Activation of specific apoptotic caspases with an engineered small-molecule-activated protease.
Apoptosis is a conserved cellular pathway that results in the activation of cysteine-aspartyl proteases, or caspases. To dissect the nonredundant roles of the executioner caspase-3, -6, and -7 in orchestrating apoptosis, we have developed an orthogonal protease to selectively activate each isoform in human cells. Our approach uses a split-tobacco etch virus (TEV) protease under small-molecule control, which we call the SNIPer, with caspase alleles containing genetically encoded TEV cleavage sites. These studies reveal that all three caspases are transiently activated but only activation of caspase-3 or -7 is sufficient to induce apoptosis. Proteomic analysis shown here and from others reveals that 20 of the 33 subunits of the 26S proteasome can be cut by caspases, and we demonstrate synergy between proteasome inhibition and dose-dependent caspase activation. We propose a model of proteolytic reciprocal negative regulation with mechanistic implications for the combined clinical use of proteasome inhibitors and proapoptotic drugs.
Gray
Daniel C
DC
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158, USA.
Mahrus
Sami
S
Wells
James A
JA
D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism*; D053178:Caspase 6 / Q000378:metabolism; D053179:Caspase 7 / Q000378:metabolism*; D002460:Cell Line; D015195:Drug Design; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D061988:Proteasome Inhibitors; D015202:Protein Engineering
D007527:Isoenzymes; D007976:Leupeptins; D061988:Proteasome Inhibitors; D010450:Endopeptidases; C099488:TEV protease; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde