Degradation mechanisms for inhibitor of apoptosis proteins and their antagonists

 描述（由申请人提供）：细胞凋亡或程序性细胞死亡在从苍蝇到人类的多细胞生物体中广泛保守，并在从癌症到炎症和神经变性的所有疾病中发挥关键作用。细胞凋亡抑制因子（IAP）蛋白，包括果蝇IAP 1（DIAP 1）和X-连锁IAP（XIAP），是E3泛素连接酶，其在细胞凋亡的调节中起主要作用，至少部分地通过直接抑制和/或泛素化半胱天冬酶。IAP拮抗剂，如Reaper、Hid、Grim和Smac，被认为通过从IAP中置换活性半胱天冬酶来诱导细胞死亡，从而导致半胱天冬酶活性增加和细胞死亡，但它们本身是泛素化的靶标。事实上，我们最近发现IAP拮抗剂Grim被DIAP 1泛素化;然而，靶向泛素化的赖氨酸也被半胱天冬酶去除，从而增强Grim的稳定性并启动前馈半胱天冬酶放大环，导致更大的细胞死亡。由于这项工作和额外的初步数据，我们已经开始欣赏和假设，IAP，IAP拮抗剂，和半胱天冬酶通过高度新颖的机制，影响IAP及其拮抗剂在细胞中的功能和营业额相互调节。该资助申请的主要目标是在分子细节中表征IAP和IAP拮抗剂选择性降解的生化机制，以开发在给定病理背景下抑制或促进细胞死亡的策略。最近在果蝇中的研究表明，在其N-末端的DIAP 1的半胱天冬酶切割使得DIAP 1易于通过所谓的N-末端规则途径降解。我们已经发现的证据表明，半胱天冬酶裂解DIAP 1和XIAP，在第二个网站，产生IAP片段，可以作为新的载体分子，针对IAP拮抗剂的转subiquitination的Ubr型E3连接酶。在目标#1中，我们将利用各种生物化学和遗传学方法评估N端规则途径在介导IAP拮抗剂降解中的作用。在额外的初步数据中，我们发现暴露于过量的铜会改变DIAP 1和XIAP，使它们容易受到半胱天冬酶切割、autoubiquitination和蛋白酶体周转的影响。在目标#2中，我们将阐明铜对IAP功能的生化和结构影响，并将确定铜是否通过威尔逊病的苍蝇模型中的DIAP 1降解诱导细胞死亡。最后，我们已经发现，非常不寻常的引发剂胱天蛋白酶Strica是DIAP 1裂解的主要介质。由于对这种半胱天冬酶几乎一无所知，在目标#3中，我们将在生物化学和结构细节中对其进行表征，并评估其在介导铜诱导的降解和凋亡中的作用。总的来说，拟议的研究将为IAP，IAP拮抗剂和半胱天冬酶之间存在的复杂关系提供重要的见解，并将为我们靶向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.