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

描述(申请人提供)：聚(ADP-核糖)(PADPr)是多细胞生物体中生命所需的一种独特的聚合物。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组的支架。该聚合物在基本的细胞功能中发挥作用，包括细胞分裂和细胞周期进展，以及转录和翻译的调节。PADPr还在细胞应激反应中发挥作用，如细胞凋亡、DNA损伤修复和先天免疫反应。在初步结果中，我们发现了pADPr及其聚合酶在调节应激颗粒(SG)组装和mRNAs转录后调控中的一个新功能，即pADPr聚合酶(PAPS)。这些结果以一种意想不到的方式将细胞应激、PAPS和mRNA调控领域联系在一起。他们认为，pADPr可以作为SG组装的结构支架，类似于它在纺锤体极点和DNA损伤部位的功能。我们预计我们的成果将对所有这三个领域产生很大影响，并希望将我们的工作扩大到包括机械学研究。我们的长期目标之一是了解pADPr是如何作为支架发挥作用的。SG组装和mRNA调控是研究pADPr支架功能的理想方法。在这个建议中，我们试图确定pADPr在SG组装中的功能机制，并开始了解pADPr与蛋白质结合是如何调节功能的。我们使用生化分析和细胞生物学相结合的方法来完成这项工作。在具体目标1中，我们确定了SG组装所需的pADPr修饰位点，并生成并确定了合成pADPr用于应力的机制。在目标2中，我们确定了pADPr调节mRNA结合和向SG募集的方式，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，拟议的实验将有助于阐明pADPr在细胞其他地方的支架功能。SGS具有重要的疾病相关性。已被证明对乳腺癌和卵巢癌治疗有效的PARP抑制，可能对其他应激相关疾病的治疗同样有效，如实体瘤、缺血和神经退行性疾病。 公共卫生相关性：聚(ADP-核糖)(PADPr)是多细胞生物体中生命所需的一种独特的聚合物。它是由一种称为pADPr聚合酶(PAPS)的蛋白质家族聚合而成。PADPr和PARPS在基本的细胞功能和细胞应激反应中发挥作用。这些应激反应通路是人类疾病治疗的常见靶点。我们最近发现了一种新的pADPr应力函数--应力颗粒组装。应激颗粒是一种大的多蛋白质复合体，聚集在细胞质中，对热休克、氧化应激、缺血和病毒感染等外部应激做出反应。它们被认为调节mRNAs的稳定性和翻译潜力。最近，pADPr与癌症有牵连，促使制药公司开发针对PARP家族成员的抑制剂。几种PARP-1抑制剂已经在接近纪录的时间内进入了治疗乳腺癌和卵巢癌的第三阶段试验。我们研究的长期目标是了解pADPr如何在应激颗粒中发挥作用。

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. PUBLIC HEALTH RELEVANCE: Poly(ADP-ribose) (pADPr) is a unique polymer required for life in multicellular organisms. It is polymerized by a family of proteins called pADPr polymerases (PARPs). pADPr and PARPs function in essential cellular functions and cell stress responses. These stress response pathways are common targets for human disease therapy. We recently identified a new stress function for pADPr - stress granule assembly. Stress granules are large multi-protein complexes that assemble in the cytoplasm in response to external stresses such as heat shock, oxidative stress, ischemia and viral infection. They are thought to regulate the stability and translation potential of mRNAs. Recently, pADPr has been implicated in cancer, prompting pharmaceutical companies to develop inhibitors that target members of the PARP family. Several PARP-1 inhibitors have progressed to Phase 3 trials for treatment of breast and ovarian cancers in near record time. The long-term goal of our studies is to understand how pADPr functions in the stress granule.