Engineering Ubiquitin Ligases to Investigate Protein Aggregation and Neurodegener

描述(由申请人提供) 摘要：蛋白质降解是从信号转导到细胞周期调控的生物过程的核心。从细胞中清除错误折叠的蛋白质是导致神经退行性疾病等人类疾病的主要原因。神经退行性变的一个共同主题是在大脑中积累不可溶的蛋白质聚集体。然而，蛋白质聚集在神经退行性变的病理生理学中的作用仍然存在争议。从受影响的神经元中去除蛋白质聚集体仍然是一项艰巨的任务。在这项提议中，我们采取了一种大胆和创新的方法来解决这个极其困难的问题：利用泛素/蛋白酶体系统来研究蛋白质聚集和神经退化。这一建议建立在我们之前开发的设计单链泛素连接酶芯片的方法基础上。我们成功地建立了一种新的策略，使我们能够在不影响连接酶活性的情况下改变CHIP的底物结合专一性。以亨廷顿病(HD)为模型，我们建议创建针对疾病蛋白Huntingtin(Htt)的重组泛素连接酶以实现泛素化。我们的长期目标是确定决定工程泛素连接酶连接酶活性的结构特征，阐明蛋白质聚集和神经变性的分子机制，并评估Htt特异性泛素连接酶的治疗潜力。如果成功，设计的泛素连接酶将代表着对体细胞中蛋白质功能的前所未有的控制水平，这将通过引入新的蛋白质敲除工具对蛋白质组学研究产生直接影响。此外，该项目的结果将为蛋白质质量控制的基本细胞和分子机制以及神经退化的病理生理提供独特的见解。应用这些发现可能有助于延缓或逆转神经退行性变的有害影响。最终，它将成为治疗其他神经退行性疾病以及非神经性人类疾病的原型。 与公共卫生的相关性：随着我们的人口继续老龄化，神经退行性疾病的流行率将增加，从而对全球公共卫生构成艰巨的挑战。这些真正灾难性的疾病包括阿尔茨海默氏症、亨廷顿氏症、帕金森氏症、肌萎缩侧索硬化症和额叶颞叶痴呆。这项建议旨在开发新的工具来针对神经退行性变的罪魁祸首，阐明潜在的机制，并提供治疗潜力，以改善这些患者的健康。

DESCRIPTION (Provided by the applicant) Abstract: Protein degradation lies at the heart of biological processes from signal transduction to cell cycle regulation. Compromised clearance of misfolded proteins from cells is the leading cause of human diseases such as neurodegenerative disorders. A common theme manifested in neurodegeneration is the accumulation of insoluble protein aggregates in the brain. However, the role of protein aggregation in the pathophysiology of neurodegeneration remains controversial. It remains a formidable task to remove protein aggregates from the affected neurons. In this proposal, we are taking a bold and innovative approach to attacking this exceedingly difficult problem: harnessing the ubiquitin/proteasome system to investigate protein aggregation and neurodegeneration. This proposal builds upon our previous development of methods to engineer single chain ubiquitin ligase CHIP. We successfully established a novel strategy that enables us to alter the substrate binding specificity of CHIP without affecting its ligase activity. Using Huntington's disease (HD) as a model, we propose herein to create recombinant ubiquitin ligases targeting the disease protein huntingtin (Htt) for ubiquitination. Our long-term goal is to define the structural features that determine the ligase activity of engineered ubiquitin ligases, elucidate the molecular mechanisms underlying protein aggregation and neurodegeneration, and evaluate the therapeutic potential of Htt-specific ubiquitin ligases. If successful, the engineered ubiquitin ligases will represent an unprecedented level of control over protein function in somatic cells, which would have direct impact on proteomic research by introducing novel "protein knockout" tools. In addition, the results of this project will provide unique insights into the fundamental cellular and molecular mechanisms underlying protein quality control and the pathophysiology of neurodegeneration. Application of these findings may help to delay or reverse the detrimental effects of neurodegeneration. Ultimately, it will serve as a prototype for the treatment of other neurodegenerative disorders, as well as non-neuronal human diseases. Public Health Relevance: As our population continues to age, neurodegenerative diseases will increase in prevalence and thus pose a daunting challenge to public health worldwide. These truly disastrous disorders include Alzheimer's, Huntington's, Parkinson's, amyotrophic lateral sclerosis and the frontal temporal dementias. This proposal aims to develop novel tools to target the culprits of neurodegeneration, elucidate underlying mechanisms, and provide therapeutic potential to improve the health of these patients.