Understanding Mycobacterium tuberculosis 20S proteasome assembly

项目总结 在美国，每年有超过8000人感染结核病，结核分枝杆菌 (MTB)能够部分通过蛋白酶体功能抵抗免疫系统。蛋白酶体是 与细胞中错误折叠或短寿命蛋白质的降解有关的大分子结构， 由桶状结构中的四个堆叠的七聚体环组成。这项工作的长期目标是帮助 了解在MTB系统中调节蛋白酶体组装的机制 建模和实验分析。本申请的总体目标是(1)阐明 装配动力学调节蛋白酶体形成的机制(2)确定它们在结核分枝杆菌中的作用 蛋白酶体组装。中心假设是Mtb蛋白酶体已经进化出一套机制， 最大限度地提高产量，从而提高细菌的免疫抵抗力。这个项目的基本原理是确定 调节Mtb蛋白酶体产量的机制可能提供一个强有力的科学框架，使新的 可以开发针对患者的结核病治疗策略。核心假设将通过以下方式进行检验 追求三个具体目标：(1)用数学方法评价mtb蛋白酶体的组装动力学 和实验分析，(2)开发一个生物物理框架来理解 蛋白酶体组装中的中间环以及(3)分析Mtb蛋白酶体中中间环的结构 集合。在第一个目标中，将使用一个数学模型来确定动力学参数在 环的形成和最终蛋白酶体的组装。此外，Mtb单体将用于实验 测量组装的动力学。对于第二个目标，将开发一个生物物理框架来研究 单体和中间环之间的相互作用取决于它们的大小和结构。此外，a 将使用质谱学方法来鉴定形成的中间环的大小和组成 在Mtb蛋白酶体组装过程中。在第三个目标中，将使用冷冻电子显微镜方法来 用原子分辨分析组装中间体的结构。这项研究中提出的 应用是创新的，因为它专注于Mtb蛋白酶体，而Mtb蛋白酶体还不够充分 它的特点是，因为它结合了数学建模和实验方法。 这项拟议的研究具有重要意义，因为它有望为发展和 新型结核分枝杆菌蛋白酶体组装抑制剂的临床应用前景。归根结底，这种知识具有 为开发治疗结核病和结核病的创新疗法提供新的机会 其他细菌感染。此外，这项奖学金是由Eric J.Deeds博士和Joseph A.Loo博士赞助的，他们 在各自的计算生物学和质谱学领域处于领先地位。拟议中的培训 计划包括一个强大的研究环境和指导团队，有助于申请者成长为 非常成功的独立研究人员。

PROJECT SUMMARY Tuberculosis affects over 8,000 individuals in the United States every year, with Mycobacterium tuberculosis (Mtb) able to resist the immune system in part through proteasome function. The proteasome is the macromolecular structure responsible for the degradation of misfolded or short-lived proteins in cells, and is composed of four stacked heptameric rings in a barrel-like structure. The long-term goal of this work is to help understand the mechanisms that regulate proteasome assembly in the Mtb system using both mathematical modeling and experimental analyses. The overall objectives in this application are to (1) elucidate the mechanism(s) by which assembly dynamics regulate proteasome formation and (2) determine their role in Mtb proteasome assembly. The central hypothesis is that Mtb proteasome has evolved a set of mechanisms that maximize yield and thus bacterial immune resistance. The rationale for this project is that determination of the mechanisms that regulate Mtb proteasome yield is likely to offer a strong scientific framework whereby new strategies for tuberculosis therapies in patients can be developed. The central hypothesis will be tested by pursuing three specific aims: (1) Evaluate the assembly kinetics of the Mtb proteasome using mathematical and experimental analyses, (2) Develop a biophysical framework to understand interactions between intermediate rings in proteasome assembly and (3) Analyze structures of intermediate rings in Mtb proteasome assembly. In the first aim, a mathematical model will be used to determine the role kinetic parameters play in ring formation and ultimately proteasome assembly. Additionally, Mtb monomers will be used to experimentally measure the kinetics of assembly. For the second aim, a biophysical framework will be developed to study the interactions between monomers and intermediate rings based on their size and structure. Furthermore, a mass-spectrometry approach will be used to identify the size and composition of intermediate rings formed during Mtb proteasome assembly. In the third aim, a cryo-Electron Microscopy approach will be used to analyze the structures of assembly intermediates with atomic resolution. The research proposed in this application is innovative because it focuses on the Mtb proteasome, which has not been sufficiently characterized to date, and because it incorporates both mathematical modeling and experimental methods. The proposed research is significant because it is expected to provide a foundation for the development and future clinical applications of novel Mtb proteasome assembly inhibitors. Ultimately, such knowledge has the potential of offering new opportunities for the development of innovative therapies to treat tuberculosis and other bacterial infections. Moreover, this fellowship is sponsored by Drs. Eric J. Deeds and Joseph A. Loo, who are leaders in their respective fields of computational biology and mass spectrometry. The proposed training plan includes a strong research environment and mentoring team conducive to the applicant’s growth into a highly successful independent researcher.