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

描述（由申请人提供）：聚（ADP-核糖）（PAR）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控相关的生物调控事件。游离PAR聚合物的过度产生与细胞凋亡相关，并且被认为在其中聚（ADP-核糖）聚合酶-1（PARP-1）活性增加的人类疾病（包括神经变性、心血管疾病和中风）中起作用。游离PAR聚合物与线粒体凋亡诱导因子（AIF）的结合从线粒体内膜释放AIF，以实现核DNA的大规模降解和细胞死亡，这一途径称为“parthanatos”。“目前还不清楚PAR结合如何使AIF的线粒体跨膜锚凋亡裂解，以允许从线粒体逃逸。我的初步数据表明，PAR结合到AIF的原因曝光的一个假定的钙蛋白酶-I招聘（PEST）基序，我建议PAR结合促进AIF的释放，通过增加与钙蛋白酶-1蛋白酶。我将研究PAR刺激的AIF从线粒体释放的机制，通过（1）表征PAR聚合物对AIF的结构重塑和（2）评估AIF的PEST基序在钙蛋白酶-1刺激AIF蛋白水解中的相关性。目的1将建立AIF和PAR链之间相互作用的亲和力和特异性，使用X射线晶体学描绘AIF和PAR配体之间的结合界面，并使用小角X射线散射（SAXS）表征在PAR存在下AIF的大分子重塑。目的2将通过使用时间分辨FRET分析测量PEST突变体的切割速率来确定PEST基序是否参与AIF的钙蛋白酶-1切割。在Aim 3中，将测试PAR结合或钙蛋白酶-I加工缺陷的AIF突变体在受损细胞中经历线粒体释放和核转位或在暴露于PAR链后离开分离的线粒体的能力。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开发提供信息，并为其他半胱天冬酶非依赖性死亡途径如何潜在地调节AIF释放提供重要的见解。

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.