Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Affinity purification – mass spectrometry of AFF4 WT or S829/S831/3/5/8A mutant

ABSTRACT: The S831 phosphosite on AFF4 lies within a disordered region adjacent to the ENL/AF9 binding site. We therefore hypothesized that phosphorylation may regulate protein:protein interactions within the Super Elongation Complex. To investigate this, HEK293T cells expressing RFP negative control, FLAG-tagged AFF4-WT or FLAG-tagged AFF4-A-mutant were stimulated with insulin for 30 min followed by immunoprecipitation and analysis of interacting proteins by LC-MS/MS.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Hek-293t Cell

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD054250 | Pride | 2025-12-16

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	220816_Lmos_BenParker_BP-4832_A1.raw	Raw
	220816_Lmos_BenParker_BP-4832_A2.raw	Raw
	220816_Lmos_BenParker_BP-4832_A3.raw	Raw
	220816_Lmos_BenParker_BP-4832_A4.raw	Raw
	220816_Lmos_BenParker_BP-4832_RFP1.raw	Raw

Items per page:

1 - 5 of 13

Similar Datasets

Analysis of insulin-regulated phosphorylation of AFF4 with Akti or S6ki and S6K invitro kinase

Project description:Aff4 was recently identified as an insulin-regulated phosphoprotein. To investigate upstream kinases, we analysed immunoprecipitated Aff4 treated with our without insulin and treated with either an Akti or a P70S6Ki. These results showed that AFF4 is dependent on P70S6K and therefore an in vitro kinase assay was performed to validate direct phosphorylation.

2025-12-16 | PXD054247 | Pride

AP-MS of C18ORF25 with AMPK- and S67 phosphorylation-depedent interactions

Project description:C18ORF25 is a homolog of Arkadia (RNF111), an E3 ubiquitin ligase with SUMO-interaction motifs (SIMs) (PMID: 31417085). However, C18ORF25 lacks the entire C-terminal RING domain of RNF111 which is required for ubiquitin binding suggesting it lacks ubiquitination activity and may therefore act as an adaptor or signalling scaffold (PMID: 26283374). We have previously shown that mice lacking C18Orf25 throughout the entire body have increased adiposity, decreased lean mass, lower exercise capacity and significantly reduced ex vivo skeletal muscle force production (PMID: 35882232). Skeletal muscle isolated from C18Orf25 knockout (KO) mice have reduced cAMP-dependent protein kinase A (PKA) levels, and reduced phosphorylation of several contractile proteins and proteins involved in calcium handling. Furthermore, analysis of single muscle fibres from C18Orf25 KO mice revealed impaired SR calcium cycling in fast-twitch fibres only (PMID: 35882232). We also previously showed that the exercise-regulated phosphorylation of S67 on C18ORF25 is directly catalysed by AMPK. This phosphorylation site plays an important role in the function of C18ORF25 as re-expression of a phospho-mimetic S66/67D but not phospho-dead S66/67A in C18Orf25 KO mice is able to rescue skeletal muscle contractile defects PMID: 35882232). In the present study, we further investigated the potential molecular functions of C18ORF25. We performed affinity purification coupled to tandem mass spectrometry (AP-MS) to probe the protein:protein interaction (PPI) landscape of C18ORF25. Included in this analysis was also an investigation of potential S67 phosphorylation-dependent PPIs.

2024-01-03 | PXD045629 | Pride

Proximity proteomics reveals a mechanism of fatty acid transfer at lipid droplet-mitochondria- endoplasmic reticulum contact sites

Project description:Membrane contact sites between organelles are critical for transfer of biomolecules. Lipid droplets store fatty acids and form contacts with mitochondria, which regulate fatty acid oxidation and adenosine triphosphate production. Protein compartmentalization at lipid droplet-mitochondria contact sites and their effects on biological processes are poorly described. Using proximity-dependent biotinylation methods, we identify 71 proteins at lipid droplet-mitochondria contact sites, including a multimeric complex containing extended synaptotagmin (ESYT) 1, ESYT2, and VAMP Associated Protein B and C (VAPB). High resolution imaging confirms localization of this complex at the interface of lipid droplet-mitochondria-endoplasmic reticulum where it likely transfers fatty acids to enable -oxidation. Deletion of ESYT1, ESYT2 or VAPB limits lipid droplet-derived fatty acid oxidation, resulting in depletion of tricarboxylic acid cycle metabolites, remodeling of the cellular lipidome, and induction of lipotoxic stress. These findings were recapitulated in Esyt1 and Esyt2 deficient mice. Our study uncovers a fundamental mechanism that is required for lipid droplet-derived fatty acid oxidation and cellular lipid homeostasis, with implications for metabolic diseases and survival.

2025-04-28 | PXD060912 | Pride

Scalable purification enables high-quality virus-like particles for therapeutic translation

Project description:Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.

2025-11-26 | PXD070195 | Pride

Low glucose culture conditions alter neuronal energetics, mimicking in vivo studies

Project description:Neurons are almost exclusively cultured in media containing glucose at much higher concentration than found in the brain. To test whether “standard” hyperglycaemic culture conditions affect neuronal respiration compared to near- euglycemic conditions, we grew neuronal cultures with minimal glial contamination from hippocampus and cortex of neonatal C57bl6 mice in standard commercially available media (25 mM Glucose) and in identical media with 5 mM glucose. Neurons grew well in both glucose concentrations until at least 14 days in vitro, with similar morphology and synaptogenesis. Neurons grown in high glucose were more dependent on glycolysis as their primary source of ATP, measured using ATP luminescence and cellular respirometry assays. In contrast, neurons grown in 5 mM glucose showed a more balanced dependence on glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) and greater reserve mitochondrial respiration capacity, relative to standard media. Changes in gene and protein expression levels corroborate these changes in function. Our results suggest that neurons cultured in high glucose media preferentially use glycolysis, opposite to what is known for neurons in vivo as the primary pathway for ATP maintenance. We suggest using neuronal culture systems in 5 mM glucose to better represent physiologically relevant neuronal respiration.

2025-06-30 | PXD059008 | Pride

AP-MS analysis of IFIT1 and 5 interactions in interferon-stimulated human cells

Project description:This project sought to identify interactions of InterFeron Induced Tetratricopeptide repeat (IFIT) proteins 1 ad 5 in human cells. SILAC-labelled human HEK293T cells were transfected with FLAG-tagged human IFIT1 and IFIT5 pulldowns. After transfection, the cells were treated with human interferon 2-alpha and FLAG-immunoprecipitations performed to identify IFIT-binding proteins. Cells were then subject to LS/MS-MS analysis on a Orbitrap Fusion and data analysed in Maxquant v1.5.7.4

2018-03-16 | PXD007584 | Pride

Huntingtin contains an ubiquitin-binding domain and regulates lysosomal targeting of mitochondrial and RNA-binding proteins

Project description:Evaluating the identity of ubiquitinated and ubiquitin-associated proteins tandem co-purifying non-covalently with wild-type (17 glutamine) human Huntingtin fragment, amino acids 1-502 C-terminally tagged with the HA peptide epitope, basally expressed from the CMV promoter within vector pcDNA3.1 transiently transfected into striatal precursor cell line St14A, significantly elevated above cells transfected with empty pcDNA3.1 vector negative control, in cells co-transfected with human FLAG-ubiquitin.

2024-07-31 | PXD053980 | Pride

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism

Project description:Macroautophagy/autophagy is a catabolic process that targets a wide variety of cellular components including proteins, lipids, damaged organelles, and pathogens. The autophagy protein ATG7 is involved in autophagy initiation by facilitating the lipidation of LC3/GABARAPs at the growing autophagosomal membrane. ATG7 is important for normal cellular homeostasis as well as diseased state such as tumor initiation and progression in various tumors. A short isoform of ATG7, termed ATG7(2) in contrast to the full-length ATG7(1), is unable to perform the characterized ATG7 mediated autophagy lipidation activity. In this study, we found that ATG7(1) and ATG7(2) are not correlated in expression in human tissues. They have different patterns of expression indicating that these isoforms are involved in diverse cellular mechanisms. To address the functional role of the short ATG7(2) isoform, we performed mass spectrometry analysis of the protein interactome of ATG7(1) and ATG7(2). This revealed that whereas ATG7(1) interacts with the autophagy machinery, ATG7(2) interacts with key glycolysis and mitochondrial membrane proteins. Functional experiments showed that ATG7(2) expression mediates a decrease in glycolytic activity, opposite to the effects mediated by ATG7(1) expression. These findings suggest a molecular switch between main catabolic processes through isoform dependent expression of a key autophagy gene.

2024-04-15 | PXD040548 | Pride

Interactome analysis of ACAD11 in HEK293 cells

Project description:Affinity purification - mass spectrometry analysis of N-terminally FLAG-tagged ACAD11 transietly expressed in HEK293 cells.

2019-03-14 | PXD010788 | Pride

Proteomics reveals the regulation of lentivirus-mediated THBS1 overexpression in ALG1 and its mechanism in meibomian gland carcinoma

Project description:Meibomian gland carcinoma (MGC) is an aggressive and poorly prognostic eyelid malignancy. Previous studies have demonstrated that miR-3907 is highly expressed in MGC, accelerating tumor progression by negatively regulating THBS1. This study delves into the downstream regulatory mechanisms of THBS1. Using 4D-label-free proteomics, ALG1 was identified as the downstream protein with the highest differential expression and consistent sequencing results following lentivirus-mediated THBS1 overexpression. First, protein-protein docking models predicted that THBS1 interacted with ALG1 through hydrogen bonds formed by key amino acid residues such as ASN-991 and MET-58, creating a stable docking structure. Immunofluorescence and co-immunoprecipitation assays further confirmed the co-localization and interaction between THBS1 and ALG1. In MGC cells, the mRNA expression of ALG1 was notably higher than that in normal meibomian gland cells. Functional assays demonstrated that knockdown of ALG1 evidently inhibited cell malignant properties. Additionally, ALG1 knockdown modulated the expression of epithelial-mesenchymal transition-related genes, which are key modulators of tumor metastasis. Conversely, ALG1 overexpression produced the opposite effects. Therefore, ALG1, as part of the miR-3907/THBS1/ALG1 axis, can serve as a biomarker for targeted diagnosis and treatment of MGC. This study provides valuable insights into potential targeted therapies and personalized treatment strategies for MGC.

2025-09-29 | PXD059655 | 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