Activity-Based Probes that Target Diverse Cysteine Protease Families

Daisuke Kato, Kelly M. Boatright, Alicia B. Berger, Tamim Nazif, Galia Blum, Ciara Ryan, Kareem A.H. Chehade, Guy S. Salvesen, Matthew Bogyo*

*Corresponding author for this work

Research output: Contribution to journalLetterpeer-review

302 Scopus citations

Abstract

Proteases are one of the largest and best-characterized families of enzymes in the human proteome. Unfortunately, the understanding of protease function in the context of complex proteolytic cascades remains in its infancy. One major reason for this gap in understanding is the lack of technologies that allow direct assessment of protease activity. We report here an optimized solid-phase synthesis protocol that allows rapid generation of activity-based probes (ABPs) targeting a range of cysteine protease families. These reagents selectively form covalent bonds with the active-site thiol of a cysteine protease, allowing direct biochemical profiling of protease activities in complex proteomes. We present a number of probes containing either a single amino acid or an extended peptide sequence that target caspases, legumains, gingipains and cathepsins. Biochemical studies using these reagents highlight their overall utility and provide insight into the biochemical functions of members of these protease families.

Original languageAmerican English
Pages (from-to)33-38
Number of pages6
JournalNature Chemical Biology
Volume1
Issue number1
DOIs
StatePublished - Jun 2005
Externally publishedYes

Bibliographical note

Funding Information:
We thank C. Watts (University of Dundee) for legumain antibodies, C. Peters and T. Reinheckel (Albert-Ludwigs-Universität Freiburg) for legumain-knockout mouse tissues, H. Chapman and J. Lee (University of California, San Francisco) for recombinant mouse legumain, V. Turk and B. Turk (J. Stefan Institute) for recombinant human cathepsin L, J. Potempa (University of Georgia) for P. gingivalis cell extracts and purified gingipains, and T. Burster (Stanford University) for cell lines. We thank A. Price and S. Snipas for technical assistance, L. Carpino (University of Massachusetts) for critical advice on the removal of the Fmoc protecting group and J. Ellman, A. Lee, L. Huang and W. Wood for helpful discussions. This work was supported by a Turman Fellowship at Stanford University (to M.B.), a National Institutes of Health National Technology Center for Networks and Pathways grant U54 RR020843 (to M.B. and G.S.S.) and a Department of Defense Breast Cancer Center of Excellence grant DAMD-17-02-0693 (to B. Sloane—M.B. subcontract). A.B. was funded by a National Human Genome Research Institute training grant 5T32 HG00044.

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