%0 Journal Article %A Bai, Bing %A Chen, Ping-Chung %A Hales, Chadwick M. %A Wu, Zhiping %A Pagala, Vishwajeeth %A High, Anthony A. %A Levey, Allan I. %A Lah, James J. %A Peng, Junmin %D 2015 %T Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer’s Disease %U https://acs.figshare.com/articles/journal_contribution/Integrated_Approaches_for_Analyzing_U1_70K_Cleavage_in_Alzheimer_s_Disease/2043492 %R 10.1021/pr5003593.s001 %2 https://acs.figshare.com/ndownloader/files/3614808 %K N 40K %K cleavage site %K AD %K Alzheimer %K N 40K share %K MS %K N 40K fragment %K N 40K expression %K RNA %K N 40K abundance %X 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 ∼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. %I ACS Publications