ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

项目摘要 蛋白质的降解受到AAA+家族依赖于ATP的间隔性蛋白的严格调控。这个 真核细胞中主要的AAA+蛋白酶是26S蛋白酶体，它是一种降解蛋白质的35亚基复合体 以多泛素链为标志，控制蛋白质的动态平衡以及许多重要的过程。 尽管蛋白酶体对细胞活力非常重要，但它对底物选择的详细机制 以及加工，特别是其调节和微调，例如通过底物连接的泛素 链条，在很大程度上仍然难以捉摸。在过去的授权期内，我们能够显著提前我们的 了解蛋白酶体的结构和功能。我们解决了衬底的高分辨率结构- 在ATP-水解循环的不同阶段参与的蛋白酶体，建立了第一个完整的动力学 底物降解图，揭示了蛋白酶体的主要构象变化是如何耦合的 底物处理的各个步骤，并揭示了这些构象转变是如何在一定程度上 受蛋白酶体亚复合体之间的相互作用调节。我们的生化工具，重组表达 系统和特定部位的荧光标记策略使我们处于独特的地位来处理众多 泛素介导的蛋白质周转、26S的分子机制等悬而未决的问题 蛋白酶体和其他AAA+马达，以及与泛素-蛋白酶体连接的通路的调节 系统。特别是我们新建立的基于FRET的单分子检测方法，可以进行前所未有的研究 底物的相互作用和通过蛋白酶体调节颗粒的进展，以及 蛋白酶体的构象动力学。令人兴奋的初步数据表明，底物结合泛素 链影响构象转换，底物结合和降解的动力学，以及 蛋白酶体的展开能力取决于链的长度和连接类型。一个主要目标是 研究蛋白酶体如何利用其三个主要的泛素受体和变构网络 蛋白酶体亚复合体，读出这个“泛素密码”，并微调其活动。我们将聘请一名 多学科方法，包括体外生化、单分子结构和原子分辨结构 学习。26S蛋白酶体上游的一条途径是AAA+蛋白解折叠酶CDC48(p97/VCP in 人类)。在一个新的研究方向，我们将使用基于荧光和FRET的分析结合 一系列差异泛素化和标记的模型蛋白研究CDC48与其复合体 适配器Ufd1/Npl4与其底物接合并展开，以及CDC48-适配器相互作用的动力学 确定底物递送、展开和去泛素化。除了增进我们对 泛素依赖的蛋白质的展开和降解，我们的研究也具有重大的医学意义 为新的小分子药物的开发提供了巨大的潜力，如26S蛋白酶体和 P97在所有细胞中发挥多种调节功能，在人类多种疾病中发挥重要作用。

Project Summary Protein degradation is tightly regulated by ATP-dependent compartmental proteases of the AAA+ family. The major AAA+ protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and controls protein homeostasis as well as numerous vital processes. Despite the proteasome’s great importance for cell viability, its detailed mechanisms for substrate selection and processing, and in particular its regulation and fine-tuning, for instance by substrate-attached ubiquitin chains, remain largely elusive. During the past granting period, we were able to significantly advance our understanding of proteasome structure and function. We solved high-resolution structures of the substrate- engaged proteasome at different stages of the ATP-hydrolysis cycle, established the first complete kinetic picture of substrate degradation, revealed how major conformational changes of the proteasome are coupled to individual steps of substrate processing, and uncovered how these conformational transitions are in part regulated by interactions between proteasomal subcomplexes. Our biochemical tools, recombinant expression systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S proteasome and other AAA+ motors, and the regulation of pathways connected to the ubiquitin-proteasome system. Especially our newly established single-molecule FRET-based assays allow unprecedented studies of substrate interactions and progression through the proteasome regulatory particle, as well as the conformational dynamics of the proteasome. Exciting preliminary data indicate that substrate-attached ubiquitin chains affect the conformational switching, the kinetics of substrate engagement and degradation, and the unfolding power of the proteasome depending on the chain length and linkage type. A primary goal is to investigate how the proteasome utilizes its three main ubiquitin receptors and allosteric networks between proteasomal subcomplexes to read out this “ubiquitin code” and fine-tune its activities. We will employ a multidisciplinary approach that includes in-vitro biochemical, single-molecule, and atomic-resolution structural studies. A pathway upstream of the 26S proteasome is the AAA+ protein unfoldase Cdc48 (p97/VCP in human). In a new research direction, we will use fluorescence- and FRET-based assays combined with a series of differentially ubiquitinated and labeled model proteins to investigate how Cdc48 in complex with its adaptor Ufd1/Npl4 engages and unfolds its substrates, and how the dynamics of Cdc48-adaptor interactions determine substrate delivery, unfolding, and deubiquitination. Besides advancing our general understanding of ubiquitin-dependent protein unfolding and degradation, our research also has substantial medical relevance and offers great potential for the development of new small-molecule drugs, as both the 26S proteasome and p97 fulfill numerous regulatory functions in all cells and play important roles in various human diseases.