title: Determining the contributions of caspase-2, caspase-8 and effector caspases to intracellular VDVADase activities during apoptosis initiation and execution.
abstract: Apoptosis signaling crucially depends on caspase activities. Caspase-2 shares features of both initiator and effector caspases. Opinions are divided on whether caspase-2 activity is established during apoptosis initiation or execution in response to DNA damage, death receptor stimulation, or heat shock. So far, approaches towards measuring caspase-2 activity were restricted to analyses in cell homogenates and extracts, yielded inconsistent results, and were often limited in sensitivity, thereby contributing to controversies surrounding the role of caspase-2 during apoptosis. Furthermore, caspases overlap in substrate specificities, and caspase-8 as well as effector caspases may cleave the optimal VDVAD recognition motif as well. We therefore generated a highly sensitive Förster resonance energy transfer (FRET) substrate to determine the relative contribution of these caspases to VDVADase activity non-invasively inside living cells. We observed limited proteolysis of the substrate during apoptosis initiation in response to death receptor stimulation by FasL, TNFα and TRAIL. However, this activity was attributable to caspase-8 rather than caspase-2. Likewise, no caspase-2-specific activity was detected during apoptosis initiation in response to genotoxic stress (cisplatin, 5-FU), microtubule destabilization (vincristine), or heat shock. The contribution of caspase-2 to proteolytic activities during apoptosis execution was insignificant. Since even residual, ectopically introduced caspase-2 activity could readily be detected inside living cells in our measurements, we conclude, in contrast to several previous studies, that caspase-2 activity does not contribute to apoptosis in the scenarios investigated, and that instead caspase-8 and effector caspases are the most significant VDVADases during canonical apoptosis signaling.
authors.affiliation: Centre for Systems Medicine, Royal College of Surgeons, Dublin, Ireland.
authors.forename: M Eugenia
authors.initials: ME
authors.lastname: Delgado
authors.forename: Magnus
authors.initials: M
authors.lastname: Olsson
authors.forename: Frank A
authors.initials: FA
authors.lastname: Lincoln
authors.forename: Boris
authors.initials: B
authors.lastname: Zhivotovsky
authors.forename: Markus
authors.initials: M
authors.lastname: Rehm
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology; D053143:Caspase 2 / Q000737:chemistry / Q000378:metabolism*; D053181:Caspase 8 / Q000737:chemistry / Q000378:metabolism*; D061945:Caspase Inhibitors / Q000494:pharmacology*; D053470:Caspases, Effector / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D061945:Caspase Inhibitors; D053143:Caspase 2; D053181:Caspase 8; D053470:Caspases, Effector
keywords: Apoptosis; Caspase-2; Caspase-8; Förster Resonance Energy Transfer (FRET); Single cell analysis