Degradation mechanisms for inhibitor of apoptosis proteins and their antagonists

凋亡蛋白抑制剂及其拮抗剂的降解机制

• 批准号: 9274332
• 负责人: Shawn B Bratton
• 金额: $ 32.43万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9274332
• 关键词: Adaptor Signaling Protein; Address; Apoptosis; Apoptosis Inhibitor; Apoptosis Regulation Gene; Applications Grants; BIRC4 gene; Baculoviridae; Baculoviruses; Binding; Biochemical; Biochemical Genetics; Biological; CASP5 gene; Caspase; Caspase Inhibitor; Cell Death; Cells; Complex; Copper; Cysteine Proteinase Inhibitors; Data; Degenerative Disorder; Development; Disease; Drosophila genus; Drosophila inhibitor of apoptosis 1; Excision; Exposure to; Feedback; Genetic Models; Goals; Growth Factor; Hepatolenticular Degeneration; Human; In Vitro; Inflammation; Injury; Link; Lysine; Malignant Neoplasms; Mammals; Mediating; Mediator of activation protein; Modeling; Molecular; Mutation; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Injury; Neurons; Organism; Pathologic; Pathway interactions; Patients; Play; Post-Translational Protein Processing; Predisposition; Process; Protease Inhibitor; Proteins; Radio; Regulation; Resistance; Role; Site; Source; Therapeutic; Ubiquitination; Wilson disease protein; Withdrawal; Work; biochemical model; cancer cell; cancer therapy; chemotherapy; feeding; fly; gain of function; genetic approach; human disease; in vivo; inhibitor-of-apoptosis protein; insight; loss of function; multicatalytic endopeptidase complex; novel; overexpression; protein function; public health relevance; tumor; ubiquitin-protein ligase; virtual

 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.

 描述（由适用提供）：凋亡或程序性细胞死亡在从苍蝇到人类的多细胞生物中广泛保守，并且在从癌症到炎症和神经变性的所有事物中都起着至关重要的作用。凋亡（IAP）蛋白的抑制剂，包括果蝇IAP1（DIAP1）和X-C-链IAP（XIAP），是E3泛素连接酶，它们在调节凋亡中起主要作用，至少通过直接抑制和/或或/或或/或或/或或/或泛液化在凋亡中起着重要作用。 IAP拮抗剂（例如收割机，HID，Grim和Smac）被认为是通过将活性caspase从IAP中置换而诱导细胞死亡的，从而导致caspase活性和细胞死亡增加，但本身是泛素化的靶标。确实，我们最近发现，IAP拮抗剂的严峻被DIAP1泛滥。然而，针对泛素化的赖氨酸也可能会被胱天蛋白酶消除，从而增强了Grim的稳定性，并启动馈送前向胱天蛋白酶放大环，从而导致更大的细胞DEAH。由于这项工作和其他初步数据，我们已经开始欣赏并假设IAP，IAP拮抗剂和caspases通过高度新颖的机制相互调节，这些机制会影响IAPS及其拮抗剂的功能和转移。该赠款应用的一个主要目标是用分子细节表征生物化学机制，通过这些机制，IAP和IAP拮抗剂被选择性地降解，以制定策略以抑制或促进给定病态背景下的细胞死亡。最近对苍蝇的研究表明，DIAP1在其N末端的胱天蛋白酶切割使DIAP1易于通过所谓的N端规则途径降解。我们发现了证据表明，在第二个位点，DIAP1和XIAP的caspase裂解产生了可以用作新型载体分子的IAP片段，靶向IAP拮抗剂，以通过UBR型E3连接酶进行跨泛素化。在AIM＃1中，我们将使用多种生化和遗传方法评估N端规则途径在介导IAP拮抗剂降解中的作用。在其他初步数据中，我们发现暴露于多余的铜修饰剂DIAP1和XIAP，使它们容易受到蛋白酶体的胱天蛋白酶裂解，自相传制和周转的影响。在AIM＃2中，我们将阐明铜对IAP功能的生化和结构效应，并将确定铜是否在威尔逊氏病的蝇模模型中通过DIAP1降解而诱导细胞死亡。最后，我们发现高度不寻常的引发剂caspase Strica是DIAP1裂解的主要介体。对于此caspase几乎一无所知，在AIM＃3中，我们将以生化和结构细节来表征它，并将评估其在介导铜诱导的降解和凋亡中的作用。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大洞察力，并将为我们旨在针对这些蛋白质治疗癌症和各种神经退行性疾病的努力。