Native alpha synuclein crosslinking
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos, Orbitrap Fusion
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: 
Nicholas Brodie
LAB HEAD: Christoph Borchers
PROVIDER: PXD011645 | Pride | 2019-02-28
REPOSITORIES: Pride
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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
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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.
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Project description:Abnormal accumulation of aggregated proteins and sustained microglial activation are important contributors of neurodegenerative process in neurological diseases. Recent studies have shown that aggregation-prone proteins, such as a-synuclein, the protein implicated in Parkinson’s disease (PD), are released from neuronal cells and thus present in the extracellular fluid, pointing to the possible paracrine effects of these proteins on microglial immune responses. However, the mechanism underlying the disease-associated microglial activation and the role of neuronal proteins in this process remain unknown. Here, we show that extracellular a-synuclein released from neuronal cells is an endogenous ligand of toll-like receptor 2 (TLR2) and activates microglia, which in turn induces neurodegeneration. Interaction between neuron-released a-synuclein and TLR2 and subsequent activation of the TLR2 signaling were demonstrated comprehensively by using computational modeling of signaling network and by the experimental validation in TLR2-deficient microglia both in vitro and in vivo. In contrast to the neuron-released a-synuclein, recombinant a-synuclein proteins, including monomer, oligomer, fibril, or nitrated forms, were not able to interact or activate TLR2, suggesting that neuronal cells have a mechanism of enriching specific forms of a-synuclein capable of activating TLR2 during the process of releasing this protein. Taken together, the results suggest that both neuron-released extracellular a-synuclein and TLR2 might be novel therapeutic targets for modifying neuroinflammation in PD and related neurodegenerative diseases. We collected culture media from differentiated SH-SY5Y cells overexpressing either human a-synuclein (alpha-SCM) or beta-galactosidase (LZCM) and treat these media to primary rat microglia at the concentration of a-synuclein of 1.1M. Transcriptome analyses with microglial cells treated with either aSCM or LZCM at two different time points, 6 h and 24 h.
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Project description:Coexpression of alpha-synuclein and p25alpha in an oligodendroglial cell line elicites a degenerative response that relies on aggregation and phosphorylation of alpha-synuclein at Ser129 We used microarray analysis to detail the changes in gene expression caused by alpha-synuclein aggregation followed by cellular degeneration Alpha-synuclein was coexpressed with p25alpha in an oligodendroglial cell line and mRNA was isolated at 8, 12, and 16 hrs after transfection. Gene expression was analyzed by two identical microarray set-ups
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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.
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Project description:α-Synuclein is an abundant presynaptic protein that when aggregated and dyslocated from the synapse is associated to Parkinson’s disease and dementia. The normal presynaptic function of α-synuclein is unclear as well as the disease triggering mechanism. In order to extend the knowledge of the α-synuclein interactome during normal function and disease, we have used porcine brain synaptosomes as a source of ligands and purified α-synuclein monomers or oligomers as bait in co-immunoprecipitation experiments. The isolated binding synaptosomal proteins were identified with LC-LTQ-orbitrap tandem mass spectrometry and quantified by peak area using the freely-available Windows client application, Skyline Targeted Proteomic Environment. To compare proteins binding to a-synuclein monomer with proteins binding to a-synuclein oligomer, quantifications were log transformed, normalized to buffer-background, and compared by students t-test. Furthermore, to specify the preferential binding an average fold increase was calculated by comparing binding to monomer and oligomer. 10 α-synuclein preferential monomer binding proteins were identified and among those, we successfully validated Abl interactor 1, and myelin proteolipid protein. 76 α-synuclein preferential oligomer binding proteins were found, including glutamate decarboxylase 2, synapsin 1, and glial fibrillary acidic protein, which were positively validated. We identified 92 proteins binding to α-synuclein, for which we were not able to detect any conformational preferences among. This study presents a catalog of proteins interacting with α-synuclein in its non-aggregated and aggregated oligomeric state, which can be used to investigate the normal and pathological roles of α-synuclein.
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