Protein unfolding in a physiological system

描述(申请人提供)：蛋白酶体位于真核细胞的胞浆和胞核中，通过控制数百种调节蛋白的浓度，去除错误折叠的蛋白，并产生一些在抗原呈递中显示的多肽，在细胞调节中发挥核心作用。这大约是个数字。2MDA是一种圆柱形颗粒，其蛋白分解活性部位深埋在结构内部，只有通过狭窄的通道才能到达。底物蛋白质必须展开以适应通道并被降解。这项研究的长期目标是结合现代生化、计算、遗传和细胞生物学方法，了解蛋白酶体展开的蛋白质。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。目前的目标是了解展开步骤如何影响蛋白酶体对其底物的专一性，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与底物结合后如何启动降解。通过泛素标签的共价连接，蛋白质被靶向蛋白酶体，但降解在底物中的不同位置开始。启动步骤有助于降解的特异性，有效启动的序列和空间要求将在体外确定。这些规则对天然蛋白质降解的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仅降解蛋白质的一部分。蛋白酶体可以将大多数蛋白质完全降解为小肽，但一种新型的信号可以导致蛋白质的部分降解。将对处理信号和机制进行表征，并将通过生物信息学搜索和遗传筛选来检测处理的生理示例。这项工作与人类疾病有关；蛋白酶体降解的许多蛋白质调节细胞的生长和分裂。由于蛋白质必须在被蛋白酶体切割之前展开，了解降解过程中的展开可能会开辟抗癌的新途径，例如通过稳定特定的蛋白质以防止展开和降解。一些神经退行性疾病与蛋白质聚集体的积累有关，但由于未知原因，蛋白酶体无法清除这些聚集体。对蛋白质部分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.