Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities

Youngchool Choe, Francesco Leonetti, Doron C. Greenbaum, Fabien Lecaille, Matthew Bogyo, Dieter Brömme, Jonathan A. Ellman, Charles S. Craik

Research output: Contribution to journalArticlepeer-review

342 Scopus citations

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.

Original languageEnglish
Pages (from-to)12824-12832
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number18
DOIs
StatePublished - 5 May 2006

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