Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Proteomic investigation of whole cortex and synaptosomes from rats with a familial Parkinson’s G51D mutation in the Snca gene.

ABSTRACT: The glycine-51 (G51) codon in exon 3 of the rat Snca gene, encoding alpha-synuclein, was mutated to aspartic acid using CRISPR/Cas9 to generate the SncaG51D rat model of Parkinson’s (Morley et al. 2023). The G51D mutation causes an aggressive form of Parkinson’s and dementia with Lewy bodies in humans. The brains from four 6-month old rats of wild-type, heterozygous, and homozygous genotypes (Snca+/+, SncaG51D/+ and SncaG51D/G51D) were harvested and all 12 brains were bisected down the midline. Frontal cortex was isolated from one half, and synaptosomes were prepared from the other half. Following quality control the samples were processed for TMT-mass spectrometry by the Fingerprints Facility, University of Dundee. MaxQuant Version 1.6.0.16 was used for the assignment of peptides to protein using the uniport-rat-jan2018.fasta file. Pairwise comparisons were made between the different mutant samples and Snca+/+ for both the cortical and synaptosome samples. Morley V, Dolt KS, Alcaide-Corral CJ, Walton T, Lucatelli C, Mashimo T, Tavares AAS & Kunath T (2023) In vivo18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation. Front. Neurosci. 17, 1095761.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Brain

DISEASE(S): Parkinson's Disease

SUBMITTER: Douglas Lamont

LAB HEAD: Professor Tilo Kunath

PROVIDER: PXD044776 | Pride | 2024-09-04

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	179-2019-EDI-SW-Analysis-Results.xlsx	Xlsx
	SW-TMT_Rpt-Frac1.raw	Raw
	SW-TMT_Rpt-Frac10.raw	Raw
	SW-TMT_Rpt-Frac11.raw	Raw
	SW-TMT_Rpt-Frac12.raw	Raw

Items per page:

1 - 5 of 24

Similar Datasets

Pseudolycoriella hygida saliva proteome

Project description:This experiment aimed at indentifying secreted proteins/enzymes from saliva of the fly Pseudolycoriella hygida. To do this, twelve-day-old Psl. hygida larvae were selected and the last segment of the larvae was tied with elastane fishing line with the help of a stereomicroscope. This was performed to avoid contamination of saliva samples with feaces. Groups of 20 tied larvae were transferred to 1.5-mL microtubes and a few holes were made in the microtube cap. Then, the microtubes were incubated at 22 °C for 3 h. After removing the larvae, the microtubes were briefly centrifuged at 15,000 g for 30 s, and the secreted saliva, usually 2-3 ul per tube, was stored at -20 °C until gel electrophoresis.

2023-04-07 | PXD033046 | Pride

tRNA 3' shortening by LCCR4 as a response to stress in Trypanosoma brucei

Project description:Sensing of environmental cues is crucial for cell survival. To adapt to changes in their surroundings cells need to tightly control the repertoire of genes expressed at any time. Regulation of translation is key, especially in organisms in which transcription is hardly controlled, like Trypanosoma brucei. In this study we describe the shortening of the bulk of the cellular tRNAs during stress at the expense of the conserved 3’ CCA-tail. This tRNA shortening is specific for nutritional stress and renders tRNAs unsuitable substrates for translation. We uncovered the nuclease LCCR4 (Tb927.4.2430), a homologue of the conserved deadenylase Ccr4, as being responsible for tRNA trimming. Once optimal growth conditions are restored tRNAs are rapidly repaired by the trypanosome tRNA nucleotidyltransferase thus rendering the recycled tRNAs amenable for translation. This mechanism represents a fast and efficient way to repress translation during stress, allowing quick reactivation with a low energy input.

2021-01-13 | PXD021540 | Pride

The temporal profile of activity-dependent presynaptic phospho-signalling reveals a PP1-dependent reduction in vesicle release

Project description:Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic proteins undergoing post-stimulus phospho-signalling to reveal long-lasting changes, key substrates and master regulators. A total of 5,715 unique phosphopeptides from 1,817 proteins were profiled and 1,917 phosphopeptides had activity-dependent phosphorylation sites. A new computational method, KinSwing, combining protein kinase substrate prediction and activity across time, enabled the prediction of effector protein kinase activity. CaMKII responded rapidly to depolarization. MAPK, CDK5 and GSK3β had a post-stimulus role in compensating for initial dephosphorylation. Despite this, the post-stimulus period was dominated by down-regulation of phosphorylation and was exacerbated by conditions stimulating increased calcium influx. Down-regulated phosphorylation correlated with perturbed phospho-signalling to protein phosphatase 1 (PP1) regulatory molecules and reduced glutamate and fluorescent dye release. Inhibition PP1 prevented reduction in dye release, identifying PP1 as a major signalling target and mediator of post-stimulus presynaptic phospho-signalling.

2019-01-24 | PXD008495 | Pride

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long lasting patterns of post-stimulus regulation

Project description:Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the post-stimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. CaMKIIα responded rapidly, scaled with stimulus strength and had long-lasting activity. MAPK/ERK was the main protein kinase predicted to control a distinct and significant pattern of post-stimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

2019-01-28 | PXD010007 | Pride

METTL21C methylates Alanine tRNA synthetase at lysine 943 in muscle tissue

Project description:Protein lysine methylation is a common post-translational modification (PTM) detected throughout the human proteome that plays important roles in diverse biological processes. In the human genome, there are greater than one hundred known and candidate protein lysine methyltransferases (PKMTs), many with important links to human disease. METTL21C (methyltransferase-like protein 21C) is a PKMT implicated in muscle biology and reported to methylate VCP (valosin-containing protein/p97) and HSPA8 (heat shock 70kDa protein 8). However, a clear in vitro methyltransferase activity for METTL21C is yet to be demonstrated. Thus, whether METTL21C is indeed an active enzyme that directly methylates substrate/s is unclear. Here, we use an unbiased biochemical-based screening assay coupled to mass spectrometry to identify Alanine-tRNA-Synthetase 1 (AARS1) as a direct substrate of METTL21C. METTL21C catalyzes mono-, di- and tri-methylation of lysine 943 of AARS1 (AARS1-K943me) in vitro and in vivo. In vitro methylation of AARS1 by METTL21C is independent of ATP or tRNA molecules. In contrast to AARS1, and in conflict with previous reports, we do not detect METTL21C methylation activity on VCP and HSPA8. Depletion of METTL21C in METTL21C-expressing cells leads to depletion of AARS1-K943 methylation. Finally, METTL2C is almost exclusively expressed in muscle tissue, and accordingly we detect the METTL21C-catalyzed methylation event of AARS1 in mouse skeletal muscle tissue. Together, our work identifies AARS1 as a bona fide substrate of METTL21C and suggests a role for the METTL21C-AARS1 axis in the regulation of protein synthesis in muscle tissue. In addition, our study describes a straightforward protocol for elucidating physiologic substrates of little characterized or uncharacterized PKMTs

2020-07-02 | PXD019886 | Pride

Isoleucyl-tRNA synthetase interactome analysis

Project description:Aminoacyl-tRNA synthetases (aaRSs) catalyze the ligation of each amino acid to the 3’ hydroxyl group of the cognate tRNA and thereby establish the genetic code for protein synthesis. AaRSs form a complicated network through assembly into a multi-synthetase complex (MSC), which is comprised of nine aaRSs (ArgRS, AspRS, GlnRS, GluProRS, IleRS, LeuRS, LysRS, and MetRS) and three auxiliary proteins (aaRS-interacting multifunctional proteins, p43, p38, and p18 or AIMP1, 2, and 3) in vertebrates. IleRS is one of the least characterized aaRSs in the MSC. It is a class 1a aaRS and has an UNE-I domain at its C-terminal end that is formed by tandem ~90 residue repeats. Systematic depletion and yeast two-hybrid (Y2H) analyses suggest that IleRS binds to the WHEP domain of GluProRS through its UNE-I domain. However, crosslinking and mass spectrometry (XL-MS) analyses revealed that IleRS interacts with ArgRS, LeuRS, and MetRS in the MSC. While such difference may be due to the different dynamic states of the MSC under various cellular environments, it remains to be resolved how IleRS associates with other components of the MSC. Therefore, we performed mass spectrometry analysis to study IleRS interactome using cells expressing wild-type IleRS, C-terminal-deleted IleRS, and the IleRS UNE-I domain alone.

2023-03-11 | PXD031261 | Pride

Enhanced Ca2+-channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease

Project description:Ca2+ overload-induced mitochondrial dysfunction is considered as a major contributing factor in the pathogenesis of alcohol-associated liver disease (ALD). However, the initiating factors that drive mitochondrial Ca2+ accumulation in ALD remain elusive. Here, we demonstrate that an aberrant increase in mitochondria-associated ER membranes (MAMs) mediates Ca2+ accumulation-induced mitochondrial dysfunction in ALD via the formation of glucose-regulated protein 75 (GRP75)-mediated MAM Ca2+-channeling (MCC) complex. Unbiased transcriptomic analysis reveals pyruvate dehydrogenase kinase 4 (PDK4) as a prominently inducible MAM kinase in ALD. Additional mass spectrometry analysis unveils the critical role of PDK4 in promoting alcohol-induced MCC complex formation and mitochondrial dysfunction by phosphorylating GRP75. Non-phosphorylatable GRP75 mutation or genetic ablation of PDK4 prevents alcohol-induced mitochondrial Ca2+ accumulation and dysfunction. Conversely, ectopic induction of MAM formation in PDK4-deficient mice abrogate the protective effect in alcohol-induced liver injury. Together, our study defines the mediatory role of PDK4 in promoting mitochondrial dysfunction in ALD.

2023-05-10 | PXD039478 | Pride

Regulation of Plant Phototropic Growth by NPH3/RPT2-like Substrate Phosphorylation and 14-3-3 Binding

Project description:Polarity underlies all directional growth responses in plants including growth towards the light (phototropism). The plasma-membrane associated protein, NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key determinant of phototropic growth which is regulated by phototropin (phot) AGC kinases. Here we demonstrate that NPH3 is directly phosphorylated by phot1 within a conserved C-terminal consensus sequence (RxS) that is necessary to promote phototropism and petiole positioning in Arabidopsis. RxS phosphorylation also triggers 14-3-3 binding combined with changes in NPH3 phosphorylation and localisation status. Mutants of NPH3 that are unable to bind or constitutively bind 14-3-3s show compromised functionality consistent with a model where phototropic curvature is established by signalling outputs arising from a gradient of NPH3 RxS phosphorylation across the stem. Our findings therefore establish that NPH3/RPT2-Like (NRL) proteins are phosphorylation targets for plant AGC kinases. Moreover, RxS phosphorylation is conserved in other members of the NRL family, suggesting a common mechanism of regulating plant growth to the prevailing light environment.

2021-11-03 | PXD028698 | Pride

Kv7.2 LC-MSMS - Updating in vivo and in vitro phosphorylation and methylation sites of voltage-gated Kv7

Project description:Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of Kv7 channels is regulated by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signalling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP2), Ca2+/Calmodulin and phosphorylation. Recent studies have reported that PIP2 sensitivity of Kv7.2 channels is affected by two PTMs, phosphorylation and methylation, harboured in the PIP2 binding domains. Here this project aimed to update phosphorylation and methylation sites on Kv7.2 from heterologous cells, the rat brain and GST-fusion Kv7.2 N- and C-terminal ends proteins.

2017-08-25 | PXD005567 | Pride

SART3 is a Novel Rme2s Reader studied by tandem MS

Project description:Arginine methylation is a ubiquitous post-translational modification involved in many biological processes such as transcription, cell signaling and RNA splicing. Tudor domains by far are the major effector domain family for arginine methylation mark recognition. Here we characterize SART3 as a novel selective reader for symmetric dimethylarginine (Rme2s) marks. SART3 harbors a series of HAT (Half-a-TPR) repeats that are aromatic-rich, and it is this region that binds Rme2s motifs. An analysis of the reported structure of the HAT repeats of SART3 identified a putative aromatic cage that potentially “read” the Rme2s-modified motifs, and the key aromatic residues required for Rme2s recognition were identified. We further confirm that this Rme2s reader ability is important for SART3-mediated RNA splicing. Together, our studies revealed that the evolutionarily conserved SART3 HAT domain was a novel reader of Rme2s mark involved in RNA splicing.

2024-10-17 | PXD046708 | Pride