Unanchored Ubiquitin Chains Do Not Lead to Marked Genetic Alterations in Drosophila melanogaster
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PROVIDER: PRJNA515202 | ENA |
REPOSITORIES: ENA
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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.
2019-05-01 | GSE125090 | GEO
Project description:The small protein modifier ubiquitin regulates various aspects of cellular biology through its chemical conjugation onto proteins. Ubiquitination of proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Ubiquitin chains can be attached onto other proteins or can exist as unanchored species, i.e. free from another protein. Unanchored ubiquitin chains are thought to be deleterious to the cell and rapidly disassembled into mono-ubiquitin. We recently examined the toxicity and utilization of unanchored poly-ubiquitin in Drosophila melanogaster We found that free poly-ubiquitin species are largely innocuous to flies and that free poly-ubiquitin can be controlled by being degraded by the proteasome or by being conjugated onto another protein as a single unit. Here, to explore whether an organismal defense is mounted against unanchored chains, we conducted RNA-Seq analyses to examine the transcriptomic impact of free poly-ubiquitin 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 essentially devoid of ubiquitin-, proteasome-, or autophagy-related components. The seeming absence of a large and multipronged response to unanchored poly-ubiquitin supports the conclusion that these species need not be toxic in vivo and underscores the need to re-examine the role of free ubiquitin chains in the cell.
| S-EPMC6550069 | biostudies-literature
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.
2021-06-28 | PXD019771 | Pride
Project description:Proteomic changes induced by linear ubiquitin chains in Drosophila
2024-09-06 | PXD052020 | Pride
Project description:SUMOylation, a protein post-translational modification present in all eukaryotes, involves the covalent attachment of SUMO (small ubiquitin-like modifier) to target proteins, modulating their function, localization or stability. One of the least understood features of SUMOylation is formation of polymeric chains and their impact on DNA metabolism. Here, using Schizosaccharomyces pombe,we demonstrated that cells devoid of SUMO chains exhibit elevated spontaneous replication stress and DNA damage. We found that SUMO chains promoted rDNA stability,controlled proper centromeric organization, and in their absence recombination factors accessed more frequently both of those loci. We directly showed increased fork stalling at rDNA and measured higher recombination rates at centromeres upon SUMO chains deficiency. Moreover, we successfully established the split-SUMO-ID proteomics approach in fission yeast to identify SUMO- and Rad52-dependent interactome. Our results suggest that SUMO chains maintain the stability of difficult-to-replicate loci by limiting the access of recombination factors to stalled replication forks.
2024-09-15 | GSE276805 | GEO
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:Met1 type ubiquitination and deubiquitination are involved in the regulation of many fundamental processes such as inflammation and innate immunity, and their interference by pathogens can suppress immune responses in human cells. However, few plant-derived deubiquitinases (DUBs) against Met1 ubiquitin chains has been reported, and the selection mechanism for linkage-type of ubiquitin chains remains elusive, which greatly limits our understanding of the functions of plant-derived DUBs. Using a dehydroalanine (DHA)-bearing Met1 diubiquitin (Met1-diUb) suicide probe, synthesized in one-pot, we captured OTUB1 from Oryza sativa (OsOTUB1) and uncovered its ability to hydrolyze Met1 ubiquitin chains (Met1 activity). Further, by resolving the apo structure of OsOTUB1 and its complex with Met1-diUb, we found that Met1-specific motifs (N-handle motif and C-handle motif) in the S1’ pocket of OsOTUB1 confers its Met1 activity. Through large-scale sequence alignment and hydrolysis experiments, Met1-specific motifs in the S1' pocket of the OTUB subfamily (OTUBs) were found to determine the Met1 ability of OTUBs, regardless of species. Furthermore, by analyzing the species distribution of OTUBs capable of hydrolyzing Met1 ubiquitin chains, we found that whereas this activity does not exist in metazoans, it is conserved in green plants (Viridiplantae). This discovery may inform studies of the differentiation between primitive plants and animals.
2022-10-15 | PXD032822 | Pride
Project description:Our understanding of the functions of DNA elements is limited by the paucity of information about the spectrum of proteins that occupy these genomic regions. Here we describe an approach to identify proteins associated with genomic regions whose chromatin is marked by specific modified histones, which we term chromatin profiling. We used chromatin immunoprecipitation followed by mass spectrometry (ChIP-MS) to identify proteins associated with genomic regions marked by histone H3K27Ac, H3K4me3, H3K79me2 and H3K36me3 in mouse embryonic stem (mES) cells. We identified 385 known and 224 novel candidate proteins associated with these histone-marked genomic segments and confirmed that several of the novel candidates are indeed associated with histone-marked segments of the genome. Future study of the novel candidates, many of which have been implicated in various diseases, should lead to an improved understanding of gene control and its dysregulation in disease. ChIP-seq for nucleosomes with modified histones and DNA-binding proteins in mouse embryonic stem cells, and DNA-binding proteins in Jurkat cells
2015-02-05 | E-GEOD-62380 | biostudies-arrayexpress
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.
2021-03-02 | PXD022621 | Pride
Project description:Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with Ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs), Ubp2 and Ubp14, and E3 ligases, Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the Ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the Intranuclear Quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with Ribosomopathies.
2023-05-16 | GSE232351 | GEO