Synthesis of novel caspase inhibitors for characterization of the active caspase proteome in vitro and in vivo.

ABSTRACT: Caspases are cysteine proteases that are essential for cytokine maturation and apoptosis. To facilitate the dissection of caspase function in vitro and in vivo, we have synthesized irreversible caspase inhibitors with biotin 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 active caspases. Using the strong affinity of avidin for biotin, we have isolated affinity-labeled caspase 6 from apoptotic cytosolic extracts of cells overexpressing procaspase 6 by treatment with 12c, which contains biotin attached to the N(epsilon)-lysine of the inhibitor by a 22.5 A linker arm, followed by affinity purification on monomeric avidin-sepharose beads. C

SUBMITTER: Henzing AJ 

PROVIDER: S-EPMC2564993 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature