Analysis of poly(ADP-ribose) function in the cytoplasmic stress response

描述（由申请人提供）：聚（adp -核糖）（pADPr）是多细胞生物生命所需的独特聚合物。它既可以作为受体蛋白的共价修饰，也可以作为结合特定蛋白质的支架。这种聚合物在细胞分裂、细胞周期进程、转录和翻译调控等基本细胞功能中起着重要作用。pADPr还在细胞应激反应中起作用，如凋亡、DNA损伤修复和先天免疫反应。在初步结果中，我们发现了pADPr及其聚合酶的新功能，即pADPr聚合酶（PARPs）在调控应激颗粒（SG）组装和mrna转录后调控中的作用。这些结果以一种意想不到的方式将细胞应激、PARPs和mRNA调控领域联系起来。他们认为pADPr作为SG组装的结构支架，类似于它在纺锤杆和DNA损伤位点的功能。我们预计我们的结果将对所有三个领域产生重大影响，并希望将我们的工作扩展到包括机制研究。我们的长期目标之一是了解pADPr作为支架是如何起作用的。SG组装和mRNA调控是研究pADPr支架功能的理想过程。在本文中，我们试图确定pADPr在SG组装中的功能机制，并开始了解pADPr与蛋白质结合如何调节功能。我们使用生化分析和细胞生物学相结合的方法来做到这一点。在特定的Aim 1中，我们确定了SG组装所需的pADPr修饰位点，并确定了pADPr合成应力的机制。在目标2中，我们确定了pADPr调节mRNA结合和招募到SG的方式，在目标3中，我们研究了控制pADPr与蛋白质结合的结构-功能关系。我们相信所提出的实验将有助于阐明pADPr在细胞其他部位的支架功能。SGs具有重要的疾病相关性。PARP抑制已被证明对乳腺癌和卵巢癌治疗有效，可能对其他与压力相关的疾病如实体瘤、缺血和神经退行性疾病的治疗同样有效。

DESCRIPTION (provided by applicant): Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It functions as both a covalent modification of acceptor proteins and as a scaffold that binds specific sets of proteins. The polymer plays a role in essential cellular functions including cell division and cell cycle progression and regulation of transcription and translation. pADPr also functions in cell stress responses such as apoptosis, DNA damage repair, and innate immune responses. In preliminary results we identified a new function for pADPr and the enzymes that polymerize it, pADPr polymerases (PARPs) in regulation of stress granule (SG) assembly and the post-transcriptional regulation of mRNAs. These results connect the fields of cellular stress, PARPs and mRNA regulation in an unexpected manner. They suggest that pADPr functions as a structural scaffold for SG assembly, similar to its function at the spindle pole and at sites of DNA damage. We anticipate that our results will have a high impact on all three fields and wish to extend our work to include mechanistic studies. One of our long-term goals is to understand how pADPr functions as a scaffold. SG assembly and mRNA regulation is an ideal process to study the scaffold function of pADPr. In this proposal, we seek to determine the mechanism of pADPr function in the assembly of a SG and begin to understand how pADPr binding to proteins regulates function. We do so using a combination of biochemical assays and cell biology. In specific Aim 1 we identify the sites of pADPr modification that are required for SG assembly and generate and determine the mechanism in which pADPr is synthesized for stress. In Aim 2 we determine the manner in which pADPr regulates mRNA binding and recruitment to the SG, and in Aim 3 we examine the structure- function relationships that govern binding of pADPr to proteins. We believe the proposed experiments will help elucidate the scaffold function of pADPr elsewhere in the cell. SGs have important disease relevance. PARP inhibition, already shown to be effective for breast and ovarian cancer therapies, might be equally effective for treatment of other stress-related diseases such as solid tumors, ischemia, and neurodegenerative disease.