Caspase-mediated cleavage events hidden by secondary proteolysis during apoptosis

Caspases are cysteine proteases that cleave specific proteins to control a range of cellular processes including cell death, inflammation, and differentiation. Proteomic approaches, like N-terminomics, have been central to identifying both cleaved proteins and where they are cleaved. We recently reported N-terminomics on differentiating and dying C2C12 myoblasts, identifying many new caspase substrates and cleavage sites. Here, we present a further analysis of this dataset demonstrating that there are caspase substrates "hidden" in our and in other N-terminomics datasets. We propose that for a subset of proteins, caspase cleavage is followed by removal of two amino acids from the neo-N-terminus by a dipeptidyl peptidase (DPP). As the peptide sequence of the neo-N-terminus is used to identify the cleavage site, this hides the caspase-mediated cleavage event. The implications of this are that N-terminomics datasets can be used to reveal hitherto unidentified caspase substrates and provide more information on cell-fate decisions. In C2C12 myoblasts, we have shown that cleavage of "hidden" substrates in apoptotic myoblasts that is absent from differentiating myoblasts (which also contain active caspase-3), suggesting that cleavage may identify apoptotic cells. A better understanding of the interplay between caspases and other proteases and their sequential action on caspase substrates may allow further elucidation of cell fate decisions.