Degradation mechanisms for inhibitor of apoptosis proteins and their antagonists

 描述(申请人提供)：细胞凋亡，或程序性细胞死亡，广泛存在于从苍蝇到人类的多细胞生物体中，在从癌症到炎症和神经退化的一切过程中都发挥着关键作用。凋亡抑制蛋白(IAP)包括果蝇IAP1(DIAP1)和X-连锁IAP(XIAP)，是E3泛素连接酶，至少部分通过直接抑制和/或泛素化caspase在细胞凋亡的调控中发挥重要作用。IAP拮抗剂，如Reaper、HID、Grim和Smac，被认为通过取代IAP上活性的caspase而诱导细胞死亡，从而导致caspase活性增加和细胞死亡，但它们本身也是泛素化的靶标。事实上，我们最近发现IAP拮抗剂Grim被DIAP1泛素化；然而，泛素化的靶向赖氨酸也会被caspase移除，从而增强Grim的稳定性并启动前馈caspase扩增环，导致更大的细胞Deah。由于这项工作和更多的初步数据，我们已经开始认识到并假设IAP、IAP拮抗剂和Caspase通过非常新颖的机制相互调节，这些机制影响IAP及其拮抗剂在细胞中的功能和周转。这项赠款申请的一个主要目标是在分子细节上描述IAP和IAP拮抗剂被选择性降解的生物化学机制，以便在给定的病理背景下制定抑制或促进细胞死亡的策略。最近在果蝇上的研究表明，DIAP1在其N端的caspase裂解使DIAP1容易通过所谓的N端规则途径降解。我们发现有证据表明，在第二个位点上，DIAP1和XIAP的caspase裂解会产生IAP片段，这些片段可以作为新的载体分子，靶向IAP拮抗剂，使其被UBR型E3连接酶转位。在目标1中，我们将利用各种生化和遗传方法来评估N-末端规则通路在调节IAP拮抗剂降解中的作用。在更多的初步数据中，我们发现暴露在过量的铜中会修改DIAP1和XIAP，使它们容易受到caspase切割、自素化和蛋白酶体的转换。在第二个目标中，我们将阐明铜对IAP功能的生化和结构影响，并将确定在威尔逊病的苍蝇模型中，铜是否通过降解DIAP1而诱导细胞死亡。最后，我们发现非常不寻常的启动子caspase Strica是DIAP1切割的主要中介。由于对caspase几乎一无所知，在目标3中，我们将从生化和结构细节上对其进行表征，并将评估其在介导铜诱导的降解和细胞凋亡中的作用。总体而言，拟议的研究将对IAP、IAP拮抗剂和Caspase之间存在的复杂关系提供重要的见解，并将为我们针对这些蛋白质治疗癌症和各种神经退行性疾病的努力提供信息。

 DESCRIPTION (provided by applicant): 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.