Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Interactome of S65 phosphorylated NEDD8 and Ub

ABSTRACT: In this project we present for the first time a proteome-wide interaction study of S65 phosphorylated NEDD8 and ubiquitin (Ub). By using cell extracts and affinity enrichment coupled to mass spectrometry, we show that pNEDD8 and pUb have different interactomes that are also distinct from those of the non-modified forms. Among the preferential pNEDD8 interactors are HSP70 family members and, indeed, pNEDD8 stimulates HSP70 ATPase activity more pronouncedly than non-modified NEDD8. Our findings provide evidence that phosphorylation of Ub and NEDD8 at S65 is involved in stress response signaling and underscore the importance of studying non-covalent interactions of NEDD8 in particular and post-translational modifications of Ubls in general.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Katrin Stuber

LAB HEAD: Prof. Dr. Martin Scheffner

PROVIDER: PXD021143 | Pride | 2021-11-03

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	HEK293_SEARCH.zip	Other
	Q2002_KS_200217_1_Beads_226.raw	Raw
	Q2002_KS_200217_1_Beads_227.raw	Raw
	Q2002_KS_200217_1_Nedd8_217.raw	Raw
	Q2002_KS_200217_1_Nedd8_218.raw	Raw

Items per page:

1 - 5 of 86

Similar Datasets

NEDD8 S65 phosphorylation under different stress conditions

Project description:In this project we present the identification of NEDD8 S65 phosphorylation under mitochondrial and DNA double strand break (DSB) stress conditions. Relative comparison revealed elevated levels of pNEDD8 under mitochondrial stress and reduced levels of pNEDD8 under DSB stress.

2021-11-03 | PXD027477 | Pride

MCF-7 PTEN-Nedd8 IP-MS

Project description:We intended to elucidate the underlying mechanism of PTEN-Nedd8 in tumorigenesis. So, we firstly employed affinity purification and mass spectrometry to interrogate PTEN-Nedd8 interactome in nucleus.

2022-05-20 | PXD011395 | Pride

MCF-7 Flag-need8 IP-MS

Project description:To allow the proteomic identification of proteins modified with Nedd8, we firstly employed affinity purification and mass spectrometry to interrogate Nedd8 interactome.

2022-04-01 | PXD011368 | Pride

NEDD8 subtrates from NEDP1 knockout cells

Project description:The aim of the study was to identify proteins that are modified by NEDD8. The NEDD8 specific protease NEDP1/SENP8 was deleted from HEK 293 cells via Crispr/CAS9 gene editing to allow for the accumulation of NEDD8 modified proteins. Lysates from NEDP1 KO cells were then enriched via pulldown with a catalytically inactivated NEDP1 (C162A) fused to the HALO protein. For a negative control a mutated NEDP1 (DAGC) with reduced binding to NEDD8 was also fused to the HALO protein for pulldown. Pulldowns were resolved by SDS-PAGE and bands were excised and subjected to in gel trypsin digestion followed by mass spectrometry analysis.

2019-02-20 | PXD011928 | Pride

Identification of NEDD8 sites at endogenous levels of NEDD8 expression upon NEDP1 (SENP8, DEN1) knockout.

Project description:We developed a strategy that allows the identification of NEDP1 dependent NEDDylation sites under endogenous expression of wild type NEDD8. We combined the use of anti-diGly antibodies that recognise both ubiquitin and NEDD8 modified peptides upon trypsin digestion with short treatment of cells with the ubiquitin E1 inhibitor MLN7243 (UAEi), that dramatically reduces ubiquitin but not NEDD8 modification. By eliminating the majority of ubiquitin-derived diGly peptides upon MLN7243 treatment, we would be able to quantify NEDP1 dependent diGly peptides. Extracts from parental and NEDP1 knockout HCT116 cells both treated with UAEi were used for the isolation of diGly modified peptides and mass spectrometry analysis.

2023-05-10 | PXD040466 | Pride

Comparison of LPS-induced gene expression in NEDD8-sufficient or -deficient primary macrophages

Project description:Bone marrow-derived macrophages (BMMs) from Nedd8(flox/flox) mice or Nedd8(flox/flox; lyz2-cre) mice were stimulated with or without 100ng/ml LPS for 4 hours. The total RNA was prepared and subjected to microarray.

2020-05-26 | GSE99386 | GEO

Gene expression profiles of myeloma cells treated with NAE inhibitor

Project description:The transcriptional profile of the human multiple myeloma (MM) cell line MM.1S treated with MLN4924 vs control MM.1S cells was characterized by oligonucleotide microarray analysis, using the human U133 plus 2.0 Affymetrix GeneChip, according to previously described protocols for total RNA extraction and purification; cDNA synthesis; in vitro transcription reaction for production of biotin-labeled cRNA; hybridization of cRNA with U133plus2.0 Affymetrix gene chips; and scanning of image output files. Scanned image output files were analyzed using DNA-Chip Analyzer (dChip) (www.dchip.org), including conversion to DCP files, normalization and modeling. The gene expression profile of MM.1S cells for each time point of MLN4924 treatment was compared to the profile of control MM.1S cells. The NEDD8 activating enzyme (NAE) is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the first step in the neddylation pathway. NEDD8 modification of cullins is required for ubiquitination of cullin-ring ligases (CRLs), which regulate degradation of a distinct subset of proteins. The more targeted impact of NAE on protein degradation prompted us to study MLN4924, an investigational NAE inhibitor, in preclinical multiple myeloma (MM) models. In vitro treatment with MLN4924 led to dose-dependent decrease of viability in a panel of human MM cell lines. In this analysis, we evaluated the molecular changes triggered in MM.1S myeloma cells by their in vitro treatment with MLN4924. The transcriptional profiles of each experimental condition were characterized by oligonucleotide microarray analysis, using the human U133 plus 2.0 Affymetrix GeneChip. The human multiple myeloma (MM) cells MM.1S were cultured in vitro in the presence or absence of the NEDD8 activating enzyme (NAE) inhibitor MLN4924 for 8, 16 or 24hrs. The gene expression profiles of drug treated cells were compared with the profiles of control MM.1S cells.

2012-05-01 | E-GEOD-33577 | biostudies-arrayexpress

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles Ube2m and Ube2f are E2 enzymes that direct protein modification by NEDD8. Here we explore the specific functions of Ube2f and Ube2m by comparing gene expression profiles following knockdown of their function in NIH 3T3 cells. We used microarrays to detail the global programme of gene expression changes following knockdown of Ube2m and Ube2f in NIH 3T3 cells. NIH 3T3 cells were transduced with retroviral constructs containing shRNA directed against Ube2m or Ube2f. Three replicates of each condition were analyzed.

2009-03-23 | E-GEOD-14088 | biostudies-arrayexpress

Comparison of gene expression in NEDD8-sufficient or -deficient neonatal liver

Project description:The total RNA was prepared from the postnatal day 3 NEDD8-sufficient or -deficient liver and subjected to microarray.

2022-06-24 | GSE133215 | GEO

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

2009-03-24 | GSE14088 | GEO