Protein-Oxidized Phospholipid Interactions Determine Epithelial Cell Fate and Asthma Control

易加重哮喘的分子机制知之甚少。虽然Type-2的关键作用 (T2)细胞因子正在出现，只有20-25%的T2-Hi患者持续恶化，提示其他因素 调节风险。我们的研究小组最近表明（Cell，2017），T2酶，15脂氧合酶1 （15 L01）与支架蛋白磷脂酰乙醇胺（PE）结合蛋白（PEBP）1的结合，触发一种形式的 程序性细胞死亡，称为铁凋亡，当它切换优选的15 LO 1底物从游离 多不饱和脂肪酸（PUFA），与PE缀合的PUFA，特别是15氢过氧二十烷四酸 酸-PE（15 HpETE-PE），其驱动铁凋亡细胞死亡。谷胱甘肽过氧化物酶（GPX）4，一种高度 对氧化应激敏感，迅速将15 HpETE-PE转化为其稳定的羟基代谢物，15 羟基二十烷四酸（15 HETE）-PE防止细胞死亡。PEBP 1还结合自噬蛋白， 微管轻链-3（LC 3），限制自噬。在此基础上，我们观察到IL-13刺激的LC 3 在人气道上皮细胞（AEC）中，脂质化和线粒体数量减少， 15 LO 1/15 HpETE-PE-过程，表明伴随着线粒体自噬。这些影响与 具有高的15 LO 1依赖性细胞内氧化应激，并且也见于急性加重的气道AEC- 容易哮喘因此，在存在“T2/IL-4/-13第一次命中”的情况下，促铁促凋亡15 L0 1-PEBP途径被激活， 但可能局限于与启动细胞凋亡相关的局部破坏性线粒体过程。 自噬/线粒体自噬（无细胞死亡）。这个谷胱甘肽依赖的过程产生氧化脆弱的细胞 分泌标志物表达增加和增殖降低与细胞衰老一致。与 “氧化第二击”，GSH福尔斯下降，降低GPX 4活性，引发全身性铁凋亡，破坏上皮细胞 屏障，增加促炎因子释放和促进恶化。因此，我们假设， 15 LO 1和PEBP 1与GPX 4和LC 3一起，从根本上调节铁凋亡和铁凋亡之间的平衡。 线粒体自噬，影响细胞功能，哮喘控制和加重。使用体外和离体人类细胞 在体内动物模型中，我们将：1）确定T2相关的第一次打击”诱导“应激的机制 哮喘气道细胞内的“稳态”，及其对哮喘严重程度和控制的影响，2）定义 “氧化第二次打击”破坏“应激稳态”以诱导广泛的铁凋亡的机制 并促进哮喘恶化。因此，我们将研究基本的死亡和生存途径， 并确定15 LO 1-PEBP活性和铁凋亡是否是哮喘的可行新靶点， 其恶化。

Molecular mechanisms for exacerbation-prone asthma are poorly understood. While a critical role for Type-2 (T2) cytokines is emerging, only 20-25% of T2-Hi patients persistently exacerbate, suggesting additional factors modulate the risk. Our group recently showed (Cell, 2017) that binding of the T2-enzyme, 15 lipoxygenase 1 (15LO1) to a scaffolding protein, phosphatidyl- ethanolamine (PE) binding protein (PEBP)1, triggers a form of programmed cell death termed ferroptosis, when it switches the preferred 15LO1 substrate from free polyunsaturated fatty acids (PUFA), to PUFAs conjugated to PE, specifically 15 hydroperoxyeicosaetetranoic acid-PE (15 HpETE-PE), which drive ferroptotic cell death. Glutathione peroxidase (GPX)4, an enzyme highly sensitive to oxidative stress, rapidly converts 15 HpETE-PE to its stable hydroxy-metabolite, 15 hydroxyeicosaetetranoic acid (15 HETE)-PE preventing cell death. PEBP1 also binds the autophagy protein, microtubule light chain-3 (LC3), limiting autophagy. Expanding on this, we observed IL-13 stimulated LC3 lipidation and lowered mitochondrial numbers in human airway epithelial cells (AECs), all through 15LO1/15HpETE-PE-processes, suggesting concomitant engagement of mitophagy. These effects associate with high 15LO1-dependent intracellular oxidative stress and are also seen in airway AECs from exacerbation- prone asthma. Thus, in the presence of a “T2/IL-4/-13 1st hit”, a pro-ferroptotic 15LO1-PEBP pathway is activated, but potentially limited to a localized disruptive mitochondrial process in association with initiation of autophagy/mitophagy (without cell death). This GSH-dependent process generates oxidatively vulnerable cells with increased secretory marker expression and lower proliferation consistent with cell senescence. With an “oxidative 2nd hit”, GSH falls, lowering GPX4 activity and initiating generalized ferroptosis, disrupting epithelial barriers, increasing pro-inflammatory factor release and promoting exacerbations. Thus, we hypothesize that 15LO1 and PEBP1, with both GPX4 and LC3, fundamentally regulate the balance between ferroptosis and mitophagy, influencing cell function, asthma control and exacerbations. Using in vitro and ex vivo human cells and in vivo animal models we will: 1) Identify the mechanisms by which a T2–associated 1st hit” induces “stressed homeostasis” in asthmatic airway cells, and its implications for asthma severity and control and 2) define mechanisms by which an “oxidative 2nd hit” disrupts the “stressed homeostasis” to induce widespread ferroptosis and promote asthma exacerbations. Thus, we will examine fundamental death and survival pathways in relation to asthma and determine whether 15LO1-PEBP activity and ferroptosis are viable new targets for asthma and its exacerbations.