Determine the mechanism of recognition of ubiquitin configurations by the 26S proteasome

项目摘要/摘要 泛素-蛋白酶体系统是调节蛋白质降解的主要途径，表现为 靶标特异性水平与翻译/翻译相似，这是大多数生物功能的基础。泛素 底物分子上的模式决定了蛋白质分解的特异性、速度和结果 蛋白酶体。泛素构型的哪些特征对蛋白酶体的选择性很重要 降解以及带有多种泛素受体的26S蛋白酶体是如何识别这些特征的 人们对此仍知之甚少。蛋白酶体的降解涉及一系列步骤。我们已出版的和初步的 研究记录了降解率与长度、连接、拷贝数和 共轭泛素链的位置。这种选择性是如何实现的仍不清楚。在这里，我提出一个 系统研究以确定泛素构型特征如何控制动力学和模式 底物与蛋白酶体上泛素受体的结合以确定降解率。 我们将使用我之前开发的单分子方法来区分多个限制步骤 并测量了它们的速率常数。绕过制备蛋白质的困难 定义了泛素化的底物，我提出了一种新的方法，使用DNA支架来工程任意 尚未定义的泛素配置，并系统地改变泛素配置的特征以确定 它们如何影响底物与蛋白酶体的相互作用和降解。泛素调节失调- 蛋白酶体系统与多种疾病有关，包括癌症、神经退行性疾病、 自身免疫力和糖尿病。我们实验室的长期目标是阐明蛋白酶体如何识别其 目标，并有选择地让他们参与到过程的退化过程中，并了解如何 蛋白质降解的失调会导致病原性包涵体的形成。我们建议的研究 将提供有关泛素结合途径突变如何导致人类疾病的见解，并将 为开发新的治疗策略产生有价值的信息。

Project Summary/Abstract The ubiquitin-proteasome system is the major pathway for regulatory protein degradation, exhibiting a similar level of target specificity as translation/translation underlying most biological functions. Ubiquitin patterns on substrate molecules determine the specificity, the rate and the outcome of proteolysis by the proteasome. Which features of ubiquitin configurations are important for the selectivity of proteasomal degradation and how the 26S proteasome, with a multitude of ubiquitin receptors, recognizes these features is still poorly understood. Proteasomal degradation involves a sequence of steps. Our published and preliminary studies recorded a strong dependence of the degradation rate on the length, linkage, copy-number and position of conjugated ubiquitin chains. How this selectivity is achieved is still unclear. Here, I propose a systematic investigation to identify how the features of ubiquitin configurations control the kinetics and modes of substrates’ engagement with the ubiquitin receptors on proteasome to determine the rate of degradation. We will employ a single-molecule method I developed previously to differentiate multiple limiting steps in the degradation process and to measure their rate constants. To circumvent the difficulty with preparing protein substrates with defined ubiquitylation, I propose a novel method of using DNA scaffolds to engineer arbitrary yet defined ubiquitin configurations, and systematically vary the features of ubiquitin configuration to determine how they affect substrate’s interaction with proteasome and degradation. Dysregulation of the ubiquitin- proteasome system is implicated in numerous diseases, including cancers, neurodegenerative disorders, autoimmunity and diabetes. The long-term goal of our lab is to elucidate how the proteasome recognizes its targets and selectively engages them into the processive degradation process, and to understand how misregulation of protein degradation leads to the formation of pathogenic inclusions. Our proposed research will provide insights into how mutations in the ubiquitin conjugation pathways lead to human diseases, and will generate valuable information for developing novel therapeutic strategies.