Substrate Profiling of Cysteine Proteases Using a Combinatorial Peptide Library Identifies Functionally Unique Specificities

Abstract

The substrate specificities of papain-like cysteine proteases (clan CA, family C1) papain, bromelain, and human cathepsins L, V, K, S, F, B, and five proteases of parasitic origin were studied using a completely diversified positional scanning synthetic combinatorial library. A bifunctional coumarin fluorophore was used that facilitated synthesis of the library and individual peptide substrates. The library has a total of 160,000 tetrapeptide substrate sequences completely randomizing each of the P1, P2, P3, and P4 positions with 20 amino acids. A microtiter plate assay format permitted a rapid determination of the specificity profile of each enzyme. Individual peptide substrates were then synthesized and tested for a quantitative determination of the specificity of the human cathepsins. Despite the conserved three-dimensional structure and similar substrate specificity of the enzymes studied, distinct amino acid preferences that differentiate each enzyme were identified. The specificities of cathepsins K and S partially match the cleavage site sequences in their physiological substrates. Capitalizing on its unique preference for proline and glycine at the P2 and P3 positions, respectively, selective substrates and a substrate-based inhibitor were developed for cathepsin K. A cluster analysis of the proteases based on the complete specificity profile provided a functional characterization distinct from standard sequence analysis. This approach provides useful information for developing selective chemical probes to study protease-related pathologies and physiologies.Proteases hydrolyze amide bonds in proteins and peptides and represent one of the largest and most important protein families known. They comprise over 2% of the human genome and play diverse physiological roles (merops.sanger.ac.uk) (1). The substrate specificity of a protease enables the enzyme to preferentially cleave its substrates in the presence of other peptides or proteins. Therefore, specificity information can provide clues about the biological function of the protease and aid in the design of efficient substrates and potent, selective inhibitors. Various methods including both biological and chemicalbased approaches to study protease specificity have been developed and were recently reviewed (2).Positional scanning synthetic combinatorial libraries (PS-SCLs) 2 of fluorogenic substrates have emerged as useful reagents for the rapid and exhaustive determination of protease specificity (3). A peptide-based PS-SCL is composed of sublibraries in which one peptide position is fixed with an amino acid, whereas the remaining positions contain an equimolar mixture of amino acids. Assaying proteases with these sublibraries rapidly establishes the amino acid preferences at the defined position. Initially, the substrate specificities of caspases and granzyme B were profiled using PSSCLs with the P1 position fixed as an aspartic acid.The limitations of the original P1 fixed libraries were overcome through the development of a modified coumarin, 7-amino-...

Enzymatic and Structural Characterization of the Major Endopeptidase in the Venus Flytrap Digestion Fluid

“…The identification of Leu/Ile at P 2 and Arg/Lys at P 1 is consistent with the papain-like preference for hydrophobic residues at P 2 and a positively charged P 1 (54). In addition, cleavage also occurred with Arg/Lys in the P 2 position, which has not been observed for papain (55) (Fig. 5B).…”

Coordinate expression and functional profiling identify an extracellular proteolytic signaling pathway

“…Limited information on MT-SP1 substrate specificity was collected by using a complete diverse positionally scanned synthetic combinatorial library (PS-SCL) of synthetic substrates. The method can be used to identify consensus, nonprime side cleavage motifs for proteases (15). Our functional characterization of the binding specificity of MT-SP1 at the substrate-binding cleft is in accord with the information obtained from structural studies revealing trypsinlike specificity at the S1 position, a shallow pocket for small, hydrophobic residues at the S2 position, and an open negatively charged cavity at the S4 position, allowing for binding of a basic residue at P3 or P4 (16).…”

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

“…2D). Cat-S cleavage of PAR 2 at E 56 2T 57 would expose a potential tethered ligand domain beginning 57 TVFSV. Incubation of cells with the decapeptide TVFSVDEFSA (50 M, 30 min), which corresponds to the putative tethered ligand and is hereafter referred to as Cat-S activating peptide (AP), did not affect the subcellular localization of Flag or HA11, which remained at the plasma membrane (Fig.…”