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Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins

Mulcahy, Matthew J. and Paulo, Joao A. and Hawrot, Edward (2018) Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins. Journal of Proteome Research, 17 (11). pp. 3959-3975. ISSN 1535-3893. PMCID PMC6301012. http://resolver.caltech.edu/CaltechAUTHORS:20181005-093937534

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Abstract

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/acs.jproteome.8b00618DOIArticle
https://pubs.acs.org/doi/suppl/10.1021/acs.jproteome.8b00618PublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301012PubMed CentralArticle
ORCID:
AuthorORCID
Mulcahy, Matthew J.0000-0001-5588-8762
Paulo, Joao A.0000-0002-4291-413X
Hawrot, Edward0000-0002-7333-6555
Additional Information:© 2018 American Chemical Society. Received: August 12, 2018; Published: October 4, 2018. Work presented here was performed in part to fulfill requirements for a Ph.D. degree (M.J.M.). This research is based in part upon work conducted using the Rhode Island NSF/EPSCoR Proteomics Share Resource Facility, which is supported in part by the National Science Foundation EPSCoR Grant No. 1004057, National Institutes of Health Grant No. 1S10RR020923, S10RR027027, a Rhode Island Science and Technology Advisory Council grant, and the Division of Biology and Medicine, Brown University. We thank Dr. James Clifton for his technical assistance in mass spectrometry sample preparation and analysis. We also thank Dr. Steven P Gygi and the Taplin Mass Spectrometry Facility at Harvard Medical School for use of their mass spectrometers. This research was supported by NIH 1R21AG038774 (E.H.), NIH 1S10RR027027 (E.H.), NSF EPS-1004057 (E.H. and M.J.M.), and NIH K01DK098285 (J.A.P.) Author Contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. M.J.M, J.A.P., and E.H. conceived and designed experiments; M.J.M. and J.A.P. performed the experiments; J.A.P. and E.H. contributed reagents/materials/analysis tools; M.J.M. wrote the manuscript; and M.J.M., J.A.P., and E.H. edited the manuscript. The authors declare no competing financial interest.
Funders:
Funding AgencyGrant Number
NSFOIA-1004057
NIH1S10RR020923
NIHS10RR027027
Rhode Island Science and Technology Advisory CouncilUNSPECIFIED
Brown UniversityUNSPECIFIED
NIH1R21AG038774
NIH1S10RR027027
NSFEPS-1004057
NIHK01DK098285
Subject Keywords:nicotine, Orbitrap Fusion, mass spectrometry, interactome, proteomics, nAChR, α-bungarotoxin
PubMed Central ID:PMC6301012
Record Number:CaltechAUTHORS:20181005-093937534
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20181005-093937534
Official Citation:Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins. Matthew J. Mulcahy, Joao A. Paulo, and Edward Hawrot. Journal of Proteome Research 2018 17 (11), 3959-3975 DOI: 10.1021/acs.jproteome.8b00618
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:90133
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:05 Oct 2018 21:37
Last Modified:21 Dec 2018 15:57

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