ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

项目摘要 蛋白质降解受AAA+家族的ATP依赖性区室蛋白酶的严格调控。的 真核细胞中主要的AAA+蛋白酶是26 S蛋白酶体，一种降解蛋白质的35亚基复合物 标记有多聚泛素链，控制蛋白质稳态以及许多重要过程。 尽管蛋白酶体对细胞活力非常重要，但其底物选择的详细机制 和加工，特别是它的调节和微调，例如通过底物附着的泛素 链，在很大程度上仍然难以捉摸。在过去的拨款期间，我们能够大大提高我们的 了解蛋白酶体的结构和功能。我们解出了基底的高分辨率结构- 在ATP水解循环的不同阶段参与蛋白酶体，建立了第一个完整的动力学 底物降解的图片，揭示了蛋白酶体的主要构象变化是如何耦合的 基质处理的各个步骤，并揭示了这些构象转变是如何部分地 受蛋白酶体亚复合物之间相互作用的调节。我们的生化工具，重组表达 系统和位点特异性荧光标记策略使我们处于一个独特的位置，以解决众多的 关于泛素介导的蛋白质周转的悬而未决的问题，26 S 蛋白酶体和其他AAA+马达，以及与泛素-蛋白酶体连接的途径的调节 系统特别是我们新建立的基于单分子FRET的检测方法， 底物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，底物连接的泛素 链影响构象转换，底物接合和降解的动力学， 蛋白酶体的解折叠能力取决于链长和连接类型。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之间 蛋白酶体亚复合物来读出这种“泛素密码”并微调其活性。我们将雇用一名 多学科方法，包括体外生化，单分子和原子分辨率结构 问题研究26 S蛋白酶体上游的途径是AAA+蛋白解折叠酶Cdc 48（在大肠杆菌中p97/VCP）。 人类）。在一个新的研究方向，我们将使用荧光和FRET为基础的测定结合 一系列差异泛素化和标记的模型蛋白，以研究Cdc 48如何与其 适配器Ufd 1/Np 14接合并展开其底物，以及Cdc 48-适配器相互作用的动力学 确定底物传递、解折叠和去泛素化。除了增进我们对 泛素依赖的蛋白质解折叠和降解，我们的研究也具有实质性的医学意义 并为开发新的小分子药物提供了巨大的潜力，因为26 S蛋白酶体和 p97在所有细胞中具有多种调节功能，并在各种人类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.