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Analysis of Ataxin-2 interactome using LC-MS/MS

ABSTRACT: We analyzed Ataxin-2 interactome using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). HEK293T cell lysate was subjected to immunoprecipitation (IP) with either anti-Ataxin-2 antibody or control IgG. The interaction between Ataxin-2 and the copurified proteins do not appear to be mediated by RNA, as the immunoprecipitation experiments were performed under buffer conditions that include RNase A and RNase I. The proteins purified using anti-Ataxin-2 and control IgG were separated by SDS-PAGE. CBB-stained gel was divided into three parts per lane in order to increase the detection sensitivity. Proteins extracted from the gels were analyzed by liquid chromatography coupled to tandem mass spectrometry to identify their protein content.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Cell Culture

SUBMITTER: Hiroto Inagaki

LAB HEAD: Shin-ichi Hoshino

PROVIDER: PXD024350 | Pride | 2021-03-22

REPOSITORIES: Pride

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Project description:Machado-Joseph disease (MJD) is a fatal neurodegenerative disease caused by expansion of the trinucleotide repeat region within the ATXN3/MJD gene. Mutation of ATXN3 causes formation of neurotoxic ataxin-3 protein aggregates, neurodegeneration and motor deficits. Here we investigated the therapeutic potential and mechanistic activity of sodium butyrate (SB), the sodium salt of butyric acid, a metabolite naturally produced by gut microbiota, on cultured SH-SY5Y cells and transgenic zebrafish expressing human ataxin-3 containing 84 glutamine (Q) residues to model MJD. MJD SH-SY5Y cells were found to contain ataxin-3 oligomeric species and protein aggregates. Treatment with SB increased activity of the autophagy protein quality control pathway in the MJD cells, decreased presence of ataxin-3 aggregates and presence of ataxin-3 oligomers in an autophagy-dependent manner. Treatment with SB was also beneficial in vivo, improving swimming performance, increasing autophagy activity and decreasing presence of insoluble ataxin-3 protein species in the transgenic MJD zebrafish. Co-treating the MJD zebrafish with SB and chloroquine, an autophagy inhibitor, prevented the beneficial effects of SB on the zebrafish swimming, suggesting that the improved swimming performance was autophagy-dependent. To identify mechanism of the induction of autophagy by SB, we performed proteomic analysis of protein lysates from the SB treated and untreated MJD SH-SY5Y cells. We found that SB treatment had increased activity of Protein Kinase A and AMPK signaling. Immunoblot analysis confirmed that SB treatment had increased levels of acetylated FOXO1 protein. Further, co-treatment with an inhibitor of FOXO1 transcriptional activity (AS1842856) prevented the increase in autophagosome formation (LC3II/I) usually produced by SB treatment. Together our findings indicate that treatment with SB can increase activity of the autophagy pathway through a FOXO1-dependent process and that this has beneficial effects in vitro and in vivo. We propose that treatment with sodium butyrate warrants further investigation for the treatment of neurodegenerative diseases underpinned by proteinopathy mechanisms, including MJD.

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Analysis of Ataxin-2 interactome using LC-MS/MS

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