Elsevier

Analytical Biochemistry

Volume 335, Issue 2, 15 December 2004, Pages 218-227
Analytical Biochemistry

An activity-based probe for the determination of cysteine cathepsin protease activities in whole cells

https://doi.org/10.1016/j.ab.2004.09.005Get rights and content

Abstract

We describe a novel diazomethylketone-containing irreversible inhibitor (BIL-DMK) which is specific for a subset of pharmaceutically important cysteine cathepsin proteases. BIL-DMK rapidly inactivates cathepsins B, F, K, L, S, and V in isolated enzyme assays and labels cathepsins in whole cells. The presence of catalytically active cathepsins B, L, and K or S was demonstrated using radioiodinated BIL-DMK in HepG2 (hepatoma), HIG82 (rabbit synoviocyte), and Ramos (B lymphoma) cell lines, respectively. The identity of each protein labeled was confirmed from the isoelectric point and molecular mass of the radioactive spots on two-dimensional gel and by comigration with each cathepsin as identified by immunoblotting. These cell lines were used to establish whole-cell enzyme occupancy assays to determine the potency of both irreversible and reversible inhibitors against each cathepsin in their native cellular lysosomal or endosomal environment. These whole-cell enzyme occupancy assays are useful to determine the cellular permeability of competing inhibitors and have the advantage of not requiring specific substrates for each cathepsin of interest.

Section snippets

Methods

Recombinant rabbit Cat K and human Cat F, L, S, and V were prepared as previously described [9], [12], [13]. Human liver Cat B was from Sigma, while recombinant human Cat C and Z were from R&D Systems. Cathepsin H was from Biomol. All protease substrates were from Bachem. Cbz-Y-A-DMK was from Enzyme Systems Probes and was radioiodinated by Amersham Pharmacia Biotech or with nonradioactive iodine using NaI and chloramine-T in dimethylformamide [14]. Dipeptidyl nitrile A was prepared by the

Inactivation of cathepsin activities by BIL-DMK

The irreversible inhibitor Cbz-Y(I)-A-DMK is a potent inhibitor of cathepsin L and V [1] but shows significantly weaker inhibition of cathepsins B, F, S, and K (Table 1). When cells from the human hepatoma cell line HepG2 were treated for 60 min with 5 nM radioiodinated form of Cbz-Y(I)-A-DMK and the samples analyzed by 2D gel electrophoresis and autoradiography, labeling of Cat B and L was readily detected (results not shown). In contrast, a relatively low amount of Cat S labeling was observed

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