Branched-K29/K48 and K48/K29 polyUb_LC-MSMS
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human) Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Xiangwei Wu
LAB HEAD: Xiangwei Wu
PROVIDER: PXD062799 | Pride | 2025-04-10
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| K29_48-TRIP12-PRM.raw | Raw | |||
| K29_48-TRIP12.raw | Raw | |||
| K29_48-TRIP12.xlsx | Xlsx | |||
| K48-K29_Ufd2.raw | Raw | |||
| K48-K29_Ufd2.xlsx | Xlsx |
Items per page: 5 1 - 5 of 6 |
Similar Datasets
Project description:The protein ubiquitylation is under the equilibrium between ubiquitin conjugation and deconjugation. How substrates stabilized by deubiquitylation are directed for degradation remains unclear. Branched ubiquitin chains promote substrate degradation through the proteasome, but the underlying mechanisms are not fully understood. TRIP12 and UBR5 are HECT-type E3s specific for the K29 and K48 linkages, respectively. Here, we show that the deubiquitylase (DUB) OTUD5 is cooperatively modified by TRIP12 and UBR5, resulting in the conjugation of K29/K48 branched ubiquitin chains and accelerated proteasomal degradation. The TRIP12–OTUD5 antagonism regulates TNF-–induced NF-B signaling. Mechanistically, although 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. This mechanism is applicable to other TRIP12 substrates associated with OTUD5. These results reveal a unique cellular strategy in which the combination of DUB-resistant and proteasome-targeting ubiquitin linkages efficiently promotes the degradation of substrates protected by deubiquitylation, underscoring the role of branched ubiquitin chains in shifting the ubiquitin conjugation/deconjugation equilibrium.
2025-02-19 | GSE286567 | GEO
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:The OTU deubiquitinase TRABID is highly tuned for the recognition and cleavage of K29 and K33-linked ubiquitin chains. Yet the cellular functions of these atypical ubiquitin signals remain unclear. Here, we compared the interactome of two different catalytically-dead TRABID constructs, one that lacks the OTU domain and the other a single point mutant that is catalytically inactive. Since both constructs also act as ubiquitin trapping mutants, this approach was used to differentiate between interactors and substrates. The E3 ubiquitin ligase HECTD1 was confirmed as a TRABID substrate, and using UbiCREST and Ubiquitin-AQUA proteomics, we determined that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that in contrast to UBE3C, HECTD1 requires the formation of branched K29/K48-linked chains for its full activity. Finally, we used transient knockdown and genetic knock out of TRABID in mammalian cells to show that TRABID stabilises HECTD1 protein levels. This study identifies TRABID-HECTD1 as a K29-specific DUB/E3 pair and provides novel insights on the assembly of branched ubiquitin signals.
2021-06-23 | PXD022703 | Pride
Project description:Analysis of E14 embryonic stem cells lacking Trip12 ubiquitin ligase activity. We used microarrays to detail the global gene expression profiles affected by Trip12 ubiquitin ligase activity in mouse embryonic stem cell. Total RNA was extracted from wild-type and Trip12 mutant mouse embryonic stem cells and hybridized on Affymetrix microarrays.
2014-01-01 | E-GEOD-27527 | biostudies-arrayexpress
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.
2024-05-15 | PXD051890 | Pride
Project description:Linkage-specific ubiquitin (Ub) chains dictate the functional outcome of many critical Ub-dependent signaling processes. However, the functions and targets of several poly-Ub topologies remain poorly understood due to a lack of tools for their specific detection and manipulation. To remedy this knowledge gap, we applied a cell-based Ub replacement strategy enabling specific, conditional abrogation of each of the seven lysine-based Ub chain types in human cells to comprehensively profile global ubiquitylation changes resulting from disabling formation of individual Ub chain types. Focusing on K29-linked ubiquitylation, we found that this linkage type is enriched among chromatin-associated proteins, and that the H3K9me3 methyltransferase SUV39H1 is a prominent cellular target of this modification. We demonstrate that K29-linked ubiquitylation is essential for the proteasomal degradation of SUV39H1 despite its extensive modification by K48-linked ubiquitylation, and that K29-linked ubiquitylation of SUV39H1 is catalyzed and reversed by TRIP12 and TRABID, respectively. Importantly, preventing K29-linked ubiquitylation of SUV39H1 markedly increases H3K9me3 levels, but not other histone marks. Collectively, our Ub replacement cell line panel and datasets provide valuable resources for illuminating cellular functions of linkage-specific ubiquitylation processes and reveal a key role for K29-linked ubiquitylation in epigenome maintenance.
2025-09-18 | PXD056493 | Pride
Project description:Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.
| S-EPMC7948964 | biostudies-literature
Project description:Analysis of E14 embryonic stem cells lacking Trip12 ubiquitin ligase activity. We used microarrays to detail the global gene expression profiles affected by Trip12 ubiquitin ligase activity in mouse embryonic stem cell.
2014-01-01 | GSE27527 | GEO
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.
2024-05-07 | PXD046025 | Pride
Project description:Ubiquitin chains encode signals defining the fates of their modified proteins. Ubiquitin chain-mediated proteasomal degradation is a widespread eukaryotic regulatory mechanism. Yet, heterogeneity of intracellular ubiquitination has precluded systematically comparing degradation capacities of different ubiquitin chains. Here, we monitor cellular degradation and deubiquitination at high temporal resolution after delivery of bespoke ubiquitinated proteins into human cells. Comparing degradation of a model substrate modified with various K48, K63 or K48/K63-branched ubiquitin chains revealed fundamental differences in their intracellular degradation capacities. K48 chains with three or more ubiquitins triggered degradation within minutes. K63-ubiquitinated substrate was rapidly deubiquitinated rather than degraded. Surprisingly, degradation and deubiquitination behaviour of K48/K63-branched chains was determined by the identity of the substrate-anchored chain, establishing that branched chains are not the sum of their parts. Overall, our work reveals a degradation code for ubiquitin chains varying by linkage, length and topology and a functional hierarchy within branched ubiquitin chains.
2025-06-23 | PXD060731 | Pride