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

Abstract

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 proteomic identification of protease cleavage sites (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.

Activation of the Nipah Virus Fusion Protein in MDCK Cells Is Mediated by Cathepsin B within the Endosome-Recycling Compartment

“…The major difference in the structures of cathepsins L and B is that the latter contains a so-called occluding loop that covers part of the catalytic center, thereby limiting access of substrate proteins for endoproteolytic cleavage. Recently, the cleavage specificities of cathepsins B and L on naturally occurring peptides have been reassessed by the proteomic approach PICS (proteomic identification of protease cleavage sites [5,63]). Remarkably, the PICS cleavage site logo at pH 6 for cathepsin B contains 6 of the 8 amino acids present in the P4-P4= NiV F cleavage site.…”

Cathepsin S Causes Inflammatory Pain via Biased Agonism of PAR2 and TRPV4

“…We first determined whether Cat-S cleaved a decapeptide in which the P2, P1, and P1Ј positions were substituted: V 55 3 S, E 56 3 P, and T 57 3 K ( 52 GVTSPKVFSVD 62 ). As expected, Cat-S was unable to cleave this peptide at concentration up to 880 M (Fig.…”

“…In experiments in HEK293 cells expressing PAR 2 with N-terminal Flag and C-terminal HA11 epitopes, we observed that Cat-S removed the extracellular Flag epitope, which indicates that Cat-S can cleave intact PAR 2 at the cell surface as well as receptor fragments. To ascertain the importance of cleavage at the E 56 2T 57 for Cat-S activation of PAR 2 , we studied the capacity of Cat-S to cleave and activate a mutant receptor in which the P2, P1 and P1Ј positions were replaced: V 55 Cat-S cleaves within the N terminus of PAR 2 is consistent with the known requirements for Cat-S substrate recognition (56,57). Aliphatic residues at the P2 position, including valine and leucine, direct Cat-S selectivity.…”

Out-of-frame start codons prevent translation of truncated nucleo-cytosolic cathepsin L in vivo

“…They are potent ubiquitously expressed endopeptidases with broad substrate specificity that work best at acidic pH and reducing redox conditions 8,9 . Cathepsin L-deficient (Ctsl À / À ) mice reproduce normally, although pups showed a slightly increased mortality during the weaning period 10 .…”