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Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells. / Ian Richard Gilmore

Swansea University Author: Ian Richard Gilmore

Abstract

Oxysterols are oxygenated derivatives of cholesterol or its precursors. One oxysterol, 24(S),25-epoxycholesterol (24(S),25-EC), which results from a shunt in the cholesterol synthesis pathway has been found at higher than expected levels in embryonic murine brain. Interestingly, the receptor that 24...

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Published: 2013
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa42712
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spelling 2018-08-29T14:39:29.6823294 v2 42712 2018-08-02 Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells. 2d177abedaba15109fe080d586294f84 NULL Ian Richard Gilmore Ian Richard Gilmore true true 2018-08-02 Oxysterols are oxygenated derivatives of cholesterol or its precursors. One oxysterol, 24(S),25-epoxycholesterol (24(S),25-EC), which results from a shunt in the cholesterol synthesis pathway has been found at higher than expected levels in embryonic murine brain. Interestingly, the receptor that 24(5),25-EC is a ligand for, Liver X Receptor (LXR), has been implicated in neurogenesis in the ventral mid brain region of embryonic brain; an area with a high density of dopaminergic neurons. The mechanism by which LXR induces this effect is unclear. Therefore, proteomic and phosphoproteomic studies were performed using a stable isotope labelled in amino acid in cell culture (SILAC) approach in order to quantify changes in the proteome between different treatment groups in a mouse substantia nigra dopaminergic cell line (SN4741) SN4741 cells were cultured in SILAC media containing differentially isotope labelled arginine and lysine. For protein expression studies SN4741 cells were treated in serum free media with vehicle, 10muM 24(S),25-EC, or 1muM GW3965, a synthetic ligand of LXR, for 24 hours. For analysis of changes in the phosphoproteome SN4741 cells were treated in serum free media with vehicle, 10muM 24(5),25-EC, or 30muM 25- hydroxycholesterol for 6 hours. Cells were lysed and protein combined in a 1:1 ratio before trypsin digestion and peptide separation via strong cation exchange chromatography. Phosphopeptides were enriched using immobilised metal affinity chromatography (IMAC). Resulting fractions were analysed, using a data dependent LC-MS/MS method. Data was quantified using MaxQuant software in conjunction with Mascot using an IPl mouse database. In protein expression analysis known oxysterol regulated genes, via SREBP or LXR, were differentially expressed. Oxysterol treatment induced global changes in proteins involved in lipid (cholesterol, fatty acid, phospholipid, triglyceride) synthesis. LXR? protein expression increased after GW3965 and 24(5),25-EC treatment, though no change was seen on LXRp mRNA, implying that ligand binding protects LXR? from degradation. 24(S),25-EC induced changes in expression and localisation of the membrane protein caveolin-1. Also, phosphoethanolamine cytidylyltransferase and collagen type IV alpha-3-binding protein, 2 proteins involved in phospholipid synthesis, had an altered expression after 24(S),25-EC treatment suggesting a role for oxysterols in membrane homeostasis. A cytokine, macrophage colony stimulating factor, which is required for normal neuronal development and macrophage differentiation had an LXR independent increased expression after 24(S),25-EC treatment. Quantitative RT-PCR data demonstrated that proteomic changes were due to both transcriptional and post-transcriptional effects of oxysterol. In addition, studies examining changes in the mouse phosphoproteome identified a number of novel phosphorylation sites. E-Thesis Neurosciences.;Cellular biology. 31 12 2013 2013-12-31 COLLEGE NANME Swansea University Medical School COLLEGE CODE Swansea University Doctoral Ph.D 2018-08-29T14:39:29.6823294 2018-08-02T16:24:30.1958047 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Medicine Ian Richard Gilmore NULL 1 0042712-02082018162516.pdf 10807481.pdf 2018-08-02T16:25:16.0130000 Output 20869105 application/pdf E-Thesis true 2018-08-02T16:25:16.0130000 false
title Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
spellingShingle Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
Ian Richard Gilmore
title_short Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
title_full Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
title_fullStr Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
title_full_unstemmed Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
title_sort Quantitative proteomic analysis of the effect of 24(S),25-epoxycholesterol on SN4741 neuron cells.
author_id_str_mv 2d177abedaba15109fe080d586294f84
author_id_fullname_str_mv 2d177abedaba15109fe080d586294f84_***_Ian Richard Gilmore
author Ian Richard Gilmore
author2 Ian Richard Gilmore
format E-Thesis
publishDate 2013
institution Swansea University
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Medicine{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Medicine
document_store_str 1
active_str 0
description Oxysterols are oxygenated derivatives of cholesterol or its precursors. One oxysterol, 24(S),25-epoxycholesterol (24(S),25-EC), which results from a shunt in the cholesterol synthesis pathway has been found at higher than expected levels in embryonic murine brain. Interestingly, the receptor that 24(5),25-EC is a ligand for, Liver X Receptor (LXR), has been implicated in neurogenesis in the ventral mid brain region of embryonic brain; an area with a high density of dopaminergic neurons. The mechanism by which LXR induces this effect is unclear. Therefore, proteomic and phosphoproteomic studies were performed using a stable isotope labelled in amino acid in cell culture (SILAC) approach in order to quantify changes in the proteome between different treatment groups in a mouse substantia nigra dopaminergic cell line (SN4741) SN4741 cells were cultured in SILAC media containing differentially isotope labelled arginine and lysine. For protein expression studies SN4741 cells were treated in serum free media with vehicle, 10muM 24(S),25-EC, or 1muM GW3965, a synthetic ligand of LXR, for 24 hours. For analysis of changes in the phosphoproteome SN4741 cells were treated in serum free media with vehicle, 10muM 24(5),25-EC, or 30muM 25- hydroxycholesterol for 6 hours. Cells were lysed and protein combined in a 1:1 ratio before trypsin digestion and peptide separation via strong cation exchange chromatography. Phosphopeptides were enriched using immobilised metal affinity chromatography (IMAC). Resulting fractions were analysed, using a data dependent LC-MS/MS method. Data was quantified using MaxQuant software in conjunction with Mascot using an IPl mouse database. In protein expression analysis known oxysterol regulated genes, via SREBP or LXR, were differentially expressed. Oxysterol treatment induced global changes in proteins involved in lipid (cholesterol, fatty acid, phospholipid, triglyceride) synthesis. LXR? protein expression increased after GW3965 and 24(5),25-EC treatment, though no change was seen on LXRp mRNA, implying that ligand binding protects LXR? from degradation. 24(S),25-EC induced changes in expression and localisation of the membrane protein caveolin-1. Also, phosphoethanolamine cytidylyltransferase and collagen type IV alpha-3-binding protein, 2 proteins involved in phospholipid synthesis, had an altered expression after 24(S),25-EC treatment suggesting a role for oxysterols in membrane homeostasis. A cytokine, macrophage colony stimulating factor, which is required for normal neuronal development and macrophage differentiation had an LXR independent increased expression after 24(S),25-EC treatment. Quantitative RT-PCR data demonstrated that proteomic changes were due to both transcriptional and post-transcriptional effects of oxysterol. In addition, studies examining changes in the mouse phosphoproteome identified a number of novel phosphorylation sites.
published_date 2013-12-31T03:53:30Z
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score 11.013371

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