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Enhanced RNA Interactome capture (eRIC) and RNA Interactome capture (RIC) of Jurkat cells (DSMZ, ACC-282) exposed to 0.5mM DMOG or vehicle (DMSO) for 6 hours.

ABSTRACT: 1.0 â€“ 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were incubated with 0.5mM DMOG (Cayman Chemical Company, 71210) or an equivalent volume of Dimethyl Sulfoxide (DMSO) (vehicle, Merck 1.02950.0500). The DMSO concentration in the medium was 0.023% v/v. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Cell, Cell Culture

DISEASE(S): Acute T Cell Leukemia

SUBMITTER: Frank Stein

LAB HEAD: Matthias W. Hentze

PROVIDER: PXD010942 | Pride | 2018-09-03

REPOSITORIES: Pride

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Discovery of RNA-binding proteins and characterization of their dynamic responses by enhanced RNA interactome capture.

Perez-Perri Joel I JI Rogell Birgit B Schwarzl Thomas T Stein Frank F Zhou Yang Y Rettel Mandy M Brosig Annika A Hentze Matthias W MW

Nature communications 20181023 1

Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal- ...[more]

PMID: 30352994

Similar Datasets

Enhanced RNA Interactome capture (eRIC) and RNA Interactome capture (RIC) of Jurkat cells (DSMZ, ACC-282).

Project description:1.0 â€“ 1.3 x 10e8 proliferating Jurkat cells at a density of about 1-1.5 x 10e6 cells/mL were employed per sample. Cells were irradiated or not with 254 nm UV light and subjected to eRIC or RIC to determine the RNA-bound proteome. Data correspond to two biologically independent experiments.

2018-09-03 | PXD010941 | Pride

Enhanced RNA interactome capture (eRIC) of human malignant bone tumor cells and normal bone/mesenchymal cells

Project description:Patient-derived bone tumor (osteosarcoma and giant cell tumor of bone) cells, and the normal mesenchymal stem cells and osteoblasts were cultured and subjected to UV crosslinking (UV) at 254 nm or without crosslinking (noUV) as negative controls. Subsequently, RNA-binding proteins (RBPs) were identified by eRIC.

2024-02-28 | PXD038185 | Pride

A simple method for developing lysine targeted covalent protein reagents

Project description:In this project we developed phosphopeptides functionalized with methacrylate ester warheads installed on a cysteine residue, which bind covalently to the protein 14-3-3 sigma. We prepared complexes of the purified protein with the peptides and used trypsin digestion and LC-MSMS to elucidate the binding site of the peptides, by comparison of compound-treated complexes to DMSO-treated protein and observing the disappearance on non-modified peptides. The file names are based on internal names used for the project and are distinct from the names given to the peptides in the final publication. Names are as follows: 4IEA - peptide 3 in the final publication 128C - peptide 8 in the final publication 132C - peptide 11 in the final publication

2024-01-26 | PXD044257 | Pride

Regulating hippocampal levels of AlzheimerÃ‚Â´s disease-related proteins with a brain-penetrating somatostatin peptide

Project description:Plaques consisting of amyloid-ÃŽÂ² (AÃŽÂ²) in the brain are characteristic for patients with AlzheimerÃ‚Â´s disease. AÃŽÂ² is enzymatically generated of the amyloid precursor protein. Furthermore, neprilysin, an AÃŽÂ²-degrading enzyme, and its main activator the neuropeptide somatostatin (SST) are downregulated in AlzheimerÃ‚Â´s disease. In the fusion protein SST-scFv8D3, SST is recombinantly fused to a blood-brain barrier transporter. The aim of this study was to study the effect of SST-scFv8D3 on the proteome of different brain regions in order to elucidate the anti-AlzheimerÃ‚Â´s disease effects of this construct. SST-scFv8D3 and phosphate buffered saline (PBS) respectively were injected into transgenic mice of an AlzheimerÃ‚Â´s disease model with the Swedish mutation (AÃŽÂ²PP KM670/671NL) every 36 hours (three times in total). Homogenates of the hippocampus, the cerebellum and the rest of the cerebrum were lysed. Afterwards, filter aided tryptic digestion was performed. The resulting peptides were analyzed using LC-UDMSE. The data was searched against a randomized mouse database and label-free quantification analysis was done. In total 1869 proteins were found. Only the proteome of the hippocampus was affected by the treatment with SST-scFv8D3. 55 proteins were significantly up and 105 down regulated compared to the PBS treated mice. Among these were mitochondrial and synaptic proteins as well as some involved in neuronÃ‚Â´s development indicating anti-Alzheimer disease effects of SST-scFv8D3 and supporting the future use of SST-scFv8D3 as a treatment option.

2021-06-28 | PXD022221 | Pride

Glycation modulates glutamatergic signaling and exacerbates ParkinsonÃ¢â‚¬â„¢s disease-like phenotypes

Project description:Alpha-synuclein (aSyn) is a central player in the pathogenesis of synucleinopathies due to its accumulation in typical protein aggregates in the brain. However, it is still unclear how it contributes to neurodegeneration. Type-2 diabetes mellitus is a risk factor for ParkinsonÃ¢â‚¬â„¢s disease (PD) and, interestingly, a common molecular alteration among these disorders is the age-associated increase in protein glycation. We hypothesized that glycation-induced neuronal dysfunction might be a contributing factor in synucleinopathies. Here, we dissected the specific impact of methylglyoxal (MGO, a glycating agent) in mice overexpressing aSyn in the brain. We found that MGO-glycation potentiates motor, cognitive, olfactory, and colonic dysfunction in aSyn transgenic (Thy1-aSyn) mice that received a single dose of MGO via intracerebroventricular (ICV) injection. aSyn accumulates in the midbrain, striatum, and prefrontal cortex, and protein glycation is increased in the cerebellum and midbrain. SWATH mass spectrometry analysis, used to quantify changes in the brain proteome, revealed that MGO mainly increase glutamatergic-associated proteins in the midbrain (NMDA, AMPA, glutaminase, VGLUT and EAAT1), but not in the prefrontal cortex, where it mainly affects the electron transport chain. Notably, the glycated proteins in the midbrain of Thy1-aSyn mice that received MGO strongly correlate with PD and dopaminergic pathways. Overall, we demonstrated that MGO-induced glycation accelerates PD-like sensorimotor and cognitive alterations and suggest that the increase of glutamatergic signaling may underly these events. Our study sheds new light into the enhanced vulnerability of the midbrain in PD-related synaptic dysfunction and suggests that glycation suppressors and anti-glutamatergic drugs may hold promise as disease-modifying therapies for synucleinopathies.

2022-05-20 | PXD032832 | Pride

Thrombin activation of the factor XI dimer is a multi-staged process for each subunit

Project description:Factor XI (FXI) is a protein in the intrinsic coagulation pathway that can be activated by two enzymes. In hemostasis, FXI is activated by thrombin, while FXIIa-mediated activation of FXI is thought to be prothrombotic. The interactions of these enzymes with FXI are poorly understood due to their transient nature which is difficult to study. Here, we applied crosslinking mass spectrometry (XLMS) and molecular dynamics simulations to investigate the binding interface between thrombin and FXI as well as the dynamics underlying the activation of FXI. From the experimental data we were able to construct the FXI homodimer as well as locate the binding interface of thrombin on the light chain of FXI. As this placed thrombin distal from both the activation site as well as the apple 1 domain that thrombin is known to interact with, we applied molecular dynamics simulations to investigate the chain of events after binding. From the resulting model, we hypothesize that the activation of FXI is a multi-staged procedure where thrombin first binds to Pro520 on FXI, after which it migrates towards the activation site by first engaging the apple 1 domain and finally Arg378. We validated the results with known mutation sites on FXI and additionally found that Pro520 is conserved in PK and enables binding of thrombin to this enzyme even though it cannot be activated by it. This interaction points a way for future interventions for bleeding and thrombosis.

2024-02-05 | PXD039489 | Pride

Quantitative and dynamic catalogs of proteins released during apoptotic and necroptotic cell death

Project description:The inflammatory functions of the cytokine tumor necrosis factor (TNF) rely on its ability to induce cytokine production and to induce cell death. Caspase dependent and independent pathways Ã¢â‚¬â€œ apoptosis and necroptosis Ã¢â‚¬â€œ respectively, regulate immunogenicity by the release of distinct sets of cellular proteins. To obtain an unbiased, systems-level understanding of this important process, we here applied mass spectrometry-based proteomics to dissect protein release during apoptosis and necroptosis. We report hundreds of proteins released from human myeloid cells in time-course experiments. Both cell death types induce receptor shedding, but only apoptotic cells released nucleosome components. Conversely, necroptotic cells release lysosomal components by activating lysosomal exocytosis at early stages of necroptosis- induced membrane permeabilisation and show reduced release of conventionally secreted cytokines.

2020-07-18 | PXD014966 | Pride

PhosphoProtein-Peptide Turnover Profiling, exploratory

Project description:We combine pulsed stable isotope labeling of amino acids (pSILAC) with enrichment of phosphorylated peptides in a method we term PhosphoProtein-Peptide Turnover Profiling (PhosphoPPToP). We apply the method to HeLa cells to find peptides whose clearance rates differ from the protein median. Using theoretical modelling, we show that readouts from PPToP are primarily defined by the wiring of the PTM-modification network and not by effects of the PTM on a protein's proteolytic stability. Thus PPToP can be used to identify PTMs occurring, e.g., preferentially early or late in a protein's lifetime. This enables identification of, e.g., protein maturation or protein complex assembly intermediates.

2022-12-02 | PXD033254 | Pride

Exoproteome of a furC-overexpressing strain: FurC tunes peptidoglycan remodeling and intercell transfer

Project description:To study the effect of FurC overexpression in the exoproteome composition of Anabaena, sp. PCC120, SWATH-MS analyses have been carried out with the isolated exoproteome of the FurC overexpressing strain, EB2770FurC (named in the present dataset "EB") comparing to the one of the wild-type strain (WT). This data togheter combined with physiological and functional studies of FurC protein revealed a potential role of this regulator in the modulation of general cell-wall biogenesis and recycling processes, as well as and in intercellular transfer.

2024-02-07 | PXD043659 | Pride

Cortisol binding sites on SARS-CoV-2 S1 identified by limited proteolysis-coupled mass spectrometry

Project description:We used a limited proteolysis-coupled mass spectrometry approach to pinpoint binding sites of cortisol on SARS-CoV-2 S1. Binding target identification is based on the principle that small-ligand binding alters (increases or decreases) the protease accessibility of the target protein37,38. The binding of the ligand (cortisol) to the target protein (SARS-CoV-2 S1) induces perturbations/conformational changes at the binding sites which can lead to either facilitating or preventing proteolysis by non-specific proteases (thermolysin and trypsin)37,38. We subjected SARS-CoV-2 S1 incubated with either cortisol or vehicle to proteolysis by thermolysin followed by trypsin digestion and mass spectrometry-based identification of the resultant peptides. We found that cortisol either facilitated or prevented S1 proteolytic cleavage at discrete sites. Six unique peptides were identified that were only present for SARS-CoV-2 S1 incubated with either cortisol or vehicle.

2022-06-10 | PXD032937 | Pride

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