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

Abstract

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.

Mechanistic insight into taxol-induced cell death

“…For instance, of the 38 sites detected in dying K562 cells, 5 were also identified in A549 cells: in reticulon-4, PTBP1, KRT8 and KRT18. Apart from cleavage of PTBP1 at Met-175 in Fasinduced apoptosis in Jurkat cells (Van Damme et al, 2005), these sites were never found in our studied models. Furthermore, five additional sites were found in KRT8 and KRT18 of K562 cells (Table 2).…”

“…Using this differential setup, levels of N termini in control and treated cells were compared and singletons or neo-N-termini in the proteome digest of detached A549 cells specifically pointed to processed proteins (Van Damme et al, 2005;Vande Walle et al, 2007) (Table 1). Two major classes were distinguished: (1) 16 processed sites C-terminal to aspartic acid (Asp), 8 of which were previously reported among others in Fastreated Jurkat cells and are considered caspase cleavage sites (Van Damme et al, 2005) and (2) 11 processed sites C-terminal to other residues, 10 of which are hitherto unreported.…”

“…Two major classes were distinguished: (1) 16 processed sites C-terminal to aspartic acid (Asp), 8 of which were previously reported among others in Fastreated Jurkat cells and are considered caspase cleavage sites (Van Damme et al, 2005) and (2) 11 processed sites C-terminal to other residues, 10 of which are hitherto unreported.…”

“…We ruled out the former, as in taxol-treated detached cells polypyrimidine tract-binding protein 1 (PTBP1) and TIF1-b, two known caspase substrates (Van Damme et al, 2005), were cleaved with the generation of correctly sized fragments (also observed in Fastreated Jurkat cells, see Figure 4b) and these cleavages were inhibited by zVADfmk. Furthermore, in lysates of taxol-treated cells, zVADfmk concentrations as low as 25 mM completely abolished general DEVDase activity (Figure 4c).…”

“…This method specifically isolates (neo-)N-terminal peptides from digested proteomes before identification by tandem mass spectrometry (Gevaert et al, 2003) and has proven to be a powerful tool for substrate degradome analysis (Lopez-Otin and Overall, 2002;Van Damme et al, 2005;Vande Walle et al, 2007): not only the protein substrates but also the exact cleavage sites are simultaneously assigned, providing links to the functional implication of the cleavage events and to the type(s) of protease(s) creating the cleavage patterns.…”