Browse
Submit Data
Databases
API
Help

Dataset Information

219 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Characterisation of protein ubiquitination using UbiSite technology

ABSTRACT: We present a novel immune-affinity-based method UbiSite for capture and further identification of ubiquitination sites by MS for any cell line or tissue sample. Implementing the UbiSite methodology for a large-scale experiment we successfully mapped 62000 unique ubiquitination sites on protein substrates for two different human cell lines.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christian Kelstrup

LAB HEAD: Blagoy Blagoev

PROVIDER: PXD009158 | Pride | 2018-07-10

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	20171221_QE7_nL3_DBJ_SA_COLLAB_BAB_DIAprot_Hep2_A15_01.raw	Raw
	20171221_QE7_nL3_DBJ_SA_COLLAB_BAB_DIAprot_Hep2_A15_02.raw	Raw
	20171221_QE7_nL3_DBJ_SA_COLLAB_BAB_DIAprot_Hep2_A15_03.raw	Raw
	20171221_QE7_nL3_DBJ_SA_COLLAB_BAB_DIAprot_Hep2_A15_04.raw	Raw
	20171221_QE7_nL3_DBJ_SA_COLLAB_BAB_DIAprot_Hep2_A15_05.raw	Raw

Items per page:

1 - 5 of 34

Similar Datasets

Characterisation of protein ubiquitination using UbiSite technology

Project description:We present a novel immune-affinity-based method UbiSite for capture and further identification of ubiquitination sites by MS for any cell line or tissue sample. Implementing the UbiSite methodology for a large-scale experiment we successfully mapped 62000 unique ubiquitination sites on protein substrates for two different human cell lines.

2018-07-03 | PXD006201 | Pride

Characterisation of protein ubiquitination using UbiSite technology

2018-07-03 | PXD006299 | Pride

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

Project description:The ubiquitin-proteasome axis has been extensively explored at a system-wide level, but the impact of deubiquitinating enzymes (DUBs) on the ubiquitinome remains largely unknown. Using UbiSite technology and inhibitors, we have compared the contributions of the proteasome and DUBs on the ubiquitinome. We uncovered large differential dynamic Ub signalling networks with substrates and sites uniquely regulated by DUBs or by the proteasome, highlighting the role of DUBs in degradation-independent ubiquitin signalling. DUBs regulate substrates via nearly 40,000 unique sites. Moreover, we found that ubiquitin conjugated to SUMO2/3 forms a unidirectional proteasomal degradation signal with strikingly rapid kinetics compared to ubiquitin polymers only. We found that PARP1, is hyper ubiquitinated in response to DUB inhibition, increasing its enzymatic activity. Our findings highlight the key regulatory roles of DUBs on ubiquitin dynamics.

2022-05-23 | PXD027328 | Pride

Deubiquitinating enzymes and the proteasome regulate unique sets of ubiquitin substrates.

2022-05-23 | PXD030644 | Pride

Mechanism of degrader-targeted protein ubiquitinability

Project description:Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. Mass spectrometry illuminates a patch of favourably ubiquitinable lysines on one face of in vitro ubiquitinated Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack highlighted by cryo-EM. In vitro ubiquitination coupled with cellular degradation and ubiquitinomics confirm the importance of Lys456 and the ‘ubiquitination zone’ lysines. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination bu UBE2R1, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization.

2024-11-08 | PXD053502 | Pride

Temporal Analysis of Cell Protein Ubiquitination during Epstein-Barr Virus Reactivation by BCR Cross-linking

Project description:Epstein-Barr virus (EBV) reactivation in latently infected B cells is essential for persistent infection and B cell receptor (BCR) activation is a physiologically relevant stimulus for EBV reactivation. Post-translational modifications, such as phosphorylation and ubiquitination, are known to be regulated by antigen binding to BCR within minutes. However, a detailed understanding of the signaling alterations at later time when EBV is being actively replicated remains elusive. To gain insights into BCR activation-mediated reprogramming of the cellular environment in both Akata-BX1 (EBV+) and Akata-4E3 (EBV-) B cells, we utilized a 3-plex stable isotope labeling by amino acid in cell culture (SILAC)-based quantitative proteomic approach to monitor the dynamic changes of protein ubiquitination during the course of immunoglobulin G (IgG) cross-linking of BCRs. We observed temporal alterations in the level of ubiquitination on approximately 150 sites in both Akata-BX1 (EBV+) and Akata-4E3 (EBV-) B cells post-IgG cross-linking compared with no cross-linking controls, with the majority of protein ubiquitination down-regulated. Our analysis revealed that IgG cross-linking plays a major role in the regulation of protein ubiquitination in both EBV+ and EBV- B cells. Bioinformatic analyses of up-regulated ubiquitination events revealed significant enrichment of proteins involved in RNA processing. Among the down-regulated ubiquitination events are proteins enriched in apoptosis and the ubiquitin-proteasome pathway. The comparative and quantitative studies provide a foundation for further understanding how BCR activation regulates cellular protein ubiquitination and how EBV utilizes or subverts BCR engagement-mediated changes to facilitate viral replication.

2017-02-02 | PXD004562 | Pride

Large-scale identification of ubiquitination sites on membrane-associated proteins in Arabidopsis thaliana seedlings

Project description:Protein phosphorylation and ubiquitination are two of the most abundant forms of post-translational modifications in eukaryotes, regulated by thousands of protein kinases, phosphatases, E3 ubiquitin ligases, and ubiquitin proteases. Although previous studies have catalogued several ubiquitinated proteins in plants (Walton et al., 2016), few membrane-localized proteins have been identified. Receptor kinases (RKs) initiate phosphorylation signal relays that regulate plant growth, development, and stress responses. While the regulatory role of phosphorylation on protein kinase function is well-documented (Couto and Zipfel, 2016), considerably less is known about the role of ubiquitination on protein kinase function, even though protein turnover is critical to their signaling competence and cellular homeostasis. Here we describe the large-scale identification of ubiquitination sites on Arabidopsis proteins associated with or integral to the plasma membrane, including over 100 protein kinases.

2021-02-01 | PXD021992 | Pride

Methylation profiling by array shows that mIDH-associated DNA hypermethylation in acute myeloid leukemia reflects differentiation blockage rather than inhibition of TET-mediated demethylation

Project description:Isocitrate dehydrogenases 1 and 2 (IDH1/2) are recurrently mutated in acute myeloid leukemia (AML), but their mechanistic role in leukemogenesis is poorly understood. The inhibition of TET enzymes by D-2-hydroxyglutarate (D-2-HG), which is produced by mutant IDH1/2 (mIDH1/2), has been suggested to promote epigenetic deregulation during tumorigenesis. In addition, mIDH also induces a differentiation block in various cell culture and mouse models. Here we analyze the genomic methylation patterns of AML patients with mIDH using Infinium 450K data from a large AML cohort and found that mIDH is associated with pronounced DNA hypermethylation at tens of thousands of CpGs. Interestingly, however, myeloid leukemia cells overexpressing mIDH, cells that were cultured in the presence of D-2-HG or TET2 mutant AML patients did not show similar methylation changes. In further analyses, we also characterized the methylation landscapes of myeloid progenitor cells and analyzed their relationship to mIDH-associated hypermethylation. Our findings identify the differentiation state of myeloid cells, rather than inhibition of TET-mediated DNA demethylation, as a major factor of mIDH-associated hypermethylation in AML. Furthermore, our results are also important for understanding the mode of action of currently developed mIDH inhibitors.

2017-09-15 | E-MTAB-6059 | biostudies-arrayexpress

Data-independent acquisition method for ubiquitinome analysis reveals non-degrative regulation of circadian biology

Project description:Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent (DIA) acquisition with regards to sensitivity in single run analysis and data completeness have not yet been explored. We developed a sensitive workflow combining diGly antibody-based enrichment, optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identified 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells â€“ double the number and quantitative accuracy of data dependent acquisition. Applied to TNF-alpha signaling, the workflow comprehensively captured known sites while adding many novel ones. A first systems-wide investigation of ubiquitination of the circadian cycle uncovered hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting novel connections between metabolism and circadian regulation.

2020-11-20 | PXD019854 | Pride

Identification of CCNF/Cyclin F targets

Project description:The founding member of the F-box protein family, Cyclin F, serves as substrate adaptor for the Ubiquitin E3 ligase Skp1-Cul1-F-box (SCF)Cyclin F which is responsible for ubiquitination of proteins involved in cell cycle progression, DNA damage and mitotic fidelity. Missense mutations in CCNF encoding for Cyclin F are associated with amyotrophic lateral sclerosis (ALS). However, it remains elusive whether CCNF mutations affect the substrate adaptor function of Cyclin F and whether altered SCFCyclin F mediated ubiquitination contributes to ALS pathogenesis in CCNF mutation carrier. To start addressing these questions, we set out to identify new SCFCyclin F targets in neuronal and patient-derived cells. Mass spectrometry-based ubiquitinome profiling of CCNF knockout and mutant cell lines as well as Cyclin F proximity and interaction proteomics converged on the HSP90 chaperone machinery as new substrate candidate. Biochemical analyses confirmed the Cyclin F dependent binding and ubiquitination of HSP90AB1 and unveiled a regulatory role of this ubiquitination in controlling the binding of a number of HSP90 clients and co-factors, thereby implying chaperone dysregulation and CCNF loss-of-function mechanism in ALS.

2022-09-13 | PXD030729 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data