Protein unfolding in a physiological system

描述（由申请人提供）：蛋白酶体位于真核细胞的细胞质和细胞核中，通过控制数百种调节蛋白的浓度、去除错误折叠蛋白和产生抗原呈递中显示的一些肽，在细胞调节中起核心作用。这是一个近似值。2MDa圆柱形粒子，其蛋白水解活性位点深埋在结构内部，只能通过狭窄的通道进入。底物蛋白必须展开以适应通道并被降解。本文提出的长期研究目标是利用现代生化、计算、遗传和细胞生物学方法的结合来理解蛋白酶体对蛋白质的展开。这些实验将提供对蛋白酶体功能的深入了解，并可能揭示意想不到的调节机制。当前的目标是了解展开步骤如何影响蛋白酶体对其底物的特异性以及它如何影响其降解产物的性质。我们将首先定义蛋白酶体结合底物后如何开始降解。蛋白质通过泛素标签的共价附着在蛋白酶体上，但降解在底物的不同位置开始。起始步骤有助于降解的特异性，有效起始的顺序和空间要求将在体外确定。这些规则对天然蛋白质降解特异性的影响将在体内进行测试。其次，我们将研究蛋白酶体如何在一个称为加工的过程中展开并降解蛋白质的一部分。蛋白酶体将大多数蛋白质完全降解为小肽，但一种新型信号可导致部分蛋白质降解。处理信号和机制将被表征，处理的生理实例将通过生物信息学搜索和遗传筛选检测。这项工作与人类疾病有关；许多被蛋白酶体降解的蛋白质调节细胞的生长和分裂。由于蛋白质在被蛋白酶体切割之前必须展开，因此了解降解过程中的展开可能会为对抗癌症开辟新的途径，例如通过稳定特定的蛋白质来防止展开和降解。一些神经退行性疾病与蛋白质聚集体的积累有关，蛋白酶体由于未知的原因无法清除。对部分蛋白质降解的研究可能有助于深入了解这个问题。

DESCRIPTION (provided by applicant): The proteasome, located in the cytosol and nucleus of eukaryotic cells, plays a central role in cellular regulation by controlling the concentrations of hundreds of regulatory proteins, removing misfolded proteins, and producing some peptides displayed in antigen presentation. It is an approx. 2MDa cylindrical particle with its active sites of proteolysis buried deep inside the structure where they are accessible only through a narrow channel. Substrate proteins must be unfolded to fit the channel and to be degraded. The long-term goals of the research proposed here are to understand protein unfolding by the proteasome using a combination of modern biochemical, computational, genetic, and cell biological methods. These experiments will provide an in-depth understanding of proteasome function and may reveal unexpected regulatory mechanisms. The immediate goal is to understand how the unfolding step affects the specificity of the proteasome for its substrates and how it affects the nature of its degradation products. We will first define how degradation is initiated after substrate binding by the proteasome. Proteins are targeted to the proteasome by the covalent attachment of a ubiquitin tag but degradation initiates at a different site in the substrate. The initiation step contributes to the specificity of degradation and the sequence and spatial requirements for efficient initiation will be determined in vitro. The effect of these rules on the specificity of degradation of natural proteins will be tested in vivo. Second, we will investigate how the proteasome unfolds and degrades only part of a protein in a process called processing. The proteasome degrades most proteins completely to small peptides but a novel type of signal can lead to partial protein degradation. The processing signal and mechanism will be characterized and physiological examples of processing will be detected by a bioinformatics search and a genetic screen. This work is relevant to human diseases; many of the proteins degraded by the proteasome regulate cell growth and division. Since proteins must be unfolded before they are cleaved by the proteasome, understanding unfolding during degradation may open new avenues to fight cancer, for example by stabilizing specific proteins against unfolding and degradation. Some neurodegenerative diseases are associated with the accumulation of protein aggregates, which the proteasome fails to remove for unknown reasons. The investigation of partial protein degradation may lend insight into this question.