Apoptosis, or programmed cell death, is broadly conserved in multicellular organisms from flies to humans and plays critical roles in everything from cancer to inflammation and neurodegeneration. Inhibitor of apoptosis (IAP) proteins, including Drosophila IAP1 (DIAP1) and X-linked IAP (XIAP), are E3 ubiquitin ligases that play major roles in the regulation of apoptosis, at least in part through direct inhibition and/or ubiquitination of caspases. IAP antagonists, such as Reaper, Hid, Grim, and Smac, are thought to induce cell death by displacing active caspases from IAPs, thereby leading to increased caspase activity and cell death, but are themselves targets of ubiquitination. Indeed, we have recently discovered that the IAP antagonist Grim is ubiquitinated by DIAP1; however, the lysine targeted for ubiquitination is also subject to removal by caspases, thereby enhancing Grim's stability and initiating a feed-forward caspase amplification loop that results in greater cell deah. As a result of this work and additional preliminary data, we have begun to appreciate and hypothesize that IAPs, IAP antagonists, and caspases reciprocally regulate one another through highly novel mechanisms that impact the function and turnover of IAPs and their antagonists in cells. A major goal of this grant application is to characterize in molecular detail the biochemica mechanisms through which IAPs and IAP antagonists are selectively degraded in order to develop strategies to inhibit or promote cell death in a given pathological context. Recent studies in flies have demonstrated that caspase cleavage of DIAP1 at its N-terminus renders DIAP1 susceptible to degradation through the so-called N-end rule pathway. We have uncovered evidence that caspase cleavage of DIAP1 and XIAP, at a second site, generates IAP fragments that can serve as novel carrier molecules, targeting IAP antagonists for transubiquitination by Ubr-type E3 ligases.
In aim #1, we will evaluate the role of the N-end rule pathway in mediating the degradation of IAP antagonists, utilizing a variety of biochemical and genetic approaches. In additional preliminary data, we have discovered that exposure to excess copper modifies DIAP1 and XIAP, rendering them susceptible to caspase cleavage, autoubiquitination, and turnover by the proteasome.
In aim #2, we will elucidate the biochemical and structural effects of copper on IAP function and will determine if copper induces cell death through degradation of DIAP1 in a fly model of Wilson's Disease. Finally, we have discovered that the highly unusual initiator caspase Strica is a primary mediator of DIAP1 cleavage. As virtually nothing is known about this caspase, in Aim #3, we will characterize it in biochemical and structural detail and will assess its role in mediating copper-induced degradation and apoptosis. Overall, the proposed studies will provide significant insight into the complex relationships that exist between IAPs, IAP antagonists, and caspases and will inform our efforts at targeting these proteins for the treatment of cancer and various neurodegenerative diseases.
Caspases (proteases), inhibitor of apoptosis (IAP) proteins, and IAP antagonists collectively regulate cell death but are often dysregulated in diseases ranging from cancer to neurodegeneration. We have discovered that novel reciprocal degradative relationships exist between these proteins, which may be exploited for therapeutic purposes.