Cancer Institute A national cancer institute
designated cancer center

Matthew Bogyo

Publication Details

  • Substrate profiling of cysteine proteases using a combinatorial peptide library identifies functionally unique specificities JOURNAL OF BIOLOGICAL CHEMISTRY Choe, Y., Leonetti, F., Greenbaum, D. C., Lecaille, F., Bogyo, M., Bromme, D., Ellman, J. A., Craik, C. S. 2006; 281 (18): 12824-12832

    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.

    View details for DOI 10.1074/jbc.M513331200

    View details for Web of Science ID 000237134700077

    View details for PubMedID 16520377

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