Molecular adn Chemical Biology of Integrated ER Stre

描述(申请人提供)：本申请的最终目标是通过高通量筛选试验开发化学和遗传探针，用于研究内质网(ER)生物学，并阐明整合的ER应激反应在人类疾病发病机制中的作用。内质网(ER)是大多数蛋白质折叠和组装的场所，目的是分泌或亚细胞隔间。反向遗传学研究表明，取消整合的内质网应激反应是致命的。因此，内质网稳态受到高度调控，对蛋白质合成的需求和内质网折叠新合成蛋白质的能力必须匹配。内质网折叠能力和对新蛋白质的需求之间的任何不匹配都会导致内质网中未折叠蛋白质的积累，并触发整合的内质网应激反应，试图通过三条不同但整合的途径之一来纠正这种不平衡。整合内质网应激反应的紊乱与许多恶性肿瘤的发病机制有关，包括I型和II型糖尿病、癌症、炎症和神经退行性疾病。尽管整合内质网应激反应的重要性和与整合内质网应激反应紊乱相关的恶性肿瘤的广泛范围，但无论是化学还是分子遗传学的整合内质网应激反应都没有得到系统的研究。因此，我们建议确定整合内质网应激反应的化学和遗传修饰物，这将使在体外和体内对内质网的研究成为可能，并将作为未来开发药物的平台，用于治疗与整合内质网应激反应失控有关的人类疾病。因此，我们建议利用我们最近开发的高通量筛选(HTS)分析，它询问集成的内质网应激反应的不同分支。具体地说，我们将筛选化学集合和小干扰RNA(SiRNA)的文库，以识别修改整合内质网应激反应的三个不同手臂的化学探针和siRNA。整合内质网应激反应的化学和遗传修饰物将在二次和反分析中得到验证。通过这一努力确定的化学探针对于阐明内质网应激在正常生理中的作用以及在涉及内质网稳态紊乱的恶性肿瘤的发生中所起的作用至关重要。这些探针也可以作为开发用于治疗人类疾病的内质网应激调节剂的先导化合物。同样，通过这一努力确定的整合内质网应激的遗传修饰物主要针对新疗法的开发。与公共卫生相关：内质网的折叠能力与折叠新蛋白质的需求之间的不匹配导致未折叠的蛋白质在内质网中积累，然后触发整合的内质网应激反应，以恢复内质网同源序列。各种生理和病理刺激以及破坏整合内质网应激反应的基因突变或其他异常与许多恶性肿瘤的发病机制有关或参与其中，包括I型和II型糖尿病、炎症以及包括阿尔茨海默病和帕金森病在内的神经退行性疾病。尽管整合的内质网应激反应在许多疾病的病理生理学中很重要，但很少有整合的内质网应激反应的化学和遗传修饰物，我们建议在我们开发的高通量整合的内质网应激反应分析中通过筛选化学和siRNA文库来补救。

DESCRIPTION (provided by applicant): The ultimate goal of this application is the development chemical and genetic probes by high throughput screening assays for the study of endoplasmic reticulum (ER) biology and elucidating the role of the integrated ER-stress response in the pathogenesis human diseases. The Endoplasmic reticulum (ER) is the site of folding and assembly for most proteins destined for secretion or sub-cellular compartments. Reverse genetic studies indicate that abrogation of the integrated ER-stress response is lethal. ER homeostasis is therefore highly regulated and the demand for protein synthesis and the capacity of ER to fold the newly synthesized proteins must be matched. Any mismatch between the folding capacity of the ER and the demand for new proteins causes the accumulation of unfolded proteins in the ER and triggers the integrated ER-stress response, which attempts to correct the imbalance by one of three distinct, but integrated routes. Disorders of integrated ER-stress response are implicated in the pathogenesis of many malignancies, including type I and type II diabetes, cancer, inflammation, and neurodegenerative disorders. Despite this importance and the broad spectrum of malignancies associated with disorders of integrated ER-stress response, neither the chemical nor molecular genetics of the integrated ER-stress response have been studied in a systematic manner. We therefore propose to identify chemical and genetic modifiers of integrated ER stress response, which will enable in vitro and in vivo studies of the ER, and will serve as a future platform for the development of pharmaceutical agents for the therapy of human disorders in which disregulation of integrated ER-stress response is implicated. We therefore propose to take advantage of our recently developed high throughput screening (HTS) assays, which interrogate different arms of the integrated ER-stress response. Specifically we will screen libraries of chemical collections and small interfering RNAs (siRNA) to identify chemical probes and siRNAs that modify three distinct arms of integrated ER-stress response. The chemical and genetic modifiers of integrated ER-stress response will be validated in secondary and counter assays. The chemical probes identified through this effort will be critical for elucidating the role of ER-stress in the normal physiology as well as in the genesis of malignancies in which perturbations of ER- homeostasis are implicated. These probes may also serve as lead compounds for the development of ER-stress modifiers for the treatment of human disorders. Similarly, the genetic modifiers of integrated ER stress identified through this effort ma b targets for the development of novel therapeutics. PUBLIC HEALTH RELEVANCE: Mismatch between the folding capacity of the ER and demand for folding new proteins causes accumulation of unfolded proteins in the ER which then triggers integrated ER-stress response to restore the ER-homeostais. Various physiological and pathological stimuli as well as genetic mutations or other aberrations that compromise the integrated ER-stress response are associated with or implicated in the pathogenesis of many malignancies including type I and type II diabetes, inflammation, and neurodegenerative disorders including Alzheimer's and Parkinson's disease. Despite the importance of integrated ER-stress response in the pathophysiology of many disorders, there are very few chemical and genetic modifiers of the integrated ER-stress response, that we propose to remedy by screening chemical and siRNA libraries in high throughput integrated ER-stress response assay we have developed.