Activation of caspase-9 within the Apaf-1 apoptosome
| dc.contributor.advisor | Mills, Edward Michael | |
| dc.contributor.advisor | Bratton, Shawn B. | |
| dc.contributor.committeeMember | Dalby, Kevin N. | |
| dc.contributor.committeeMember | Fast, Walter L. | |
| dc.contributor.committeeMember | Van Den Berg, Carla L. | |
| dc.contributor.committeeMember | Tang, Dean G. | |
| dc.creator | Wu, Chu-Chiao | |
| dc.creator.orcid | 0000-0003-2576-1079 | |
| dc.date.accessioned | 2017-05-10T20:14:02Z | |
| dc.date.available | 2017-05-10T20:14:02Z | |
| dc.date.issued | 2015-08 | |
| dc.date.submitted | August 2015 | |
| dc.date.updated | 2017-05-10T20:14:02Z | |
| dc.description.abstract | A family of cysteinyl aspartate-specific proteases (caspases) induces dramatic biochemical and morphological changes during apoptosis. Initiator caspases are thought to be activated through induced homodimerization, whereas their downstream effector caspases, are activated through cleavage of the intersubunit linkers between their large and small subunits. During cellular stress, mitochondrial outer membrane permeabilization triggers cytochrome c release and formation of the Apaf-1 apoptosome, which in turn recruits and activates the initiator procaspase-9. Mutations in the putative caspase-9 dimerization interface reduce its catalytic activity. However, whether caspase-9 dimerization actually occurs within the apoptosome—and its requirement for caspase activation—remain unclear. In this study, we utilized a novel method for incorporating unnatural, cross-linkable amino acids into the dimerization interface of procaspase-9 and have found that caspase-9 does indeed homodimerize within the apoptosome, the first such demonstration for any initiator caspase within its caspase-activating complex. Interestingly, procaspase-9 also heterodimerized with Apaf-1 through the same region, implying that caspase-9 exists within the apoptosome in two distinct states. In agreement with this finding, saturation experiments with excess Apaf-1 indicated that procaspase-9 could be activated within the apoptosome as a heterodimer. Our lab has found that cleavage of procaspase-9 leads to its dissociation from the apoptosome and a subsequent loss in activity. This study further demonstrates that proteolytic cleavage of procaspase-9 does not result in the loss of its small subunit, but rather disrupts the interaction of processed caspase-9 with itself and Apaf-1. SEC-MALS (size-exclusion chromatography-multi-angle light scattering) and SPR (surface plasmon resonance) data indicated that procaspase-9 possessed higher for itself than did processed caspase-9 for itself, and displacement assays showed that dimerization defective caspase-9 failed to displace caspase-9 from the apoptosome. Collectively, the data suggested that procaspase-9 homo-dimerization stabilizes its presence within the apoptosome, but that procaspase-9 may also be activated when it transitions from a caspase-9:caspase-9 homodimer to a caspase-9:Apaf-1 heterodimer. Cleavage of procaspase-9 to its processed form then leads to its eventual loss from the complex altogether. To understand the mechanism of caspase-9 activation in vivo, procaspase-9 and IAP un-inhibitable caspase-9 knock-in mouse models were also generated, and stress-induced phenotypes will be characterized in the future. | |
| dc.description.department | Cellular and Molecular Biology | |
| dc.format.mimetype | application/pdf | |
| dc.identifier | doi:10.15781/T2BG2HG5N | |
| dc.identifier.uri | http://hdl.handle.net/2152/46811 | |
| dc.language.iso | en | |
| dc.subject | Apoptosis | |
| dc.subject | Caspase-9 | |
| dc.subject | Apaf-1 | |
| dc.subject | Apoptosome | |
| dc.subject | Auto-catalytic cleavage | |
| dc.subject | Molecular timer | |
| dc.subject | Non-cleavable caspase-9 knock-in mouse | |
| dc.title | Activation of caspase-9 within the Apaf-1 apoptosome | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Cellular and Molecular Biology | |
| thesis.degree.discipline | Cell and Molecular Biology | |
| thesis.degree.grantor | The University of Texas at Austin | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy |