| Abstract
| - Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate thedissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors withbiotin attached via linker arms of various lengths (12a−d) and a 2,4-dinitrophenyl labeled inhibitor (13).Affinity labeling of apoptotic extracts followed by blotting reveals that these affinity probes detect activecaspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 fromapoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which containsbiotin attached to the Nε-lysine of the inhibitor by a 22.5 Å linker arm, followed by affinity purification onmonomeric avidin-sepharose beads. Compound 13 has proven sufficiently cell permeable to rescue cellsfrom apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study ofthe active caspase proteome during apoptosis both in vitro and in vivo.
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