Synthesis of novel caspase inhibitors for characterization of the active caspase proteome in vitro and in vivo.
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. Compound 13 has proven sufficiently cell permeable to rescue cells from apoptotic execution. These novel caspase inhibitors should provide powerful probes for the study of the active caspase proteome during apoptosis both in vitro and in vivo.
Henzing
Alexander J
AJ
The Wellcome Trust Centre for Cell Biology, ICB, Swann Building, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, United Kingdom.
Dodson
Helen
H
Reid
Joel M
JM
Kaufmann
Scott H
SH
Baxter
Robert L
RL
Earnshaw
William C
WC
D000345:Affinity Labels; D017209:Apoptosis / Q000187:drug effects; D053178:Caspase 6 / Q000378:metabolism; D061945:Caspase Inhibitors; D002846:Chromatography, Affinity; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects*; D004792:Enzyme Precursors / Q000737:chemistry*; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D019169:Jurkat Cells / Q000187:drug effects; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000345:Affinity Labels; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; D020543:Proteome; D053178:Caspase 6