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 结核分枝杆菌(Mtb)在宿主细胞中遇到几种类型的应激，如活性氧(ROS)， 活性氮物种(RNS)和化疗。这些压力会损害蛋白质的完整性，导致 蛋白质聚集。受损或聚集的蛋白质必须被降解和回收，否则 已解决并修复。Mtb中蛋白质的降解和再循环主要是由蛋白酶体完成的。 系统，而蛋白质拯救(即，将有毒蛋白质聚集体分解到自然折叠状态)是通过 由三磷酸腺苷驱动的ClpB/DNAK双伴侣系统。因此，蛋白酶体和ClpB/DNAK系统 在结核分枝杆菌中，细胞蛋白平衡的阴阳。有趣的是，大多数细菌中都没有蛋白酶体；它 仅在放线菌目中存在，对结核分枝杆菌在宿主内的生存是必不可少的。遗传 研究已将结核分枝杆菌蛋白酶体确立为抗结核药物开发的理想靶点。在……里面 与化学生物学实验室合作，我们建议研究几种抗结核病药物的结合姿势，这些药物 在代谢上稳定，并特异性地抑制Mtb 20S蛋白酶体核心颗粒，同时节省 人类构成和免疫蛋白酶体(目标1)。在之前的资金周期中，我们解决了水晶 依赖于ATP的蛋白酶体激活子、MPA六聚体以及不依赖于ATP的结构 蛋白酶体激活剂称为Pafe十二聚体。接下来，我们将研究如何部分展开或氧化 Mtb蛋白酶体识别蛋白质底物并将其作为降解的目标(目标2)。值得注意的是， Mtb ClpB/DNAK双伴侣系统的转录抑制因子HspR是Mtb Pafe的底物。 蛋白酶体系统；因此，Mtb蛋白酶体直接调节双伴侣系统。我们计划研究一下 ATP驱动的解聚机制的结构和功能，即Mtb ClpB六聚体(目标3)，AS 长期计划的一部分，以确定完整的ClpB/DNAK双伴侣系统的特征。描述了 蛋白质降解和蛋白质再激活两个对立的系统为发展奠定了基础 同时针对这两个系统的组合方法，导致无法弥补的中断 微生物蛋白抑制和协同杀灭非复制菌。总体而言，拟议工作将有所改善 我们对最致命细菌结核分枝杆菌蛋白质动态平衡分子机制的理解 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.