Structural biology of proteostasis in M. tuberculosis

项目摘要 结核病（TB）是细菌感染导致死亡的主要原因。在感染过程中，分枝杆菌 结核病（Mtb）在宿主细胞中遇到几种类型的应激，例如活性氧（ROS）， 活性氮（RNS）和化疗。这些应激损害蛋白质的完整性， 蛋白质聚集受损或聚集的蛋白质必须被降解和回收，否则 解决和修复。结核分枝杆菌中蛋白质的降解和循环主要由蛋白酶体完成 系统，而蛋白质拯救（即，将毒性蛋白质聚集体分解为天然折叠状态）， ATP驱动的ClpB/DnaK双分子伴侣系统。因此，蛋白酶体和ClpB/DnaK系统 是结核分枝杆菌细胞蛋白质稳态的阴阳。有趣的是，蛋白酶体在大多数细菌中不存在， 仅存在于放线菌目中，并且对于Mtb在宿主内的存活是必需的。遗传 研究已经确定Mtb蛋白酶体是开发抗TB剂的理想靶点。在 与化学生物学实验室合作，我们建议研究几种抗结核药物的结合形式， 是代谢稳定的，并且特异性抑制Mtb 20S蛋白酶体核心颗粒，同时保留Mtb 20S蛋白酶体核心颗粒。 人组成型和免疫蛋白酶体（Aim 1）。在上一个融资周期中，我们解决了晶体 ATP依赖性蛋白酶体激活剂Mpa六聚体以及ATP非依赖性蛋白酶体的结构 称为PafE十二聚体的蛋白酶体激活剂。接下来我们将研究部分展开或氧化 蛋白质底物被Mtb蛋白酶体识别并靶向降解（Aim 2）。值得注意的是， Mtb ClpB/DnaK双分子伴侣系统的转录阻遏物HspR是Mtb PafE的底物。 因此，Mtb蛋白酶体直接调节双分子伴侣系统。我们计划学习 ATP驱动的解聚机制的结构和功能，即，Mtb ClpB六聚体（Aim 3），如 长期计划的一部分，以表征完整的ClpB/DnaK双分子伴侣系统。表征 蛋白质降解和蛋白质再活化的两个对立系统为蛋白质的发展奠定了基础。 同时针对这两个系统的组合方法，导致无法弥补的破坏， 微生物蛋白质抑制和协同杀死非复制细菌。总体而言，拟议工作将有所改进 我们对结核分枝杆菌中蛋白质稳态的分子机制的理解， 病原体

Project Summary Tuberculosis (TB) is the leading cause of death from bacterial infection. During infection, Mycobacterium tuberculosis (Mtb) encounters several types of stress in the host cells, such as reactive oxygen species (ROS), reactive nitrogen species (RNS), and chemotherapy. These stresses impair protein integrity and result in protein aggregation. The impaired or aggregated proteins have to be either degraded and recycled or else resolved and repaired. Protein degradation and recycling in Mtb is mainly carried out by the proteasome system, while protein rescue (i.e., resolving toxic protein aggregates to a native folded state) is carried out by the ATP-powered ClpB/DnaK bi-chaperone system. Therefore, the proteasome and the ClpB/DnaK systems are the yin and yang of cellular proteostasis in Mtb. Interestingly, the proteasome is absent in most bacteria; it is present only in the order of Actinomycetales and is essential to Mtb's survival inside the host. Genetic studies have established the Mtb proteasome as an ideal target for the development of anti-TB agents. In collaboration with chemical biology labs, we propose to study the binding poses of several anti-TB agents that are metabolically stable and that specifically inhibit the Mtb 20S proteasome core particle while sparing the human constitutive and immunoproteasome (Aim 1). In the previous funding cycle, we solved the crystal structures of the ATP-dependent proteasomal activator, the Mpa hexamer, as well as the ATP-independent proteasomal activator called the PafE dodecamer. We will next investigate how partially unfolded or oxidized protein substrates are recognized and targeted for degradation by the Mtb proteasome (Aim 2). It is notable the transcriptional repressor HspR of the Mtb ClpB/DnaK bi-chaperone system is a substrate of the Mtb PafE- proteasome system; hence, the Mtb proteasome directly regulates the bi-chaperone system. We plan to study the structure and function of the ATP-driven disaggregation machinery, i.e., the Mtb ClpB hexamer (Aim 3), as part of the long-term plan to characterize the complete ClpB/DnaK bi-chaperone system. Characterizing the two opposing systems of protein degradation and protein reactivation lays the groundwork for the development of a combination approach that simultaneously targets both systems, leading to irreparable disruption of the microbial proteostasis and synergistic killing of nonreplicating bacteria. Overall, the proposed work will improve our understanding of the molecular mechanism of protein homeostasis in Mtb, the deadliest bacterial pathogen.