CAREER: Dissecting the Molecular Mechanisms of Substrate Selection and Degradation by the 26S Proteasome

知识价值。细胞内的蛋白质含量是不断变化的。根据细胞的需要，某些蛋白质被制造出来，而另一些则被移除或降解。蛋白质降解必须是高度特异性的，因此受到区室蛋白酶的严格调控，这种大的桶形复合物利用ATP水解产生的能量机械地解开蛋白质底物，然后将蛋白质底物转移到一个隔离的内部腔室进行降解。真核细胞中主要的atp依赖蛋白酶是26S蛋白酶体，它通过特异性降解细胞分裂、信号转导和程序性细胞死亡等调节蛋白来控制蛋白质周转和许多重要过程。大多数底物通过另一种小蛋白泛素的可逆附着来标记降解，泛素可以连续连接在一起形成多泛素链，作为蛋白酶体的靶向信号。本研究的目的是通过使用新的方法对26S蛋白酶体进行结构、定量生化和生物物理表征，阐明泛素依赖性蛋白降解的详细机制。具体目标是1)了解底物降解与泛素链先前去除之间的复杂耦合，2)获得蛋白酶体分子机器结构的详细结构和功能洞察及其底物识别，展开和泛素去除的机制，以及3)使用前所未有的单分子测量来提供对底物识别和降解的机械化学的瞥见。研究人员认为，阐明这些分子机制将有助于确定体内高度多样化蛋白质降解的控制原理，从而进一步了解真核细胞中前糖体的各种调节功能。由于蛋白酶体在蛋白质周转和关键途径的维持中无处不在的功能，通过本研究产生的结果和新工具将强烈影响其他细胞生物学研究和方法。更广泛的影响。对底物处理分子机制的详细了解将有助于开发专用蛋白酶体，用于暂时控制细胞中特定酶和其他蛋白质的消除，这在生物工程和系统生物学中的应用是非常需要的，并且可能有助于药物前体或生物燃料的合成。拟议的研究将与教学和外展相结合，以加强科学、技术、工程和数学（STEM）领域的教育。由于其强烈的合作性质，这些研究项目为生物学、物理学和化学交叉领域的本科生、博士后和研究生提供了密集的跨学科培训。此外，为了从小开始科学教育和推广，将制定一项计划，其中包括为高中生提供暑期研究实习，为本科生、社区大学学生和高中教师提供课程的实验室训练营计划，以及在当地公立特许学校组织科学博览会，这些学校有很高比例的少数族裔。

Intellectual merit. The protein content inside a cell is constantly changing. Depending on the cell's needs, certain proteins are made while others are removed or degraded. Protein degradation must be highly specific and is therefore tightly regulated by compartmental proteases, large barrel-shaped complexes that utilize the energy from ATP hydrolysis to mechanically unravel and then translocate protein substrates into a sequestered internal chamber for degradation. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, which controls protein turnover and numerous vital processes by specifically degrading regulatory proteins involved for instance in cell division, signal transduction, and programmed cell death. Most substrates are marked for degradation by the reversible attachment of another small protein, ubiquitin, which can be consecutively linked together to form poly-ubiquitin chains that act as a targeting signal to the proteasome. The goal of this research is to elucidate the detailed mechanisms of ubiquitin-dependent protein degradation by using novel approaches for the structural, quantitative biochemical, and biophysical characterization of the 26S proteasome. Specific aims are 1) to understand the intricate coupling between substrate degradation and the preceding removal of ubiquitin chains, 2) to gain detailed structural and functional insight into the architecture of the proteasome molecular machine and its mechanisms for substrate recognition, unfolding, and ubiquitin removal, and 3) to use unprecedented single-molecule measurements to provide a glimpse into the mechanochemistry of substrate recognition and degradation. It is expected that elucidating these molecular mechanisms will help to define the principles that control the degradation of highly diverse proteins in vivo, and thus further the understanding of the various regulatory functions of preoteasomes in eukaryotic cells. Due to the ubiquitous functions of the proteasome in protein turnover and maintenance of crucial pathways, the results and novel tools generated through this research will strongly impact other cell-biological studies and approaches. Broader impacts. A detailed knowledge of the molecular mechanisms for substrate processing will allow the development of dedicated proteasomes for the temporally controlled elimination of specific enzymes and other proteins in the cell, which is highly desired for applications in bioengineering and systems biology, and may aid the synthesis of drug precursors or biofuels. The proposed research will be integrated with teaching and outreach to strengthen education in the science, technology, engineering, and mathematics (STEM) fields. Due to their strongly collaborative nature, the research projects provide intense interdisciplinary training for undergraduates, postdocs, and graduate students at the intersection of biology, physics, and chemistry. Furthermore, in order to start science education and outreach already at a young age, a program will be developed that includes summer research internships for high school students, a Laboratory Boot Camp program with courses for undergraduates, community college students, and high school teachers, and the organization of science fairs at local public charter schools with high percentage of underrepresented minorities.