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

Bai B, Chen PC, Hales CM, Wu Z, Pagala V, High AA, Levey AI, Lah JJ, Peng J - J. Proteome Res. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Structural Biology and Developmental Neurobiology, ‡St. Jude Proteomics Facility, St. Jude Children's Research Hospital , Memphis, Tennessee 38105, United States.

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.

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Identification of U1-70Kand N40K peptides in AD. These peptidesare shown in different colors in the protein sequence, together withpeptides monitored in LC–SRM.
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fig2: Identification of U1-70Kand N40K peptides in AD. These peptidesare shown in different colors in the protein sequence, together withpeptides monitored in LC–SRM.

Mentions: We then sought to determine whether N40K is an alternative splicingisoform or a cleaved fragment of full-length U1-70K. In the UniProt/Swiss-Protdatabase, U1-70K has four different splicing isoforms of 437 (fulllength, Figure 2), 428, 341, and 166 residues.None of these four isoforms fit the size of N40K. To explore potentialnew U1-70K splicing isoforms in AD, we analyzed RNA-seq data setsof multiple AD brains26 but did not identifyany additional isoforms. All exon reads of U1-70K were evenly distributedalong the transcript, consistent with only one major RNA form of thefull length U1-70K. In addition, U1 SnRNP deficiency may cause prematurecleavage and polyadenylation in AD,26 leadingto shortened RNA transcripts. However, we did not find any prematurepolyadenylation in the exons of U1-70K. Therefore, N40K is most likelyproduced by the proteolysis of full length U1-70K.


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

Bai B, Chen PC, Hales CM, Wu Z, Pagala V, High AA, Levey AI, Lah JJ, Peng J - J. Proteome Res. (2014)

Identification of U1-70Kand N40K peptides in AD. These peptidesare shown in different colors in the protein sequence, together withpeptides monitored in LC–SRM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4227550&req=5

fig2: Identification of U1-70Kand N40K peptides in AD. These peptidesare shown in different colors in the protein sequence, together withpeptides monitored in LC–SRM.
Mentions: We then sought to determine whether N40K is an alternative splicingisoform or a cleaved fragment of full-length U1-70K. In the UniProt/Swiss-Protdatabase, U1-70K has four different splicing isoforms of 437 (fulllength, Figure 2), 428, 341, and 166 residues.None of these four isoforms fit the size of N40K. To explore potentialnew U1-70K splicing isoforms in AD, we analyzed RNA-seq data setsof multiple AD brains26 but did not identifyany additional isoforms. All exon reads of U1-70K were evenly distributedalong the transcript, consistent with only one major RNA form of thefull length U1-70K. In addition, U1 SnRNP deficiency may cause prematurecleavage and polyadenylation in AD,26 leadingto shortened RNA transcripts. However, we did not find any prematurepolyadenylation in the exons of U1-70K. Therefore, N40K is most likelyproduced by the proteolysis of full length U1-70K.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Departments of Structural Biology and Developmental Neurobiology, ‡St. Jude Proteomics Facility, St. Jude Children's Research Hospital , Memphis, Tennessee 38105, United States.

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.

Show MeSH
Related in: MedlinePlus