Role of NadD in Mycobacterium tuberculosis proteostasis

摘要 这项研究计划的目标是表征烟酸转移酶(NADD)的生物学特性。 结核分枝杆菌(Mtb)，结核病的病原体。在感染期间，结核分枝杆菌面临着 不同的寄主微环境和寄主化学物质可导致蛋白毒性应激，包括低氧， 轻度酸度，金属饥饿或中毒，以及活性氧和氮物种。抗菌药物治疗 结核病患者可以通过阻止核糖体上的新生蛋白或通过形成反应性蛋白来破坏结核分枝杆菌蛋白 氧或氮的中间体。这些寄主压力中的许多与防止或减缓结核分枝杆菌感染有关 复制和非遗传性、表型耐药性的发展。结核分枝杆菌可保持蛋白质完整性 蛋白质平衡网络--100多种蛋白质的集合，包括伴侣及其辅因子 (ClpB、Dna K、Dna J1、Dna J2、GRPE、GroEL/ES)和桶形蛋白水解酶(ClpP1P2和Mtb20S)。这个 蛋白平衡网络的许多成员的转录是由热等蛋白毒性应激诱导的。 蛋白质平衡网络成分的遗传和化学破坏与减少有关 巨噬细胞和小鼠结核病模型的毒力。我们试图确定靶向成分的化合物 通过使用化学筛选其活性被增强的化合物的蛋白质平衡网络 热所产生的蛋白毒性应激。一种热应激强化化合物的表征 出人意料地透露纳德是目标。NADD是一个高度优先的药物目标，位于 NAD(H)和NADP(H)生物合成的从头途径和挽救途径。野生型NADD的化学抑制作用 MTB导致菌体内蛋白质聚集增加。我们假设失去NADD会扰乱 NADPH依赖的抗氧化防御系统的功能和/或NADD发挥非常规作用 蛋白质伴侣。这项研究采取两个相辅相成的方向：在目标1中，我们将使用遗传学和 化学生物学以确定NADD在蛋白质平衡网络中的作用，特别是通过监测世代 细菌内的活性氧和氮物种、蛋白质的羰基化和蛋白质的聚集； 目的2，我们将确定NADD促进Mtb蛋白稳定的机制。这些研究将为 为了解NADD如何作为蛋白质平衡网络的非规范成员在 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.