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. 2014 Nov 7;13(11):4526-34.
doi: 10.1021/pr5003593. Epub 2014 Jun 13.

Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease

Bing Bai  1 , Ping-Chung Chen, Chadwick M Hales, Zhiping Wu, Vishwajeeth Pagala, Anthony A High, Allan I Levey, James J Lah, Junmin Peng
Affiliations

Integrated approaches for analyzing U1-70K cleavage in Alzheimer's disease

Bing Bai et al. J Proteome Res. .

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.

Keywords: Alzheimer’s disease; LC−MS/MS; RNA splicing; U1 snRNP; U1-70K; mass spectrometry; neurodegenerative disease; proteolytic cleavage; proteomics; stable isotope labeling.

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Figures

Figure 1
Figure 1
N40K occurrence is independent of the postmortem interval and correlates with the decrease of the U1-70K level in AD. (A) Identification of N40K in the detergent insoluble fraction of AD brain by 1D SDS gel and LC–MS/MS. Ctl: control case. (B) Validation of N40K only in AD (case 8 in Supporting Information Table S1) by Western blotting. (C) No statistically significant difference in postmortem intervals between the N40K negative and positive cases. (D) Western blotting to show reduced U1-70K level in the total homogenate in N40K positive cases. The numbers above the gel indicate the corresponding case numbers in Supporting Information Table S1. (E) Quantitation of the Western blotting intensity in panel D to indicate an inverse correlation of U1-70K in total homogenate with insoluble N40K. Relative intensities of U1-70K and N40K were normalized by setting the maximal values to 100, respectively.
Figure 2
Figure 2
Identification of U1-70K and N40K peptides in AD. These peptides are shown in different colors in the protein sequence, together with peptides monitored in LC–SRM.
Figure 3
Figure 3
Analysis of the N40K C-terminus by LC–SRM. (A) Work flow of the method. (B) Identification and size confirmation of purified recombinant U1-70K by Western blotting, followed by band excision and in-gel digestion. (C) Identification of a N40K–containing gel band by Western blotting. The 10 kDa ladder was also used for precise alignment of immunoblotting images with stained gel. (D) The intensity ratios of light/heavy (L/H) of U1-70K peptide pairs, leading to the predicted cleavage region.
Figure 4
Figure 4
Analysis of the N40K N-terminus by MS. (A–C) MS/MS spectra for identifying the N-terminal peptide with methionine removal and threonine acetylation in U1-70K, N40K, and purified U1-70K in HEK293 cells, respectively. Matched ions were assigned by arrows indicating specific b or y ions.
Figure 5
Figure 5
Determination of N40K C-terminus by Western blotting. (A) Expression of U1-70K N-terminal truncations in HEK293 cells. (B) Analysis of N40K in AD and three recombinant truncations. (C) Diagram of U1-70K domains and predicted N40K cleavage region. (D) N40K truncations covering all possible cleavage products with different C-terminal residues.
Figure 6
Figure 6
Toxicity analysis of N40K in rat hippocampal primary neurons. (A) Restriction enzyme digestion at the cloning sites by BamHI and EcoRI to show equal levels of the plasmids of U1-70K and six N40K truncations. (B) Western blotting to show similar protein expression in HEK293 cells. (C) Co-transfection of nine plasmids each with RFP by the calcium phosphate method. (D) Quantitative analysis by counting degenerating neurons. Error bars represent standard errors of mean (SEM) of analyzed neurons in duplicated plates of the same batch of neuronal culture. The experiments were repeated three times with different batches of culture, all showing similar results. The asterisks indicate p < 0.05 by the ANOVA and Tukey range test. (E) The MTT assay of neuronal viability upon Lentiviral N40K transfection. Error bars represent SEM. The double asterisks indicate p < 0.01 by Student’s t-test.
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