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Subcellular proteomics of murine ESC-derived neurons

ABSTRACT: We studied the effects of different stimuli on on the subcellular proteome of neurons differentiated from murine embryonic stem cells (ESC). We focused on the changes in the chromatin-bound fraction to elucidate specific differences in the gene regulatory machinery activated upon stimulation with Brain-derived neurotrophic factor (BDNF) or a general membrane-depolarization stimulus, potassium chloride (KCl).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Neuron, Cell Culture

SUBMITTER: Henrik Hammarén

LAB HEAD: Mikhail M Savitski

PROVIDER: PXD022378 | Pride | 2022-09-15

REPOSITORIES: pride

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			Action	DRS
	Bert_S1582_HH_Exp8_2_Set1_R1_F1.raw	Raw
	Bert_S1582_HH_Exp8_2_Set1_R1_F10.raw	Raw
	Bert_S1582_HH_Exp8_2_Set1_R1_F11.raw	Raw
	Bert_S1582_HH_Exp8_2_Set1_R1_F12.raw	Raw
	Bert_S1582_HH_Exp8_2_Set1_R1_F2.raw	Raw

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Publications

Comparative chromatin accessibility upon BDNF stimulation delineates neuronal regulatory elements.

Ibarra Ignacio L IL Ratnu Vikram S VS Gordillo Lucia L Hwang In-Young IY Mariani Luca L Weinand Kathryn K Hammarén Henrik M HM Heck Jennifer J Bulyk Martha L ML Savitski Mikhail M MM Zaugg Judith B JB Noh Kyung-Min KM

Molecular systems biology 20220801 8

Neuronal stimulation induced by the brain-derived neurotrophic factor (BDNF) triggers gene expression, which is crucial for neuronal survival, differentiation, synaptic plasticity, memory formation, and neurocognitive health. However, its role in chromatin regulation is unclear. Here, using temporal profiling of chromatin accessibility and transcription in mouse primary cortical neurons upon either BDNF stimulation or depolarization (KCl), we identify features that define BDNF-specific chromatin ...[more]

PMID: 35996956

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2022-12-07 | PXD029988 | Pride

PhosphoProtein-Peptide Turnover Profiling, validation

Project description:We combined pulsed stable isotope labeling of amino acids (pSILAC) with enrichment of phosphorylated peptides in a method we term PhosphoProtein-Peptide Turnover Profiling (PhosphoPPToP). We applied 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. This dataset covers experimental testing of identified phosphosites with PPToP. Here, proteins of interest are expressed as GFP-fusion constructs in HeLa cells and subjected to a SILAC pulse. Thereafter, proteins are pulled-down with anti-GFP beads to isolate the exogenously expressed constructs. We multiplex SILAC timepoints from wild-type and mutant constructs for every given protein of interest using TMT16.

2022-12-02 | PXD032945 | Pride

Delineation of the pH-responsive regulon controlled by the Helicobacter pylori ArsRS two2 component system

Project description:Helicobacter pylori encounters a wide range of pH within the human stomach. In a comparison of H. pylori cultured in vitro under neutral or acidic conditions, about 15% of genes are26 differentially expressed, and corresponding changes are detectable for many of the encoded proteins. The ArsRS two-component system (TCS), comprised of the sensor kinase ArsS and its cognate response regulator ArsR, has an important role in mediating pH-responsive changes in H. pylori gene expression. In this study, we sought to delineate the pH-responsive ArsRS regulon and further define the role of ArsR in pH-responsive gene expression. We compared H. pylori strains containing an intact ArsRS system with an arsS null mutant or strains containing site32 specific mutations of a conserved aspartate residue (D52) in ArsR, which is phosphorylated in response to signals relayed by the cognate sensor kinase ArsS. We identified 178 genes that were pH-responsive in strains containing an intact ArsRS system but not in ∆arsS or arsR mutants. These constituents of the pH-responsive ArsRS regulon include genes involved in acid acclimatization (ureAB, amidases), oxidative stress responses (katA, sodB), transcriptional regulation related to iron or nickel homeostasis (fur, nikR), and genes encoding outer membrane proteins [including sabA, alpA, alpB, hopD (labA), and horA]. When comparing H. pylori strains containing an intact ArsRS TCS with arsRS mutants, each cultured at neutral pH, relatively few genes are differentially expressed. Collectively, these data suggest that ArsRS41 mediated gene regulation has an important role in H. pylori adaptation to changing pH conditions.

2022-02-16 | PXD023679 | Pride

Complexome Profiling of the mitochondrial proteome from Viscum album

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2023-10-24 | PXD041061 | Pride

Whole-cell differential proteomics analysis of MG1655 Escherichia coli (Wild-type and mutants lacking the small regulatory RNA SdsR, OxyS or ZbiJ, or the RNA Chaperone Hfq) experiencing long-term nitrogen starvation in Gutnick minimal media.

Project description:Proteomics was carried out to compare the proteomes of wild-type MG1655 E coli with mutants lacking either prominent sRNA, SdsR, OxyS or ZbiJ, or lacking the ubiquitous RNA chaperone proteins hfq, in bacteria that had been exposure to long-term (24hr) nitrogen starvation in Gutnick minimal media. The aim of this was to understand what the regulatory contribution of these three sRNA was to bacteria experiencing long-term nitrogen starvation.

2024-05-24 | PXD045656 | Pride

Proteomic analysis of primary human microvascular endothelial cells (HMVECs) stimulated for 24â€‰h with either 50 ï�g/mL recombinant Fibrillin-1 C-terminal fragment or 25â€‰ng/ml VEGF-A compared to unstimulated cells.

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2023-05-31 | PXD029834 | Pride

Trifunctional lipid derivatives: PEs mi-tochondrial interactome

Project description:In this study, we employed chemically synthesized trifunctional lipid probes to identify putative protein partners that interact with lipids in HeLa cells. These probes incorporate three distinct chemical functionalities. First, a coumarin-based “caging” moiety is added to the lipid head group, which shields the lipid from metabolic degradation and facilitates its uptake by cells. Second, a diazirine group is embedded within the acyl chain. Upon photoactivation (e.g., exposure to UV light), this diazirine crosslinks the lipid probe to nearby proteins. Finally, an alkyne group is introduced at the tail of the acyl chain, enabling click chemistry conjugation to azide-functionalized beads.This permits the selective enrichment of crosslinked protein complexes for downstream isolation and mass spectrometry–based proteomic analysis. This strategy allows the identification of native lipid–protein interactions while minimally perturbing cellular systems. Using HeLa cells as our experimental model, we were able to detect and enrich candidate lipid-binding proteins for subsequent in-depth proteomic characterization.

2025-01-21 | PXD059960 | 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.

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BioID with P. falciparum protein PhiL1

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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.

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