title: A designed redox-controlled caspase.
abstract: Caspases are a powerful class of cysteine proteases. Introduction of activated caspases in healthy or cancerous cells results in induction of apoptotic cell death. In this study, we have designed and characterized a version of caspase-7 that can be inactivated under oxidizing extracellular conditions and then reactivated under reducing intracellular conditions. This version of caspase-7 is allosterically inactivated when two of the substrate-binding loops are locked together via an engineered disulfide. When this disulfide is reduced, the protein regains its full function. The inactive loop-locked version of caspase-7 can be readily observed by immunoblotting and mass spectrometry. The reduced and reactivated form of the enzyme observed crystallographically is the first caspase-7 structure in which the substrate-binding groove is properly ordered even in the absence of an active-site ligand. In the reactivated structure, the catalytic-dyad cysteine-histidine are positioned 3.5 Å apart in an orientation that is capable of supporting catalysis. This redox-controlled version of caspase-7 is particularly well suited for targeted cell death in concert with redox-triggered delivery vehicles.
authors.affiliation: Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, USA.
authors.forename: Witold A
authors.initials: WA
authors.lastname: Witkowski
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
mesh_terms: D001665:Binding Sites; D053179:Caspase 7 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D015202:Protein Engineering; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004220:Disulfides; D011994:Recombinant Proteins; D053179:Caspase 7