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The interactome of stabilized α-synuclein oligomers and neuronal proteins

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The interactome of stabilized α-synuclein oligomers and neuronal proteins. / van Diggelen, Femke; Frank, Signe Andrea; Somavarapu, Arun Kumar; Scavenius, Carsten; Apetri, Mihaela M; Nielsen, Janni; Tepper, Armand W J W; Enghild, Jan J; Otzen, Daniel E.

In: The FEBS Journal, Vol. 287, No. 10, 2020, p. 2037-2054.

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@article{c5452f9c32b240e9827412cffa586b26,
title = "The interactome of stabilized α-synuclein oligomers and neuronal proteins",
abstract = "While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium–potassium pump (Na +/K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs. ",
keywords = "DHA stabilization, Rab-3A, co-immunoprecipitation, computational binding site prediction, proteomics, DEPENDENT ANION CHANNEL, RAB3A, ALZHEIMERS-DISEASE, PARKINSON DISEASE, TOXICITY, SYNAPTIC PLASTICITY, IN-VIVO, ELECTROSTATICS, BINDING, SUBUNIT",
author = "{van Diggelen}, Femke and Frank, {Signe Andrea} and Somavarapu, {Arun Kumar} and Carsten Scavenius and Apetri, {Mihaela M} and Janni Nielsen and Tepper, {Armand W J W} and Enghild, {Jan J} and Otzen, {Daniel E}",
note = "{\textcopyright} 2019 Federation of European Biochemical Societies.",
year = "2020",
doi = "10.1111/febs.15124",
language = "English",
volume = "287",
pages = "2037--2054",
journal = "F E B S Journal",
issn = "1742-464X",
publisher = "Wiley-Blackwell Publishing Ltd.",
number = "10",

}

RIS

TY - JOUR

T1 - The interactome of stabilized α-synuclein oligomers and neuronal proteins

AU - van Diggelen, Femke

AU - Frank, Signe Andrea

AU - Somavarapu, Arun Kumar

AU - Scavenius, Carsten

AU - Apetri, Mihaela M

AU - Nielsen, Janni

AU - Tepper, Armand W J W

AU - Enghild, Jan J

AU - Otzen, Daniel E

N1 - © 2019 Federation of European Biochemical Societies.

PY - 2020

Y1 - 2020

N2 - While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium–potassium pump (Na +/K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs.

AB - While it is generally accepted that α-synuclein oligomers (αSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes αSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative αSO binding partners in rat primary neurons using DHA-stabilized human αSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the αSOs-binding partners. We identify in total 29 proteins associated with DHA-αSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium–potassium pump (Na +/K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified αSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between αSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic αSOs.

KW - DHA stabilization

KW - Rab-3A

KW - co-immunoprecipitation

KW - computational binding site prediction

KW - proteomics

KW - DEPENDENT ANION CHANNEL

KW - RAB3A

KW - ALZHEIMERS-DISEASE

KW - PARKINSON DISEASE

KW - TOXICITY

KW - SYNAPTIC PLASTICITY

KW - IN-VIVO

KW - ELECTROSTATICS

KW - BINDING

KW - SUBUNIT

UR - http://www.scopus.com/inward/record.url?scp=85075445678&partnerID=8YFLogxK

U2 - 10.1111/febs.15124

DO - 10.1111/febs.15124

M3 - Journal article

C2 - 31686426

VL - 287

SP - 2037

EP - 2054

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

IS - 10

ER -