Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein
Application of high-throughput, capillary-based Western analysis to modulated cleavage of the cellular prion protein
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DOI：
10.1074/jbc.ra118.006367
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发表时间：
2019-02-22
影响因子：
4.8
通讯作者：
Westaway, David
中科院分区：
文献类型：
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作者：
Castle, Andrew R.;Daude, Nathalie;Westaway, David
细胞蛋白(PrPC)是一种糖蛋白，通过几种蛋白分解途径进行加工。PrPC蛋白水解酶的调节剂之所以引起人们的兴趣，是因为全长PrPC及其切割片段在错误折叠的倾向上有所不同，这一过程在Prion疾病的发病机制中发挥了关键作用。PrPC也可以作为与神经退行性变有关的其他蛋白质的神经毒性、寡聚体的受体。重要的是，PrPC C-末端片段C1不包含已报道的这些寡聚体的结合位点。Western blotting将是一种简单的终点检测方法，用于基于细胞的化合物文库筛选对PrPC蛋白分解或整体表达水平的影响。然而，传统的Western blotting方法提供的定量结果并不可靠，而且吞吐量很低。因此，我们探索了基于毛细管的西方技术作为一种潜在的替代方案；我们相信这项研究是第一次报告使用这种方法进行PrPC的分析。我们成功地优化了全长PrPC及其C端切割片段C1和C2的脱糖基化形式的检测和定量，包括同时定量β-微管蛋白水平以控制加载误差。我们还开发和测试了一种方法，用于在毛细管蛋白质分析之前，在96孔微孔板中执行所有细胞培养、裂解和脱糖步骤。这些进展代表了在开发自动化、高通量筛选管道以识别PrPC表达水平或蛋白质降解的调节器的过程中所采取的步骤。
The cellular prion protein (PrPC) is a glycoprotein that is processed through several proteolytic pathways. Modulators of PrPC proteolysis are of interest because full-length PrPC and its cleavage fragments differ in their propensity to misfold, a process that plays a key role in the pathogenesis of prion diseases. PrPC may also act as a receptor for neurotoxic, oligomeric species of other proteins that are linked to neurodegeneration. Importantly, the PrPC C-terminal fragment C1 does not contain the reported binding sites for these oligomers. Western blotting would be a simple end point detection method for cell-based screening of compound libraries for effects on PrPC proteolysis or overall expression level. However, traditional Western blotting methods provide unreliable quantification and have only low throughput. Consequently, we explored capillary-based Western technology as a potential alternative; we believe that this study is the first to report analysis of PrPC using such an approach. We successfully optimized the detection and quantification of the deglycosylated forms of full-length PrPC and its C-terminal cleavage fragments C1 and C2, including simultaneous quantification of beta-tubulin levels to control for loading error. We also developed and tested a method for performing all cell culture, lysis, and deglycosylation steps in 96-well microplates prior to capillary Western analysis. These advances represent steps along the way to the development of an automated, high-throughput screening pipeline to identify modulators of PrPC expression levels or proteolysis.