title: Cathepsin D primes caspase-8 activation by multiple intra-chain proteolysis.
abstract: During the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. A direct and fast activation of caspase-8 by cathepsin D was shown to be crucial in the initial steps of neutrophil apoptosis. Nevertheless, the activation mechanism of caspase-8 remains unclear. Here, by using site-specific mutants of caspase-8, we show that both cathepsin D-mediated proteolysis and homodimerization of caspase-8 are necessary to generate an active caspase-8. At acidic pH, cathepsin D specifically cleaved caspase-8 but not the initiator caspase-9 or -10 and significantly increased caspase-8 activity in dimerizing conditions. These events were completely abolished by pepstatin A, a pharmacological inhibitor of cathepsin D. The cathepsin D intra-chain proteolysis greatly stabilized the active site of caspase-8. Moreover, the main caspase-8 fragment generated by cathepsin D cleavage could be affinity-labeled with the active site probe biotin-VAD-fluoromethyl ketone, suggesting that this fragment is enzymatically active. Importantly, in an in vitro cell-free assay, the addition of recombinant human caspase-8 protein, pre-cleaved by cathepsin D, was followed by caspase-3 activation. Our data therefore indicate that cathepsin D is able to initiate the caspase cascade by direct activation of caspase-8. As cathepsin D is ubiquitously expressed, this may represent a general mechanism to induce apoptosis in a variety of immune and nonimmune cells.
authors.affiliation: Institute of Pharmacology, University of Bern, CH-3010 Bern, Switzerland. sebastien.conus@pki.unibe.ch
authors.forename: Sébastien
authors.initials: S
authors.lastname: Conus
authors.forename: Cristina
authors.initials: C
authors.lastname: Pop
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Snipas
authors.forename: Guy S
authors.initials: GS
authors.lastname: Salvesen
authors.forename: Hans-Uwe
authors.initials: HU
authors.lastname: Simon
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D053455:Caspase 10 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053181:Caspase 8 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053453:Caspase 9 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002402:Cathepsin D / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D006801:Humans; D008297:Male; D009504:Neutrophils / Q000166:cytology / Q000201:enzymology*; D055503:Protein Multimerization / Q000502:physiology*; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053455:Caspase 10; D053181:Caspase 8; D053453:Caspase 9; C506438:CASP10 protein, human; C533922:CTSD protein, human; D002402:Cathepsin D