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。虽然类型2的关键作用是 (T2)细胞因子正在出现，只有20%-25%的T2-Hi患者持续恶化，这表明还有其他因素 调节风险。我们的小组最近(Cell，2017)发现T2酶，15脂氧合酶1的结合 (15LO1)到支架蛋白，磷脂酰乙醇胺(PE)结合蛋白(PEBP)1，触发一种形式的 程序性细胞死亡称为铁性下垂，当它将首选的15LO1底物从游离切换到 多不饱和脂肪酸(PUFA)，到与PE结合的PUFA，特别是15羟基过氧二十烷酸 酸-PE(15HpETE-PE)，导致铁链细胞死亡。谷胱甘肽过氧化物酶(GPX)4，一种高度敏感的酶 对氧化应激敏感，能迅速将15HpETE-PE转化为稳定的羟基代谢物 羟基二十碳四酸(15HETE)-PE防止细胞死亡。PEBP1还与自噬蛋白结合， 微管轻链-3(LC3)，限制自噬。在此基础上，我们观察到IL-13刺激了LC3 人呼吸道上皮细胞(AECs)中的脂肪氧化和线粒体数量减少 15LO1/15HpETE-PE-过程，提示伴随有丝分裂。这些效果与 具有高15LO1依赖的细胞内氧化应激，也可在恶化的气道AECs中看到- 易患哮喘。因此，在存在“T2/IL-4/-13第一次命中”的情况下，前铁链15LO1-PEBP通路被激活， 但可能仅限于局部破坏性线粒体过程，与启动 自噬/有丝分裂(无细胞死亡)。这种依赖谷胱甘肽的过程会产生易氧化的细胞 分泌标志物表达增加，增殖降低，与细胞衰老相一致。带着一个 “氧化二次打击”，GSH下降，降低Gpx4活性，引发全身性铁性下垂，扰乱上皮 屏障，增加促炎因子的释放，促进病情恶化。因此，我们假设 15LO1和PEBP1与Gpx4和Lc3一起，从根本上调节铁下垂和 有丝分裂、影响细胞功能、哮喘控制和加重。利用体外和体外的人类细胞 在活体动物模型中，我们将：1)确定与T2相关的第一次打击“诱导”应激的机制 哮喘呼吸道细胞的“动态平衡”及其对哮喘严重程度和控制的意义 “氧化二次打击”破坏“应激内稳态”导致大范围铁下垂的机制 并促进哮喘加重。因此，我们将研究基本的死亡和生存途径之间的关系 并确定15LO1-PEBP活性和铁下垂是否是治疗哮喘和 它的恶化。

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.