Role of NadD in Mycobacterium tuberculosis proteostasis

总结 这项研究计划的目标是表征来自人的NadD（烟酸腺苷酰转移酶）的生物学特性。 结核分枝杆菌（Mycbobacterium tuberculosis，Mtb）是结核病（TB）的病原体。在感染过程中，结核病面临着 多种多样的宿主微环境和宿主化学物质可导致蛋白毒性应激，包括缺氧， 温和的酸度、金属缺乏或中毒以及活性氧和氮物质。抗生素治疗 结核病患者可以通过阻止核糖体上的新生蛋白质或通过形成反应性蛋白质来破坏结核分枝杆菌蛋白质。 氧或氮中间体。这些宿主压力中的许多与预防或减缓结核分枝杆菌相关。 复制和发展的非遗传性，表型耐药性。MTB保持蛋白质的完整性， 蛋白质稳态网络-超过100种蛋白质的集合，包括分子伴侣及其辅因子 (ClpB、DnaK、DnaJ 1、DnaJ 2、GrpE、GroEL/ES）和桶形蛋白酶（ClpP 1 P2和Mtb 20 S）。的 蛋白质稳态网络的许多成员的转录由蛋白毒性应激如热诱导。 蛋白质稳定网络的组分的遗传和化学破坏与蛋白质稳定性降低有关。 在巨噬细胞和TB小鼠模型中的毒力。我们试图确定化合物的目标成分， 通过使用化学筛选其活性被以下物质增强的化合物， 热引起的蛋白毒性应激。我们对一种热应激增强化合物的表征 意外地发现NadD是目标。NadD是一个高度优先的药物靶点，位于以下关键交叉点： NAD（H）和NADP（H）生物合成的从头和补救途径。野生型中NadD的化学抑制 结核分枝杆菌导致细菌内蛋白聚集增加。我们假设NadD的缺失破坏了 NADPH依赖性抗氧化防御系统和/或NadD的功能作为一种非常规的抗氧化防御系统发挥作用。 蛋白伴侣这项研究有两个互补的方向：在目标1中，我们将使用遗传学， 化学生物学，以表征NadD在蛋白质稳定网络中的作用，特别是通过监测生成 细菌内活性氧和氮物质、蛋白质羰基化和蛋白质聚集;以及 目的2，我们将确定NadD有助于Mtb蛋白质稳态的机制。这些研究将奠定 基础，以了解NadD如何作为蛋白质稳态网络的非经典成员， 感染和抗生素治疗。

Summary The goal of this research program is to characterize the biology of NadD (nicotinate adenylyltransferase) from Mycbobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). During infection, Mtb faces a diverse set of host microenvironments and host chemistries that can lead to proteotoxic stress, including hypoxia, mild acidity, metal starvation or intoxication, and reactive oxygen and nitrogen species. Antibiotic treatment of TB patients can damage Mtb proteins by stalling nascent proteins on the ribosome or via the formation of reactive oxygen or nitrogen intermediates. Many of these host stresses are associated with preventing or slowing of Mtb replication and the development of non-genetic, phenotypic drug resistance. Mtb maintains protein integrity with the proteostasis network – a collection of over one hundred proteins, including chaperones and their cofactors (ClpB, DnaK, DnaJ1, DnaJ2, GrpE, GroEL/ES) and barrel-shaped proteases (ClpP1P2 and Mtb20S). The transcription of many members of the proteostasis network is induced by proteotoxic stresses such as heat. Genetic and chemical disruption of components of the proteostasis network is associated with decreased virulence in macrophage and murine models of TB. We sought to identify compounds targeting components of the proteostasis network by using a chemical screen for compounds whose activity was potentiated by a proteotoxic stress generated by heat. Our characterization of one heat-stress potentiated compound unexpectedly revealed NadD as the target. NadD is a high priority drug target that lies at a critical intersection of de novo and salvage pathways for NAD(H) and NADP(H) biosynthesis. Chemical inhibition of NadD in wild-type Mtb led to an increase in intrabacterial protein aggregation. We hypothesize that loss of NadD disrupts the function of NADPH-dependent antioxidant defense systems and/or NadD plays a role as an unconventional protein chaperone. This research takes two complementary directions: in Aim 1, we will use genetics and chemical-biology to characterize NadD’s role in the proteostasis network, in particular by monitoring generation of intrabacterial reactive oxygen and nitrogen species, protein carbonylation, and protein aggregation; and in Aim 2, we will define the mechanism by which NadD contributes to Mtb proteostasis. These studies will lay the groundwork to understand how NadD contributes as a noncanonical member of the proteostasis network during infection and antibiotic treatment.