for CYLD at the PSD complex in approximately one in four synapses. Following depolarization by exposure to high K+, the proportion of CYLD-labeled PSDs as well as the labeling intensity of CYLD at the PSD increased by more than eighty percent, indicating that neuronal activity promotes accumulation of CYLD at the PSD. An increase in postsynaptic CYLD following activity, would promote removal of lysine63-polyubiquitins from PSD proteins and thus could regulate their trafficking and prevent their autophagic degradation.
PMCID: PMC3545086  PMID: 23146630
Ubiquitin; CYLD; Deubiquitinase; K63-linked; PSD; postsynaptic density
10.  Polyubiquitin chain assembly and organization determine the dynamics of protein activation and degradation 
Protein degradation via ubiquitination is a major proteolytic mechanism in cells. Once a protein is destined for degradation, it is tagged by multiple ubiquitin (Ub) molecules. The synthesized polyubiquitin chains can be recognized by the 26S proteosome where proteins are degraded. These chains form through multiple ubiquitination cycles that are similar to multi-site phosphorylation cycles. As kinases and phosphatases, two opposing enzymes (E3 ligases and deubiquitinases DUBs) catalyze (de)ubiquitination cycles. Although multi-ubiquitination cycles are fundamental mechanisms of controlling protein concentrations within a cell, their dynamics have never been explored. Here, we fill this knowledge gap. We show that under permissive physiological conditions, the formation of polyubiquitin chain of length greater than two and subsequent degradation of the ubiquitinated protein, which is balanced by protein synthesis, can display bistable, switch-like responses. Interestingly, the occurrence of bistability becomes pronounced, as the chain grows, giving rise to “all-or-none” regulation at the protein levels. We give predictions of protein distributions under bistable regime awaiting experimental verification. Importantly, we show for the first time that sustained oscillations can robustly arise in the process of formation of ubiquitin chain, largely due to the degradation of the target protein. This new feature is opposite to the properties of multi-site phosphorylation cycles, which are incapable of generating oscillation if the total abundance of interconverted protein forms is conserved. We derive structural and kinetic constraints for the emergence of oscillations, indicating that a competition between different substrate forms and the E3 and DUB is critical for oscillation. Our work provides the first detailed elucidation of the dynamical features brought about by different molecular setups of the polyubiquitin chain assembly process responsible for protein degradation.
PMCID: PMC3901042  PMID: 24478717
ubiquitin; polyubiquitin chain; ubiquitination dynamics; bistability; oscillations; protein degradation; protein lifetime
11.  Unique structural, dynamical, and functional properties of K11-linked polyubiquitin chains 
Structure (London, England : 1993)  2013;21(7):1168-1181.
K11-linked polyubiquitin chains play important signaling and regulatory roles in both degradative and non-proteolytic pathways in eukaryotes. To understand the structural basis of how these chains are recognized and distinguished from other polyubiquitins, we determined solution structures of K11-linked di-ubiquitin (K11-Ub2) in the absence and presence of salt. These structures reveal that K11-Ub2 adopts conformations distinct from those of K48-linked or K63-linked chains. Importantly, our solution NMR and SANS data are inconsistent with published crystal structures of K11-Ub2. We found that increasing salt concentration compacts K11-Ub2 and strengthens interactions between the two Ub units. Binding studies indicate that K11-Ub2 interacts with ubiquitin-receptor proteins from both proteasomal and non-proteasomal pathways, but with intermediate affinity and different binding modes than either K48-linked or K63-linked di-ubiquitin. Our data support the hypothesis that polyubiquitin chains of different linkages possess unique conformational and dynamical properties allowing them to be recognized differently by downstream receptor proteins.
PMCID: PMC3802530  PMID: 23823328
12.  Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3 
eLife  2013;2:e00828.
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.
eLife digest
Ubiquitin is a small protein that can be covalently linked to other, ‘target’, proteins in a cell to influence their behavior. Ubiquitin can be linked to its targets either as single copies or as polyubiquitin chains in which several ubiquitin molecules are bound end-on-end to each other, with one end of the chain attached to the target protein. A multi-step cascade involving enzymes known as E1, E2, and E3 adds ubiquitin to its targets. These enzymes function in a manner like runners in a relay, with ubiquitin a baton that is passed from E1 to E2 to E3 to the target.
The E3 enzyme is a ligase that catalyzes the formation of a new chemical bond between a ubiquitin and its target. There are approximately 600 different E3 enzymes in human cells that regulate a wide variety of target proteins. A major class of E3 enzymes, called HECT E3s, attaches ubiquitin to its targets in a unique two-step mechanism: the E2 enzymes covalently link a ubiquitin to a HECT E3 to form a complex that subsequently transfers the ubiquitin to its target protein. The ubiquitin is typically added to a particular amino acid, lysine, on the target protein, but the details of how HECT E3s execute this transfer are not well understood. To address this issue, Kamadurai et al. investigate how Rsp5, a HECT E3 ligase in yeast, attaches ubiquitin to a target protein called Sna3.
All HECT E3s have a domain—the HECT domain—that catalyzes the transfer of ubiquitin to its target protein. This domain consists of two sub-structures: the C-lobe, which can receive ubiquitin from E2 and then itself become linked to ubiquitin, and the N-lobe. These lobes were previously thought to adopt various orientations relative to each other to deliver ubiquitin to sites on different target proteins (including to multiple lysines on a single target protein). Unexpectedly, Kamadurai et al. find that in order to transfer the ubiquitin to Sna3, Rsp5 adopts a discrete HECT domain architecture that creates an active site in which parts of the C-lobe and the N-lobe, which are normally separated, are brought together with a ubiquitin molecule. This architecture also provides a mechanism that dictates which substrate lysines can be ubiquitinated based on how accessible they are to this active site.
The same regions of Rsp5 transfer ubiquitin to targets other than Sna3, suggesting that a uniform mechanism—which Kamadurai et al. show is conserved in two related human HECT E3 ligases—might transfer ubiquitin to all its targets. These studies therefore represent a significant step toward understanding how a major class of E3 enzymes modulates the functions of their targets.
PMCID: PMC3738095  PMID: 23936628
ubiquitin; HECT; E3 ligase; E2 conjugating enzyme; NEDD4; Rsp5; S. cerevisiae
13.  The Structure and conformation of Lys-63 linked tetraubiquitin 
Journal of molecular biology  2009;392(5):1117-1124.
Ubiquitination involves the covalent attachment of the ubiquitin C-terminus to the lysine sidechain of a substrate protein by an isopeptide bond. The modification can comprise a single ubiquitin moiety or a chain of ubiquitin molecules joined by isopeptide bonds between the C-terminus of one ubiquitin with one of the seven lysine residues in the next ubiquitin. Modification of substrate proteins with Lys63-linked polyubiquitin plays a key non-degradative signaling role in many biological processes including DNA repair and NF-κB activation, whereas substrates modified by lysine-48 (Lys48) linked chains are targeted to the proteasome for degradation. The distinct signaling properties of alternatively linked ubiquitin chains presumably stems from structural differences that can be distinguished by effector proteins. We have determined the crystal structure of Lys63 tetraubiquitin at a resolution of 1.96 Å and performed Small Angle X-ray scattering (SAXS) experiments and molecular dynamics (MD) simulations to probe the conformation of Lys63 tetraubiquitin in solution. The chain adopts a highly extended conformation in the crystal, in contrast with the compact globular fold of Lys48 Ub4. Small Angle X-ray scattering (SAXS) experiments show that the tetraubiquitin chain is dynamic in solution, adopting an ensemble of conformations that are more compact than the extended form in the crystal. The results of these studies provide a basis for understanding the differences in the behavior and recognition of Lys63 polyubiquitin chains.
PMCID: PMC2762427  PMID: 19664638
14.  Using Antiubiquitin Antibodies to Probe the Ubiquitination State Within rhTRIM5α Cytoplasmic Bodies 
AIDS Research and Human Retroviruses  2013;29(10):1373-1385.
The first line of defense protecting rhesus macaques from HIV-1 is the restriction factor rhTRIM5α, which recognizes the capsid core of the virus early after entry and normally blocks infection prior to reverse transcription. Cytoplasmic bodies containing rhTRIM5α have been implicated in the ubiquitin–proteasome pathway, but the specific roles these structures play remain uncharacterized. Here, we examine the ubiquitination status of cytoplasmic body proteins. Using antibodies specific for different forms of ubiquitin, we show that ubiquitinated proteins are present in cytoplasmic bodies, and that this localization is altered after proteasome inhibition. A decrease in polyubiquitinated proteins localizing to cytoplasmic bodies was apparent after 1 h of proteasome inhibition, and greater differences were seen after extended proteasome inhibition. The decrease in polyubiquitin conjugates within cytoplasmic bodies was also observed when deubiquitinating enzymes were inhibited, suggesting that the removal of ubiquitin moieties from polyubiquitinated cytoplasmic body proteins after extended proteasome inhibition is not responsible for this phenomenon. Superresolution structured illumination microscopy revealed finer details of rhTRIM5α cytoplasmic bodies and the polyubiquitin conjugates that localize to these structures. Finally, linkage-specific polyubiquitin antibodies revealed that K48-linked ubiquitin chains localize to rhTRIM5α cytoplasmic bodies, implicating these structures in proteasomal degradation. Differential staining of cytoplasmic bodies seen with different polyubiquitin antibodies suggests that structural changes occur during proteasome inhibition that alter epitope availability. Taken together, it is likely that rhTRIM5α cytoplasmic bodies are involved in recruiting components of the ubiquitin–proteasome system to coordinate proteasomal destruction of a viral or cellular protein(s) during restriction of HIV-1.
PMCID: PMC3785812  PMID: 23799296
15.  NEMO oligomerization and its ubiquitin-binding properties 
Biochemical Journal  2009;421(Pt 2):243-251.
The IKK [IκB (inhibitory κB) kinase] complex is a key regulatory component of NF-κB (nuclear factor κB) activation and is responsible for mediating the degradation of IκB, thereby allowing nuclear translocation of NF-κB and transcription of target genes. NEMO (NF-κB essential modulator), the regulatory subunit of the IKK complex, plays a pivotal role in this process by integrating upstream signals, in particular the recognition of polyubiquitin chains, and relaying these to the activation of IKKα and IKKβ, the catalytic subunits of the IKK complex. The oligomeric state of NEMO is controversial and the mechanism by which it regulates activation of the IKK complex is poorly understood. Using a combination of hydrodynamic techniques we now show that apo-NEMO is a highly elongated, dimeric protein that is in weak equilibrium with a tetrameric assembly. Interaction with peptides derived from IKKβ disrupts formation of the tetrameric NEMO complex, indicating that interaction with IKKα and IKKβ and tetramerization are mutually exclusive. Furthermore, we show that NEMO binds to linear di-ubiquitin with a stoichiometry of one molecule of di-ubiquitin per NEMO dimer. This stoichiometry is preserved in a construct comprising the second coiled-coil region and the leucine zipper and in one that essentially spans the full-length protein. However, our data show that at high di-ubiquitin concentrations a second weaker binding site becomes apparent, implying that two different NEMO–di-ubiquitin complexes are formed during the IKK activation process. We propose that the role of these two complexes is to provide a threshold for activation, thereby ensuring sufficient specificity during NF-κB signalling.
PMCID: PMC2708934  PMID: 19422324
analytical ultracentrifugation (AUC); IKK (inhibitory κB kinase) complex; isothermal titration calorimetry (ITC); multi-angle light scattering (MALS); protein–protein interaction; ubiquitin; AUC, analytical ultracentrifugation; CoZi, C-terminal portion of NEMO encompassing the second coiled-coil region and the leucine zipper domain; IκB, inhibitory κB; IKK, IκB kinase; IKKNBD, IKKβ NEMO-binding domain; ITC, isothermal titration calorimetry; LZ, leucine zipper; MALS, multi-angle laser light scattering; NF-κB, nuclear factor κB; NEMO, NF-κB essential modulator; NEMO355, NEMO residues 1–355, with mutations C54S and K285N; SEC, size-exclusion chromatography; TCEP, tris-(2-carboxyethyl)phosphine; RI, refractive index; RIP1, receptor interaction protein kinase; vFLIP, viral FLICE [FADD (Fas-associated death domain)-like interleukin 1β-converting enzyme]-inhibitory protein; ZF, zinc finger
16.  Mechanism of Recruitment and Activation of the Endosome-associated Deubiquitinase AMSH 
Biochemistry  2013;52(44):7818-7829.
AMSH, a deubiquitinating enzyme (DUB) with exquisite specificity for Lys63-linked polyubiquitin chains, is an endosome-associated DUB that regulates sorting of activated cell-surface signaling receptors to lysosome, a process mediated by the members of the endosomal sorting complexes required for transport (ESCRT) machinery. Whole-exome sequencing of DNA samples from children with microcephaly capillary malformation (MIC-CAP) syndrome identified recessive mutations encoded in the AMSH gene causatively linked to the disease. Herein, we report a number of important observations that significantly advance our understanding of AMSH within the context of the ESCRT machinery. First, we performed mutational and kinetic analysis of the putative residues involved in diubiquitin recognition and catalysis with a view to better understanding the catalytic mechanism of AMSH. Our mutational and kinetic analysis reveals that recognition of the proximal ubiquitin is imperative for the linkage specificity and catalytic efficiency of the enzyme. The MIC-CAP disease mutation, Thr313Ile, shows a substantial loss of catalytic activity without any significant change in thermodynamic stability of the protein, indicating that its perturbed catalytic activity is the basis of the disease. The catalytic activity of AMSH is stimulated upon binding to the ESCRT-0 member STAM, however, the precise mechanism and its significance are not known. Based on a number of biochemical and biophysical analysis, we are able to propose a model for activation according to which activation of AMSH is enabled by facile, simultaneous binding to two ubiquitin groups in a polyubiquitin substrate, one by the catalytic domain of the DUB (binding to the distal ubiquitin) and the other (the proximal ubiquitin) by the ubiquitin interacting motif (UIM) from STAM. Such a mode of binding would stabilize the ubiquitin chain in a productive orientation, resulting in an enhancement of the activity of the enzyme. These data together provide a mechanism for understanding the recruitment and activation of AMSH at ESCRT-0, providing biochemical and biophysical evidence in support of a role for AMSH when it is recruited to the initial ESCRT complex: it functions to facilitate transfer of ubiquitinated receptors (cargo) from one ESCRT member to the next by disassembling the polyubiquitin chain while leaving some ubiquitin groups still attached to the cargo.
PMCID: PMC3972757  PMID: 24151880
17.  Deciphering Tissue-specific Ubiquitination by Mass Spectrometry 
Protein ubiquitination is a highly conserved, central mechanism to regulate cellular events in all eukaryotes, such as proteasomal degradation, protein trafficking, DNA repair, synaptic plasticity and immune response. The consequence of protein ubiquitination is modulated by the structure of ubiquitin moiety attached on the substrates, including ubiquitin monomer and diverse polyubiquitin chains with different linkages (N-terminus, K6, K11, K27, K29, K33, K48 and K63). The development of ubiquitin-enrichment strategies coupled with sensitive mass spectrometry enables direct analysis of ubiquitinated proteins in cells, providing an invaluable tool for ubiquitin research. In this chapter we describe recent technology updates for analyzing tissue-specific ubiquitin conjugates in transgenic models, as well as targeted proteomics methods for quantifying different polyubiquitin chain linkages in any type of samples, including human tissues.
PMCID: PMC3475722  PMID: 22350876
ubiquitin; proteomics; mass spectrometry; tissue
18.  Mechanisms of Generating Polyubiquitin Chains of Different Topology 
Cells  2014;3(3):674-689.
Ubiquitination is an important post-translational process involving attachment of the ubiquitin molecule to lysine residue/s on a substrate protein or on another ubiquitin molecule, leading to the formation of protein mono-, multi- or polyubiquitination. Protein ubiquitination requires a cascade of three enzymes, where the interplay between different ubiquitin-conjugating and ubiquitin-ligase enzymes generates diverse ubiquitinated proteins topologies. Structurally diverse ubiquitin conjugates are recognized by specific proteins with ubiquitin-binding domains (UBDs) to target the substrate proteins of different pathways. The mechanism/s for generating the different ubiquitinated proteins topologies is not well understood. Here, we will discuss our current understanding of the mechanisms underpinning the generation of mono- or polyubiquitinated substrates. In addition, we will discuss how linkage-specific polyubiquitin chains through lysines-11, -48 or -63 are formed to target proteins to different fates by binding specific UBD proteins.
PMCID: PMC4197637  PMID: 24987835
protein ubiquitination; ubiquitin chain topologies; polyubiquitination
19.  Stabilization of an unusual salt bridge in ubiquitin by the extra C-terminal domain of the proteasome-associated deubiquitinase UCH37 as a mechanism of its exo specificity 
Biochemistry  2013;52(20):3564-3578.
Ubiquitination is countered by a group of enzymes collectively called deubiquitinases (DUBs) - about 100 of them can be found in the human genome. One of the most interesting aspects of these enzymes is the ability of some members to selectively recognize specific linkage types between ubiquitin in polyubiquitin chains and their endo and exo specificity. The structural basis of exo-specific deubiquitination catalyzed by a DUB is poorly understood. UCH37, a cysteine DUB conserved from fungi to humans, is a proteasome-associated factor that regulates the proteasome by sequentially cleaving polyubiquitin chains from their distal ends, i.e., by exo-specific deubiquitination. In addition to the catalytic domain, the DUB features a functionally uncharacterized UCH37-like domain (ULD), presumed to keep the enzyme in an inhibited state in its proteasome-free form. Herein we report the crystal structure of two constructs of UCH37 from Trichinella spiralis in complex with a ubiquitin-based suicide inhibitor, ubiquitin vinyl methyl ester (UbVME). These structures show that the ULD makes direct contact with ubiquitin stabilizing a highly unusual intra-molecular salt bridge between Lys48 and Glu51 of ubiquitin, an interaction that would be favored only with the distal ubiquitin but not with the internal ones in a Lys48-linked polyubiquitin chain. An inspection of 39 DUB-ubiquitin structures in the protein data bank reveals the uniqueness of the salt bridge in ubiquitin bound to UCH37, an interaction that disappears when the ULD is deleted, as revealed in the structure of the catalytic domain alone bound to UbVME. The structural data are consistent with previously reported mutational data on the mammalian enzyme, which, together with the fact that the ULD residues that bind to ubiquitin are conserved, points to a similar mechanism behind the exo specificity of the human enzyme. To the best of our knowledge, these data provide the only structural example so far of how the exo specificity of a DUB can be determined by its non-catalytic domain. Thus, our data show that, contrary to its proposed inhibitory role, the ULD actually contributes to substrate recognition and could be a major determinant of proteasome-associated function of UCH37. Moreover, our structures show that the unproductively oriented catalytic cysteine in the free enzyme is aligned correctly when ubiquitin binds, suggesting a mechanism for ubiquitin selectivity.
PMCID: PMC3898853  PMID: 23617878
20.  Dual roles for ubiquitination in the processing of sperm organelles after fertilization 
The process of fertilization involves a cell fusion event between the sperm and oocyte. Although sperm contain mitochondria when they fuse with the oocyte, paternal mitochondrial genomes do not persist in offspring and, thus, mitochondrial inheritance is maternal in most animals. Recent evidence suggests that paternal mitochondria may be eliminated via autophagy after fertilization. In C. elegans, sperm-specific organelles called membraneous organelles (MO) cluster together with paternal mitochondria immediately after fertilization. These MOs but not the mitochondria become polyubiquitinated and associated with proteasomes. The current model for the elimination of paternal mitochondria in C. elegans is that ubiquitination of the MOs induces the formation of autophagosomes which also capture the mitochondria and cause their degradation.
Sperm-derived mitochondria and MOs show a sharp decrease in number during the time between sperm-oocyte fusion and the onset of mitosis. During this time, paternal mitochondria remain closely clustered with the MOs. Two types of polyubiquitin chains are observed on the MOs: K48-linked ubiquitin chains which are known to lead to proteasomal degradation and K63-linked ubiquitin chains which have been linked to autophagy. K48-linked ubiquitin chains and proteasomes show up on MOs very soon after sperm-oocyte fusion. These are present on MOs for only a short period of time. Maternal proteasomes localize to MOs and sperm proteasomes localize to structures that are at the periphery of the MO cluster suggesting that these two proteasome populations may have different roles in degrading paternal material. K63-linked ubiquitin chains appear on MOs early and remain throughout the first several cell divisions.
Since there are two different types of polyubiquitin chains associated with sperm organelles and their timing differs, it suggests that ubiquitin has two or more roles in the processing of sperm components after fertilization. The K63 chains potentially provide a signal for autophagy of paternal organelles, whereas the K48 chains and proteasomes may be involved in degradation of specific proteins.
PMCID: PMC3937010  PMID: 24528894
21.  ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination 
Cancer research  2010;70(9):3709-3717.
The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185 kDa ErbB2 receptor is rapidly (2–6 h) induced by the HSP90 chaperone inhibitor, geldanamycin (GA), while its downregulation and lysosomal degradation are more slowly (24 h) induced by the proteasome inhibitor, bortezomib/PS341. In PS341 treated SK-BR-3 cells, overexpressed ErbB2 co-precipitates with the E3 ubiquitin ligase, c-Cbl, and also with the deubiquitinating enzyme, USP9x; moreover, siRNA downregulation of USP9x enhances PS341 induced ErbB2 down-regulation. Since polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM) mass spectrometry (MS) and polyubiquitin linkage-specific antibodies were used to quantitatively track K48 and K63 linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4- to 8-fold) and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20- to 40-fold) sequential rise in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces co-localization of K48 and K63 linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling appear to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms.
PMCID: PMC2862137  PMID: 20406983
ErbB2; breast cancer; lysine (K48-K63)-linked polyubiquitin; lysosome; proteasome; HSP90
22.  The loop-less tmCdc34 E2 mutant defective in polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2 
Cell Division  2011;6:7.
The S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown.
tmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tmCDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tmCDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure.
The catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.
PMCID: PMC3080790  PMID: 21453497
23.  Polyubiquitin chains: functions, structures, and mechanisms 
Ubiquitin is a highly conserved 76-amino acid polypeptide that is found throughout the eukaryotic kingdom. The covalent conjugation of ubiquitin (often in the form of polymers) to substrates governs a variety of biological processes ranging from proteolysis to DNA damage tolerance. The functional flexibility of this post-translational modification roots in the existence of a large number of ubiquitinating enzymes that catalyze the formation of distinct ubiquitin polymers, which in turn encode different signals. This review summarizes the recent advances in the field with an emphasis on the non-canonical functions of polyubiquitination. We also discuss the potential mechanism of chain linkage specification as well as how structural disparity in ubiquitin polymers may be distinguished by ubiquitin receptors to translate the versatile ubiquitin signals into various cellular functions.
PMCID: PMC2700825  PMID: 18438605
ubiquitin; polyubiquitination; p97; UBA; proteasome; autophagy
24.  Molecular Basis and Regulation of OTULIN-LUBAC Interaction 
Molecular Cell  2014;54(3):335-348.
The linear ubiquitin (Ub) chain assembly complex (LUBAC) generates Met1-linked “linear” Ub chains that regulate the activation of the nuclear factor κB (NFκB) transcription factor and other processes. We recently discovered OTULIN as a deubiquitinase that specifically cleaves Met1-linked polyUb. Now, we show that OTULIN binds via a conserved PUB-interacting motif (PIM) to the PUB domain of the LUBAC component HOIP. Crystal structures and nuclear magnetic resonance experiments reveal the molecular basis for the high-affinity interaction and explain why OTULIN binds the HOIP PUB domain specifically. Analysis of LUBAC-induced NFκB signaling suggests that OTULIN needs to be present on LUBAC in order to restrict Met1-polyUb signaling. Moreover, LUBAC-OTULIN complex formation is regulated by OTULIN phosphorylation in the PIM. Phosphorylation of OTULIN prevents HOIP binding, whereas unphosphorylated OTULIN is part of the endogenous LUBAC complex. Our work exemplifies how coordination of ubiquitin assembly and disassembly activities in protein complexes regulates individual Ub linkage types.
Graphical Abstract
•OTULIN binds the HOIP PUB domain via a conserved N-terminal PUB-interacting motif•Structural studies reveal specificity determinants for the binary interaction•Loss of HOIP-OTULIN interaction causes deregulated accumulation of Met1-polyUb•OTULIN binding to LUBAC is regulated by phosphorylation
Elliott et al. show how the linear polyubiquitin-specific deubiquitinase OTULIN binds the linear ubiquitin chain assembly complex (LUBAC). The highly specific nanomolar interaction is mediated by the PUB domain of HOIP and an internal PUB-interacting motif (PIM) in OTULIN. Phosphorylation of the Tyr in the PIM regulates endogenous complex formation.
PMCID: PMC4017264  PMID: 24726323
25.  Molecular Basis for the Unique De-ubiquitinating Activity of the NF-κB Inhibitor A20 
Journal of molecular biology  2007;376(2):526-540.
NF-κB activation in the TNF, IL-1 and Toll-like receptor pathways requires Lys63-linked non-degradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways and possesses dual ubiquitin editing functions. While the N-terminal domain of A20 is a de-ubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the TNF, IL-1 and Toll-like receptor pathways.
PMCID: PMC2346432  PMID: 18164316
A20; crystal structure; de-ubiquitination; DUB; TRAF6

Results 1-25 (1331253)