Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

K48-K63-Ubiquitin chain pulldown from U2OS cell lysates

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Frederic Lamoliatte

LAB HEAD: Dr Yogesh Kulathu

PROVIDER: PXD046025 | Pride | 2024-05-07

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	DiaNN_Search.zip	Other
	FL_20220411_Sven_YK_01.raw	Raw
	FL_20220411_Sven_YK_02.raw	Raw
	FL_20220411_Sven_YK_03.raw	Raw
	FL_20220411_Sven_YK_04.raw	Raw

Items per page:

1 - 5 of 18

Similar Datasets

The UBA domain of Ubc1 (Ube2K) Facilitates the Assembly of K48/K63-Branched Ubiquitin Chains

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

The UBA-domain of Ubc1/Ube2K Facilitates the Assembly of K48-/K63-Branched Ubiquitin Chains

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

Ubiquitination profile of polarized macrophages

Project description:Previous studies linked mac activation with the ubiquitination (ub) status of key proteins in inflammatory signal pathways. Lysine-48 ub (K48-ub) of the NF-κB inhibitor (IκB), the final checkpoint of the NFκB pathway, leads to IκB degradation and consequent NF-κB-associated inflammation. In contrast, TRAF6, an upstream positive regulator of the NF-κB pathway, is modulated by K63-ub, leading to its conformational change and activation. However, a comprehensive analysis of ub profiles of polarized macs has not been reported. If a unique ub signature is associated with individual mac subsets is not known.

2020-08-06 | PXD018743 | Pride

K48- and K63-linked ubiquitin chain interactome reveals branch- and length-specific ubiquitin interactors

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

UbiREAD deciphers proteasomal degradation code of homotypic and branched K48 and K63 ubiquitin chains

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

Covalent inhibition of Plasmodium falciparum Ubc13 impairs global protein synthesis

Project description:The ubiquitin-conjugating enzyme 13 (Ubc13) has an essential function and putative role in artemisinin activity against Plasmodium falciparum. Ubc13 conjugates lysine 63-linked ubiquitin (K63-Ub) to proteins, but the role of this modification in Plasmodium remains largely unknown. Herein, we characterize and deploy NSC697923 to interrogate PfUbc13 function. We demonstrate that NSC697923 covalently targets the PfUbc13 active site residue and exhibits nanomolar inhibitory potency. NSC697923 displays anti-Plasmodium activity against drug-sensitive and drug-resistant asexual strains, gametocytes, and liver stage parasites, and synergizes with the malaria drug dihydroartemisinin. NSC697923 specifically reduces K63-Ub levels in asexual blood stage parasites, and subsequent ubiquitin pull-down assays and proteomic analyses identified putative PfUbc13 substrates. We highlight 37 proteins that overlapped with two previous ubiquitin datasets and were enriched in translation, transcription, and proteasome processes. A cellular puromycin-incorporation study demonstrated reduction of P. falciparum nascent protein synthesis following NSC697923 exposure, supporting a role for PfUbc13 and K63-Ub in mediating parasite protein translation. These findings expand our knowledge of the PfUbc13-dependent processes in these pathogenic parasites and highlight this enzyme as a potential antimalarial drug target.

2025-04-29 | PXD059643 | Pride

The RNA Helicase DHX16 Provides Specific Viral RNA for RIG-I to Trigger Innate Antiviral Immunity

Project description:Type-I interferon (IFN-I) is essential to establish antiviral innate immunity. Unanchored (or free) polyubiquitin (poly-Ub) has been shown to regulate IFN-I responses, however few unanchored poly-Ub interactors are known. To identify factors regulated by unanchored poly-Ub in a physiological setting, we developed an approach to isolate unanchored poly-Ub from lung tissue and identified the DEAH-box RNA helicase DHX16 as a factor that enhances IFN-I production. Silencing of DHX16 in cells and in vivo diminished IFN-I responses against influenza virus. DHX16-dependent IFN-I production requires RIG-I and unanchored K48-poly-Ub synthesized by the E3 Ub ligase TRIM6. DHX16 recognizes a signal in influenza segments that undergo splicing and requires its intact RNA helicase motif for direct, high-affinity interactions with specific viral RNAs. Our study establishes DHX16 as a potentiator of RIG-I-mediated IFN-I production and recognizes a mechanism for activation of antiviral immunity requiring unanchored poly-Ub and the viral RNA recognition activity of DHX16

2022-03-08 | GSE168255 | GEO

Transcription profiling of wild type and trienoic fatty acid deficient mutant fad3-2/fad7-2/fad8 Arabidopsis plants in control conditions

Project description:comparison of the transcriptome of Columbia-0 WT and the trienoic fatty acid deficient mutant fad3-2/fad7-2/fad8 in control conditions.

2008-02-01 | E-ATMX-37 | biostudies-arrayexpress

Transcription profiling of wild type and jasmonate deficient mutant aos Arabidopsis plants in control conditions

Project description:comparison of the transcriptome of Columbia WT and the jasmonate deficient mutant aos (allene oxide synthase)in control conditions.

2008-02-01 | E-ATMX-36 | biostudies-arrayexpress

Profiling linkage-specific ubiquitin chain functions reveals a key role of K29-linked ubiquitylation in epigenome integrity

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 integrity.

2025-09-17 | GSE279218 | GEO