Integrated Approaches for Analyzing U1-70K Cleavage in Alzheimer's Disease

• Type of Material: Article
• Authors: Bing Bai, St. Jude Children’s Research Hospital; Ping-Chung Chen, St. Jude Children’s Research Hospital; Chadwick Hales, Emory University; Zhiping Wu, St. Jude Children’s Research Hospital; Vishwajeeth Pagala, St. Jude Children’s Research Hospital; Anthony A. High, St. Jude Children’s Research Hospital; Allan Levey, Emory University; James Lah, Emory University; Junmin Peng, St. Jude Children’s Research Hospital
• Language: English
• Date: 2014-11-01
• Publisher: American Chemical Society
• Publication Version: Final Published Version
• Copyright Statement: © 2014 American Chemical Society., ACS AuthorChoice - This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.
• Final Published Version (URL): http://dx.doi.org/10.1021/pr5003593
• Title of Journal or Parent Work: Journal of Proteome Research
• ISSN: 1535-3893
• Volume: 13
• Issue: 11
• Start Page: 4526
• End Page: 4534
• Grant/Funding Information: This work was partially supported by NIH grants P50AG005136, and American Academy of Neurology Foundation Clinical Research Training Fellowship to C.M.H. J.P. is supported by ALSAC (American Lebanese Syrian Associated Charities).
• Keywords: RNA splicing; Science & Technology; Alzheimers disease; AMYOTROPHIC-LATERAL-SCLEROSIS; FRONTOTEMPORAL LOBAR DEGENERATION; proteomics; NEURODEGENERATIVE DISEASE; PROTEIN; DEATH; U1-70K; TDP-43; TAU; U1 snRNP; Life Sciences & Biomedicine; neurodegenerative disease; mass spectrometry; Biochemistry & Molecular Biology; MASS-SPECTROMETRY; SPECTROMETRY-BASED PROTEOMICS; BRAIN; LC-MS/MS; proteolytic cleavage; stable isotope labeling; Biochemical Research Methods
• Research Categories: Biology, Molecular; Biology, Neuroscience

Abstract

The accumulation of pathologic protein fragments is common in neurodegenerative disorders. We have recently identified in Alzheimers 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 Alzheimers disease.