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Caspase-specific and nonspecific in vivo protein processing during Fas-induced apoptosis


We generated a comprehensive picture of protease substrates in anti-Fas–treated apoptotic human Jurkat T lymphocytes. We used combined fractional diagonal chromatography (COFRADIC) sorting of protein amino-terminal peptides coupled to oxygen-16 or oxygen-18 differential labeling. We identified protease substrates and located the exact cleavage sites within processed proteins. Our analysis yielded 1,834 protein identifications and located 93 cleavage sites in 71 proteins. Indirect evidence of apoptosis-specific cleavage within 21 additional proteins increased the total number of processed proteins to 92. Most cleavages were at caspase consensus sites; however, other cleavage specificities suggest activation of other proteases. We validated several new processing events by immunodetection and by an in vitro assay using recombinant caspases and synthetic peptides containing presumed cleavage sites. The spliceosome complex appeared a preferred target, as 14 of its members were processed. Differential isotopic labeling further revealed specific release of nucleosomal components from apoptotic nuclei.

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The authors thank P. Vandenabeele for his critical reading of the manuscript, for the suggestions made and for providing the recombinant caspases. K.G. is a postdoctoral fellow and L.M. is a research assistant of the Fund for Scientific Research-Flanders (Belgium; F.W.O.-Vlaanderen). The project was supported by research grants from the Fund for Scientific Research-Flanders (Belgium; project number G.0008.03), the Inter University Attraction Poles (IUAP, project number P5/05), the GBOU research initiative (project number 20204) of the Flanders Institute of Science and Technology (IWT) and the European Union Interaction Proteome (6th Framework Program).

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Correspondence to Kris Gevaert.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Histogram showing the distribution of ratios of isotopic peptide couples measured following COFRADIC isolation. (PDF 108 kb)

Supplementary Fig. 2

Analysis of the apoptotic-specific cleavage sites within known or predicted domains and motifs of the identified processed Jurkat proteins. (PDF 1648 kb)

Supplementary Table 1

N-termini of mitochondrial proteins. (PDF 89 kb)

Supplementary Table 2

Internally located α-N-acetylated peptides. (PDF 171 kb)

Supplementary Table 3

Identification features of peptides linked to proteins processed in apoptotic Jurkat cells. (PDF 149 kb)

Supplementary Table 4

Peptides only present in lysates of living cells. (PDF 128 kb)

Supplementary Table 5

Summary of proteins that are specifically cleaved in Fas-stimulated Jurkat T-lymphocytes. (PDF 113 kb)

Supplementary Table 6

Peptides exclusively found in the proteome digest of apoptotic cells. (PDF 102 kb)

Supplementary Methods (PDF 53 kb)

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Figure 1: Scenarios leading to different categories of N-blocked peptides in apoptotic lysates.
Figure 2: Identification of cleavage sites in death substrates.
Figure 3: Validation of protein processing in apoptotic Jurkat cells.
Figure 4: Location of the apoptotic-specific cleavage sites within the predicted domain organizations of affected spliceosomal proteins.