Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer鈥檚 Disease
详细信息 查看全文
- 作者：Bing Bai ; Ping-Chung Chen ; Chadwick M. Hales ; Zhiping Wu ; Vishwajeeth Pagala ; Anthony A. High ; Allan I. Levey ; James J. Lah ; Junmin Peng
- 刊名：Journal of Proteome Research
- 出版年：2014
- 出版时间：November 7, 2014
- 年：2014
- 卷：13
- 期：11
- 页码：4526-4534
- 全文大小：457K
- ISSN：1535-3907
文摘
The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer鈥檚 disease (AD) the aggregation of the U1-70K splicing factor and abnormal RNA processing. Here, we present that U1-70K can be cleaved into an N-terminal truncation (N40K) in 鈭?0% of AD cases, and the N40K abundance is inversely proportional to the total level of U1-70K. To map the cleavage site, we compared tryptic peptides of N40K and stable isotope labeled U1-70K by liquid chromatography鈥搕andem mass spectrometry (MS), revealing that the proteolysis site is located in a highly repetitive and hydrophilic domain of U1-70K. We then adapted Western blotting to map the cleavage site in two steps: (i) mass spectrometric analysis revealing that U1-70K and N40K share the same N-termini and contain no major modifications; (ii) matching N40K with a series of six recombinant U1-70K truncations to define the cleavage site within a small region (Arg300 卤 6 residues). Finally, N40K expression led to substantial degeneration of rat primary hippocampal neurons. In summary, we combined multiple approaches to identify the U1-70K proteolytic site and found that the N40K fragment might contribute to neuronal toxicity in Alzheimer鈥檚 disease.