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Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer’s Disease
journal contribution
posted on 2015-12-17, 05:41 authored by Bing Bai, Ping-Chung Chen, Chadwick
M. Hales, Zhiping Wu, Vishwajeeth Pagala, Anthony A. High, Allan I. Levey, James
J. Lah, Junmin PengThe
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.