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Author Notes:

Corresponding Author: Department of Neurology, Center for Neurodegenerative Diseases, Emory University, Atlanta, Georgia 30322, United States J. Peng. Tel.: 901-336-1083. E-mail: junmin.peng@stjude.org.

The authors thank all of the lab members for helpful discussion.

Subjects:

Research Funding:

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:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemical Research Methods
  • Biochemistry & Molecular Biology
  • mass spectrometry
  • LC-MS/MS
  • proteomics
  • neurodegenerative disease
  • Alzheimers disease
  • stable isotope labeling
  • proteolytic cleavage
  • U1 snRNP
  • U1-70K
  • RNA splicing
  • FRONTOTEMPORAL LOBAR DEGENERATION
  • AMYOTROPHIC-LATERAL-SCLEROSIS
  • SPECTROMETRY-BASED PROTEOMICS
  • MASS-SPECTROMETRY
  • NEURODEGENERATIVE DISEASE
  • PROTEIN
  • TDP-43
  • BRAIN
  • DEATH
  • TAU

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

Tools:

Journal Title:

Journal of Proteome Research

Volume:

Volume 13, Number 11

Publisher:

, Pages 4526-4534

Type of Work:

Article | Final Publisher PDF

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.

Copyright information:

© 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.

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