EPIC-XS H2020 alpha synuclein IMIC
Ontology highlight
ABSTRACT:
INSTRUMENT(S): solariX
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Tissue Not Applicable To Dataset
DISEASE(S): Not Available
SUBMITTER: 
Zdenek Kukacka
LAB HEAD: Petr Novak
PROVIDER: PXD019659 | Pride | 2020-08-03
REPOSITORIES: Pride
Similar Datasets
Project description:TEAD transcription factors mediate gene expression regulation through interactions with their DNA response M-CAT motif. They are active mainly during growth and development and induce gene expression of proteins involved in cell proliferation, differentiation or apoptosis prevention. TEADs and many of their target proteins are known to be upregulated in several types of cancers. To study the interaction of DNA binding domain of TEAD1 with M-CAT motifs of different orientations originating from regulatory regions of human genes (CTGF, SRF, C-MYC and GLUT1), dissociation constant of each complex was determined using fluorescence anisotropy assay. Structural mass spectrometry techniques (H/D exchange and quantitative chemical cross-linking) and molecular docking were utilized for structural characterization of the binding interface. In-vitro results were correlated with situation in living cells studied by ChIP and Q-PCR.
2020-12-08 | PXD012127 | Pride
Project description:Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to any perturbation or stimulus, protein structure may be altered by a variety of molecular events, such as post translational modification, protein-protein interaction, aggregation, allostery, or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of a variety of biological processes and pathways. Here we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale (Cappelletti et al., 2021; Piazza et al., 2020; Schopper et al., 2017). We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition (DIA) based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. In addition, we introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. Altogether, the newly proposed improvements expand the adaptability of the method and allow for its wide use in systematic functional proteomic studies and translational applications.
2023-01-10 | PXD031627 | Pride
Project description:Previous studies have demonstrated an importance of alpha-synuclein as a modulator of various mechanisms implicated in chemical neurotransmission but information about other two synuclein family members’ involvement in molecular processes taking place in presynaptic terminals is limited. Here we demonstrated that dopamine uptake by synaptic vesicles isolated from the striatum of mice lacking beta-synuclein was significantly reduced. Reciprocally, reintroduction, either in vivo or in vitro, of beta-synuclein but not alpha- or gamma-synuclein improved uptake by vesicles isolated from the striatum of triple alpha/beta/gamma-synuclein deficient mice. Proteomic analysis of synuclein-free and beta-synuclein-only-containing synaptic vesicles suggested that mechanistically, beta-synuclein potentiates vesicular monoamine transporter 2 (VMAT2)-dependent dopamine uptake by assembling specific multiprotein complexes comprised of resident vesicular proteins and transiently associated, predominantly cytosolic proteins. The increased availability of such complexes on the surface of striatal synaptic vesicles lacking other synucleins should also promote sequestration of 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which explains the resistance of dopaminergic neurons of substantia nigra lacking alpha-synuclein and/or gamma-synuclein to this neurotoxin.
2022-02-17 | PXD027786 | Pride
Project description:Structural analysis of spp2 bound to prp2 with chemical crosslinking and mass spectrometry
2020-05-27 | PXD015793 | Pride
Project description:Alpha synuclein in it's native state in solution was crosslinked using a variety of short-range crosslinking reagents. Crosslinks detected by mass spectrometry were then used as constraints in a discrete molecular dynamics simulation of the synuclein protein, and a structural ensemble of the protein was determined.
2019-02-28 | PXD011645 | Pride
Project description:Optimising the parameters for cross-link analysis using a QToF mass spectrometer. We performed triplicate analysis of 6 different collision energy ramps to determine the optimal energy for cross-link fragmentation.
2019-11-12 | PXD011704 | Pride
Project description:Proteins regulate biological processes by changing their structure or abundance to accomplish a specific function. In response to any perturbation or stimulus, protein structure may be altered by a variety of molecular events, such as post translational modification, protein-protein interaction, aggregation, allostery, or binding to other molecules. The ability to probe these structural changes in thousands of proteins simultaneously in cells or tissues can provide valuable information about the functional state of a variety of biological processes and pathways. Here we present an updated protocol for LiP-MS, a proteomics technique combining limited proteolysis with mass spectrometry, to detect protein structural alterations in complex backgrounds and on a proteome-wide scale (Cappelletti et al., 2021; Piazza et al., 2020; Schopper et al., 2017). We describe advances in the throughput and robustness of the LiP-MS workflow and implementation of data-independent acquisition (DIA) based mass spectrometry, which together achieve high reproducibility and sensitivity, even on large sample sizes. In addition, we introduce MSstatsLiP, an R package dedicated to the analysis of LiP-MS data for the identification of structurally altered peptides and differentially abundant proteins. Altogether, the newly proposed improvements expand the adaptability of the method and allow for its wide use in systematic functional proteomic studies and translational applications.
2023-01-10 | PXD031616 | Pride
Project description:Due to its high pharmaceutical relevance, α-synuclein is one of the best studied intrinsically disordered proteins to date. Its structural plasticity reaches from α-helical when bound to membranes through disordered in solution to β-sheet when forming amyloid fibrils. Recently the possible toxicity of membrane bound α-synuclein oligomers has elicited increased interest in the understanding of its membrane bound state. Both the high structural variability in the bound state and the requirement of membranes to elicit structural conversion of α-synuclein into is oligomeric membrane bound state have impeded the determination of a high-resolution oligomeric membrane-bound structure. The use of chemical crosslinking mass spectrometry (XL-MS) in this context leads to a very large set of peptide spectrum matches (PSMs), which do not permit the elucidation of a single, well defined structure based on XL-MS alone. The pattern of PSMs obtained does, however, represent possible links within the ensemble of membrane bound α-synuclein states. Here, we use fully 15N isotopically labeled α-synuclein in 1:1 mixtures with the natural abundance (14N) protein in order to allow distinction between intermolecular and intramolecular crosslinks in α-synuclein oligomers. Information obtained on the main crosslinking regions as well as changes in the observed PSM patterns is then used in conjunction with data obtained from nuclear magnetic resonance (NMR) measurements to develop high-resolution structural models of α-synuclein. We expect that the approach taken here will also prove useful in other highly flexible oligomeric systems.
2023-04-07 | PXD027349 | Pride
Project description:Eukaryotic protein homeostasis (proteostasis) is largely dependent on the action of highly conserved Hsp70 molecular chaperones. Recent evidence indicates that apart from conserved molecular allostery, Hsp70 proteins retained and adapted throughout the evolution the ability to assemble as functionally relevant ATP-bound dimers. Here we have compared the ATP-dependent dimerization of DnaK, human stress-inducible Hsp70, Hsc70 and BiP Hsp70 proteins showing that their dimerization propensities differ with stress-inducible Hsp70 being predominantly dimeric in the presence of ATP. The structural analyses using hydrogen/deuterium exchange mass spectrometry, native electrospray ionization mass spectrometry, chemical cross-linking and small-angle X-ray scattering revealed that stress-inducible Hsp70 assembles in solution as an antiparallel dimer with the intermolecular interface closely resembling the ATP-bound dimer interfaces captured in DnaK and BiP crystal structures. ATP-dependent dimerization of stress-inducible Hsp70 is necessary for its efficient interaction with Hsp40 as shown by experiments with dimerization-deficient mutants. Moreover, dimerization of ATP-bound Hsp70 is required for its participation in high molecular weight protein complexes detected ex vivo supporting its functional role in vivo. As human cytosolic Hsp70 has the ability to interact with tetratricopeptide repeat (TPR) domain containing co-chaperones, we tested the interaction of Hsp70 ATP-dependent dimer with Chip and Tomm34 co-chaperones. While Chip associates with intact Hsp70 dimer to form a larger complex, binding of Tomm34 disrupts Hsp70 dimer and this event plays an important role in Hsp70 activity regulation. In summary, this study provides structural evidence of robust ATP-dependent antiparallel dimerization of human inducible Hsp70 protein and suggests novel role of TPR domain co-chaperones in multichaperone complexes involving Hsp70 ATP-bound dimers.
2018-12-11 | PXD010069 | Pride
Project description:The recently introduced cross-linking of isotope-labelled RNA coupled with mass spectrometry (CLIR-MS) technique enables protein-RNA cross-links to be used as precisely localized distance restraints in de novo structural modelling. The novel data type requires a bespoke data analysis approach. The new RNxQuest Python package supports this approach. Here we demonstrate the performance of the new package using a mixture of novel and published datasets.
2023-09-06 | PXD039754 | Pride