Insights Into Ubiquitin Chain Architecture Using Ub-Clipping
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
David Komander
PROVIDER: MSV000083662 | MassIVE | Wed Apr 03 15:16:00 BST 2019
REPOSITORIES: MassIVE
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Project description:Mass spectrometry-based proteomics has become an integral approach for characterising ubiquitin chain-linkage compositions and architectures. In this study, we optimised sample preparation and chromatographic separation of Ubiquitin peptides for Absolute Quantification by Parallel Reaction Monitoring (Ub-AQUA-PRM). Using this refined Ub-AQUA-PRM assay, we were able to quantify all ubiquitin chain types in 10-minute LC-MS/MS runs. We used this method to determine the ubiquitin chain-linkage composition in murine bone marrow-derived macrophages (BMDMs) and different mouse tissues. We could show tissue-specific differences in ubiquitin levels in murine tissues, with polyubiquitin chain types contributing a small proportion to the total pool of ubiquitin. Interestingly, we observed enrichment of atypical ubiquitin chain types (K29 and K33) in heart and muscle. Ubiquitin chain topology profiling using tandem ubiquitin binding entities, directed at K29/K33 ubiquitin chains (TRABID), and mass spectrometry analysis identified several mitochondrial proteins as putatively associated with these atypical ubiquitin chain types. Our approach enabled high-throughput screening of ubiquitin chain-linkage composition in different murine tissues and highlighted a possible role for atypical ubiquitylation in contractile tissues. Our results contribute to our understanding of in vivo ubiquitin chain-linkage composition in murine tissues.
2020-09-28 | PXD017754 | Pride
Project description:The attachment of differently linked ubiquitin (Ub) chains of varying length to proteins is a prevalent posttranslational modification in eukaryotic cells. The fate of a modified protein is determined by Ub-binding proteins (UBPs) that interact with Ub chains in a linkage-selective manner. Therefore, proteome-wide interaction studies using differently linked Ub chains have become a focus of research activities. However, the impact and functional consequences of chain length on the binding selectivity of UBPs remain mostly elusive, due to a lack of available tools and sufficient amounts of pure, length-defined Ub chains. Here we generated linkage- and length-defined Ub chains using click-chemistry and gel-free fractionation and employed such defined polymers in affinity-based enrichment assays to identify length- and linkage-selective interactors on a proteome-wide scale. For the first time, this revealed that the length of a Ub chain has generally a major impact on its ability to be selectively recognized by UBPs.
2020-04-20 | PXD015877 | Pride
Project description:Assembly of polymeric ubiquitin (Ub) chains is an essential posttranslational protein modification that regulates widespread intracellular processes in eukaryotic cells. Factors and mechanisms that regulate the formation of Ub chains are key to the understanding of many cellular processes. The E2 enzyme Ubc1 (Ube2K) exclusively targets K48 in Ub for chain formation and has been linked to cell-cycle progression and proteostasis. Uniquely among E2 enzymes, it harbors a Ub binding UBA domain, which has been implicated in Ub chain formation but its function remained elusive. Through in vitro binding experiments, we unexpectedly found that the UBA domain enables preferential binding of K63-linked Ub chains. Based on structural modeling, extensive in vitro ubiquitination experiments and NMR binding studies, we propose a mechanism through which Ubc1 selectively forms K48/K63 branched Ub chains – an usual chain architecture, about whose prevalence and function little is known to date. Ultimately, we link UBA dependent activity of Ubc1 to its role in proteostasis through genetic experiments.
2021-09-09 | PXD018651 | Pride
Project description:Ubiquitin-interactor co-pulldown coupled with mass spectrometry used to elucidate K48- and K63-linked interactomes including those of Ub chains of varying length (Ub-Ub3) and heterotypic branched Ub chains. Ubiquitin-interactors were enriched from human HeLa and yeast s. cerevisiae cell lysate with either chloroacetamide (CAA) or N-ethylmaleimide (NEM) as DUB inhibitors.
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Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.
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Project description:Protein ubiquitination is a multi-functional post-translational modification that affects all cellular processes. Its versatility arises from architecturally complex polyubiquitin chains, in which individual ubiquitin moieties may be ubiquitinated on one or multiple residues, and/or modified by phosphorylation and acetylation1-3. Advances in mass spectrometry have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code; however, the architecture of polyubiquitin signals has remained largely inaccessible. Here we introduce Ub-clipping as a methodology by which to understand polyubiquitin signals and architectures. Ub-clipping uses an engineered viral protease, Lbpro∗, to incompletely remove ubiquitin from substrates and leave the signature C-terminal GlyGly dipeptide attached to the modified residue; this simplifies the direct assessment of protein ubiquitination on substrates and within polyubiquitin. Monoubiquitin generated by Lbpro∗ retains GlyGly-modified residues, enabling the quantification of multiply GlyGly-modified branch-point ubiquitin. Notably, we find that a large amount (10-20%) of ubiquitin in polymers seems to exist as branched chains. Moreover, Ub-clipping enables the assessment of co-existing ubiquitin modifications. The analysis of depolarized mitochondria reveals that PINK1/parkin-mediated mitophagy predominantly exploits mono- and short-chain polyubiquitin, in which phosphorylated ubiquitin moieties are not further modified. Ub-clipping can therefore provide insight into the combinatorial complexity and architecture of the ubiquitin code.
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Project description:SpyTagged branched and unbranched K48- and K63-linked Tetra-Ubiquitin chains were immobilized on SpyCatcher agarose (48Ub4; 63Ub4; [Ub]2-48,63Ub-48Ub; [Ub]2-48,63Ub-63Ub). Pulldown were performed from protease-inhibitor treated U2OS cell lines to identify specific binders of K48-K63-branched Ub4.
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Project description:In this experiments, we used several ubiquitin activity-based probes (Ub-Prg, Ub-VME, and Ub-VS) to covalently capture ubiquitin-interacting proteins in Legionella pneumophila. These enriched interactome were analyzed by LC-MS/MS to identify Legionella effectors uniquely captured by the probes.
2023-12-09 | MSV000093623 | MassIVE
Project description:The assembly of a specific polymeric ubiquitin (Ub) chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The ubiquitin-conjugating (E2) enzymes Ubc1 in yeast and Ube2K in mammals exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a Ub binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with Ub chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched Ub molecules. Genetic experiments link the activity of the UBA domain and hence the formation of this unusual Ub chain topology to the maintenance of cellular proteostasis.
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Project description:The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.
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