Proteomic Identification of Protease Cleavage Sites Characterizes Prime and Non-prime Specificity of Cysteine Cathepsins B, L, and S

Cysteine cathepsins mediate proteome homeostasis and have pivotal functions in diseases such as cancer. To better understand substrate recognition by cathepsins B, L, and S, we applied <u>p</u>roteomic <u>i</u>dentification of protease <u>c</u>leavage <u>s</u>ites (PICS) for simultaneous profiling of prime and non-prime specificity. PICS profiling of cathepsin B endopeptidase specificity highlights strong selectivity for glycine in P3′ due to an occluding loop blocking access to the primed subsites. In P1′, cathepsin B has a partial preference for phenylalanine, which is not found for cathepsins L and S. Occurrence of P1′ phenylalanine often coincides with aromatic residues in P2. For cathepsin L, PICS identifies 845 cleavage sites, representing the most comprehensive PICS profile to date. Cathepsin L specificity is dominated by the canonical preference for aromatic residues in P2 with limited contribution of prime-site selectivity determinants. Profiling of cathepsins B and L with a shorter incubation time (4 h instead of 16 h) did not reveal time-dependency of individual specificity determinants. Cathepsin S specificity was profiled at pH 6.0 and 7.5. The PICS profiles at both pH values display a high degree of similarity. Cathepsin S specificity is primarily guided by aliphatic residues in P2 with limited importance of prime-site residues