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.
Van Damme
Petra
P
Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Belgium.
Martens
Lennart
L
Van Damme
Jozef
J
Hugelier
Koen
K
Staes
An
A
Vandekerckhove
Joël
J
Gevaert
Kris
K
D000595:Amino Acid Sequence; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D019014:fas Receptor / Q000378:metabolism
D010455:Peptides; D011506:Proteins; D019014:fas Receptor; D020169:Caspases