Structural mechanisms of AIF release during poly(ADP-ribose)-induced cell death

聚（ADP-核糖）诱导细胞死亡过程中 AIF 释放的结构机制

• 批准号: 8788619
• 负责人: Chris A. Brosey
• 金额: $ 5.6万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8788619
• 关键词: Affinity; Animal Model; Apoptosis; Apoptotic; Binding; Binding Sites; Biochemistry; Biological; Biological Assay; Brain Injuries; C-terminal; Calpain; Calpain I; Cardiovascular Diseases; Caspase; Cell Death; Cell Nucleus; Cells; Cellular Assay; Cessation of life; Complex; Crystallization; Crystallography; DNA; DNA Damage; Data; Degenerative Disorder; Dependence; Development; Disease; Distant; Epitopes; Event; Exhibits; Exposure to; Fluorescein; Fluorescence Resonance Energy Transfer; Health; Hela Cells; Image; In Vitro; Inner mitochondrial membrane; Knowledge; Label; Length; Ligands; Link; Measurement; Measures; Mediating; Membrane; Mitochondria; Molecular; Monitor; N-terminal; Nerve Degeneration; Neurodegenerative Disorders; Nuclear; Nuclear Translocation; Outcome Study; Pathway interactions; Peptide Hydrolases; Physiological; Play; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerases; Polymerase; Polymers; Post-Translational Protein Processing; Process; Proteins; Proteolysis; Proteolytic Processing; Role; Site; Solutions; Specificity; Stroke; Structural Models; System; Testing; Therapeutic Intervention; Time; Transcriptional Regulation; apoptosis inducing factor; base; cell injury; cellular imaging; chromatin remodeling; design; human disease; in vitro Model; insight; mutant; respiratory; response; small molecule; stoichiometry; tool

DESCRIPTION (provided by applicant): Protein post-translational modification by poly(ADP-ribose) (PAR) is implicated in a growing number of biological regulatory events associated with DNA damage responses, chromatin remodeling, and transcriptional regulation. Hyperproduction of free PAR polymers is linked to cellular commitment to apoptosis and is suggested to play a role in human diseases in which poly(ADP-ribose) polymerase-1 (PARP-1) activity is increased, including neurodegeneration, cardiovascular disease, and stroke. Binding of free PAR polymers to mitochondrial apoptosis-inducing factor (AIF) releases AIF from the mitochondrial inner membrane to effect large-scale degradation of nuclear DNA and cell death, a pathway termed "parthanatos." It is unclear how PAR binding enables apoptotic cleavage of AIF's mitochondrial transmembrane anchor to allow escape from mitochondria. My preliminary data indicate that PAR binding to AIF causes exposure of a putative calpain-I recruitment (PEST) motif, and I propose that PAR binding promotes AIF release by increasing association with calpain-1 protease. I will investigate this mechanism for PAR-stimulated release of AIF from mitochondria by (1) characterizing structural remodeling of AIF by PAR polymers and (2) evaluating the relevance of AIF's PEST motif in stimulating AIF proteolysis by calpain-1. Aim 1 will establish the affinity and specificity of interaction between AIF and PAR chains, delineate the binding interface between AIF and PAR ligands using x-ray crystallography, and characterize macromolecular remodeling of AIF in the presence of PAR using small- angle x-ray scattering (SAXS). Aim 2 will determine whether the PEST motif participates in calpain-1 cleavage of AIF by measuring cleavage rates of PEST mutants using a time-resolved FRET assay. AIF mutants defective in PAR binding or calpain-I processing will be tested in Aim 3 for their ability to undergo mitochondrial release and nuclear translocation in damaged cells or to exit isolated mitochondria upon exposure to PAR chains. Outcomes from this study will inform the development of small molecule tools for probing disease states associated with PAR hyperproduction and provide critical insight into how other caspase-independent death pathways potentially regulate AIF release.

描述(由申请人提供)：由聚(ADP-核糖)(PAR)进行的蛋白质翻译后修饰与越来越多的与DNA损伤反应、染色质重塑和转录调节相关的生物调节事件有关。游离PAR聚合物的高表达与细胞对细胞凋亡的承诺有关，并被认为在多聚(ADP-核糖)聚合酶-1(PARP-1)活性增加的人类疾病中发挥作用，包括神经变性、心血管疾病和中风。自由PAR聚合物与线粒体凋亡诱导因子(AIF)结合，从线粒体内膜释放AIF，影响核DNA的大规模降解和细胞死亡，这一途径被称为“天堂”。目前尚不清楚PAR结合如何使AIF的线粒体跨膜锚定的凋亡性裂解得以从线粒体逃逸。我的初步数据表明，PAR与AIF的结合导致了一个可能的Calain-I招募(PEST)基序的暴露，我认为PAR结合通过增加与Calain-1蛋白酶的结合来促进AIF的释放。我将从以下两个方面探讨PAR刺激线粒体释放AIF的机制：(1)表征PAR聚合物对AIF的结构重塑；(2)评估AIF的PEST基序与Calain-1刺激AIF蛋白降解的相关性。目的1建立AIF与PAR链相互作用的亲和力和特异性，用X射线结晶学描述AIF与PAR配体的结合界面，并用小角X射线散射(SAXS)表征PAR存在下AIF的大分子重塑。目的2将通过使用时间分辨FRET方法测量害虫突变体的裂解率来确定害虫基序是否参与了AIF的calain-1裂解。在PAR结合或calain-I处理方面有缺陷的AIF突变体将在Aim 3中接受测试，以了解它们在受损细胞中经历线粒体释放和核转位的能力，或在暴露于PAR链时退出分离的线粒体的能力。这项研究的结果将为开发小分子工具来探测与PAR过度产生相关的疾病状态提供信息，并为其他caspase非依赖性死亡途径如何潜在地调节AIF释放提供关键的见解。