Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease.
J Proteome Res
; 13(11): 4526-34, 2014 Nov 07.
Article
in En
| MEDLINE
| ID: mdl-24902715
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 â¼50% 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.Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Peptide Fragments
/
Ribonucleoprotein, U1 Small Nuclear
/
Alzheimer Disease
/
Hippocampus
/
Neurons
Type of study:
Prognostic_studies
Limits:
Animals
/
Humans
Language:
En
Journal:
J Proteome Res
Journal subject:
BIOQUIMICA
Year:
2014
Type:
Article
Affiliation country:
United States