Unanchored Ubiquitin Chains Do Not Lead to Marked Genetic Alterations in Drosophila melanogaster
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ABSTRACT:
PROVIDER: PRJNA515202 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8443370.fastq.gz | Fastqsanger.gz | |||
| SRR8443371.fastq.gz | Fastqsanger.gz | |||
| SRR8443372.fastq.gz | Fastqsanger.gz | |||
| SRR8443373.fastq.gz | Fastqsanger.gz | |||
| SRR8443374.fastq.gz | Fastqsanger.gz |
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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.
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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.
2021-06-28 | PXD019771 | Pride
Project description:STING activation by cyclic dinucleotides in mammals induces Type I Interferon- and NFkB-related gene expression, and the lipidation of LC3B at Golgi membranes. While the mechanisms of the Interferon-related responses are well understood, the mechanism of NFkB activation mediated by STING activation, and how the multi-functional responses downstream of STING signaling are related remain unknown. We report that STING activation induces K63 and linear ubiquitin chain formation, and ubiquitin co-localizes with STING and LC3B at Golgi-derived vesicles. Loss of the LUBAC E3 ubiquitin ligase subunit, HOIP, prevents formation of the linear, but not K63-linked, ubiquitin chains. The ubiquitin and LC3B binding proteins, Optineurin and TNIP1, are co-localized with STING activation-induced, perinuclear LC3B foci via their UBAN domains, however loss of HOIP does not influence this localization, indicating they may bind the K63-linked ubiquitin chains. HOIP-mediated linear ubiquitin chains are well established to mediate NFkB downstream of TNFa and IL1beta receptor stimulation. Loss of HOIP in monocytic THP-1 cells inhibits STING-induced NFkB activation and NFkB-related gene transcription, as well as Interferon-related transcription. Further, the recently reported proton channel activity of STING that blocks LC3B lipidation at Golgi membranes is also important for both K63 and Linear ubiquitin chain formation, and NFkB signaling. Thus, STING induces HOIP and linear ubiquitin chain formation to activate NFkB.
| MSV000092940 | MassIVE
Project description:Proteomic changes induced by linear ubiquitin chains in Drosophila
2024-09-06 | PXD052020 | Pride
Project description:Reversibility of protein ubiquitylation play essential roles in cellular protein homeostasis. How substrates stabilized by deubiquitylation are directed for degradation remains largely elusive. Here, we show that the branched ubiquitin chains promote the degradation of the deubiquitylase (DUB) OTUD5. OTUD5 is sequentially modified by TRIP12 and UBR5, E3s specific for the K29 and K48 linkages, respectively, resulting in the conjugation of K29/K48 branched ubiquitin chains. The TRIP12-OTUD5 antagonism regulates TNF--induced NF-B signaling. Mechanistically, while OTUD5 readily cleaves K48-linkages, K29-linkages are resistant against OTUD5 activity. Consequently, K29-linkages overcome OTUD5 DUB activity to facilitate UBR5-dependent K48-linked chain branching. Regarding generality, this mechanism adopts to other TRIP12 substrates associated with OTUD5. These results uncover a cellular unique strategy in which the sequential addition of DUB-resistant and proteasome-targeting ubiquitin linkages efficiently promote degradation of substrates protected by deubiquitylation, underscoring the role of branched ubiquitin chains in the quality control of hard-to-degrade substrates.
2025-02-19 | GSE273273 | GEO
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:AMYLOIDOGENIC LIGHT CHAINS IMPAIR PLASMA CELL SURVIVAL
| PRJNA734939 | ENA