title: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California at San Francisco, California 94143, USA.
authors.forename: Youngchool
authors.initials: Y
authors.lastname: Choe
authors.forename: Francesco
authors.initials: F
authors.lastname: Leonetti
authors.forename: Doron C
authors.initials: DC
authors.lastname: Greenbaum
authors.forename: Fabien
authors.initials: F
authors.lastname: Lecaille
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
authors.forename: Dieter
authors.initials: D
authors.lastname: Brömme
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Ellman
authors.forename: Charles S
authors.initials: CS
authors.lastname: Craik
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D056657:Cathepsin K; D002403:Cathepsins / Q000737:chemistry; D003374:Coumarins / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry*; D010802:Phylogeny; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D007659:Ketones; D010455:Peptides; C030123:coumarin; D002403:Cathepsins; C028217:cathepsin S; C534130:CTSK protein, human; D056657:Cathepsin K; D003545:Cysteine