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Proximity of SCG 10 and prion protein in membrane rafts
Author(s) -
Iwamaru Yoshifumi,
Kitani Hiroshi,
Okada Hiroyuki,
Takenouchi Takato,
Shimizu Yoshihisa,
Imamura Morikazu,
Miyazawa Kohtaro,
Murayama Yuichi,
Hoover Edward A.,
Yokoyama Takashi
Publication year - 2016
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/jnc.13488
Subject(s) - proximity ligation assay , biology , blot , immunoprecipitation , microbiology and biotechnology , western blot , lipid raft , gene isoform , colocalization , membrane protein , transfection , chemistry , biochemistry , signal transduction , membrane , gene , receptor
The conversion of normal cellular prion protein (Pr PC ) into its pathogenic isoform (Pr PS c) is an essential event in prion pathogenesis. In culture models, membrane rafts are suggested to play a critical role in Pr PS c formation. To identify the candidate molecules capable of interacting with Pr PC and facilitating Pr PS c formation in membrane rafts, we applied a novel biochemical labeling method termed enzyme‐mediated activation of radical sources. Enzyme‐mediated activation of radical sources was applied to the Lubrol WX insoluble detergent‐resistant membrane fractions from mouse neuroblastoma (N2a) cells in which the surface Pr PC was labeled with HRP ‐conjugated anti‐PrP antibody. Two‐dimensional western blots of these preparations revealed biotinylated spots of approximately 20 kD a with an isoelectric point of 8.0–9.0. Liquid chromatography–tandem mass spectrometry analysis resulted in the identification of peptides containing SCG 10, the neuron‐specific microtubule regulator. Proximity of SCG 10 and Pr PC was confirmed using proximity ligation assay and co‐immunoprecipitation assay. Transfection of persistently 22L prion‐infected N2a cells with SCG 10 small interfering RNA reduced SCG 10 expression, but did not prevent Pr PS c accumulation, indicating that SCG 10 appears to be unrelated to Pr PS c formation of 22L prion. Immunofluorescence and western blot analyses showed reduced levels of SCG 10 in the hippocampus of prion‐infected mice, suggesting a possible association between SCG 10 levels and the prion neuropathogenesis.By applying a novel biochemical labeling method against detergent‐resistant membrane fractions from mouse neuroblastoma cells, the neuron‐specific microtubule‐destabilization protein, SCG10 was identified as a novel candidate that is proximate to normal prion protein (PrP) in membrane rafts. SCG10 seemed unrelated to disease‐related PrP formation under certain conditions, while there is a possible association between SCG10 levels and prion neuropathogenesis. Cover Image for this issue: doi: 10.1111/jnc.13310 .