期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 8, 页码 2642-2650出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.006367
关键词
prion disease; neurodegeneration; proteolysis; high-throughput screening (HTS); glycoprotein; protein misfolding; capillary Western; PrPC; simple Western
资金
- Canadian Institutes of Health Research [MOP123525, MOP137094]
- Alberta Prion Research Institute [APRIEP 201600033]
- Canada Research Chairs Program (CRC Tier 1 Grant) [230404]
- Canada Foundation for Innovation [NIF21633]
- Alzheimer's Society of Alberta and Northwest Territories/University Hospital Foundation Hope for Tomorrow catalyst grant
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.
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