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

描述（由申请人提供）：通过聚（adp -核糖）（PAR）进行的蛋白质翻译后修饰涉及越来越多的与DNA损伤反应、染色质重塑和转录调节相关的生物调节事件。游离PAR聚合物的过量产生与细胞凋亡有关，并被认为在人类疾病中发挥作用，其中聚（adp -核糖）聚合酶-1 （PARP-1）活性增加，包括神经退行性疾病、心血管疾病和中风。游离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“parthanatos”。目前尚不清楚PAR结合如何使AIF的线粒体跨膜锚点发生凋亡裂解，从而使其从线粒体中逃逸。我的初步数据表明，PAR与AIF结合会导致假定的calpain-1募集（PEST）基元暴露，我提出PAR结合通过增加与calpain-1蛋白酶的关联来促进AIF释放。我将通过(1)通过PAR聚合物表征AIF的结构重塑和(2)评估AIF的PEST基元与calpain-1刺激AIF蛋白水解的相关性来研究PAR刺激线粒体释放AIF的机制。目的1将建立AIF和PAR链之间相互作用的亲和力和特异性，利用x射线晶体学描绘AIF和PAR配体之间的结合界面，并利用小角度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实验测量PEST突变体的切割率来确定PEST基序是否参与AIF的calpain-1切割。在PAR结合或calpain- 1加工缺陷的AIF突变体将在Aim 3中测试其在受损细胞中进行线粒体释放和核易位的能力，或暴露于PAR链后退出分离线粒体的能力。这项研究的结果将为探索与PAR过度产生相关的疾病状态的小分子工具的发展提供信息，并为其他caspase非依赖性死亡途径如何潜在地调节AIF释放提供重要见解。

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.