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

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 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: PXD010941 | Pride | 2018-09-03

REPOSITORIES: Pride

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			Action	DRS
	170421_P0309_MR_JP_Rep12_fr1.raw	Raw
	170421_P0309_MR_JP_Rep12_fr2.raw	Raw
	170421_P0309_MR_JP_Rep12_fr3.raw	Raw
	170421_P0309_MR_JP_Rep12_fr4.raw	Raw
	170421_P0309_MR_JP_Rep12_fr5.raw	Raw

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Publications

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

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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.

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 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.

2018-09-03 | PXD010942 | 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

Neonatal rat cardiomyocyte RBPome

Project description:Mutations or decreased expression of RNA-binding proteins (mRBPs) can lead to cardiomyopathies in humans. Here we defined RBPs in healthy and diseased primary cardiomyocytes at a system-wide level by RNA Interactome Capture. This identified 67 novel cardiomyocyte specific RBPs including several contractile proteins. Furthermore, we evaluated dynamic binding capacities of RBPs during pathologyical cardiac hypertrophy and identified Cytoplasmic polyadenylation element binding protein 4 (Cpeb4) as a dynamic RBP, regulating cardiac growth both in vitro and in vivo.

2024-07-08 | PXD019811 | 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

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

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