Soluble adenylyl cyclases in lung endothelial tauopathy

项目总结/摘要 肺泡毛细血管膜促进有效的气体交换，同时保持限制性的渗透性 屏障绿脓杆菌感染破坏肺泡-毛细血管屏障导致渗出性水肿 和氧合受损铜绿假单胞菌利用III型分泌系统及其效应物破坏屏障 完整特别地，外切酶Y被引入肺内皮，在那里它获得核苷酸酰化， 环化酶活性并产生cGMP、cAMP和cUMP。这些环核苷酸单磷酸激活 蛋白激酶A导致内皮tau蛋白磷酸化，tau蛋白从微管和微管中解离， 破坏，这共同阻碍了感染后的修复。磷酸化tau蛋白从 内皮细胞作为细胞毒性变体导致肺损伤。胞外酶的信号传导机制 Y产生细胞毒性tau的机制尚不完全清楚，但cUMP在特别高的浓度下产生 并且cUMP信号与细胞毒性tau的产生平行。cUMP的升高足以促进 产生细胞毒性tau变体。我们的初步数据表明，外切酶Y诱导的cUMP 信号还减少内皮烟酰胺腺嘌呤二核苷酸（NAD+）并增加烟酰胺， NAD+水解酶活性的产物，这可能会损害感染后的恢复。肺内皮细胞表达 不育α和TIR基序包含1（SARM 1），唯一的TIR（Toll/白细胞介素-1受体）结构域蛋白， 具有NAD+水解酶活性的哺乳动物细胞。最近的研究表明，SARM 1细菌 同源物直接被cUMP激活作为一种基本的先天免疫机制。虽然我们的研究表明 cUMP在内皮细胞对感染的反应中的重要作用，外切酶Y如何产生cUMP 导致tau磷酸化和细胞毒性tau变体的产生，以及cUMP如何降低NAD+， 阻碍内皮细胞修复的原因仍然知之甚少。为了以严格的方式解决这一知识差距， 该项目测试了铜绿假单胞菌外切酶Y产生cUMP的假设，cUMP有助于 tau蛋白磷酸化、微管断裂和SARM 1依赖性NAD+水解酶活性， 伤害和阻碍修复。

PROJECT SUMMARY/ABSTRACT The alveolar-capillary membrane facilitates efficient gas exchange while maintaining a restrictive permeability barrier. Pseudomonas aeruginosa infection disrupts the alveolar-capillary barrier leading to exudative edema and impaired oxygenation. P. aeruginosa utilizes a type III secretion system and its effectors to disrupt barrier integrity. In particular, the exoenzyme Y is introduced into lung endothelium, where it acquires nucleotidylyl cyclase activity and produces cGMP, cAMP, and cUMP. These cyclic nucleotide monophosphates activate protein kinase A resulting in endothelial tau phosphorylation, tau dissociation from microtubules, and microtubule breakdown, which collectively hinders repair following infection. Phosphorylated tau is released from endothelium as cytotoxic variants that contribute to lung injury. The signaling mechanisms used by exoenzyme Y to produce cytotoxic tau is incompletely understood, yet cUMP is produced at especially high concentrations and the cUMP signal parallels the generation of cytotoxic tau. Elevations in cUMP are sufficient to promote the production of cytotoxic tau variants. Our preliminary data demonstrate that the exoenzyme Y-induced cUMP signal also decreases endothelial nicotinamide adenine dinucleotide (NAD+) and increases nicotinamide, the product of NAD+ hydrolase activity, which may impair recovery following infection. Lung endothelium expresses sterile alpha and TIR motif containing 1 (SARM1), the only TIR (Toll/Interleukin-1 Receptor) domain protein in mammalian cells that possesses NAD+ hydrolase activity. Recent studies revealed a SARM1 bacterial homologue is directly activated by cUMP as an essential innate immune mechanism. While our studies illustrate an important role for cUMP in the endothelial cell response to infection, how exoenzyme Y generates the cUMP that leads to tau phosphorylation and production of cytotoxic tau variants, and how cUMP lowers NAD+ while hindering endothelial cell repair remains poorly understood. To address this knowledge gap in a rigorous way, this project tests the hypothesis that the P. aeruginosa exoenzyme Y generates cUMP, which contributes to the tau phosphorylation, microtubule breakdown, and SARM1-dependent NAD+ hydrolase activity that causes lung injury and hinders repair.