Activation of Specific Apoptotic Caspases with an Engineered Small-Molecule-Activated Protease

SummaryApoptosis 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.PaperClip To listen to this audio, enable JavaScript on your browser. However, you can download and play the audio by clicking on the icon belowHelp with MP3 filesOptionsDownload audio (2090 K)

Graphical AbstractFigure optionsDownload high-quality image (166 K)Download as PowerPoint slideHighlights
► An orthogonal protease was optimized to activate the executioner caspases in cells
► Activation of caspase-3 or -7 is sufficient to induce apoptosis
► Caspase-3, -6, and -7 activity is amplified with proteasome inhibitors
► At least 20 proteasome subunits are cleaved by the caspases during apoptosis