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

项目概要/摘要 泛素-蛋白酶体系统是调节蛋白降解的主要途径， 与大多数生物学功能下的翻译/翻译相似的靶特异性水平。泛素 底物分子上的模式决定了特异性，速率和蛋白水解的结果。 蛋白酶体什么样的泛素构型对蛋白酶体的选择性有重要影响 降解和如何26 S蛋白酶体，与众多的泛素受体，认识到这些特点是 仍然知之甚少。蛋白酶体降解涉及一系列步骤。我们已公布的和初步的 研究记录了降解速率对长度、连锁、拷贝数和 共轭泛素链的位置。这种选择性是如何实现的尚不清楚。在此，我提议 系统的研究，以确定如何泛素结构的特点控制动力学和模式 底物与蛋白酶体上的泛素受体的结合来确定降解速率。 我们将采用我以前开发的单分子方法来区分多个限制步骤， 降解过程，并测定其速率常数。为了解决蛋白质制备的困难 底物与定义的泛素化，我提出了一种新的方法，使用DNA支架工程任意 还定义了泛素构型，并系统地改变泛素构型的特征，以确定 它们如何影响底物与蛋白酶体相互作用和降解。泛素的失调- 蛋白酶体系统涉及许多疾病，包括癌症，神经变性疾病， 自身免疫和糖尿病。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性的退化过程中，并了解如何 蛋白质降解的失调导致致病性内含物的形成。我们提出的研究 将提供关于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提供有价值的信息。

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.