Activity-based probes that target diverse cysteine protease families

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.

Commonly used caspase inhibitors designed based on substrate specificity profiles lack selectivity

“…For these studies, we obtained three commercially available inhibitors (Z-DEVD-FMK, Z-IETD-FMK, and Z-LEHD-FMK) equipped with a fluoromethylketone (FMK) reactive group. We also synthesized three inhibitors that contained the same peptide sequences (NP-DEVD-AOMK, NP-LETD-AOMK, and NP-LEHD-AOMK) but linked to the acyloxymethylketone (AOMK) reactive group shown previously to be effective towards caspases [7,8] ( Figure 1A). To test inhibitor selectivity against caspases 3, 7, and 9 we used an apoptotic proteome in which the intrinsic apoptotic pathway is activated through addition of cytochrome c and dATP [9].…”

“…After incubation, the poly-caspase probe KMB01 (biotin-hex-EVD-AOMK) [8] was added to label residual caspase active sites for an additional 30 min. After 10-min incubation with cytochrome c and dATP, KMB01 labels the fully cleaved forms of 20 npg Selectivity of caspase inhibitors…”

Design of cell-permeable, fluorescent activity-based probes for the lysosomal cysteine protease asparaginyl endopeptidase (AEP)/legumain

“…Activity based probes (ABPs) are reagents that can specifically label active proteases, thus allowing their activity, and more importantly their regulation, to be directly monitored [6,7]. Our laboratory recently reported a synthesis strategy based on the general solid phase methods developed by Ellman and co-workers [8] for the production of peptidyl acyloxymethyl ketone ABPs for diverse cysteine protease activities [9].We have previously demonstrated that the biotinylated ABP b-hex-D-AOMK efficiently labels endogenous legumain in 816 B cell lysates [9]. However this reagent lacks cell permeability and its overall selectivity towards legumain had not been extensively examined.…”

“…Activity based probes (ABPs) are reagents that can specifically label active proteases, thus allowing their activity, and more importantly their regulation, to be directly monitored [6,7]. Our laboratory recently reported a synthesis strategy based on the general solid phase methods developed by Ellman and co-workers [8] for the production of peptidyl acyloxymethyl ketone ABPs for diverse cysteine protease activities [9].…”

“…We have previously demonstrated that the biotinylated ABP b-hex-D-AOMK efficiently labels endogenous legumain in 816 B cell lysates [9]. However this reagent lacks cell permeability and its overall selectivity towards legumain had not been extensively examined.…”