Toward computer-based cleavage site prediction of cysteine endopeptidases

Identification of relevant substrates is essential for elucidation of in vivo functions of peptidases. The recent availability of the complete genome sequences of many eukaryotic organisms holds the promise of identifying specific peptidase substrates by systematic proteome analyses in combination with computer based screening of genome databases. Currently available proteomics and bioinformatics tools are not sufficient for reliable endopeptidase substrate predictions. To address these shortcomings the bioinformatics tool PEPS (Prediction of Endopeptidase Substrates) has been developed and is presented here. PEPS uses individual rulebased endopeptidase cleavage site scoring matrices (CSSM). The efficiency of PEPS in predicting putative caspase 3, cathepsin B and cathepsin L cleavage sites is demonstrated in comparison to established algorithms. Mortalin, a member of the heat shock protein family HSP70, was identified by PEPS as a putative cathepsin L substrate. Comparative proteome analyses of cathepsin Ldeficient and wildtype mouse fibroblasts showed that mortalin is enriched in the absence of cathepsin L. These results indicate that CSSM/PEPS can correctly predict relevant peptidase substrates.