Deciphering Membrane-Associated Molecular Processes in Target Tissue of Autoimmune Uveitis by Label-Free Quantitative Mass Spectrometry
Deciphering Membrane-Associated Molecular Processes in Target Tissue of Autoimmune Uveitis by Label-Free Quantitative Mass Spectrometry
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DOI：
10.1074/mcp.m110.001073
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发表时间：
2010-10-01
影响因子：
7
通讯作者：
Ueffing, Marius
中科院分区：
文献类型：
--
作者：
Hauck, Stefanie M.;Dietter, Johannes;Ueffing, Marius
自身免疫性葡萄膜炎是一种致盲性疾病，表现为针对眼睛特异蛋白的自身抗体以及自体进行性T细胞入侵和攻击免疫特权的靶组织视网膜。使T细胞入侵和攻击组织的分子事件仍然难以捉摸。疾病阶段和健康阶段之间膜蛋白表达模式的变化特别有趣，因为疾病的起始事件很可能发生在膜上。由于疾病进展伴随着血-视网膜屏障的破坏，血清衍生蛋白掩盖了潜在的靶组织相关变化。为了克服这一局限性，我们使用了唯一可用的马复发性葡萄膜炎自发动物模型的视网膜膜富集组分，并通过基于无标记LC-MSMS的策略将表达水平与健康对照样本进行了比较。我们可以很容易地鉴定出总共893种马蛋白，其中57%归因于基因本体论项目术语“膜”。其中，179个蛋白质在马复发性葡萄膜炎组织中发现差异表达。途径浓缩分析表明，与抗原处理和提呈、肿瘤坏死因子受体信号转导、整合素细胞表面相互作用和焦点粘连相关的蛋白质增加。此外，还观察到反映视力下降的视网膜特异性蛋白的丢失，以及反映胶质细胞反应性的Muller胶质细胞特异性蛋白的增加。选定的候选蛋白(小窝蛋白1、整合素α1和粘着斑激酶)通过免疫组织化学和组织染色图谱显示，这些蛋白在作为血-视网膜外屏障一部分的外界膜水平显著增加。总之，膜浓缩与基于LC-MSMS的无标记定量相结合极大地提高了比较组织图谱的灵敏度，并导致了与马复发性葡萄膜炎相关的新的分子途径的检测。分子与细胞蛋白质组学9：2292-2305,2010。
Autoimmune uveitis is a blinding disease presenting with autoantibodies against eye-specific proteins as well as autoagressive T cells invading and attacking the immune-privileged target tissue retina. The molecular events enabling T cells to invade and attack the tissue have remained elusive. Changes in membrane protein expression patterns between diseased and healthy stages are especially interesting because initiating events of disease will most likely occur at membranes. Since disease progression is accompanied with a break-down of the blood-retinal barrier, serum-derived proteins mask the potential target tissue-related changes. To overcome this limitation, we used membrane-enriched fractions derived from retinas of the only available spontaneous animal model for the disease equine recurrent uveitis, and compared expression levels by a label-free LC-MSMS-based strategy to healthy control samples. We could readily identify a total of 893 equine proteins with 57% attributed to the Gene Ontology project term "membrane." Of these, 179 proteins were found differentially expressed in equine recurrent uveitis tissue. Pathway enrichment analyses indicated an increase in proteins related to antigen processing and presentation, TNF receptor signaling, integrin cell surface interactions and focal adhesions. Additionally, loss of retina-specific proteins reflecting decrease of vision was observed as well as an increase in Muller glial cell-specific proteins indicating glial reactivity. Selected protein candidates (caveolin 1, integrin alpha 1 and focal adhesion kinase) were validated by immunohistochemistry and tissue staining pattern pointed to a significant increase of these proteins at the level of the outer limiting membrane which is part of the outer blood-retinal barrier. Taken together, the membrane enrichment in combination with LC-MSMS-based label-free quantification greatly increased the sensitivity of the comparative tissue profiling and resulted in detection of novel molecular pathways related to equine recurrent uveitis. Molecular & Cellular Proteomics 9:2292-2305, 2010.