title: Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.
abstract: 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.
authors.affiliation: Departments of Structural Biology and Developmental Neurobiology, ‡St. Jude Proteomics Facility, St. Jude Children's Research Hospital , Memphis, Tennessee 38105, United States.
authors.forename: Bing
authors.initials: B
authors.lastname: Bai
authors.forename: Ping-Chung
authors.initials: PC
authors.lastname: Chen
authors.forename: Chadwick M
authors.initials: CM
authors.lastname: Hales
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Wu
authors.forename: Vishwajeeth
authors.initials: V
authors.lastname: Pagala
authors.forename: Anthony A
authors.initials: AA
authors.lastname: High
authors.forename: Allan I
authors.initials: AI
authors.lastname: Levey
authors.forename: James J
authors.initials: JJ
authors.lastname: Lah
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
mesh_terms: D000544:Alzheimer Disease / Q000378:metabolism* / Q000503:physiopathology; D000818:Animals; D015153:Blotting, Western; D002853:Chromatography, Liquid; D006624:Hippocampus / Q000166:cytology*; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D059748:Proteolysis; D051381:Rats; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D010446:Peptide Fragments; D017412:Ribonucleoprotein, U1 Small Nuclear; C490688:SNRNP70 protein, human
keywords: Alzheimer’s disease; LC−MS/MS; RNA splicing; U1 snRNP; U1-70K; mass spectrometry; neurodegenerative disease; proteolytic cleavage; proteomics; stable isotope labeling