期刊
JOURNAL OF PROTEOME RESEARCH
卷 13, 期 11, 页码 4526-4534出版社
AMER CHEMICAL SOC
DOI: 10.1021/pr5003593
关键词
mass spectrometry; LC-MS/MS; proteomics; neurodegenerative disease; Alzheimers disease; stable isotope labeling; proteolytic cleavage; U1 snRNP; U1-70K; RNA splicing
资金
- NIH [P50AG005136]
- American Academy of Neurology Foundation Clinical Research Training Fellowship
- ALSAC (American Lebanese Syrian Associated Charities)
- NIH Cancer Center Support Grant [P30CA021765]
The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimer's 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 similar to 50% 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-tandem 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 +/- 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's disease.
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