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

蛋白氧化磷脂相互作用决定上皮细胞命运和哮喘控制

• 批准号: 10375454
• 负责人: Valerian E Kagan
• 金额: $ 54万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10375454
• 关键词: Acids; Allergens; Animal Model; Apoptosis; Arachidonate 15-Lipoxygenase; Asthma; Automobile Driving; Autophagocytosis; Binding; Binding Proteins; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Cell Aging; Cell Death; Cell Differentiation process; Cell Membrane Permeability; Cell Survival; Cell model; Cell physiology; Cells; Cessation of life; Cultured Cells; Data; Development; Endogenous Factors; Enzymes; Epithelial; Epithelial Cells; Equilibrium; Exogenous Factors; Glutathione; Glutathione Disulfide; Homeostasis; Human; Hydroxyeicosatetraenoic Acids; In Vitro; Inflammation; Inflammatory; Interleukin-13; Interleukin-4; LIGHT protein; Lead; Light; Lipids; Lipoxygenase 1; Measures; Microtubules; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Oxidation-Reduction; Oxidative Stress; Oxides; Participant; Pathway interactions; Patients; Phenotype; Phosphatidylethanolamine; Phosphatidylethanolamine Binding Protein; Phospholipid Interaction; Phospholipids; Polyunsaturated Fatty Acids; Process; Proteins; Recycling; Risk; Role; Scaffolding Protein; Severities; Stress; Tissues; airway epithelium; asthma exacerbation; asthmatic; asthmatic airway; asthmatic patient; base; cytokine; experience; falls; glutathione peroxidase; in vivo; macrophage; mitochondrial membrane; monocyte; mouse model; novel; novel therapeutics; prevent; release factor; selenoenzyme; targeted agent

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.

较恶化的哮喘的分子机制知之甚少。而类型2的关键作用 （T2）细胞因子正在出现，只有20-25％的T2-Hi患者持续加重，提出了其他因素 调节风险。我们的小组最近显示了T2-酶的结合，15脂氧合酶1 （15LO1）到脚手架蛋白，磷脂酰乙醇胺（PE）结合蛋白（PEBP）1，触发一种形式的形式 程序性细胞死亡称为铁毒性，当它从free切换首选15lo1底物时 多不饱和脂肪酸（PUFA），偶联到PE，特别是15氢过氧乙烯乙烯酸 酸-PE（15 hpete-pe），驱动铁毒细胞死亡。谷胱甘肽过氧化物酶（GPX）4，高度酶 对氧化应激敏感，将15 hpete-pe迅速转换为稳定的羟基 - 金代谢物，15 羟基乙酸乙酸（15种HETE） - PE预防细胞死亡。 PEBP1还结合自噬蛋白， 微管轻链3（LC3），限制自噬。为此扩展，我们观察到IL-13刺激了LC3 人类气道上皮细胞（AEC）的脂质和降低线粒体数量 15lo1/15hpete-pe-processes，表明线粒体伴随着互动。这些效果关联 具有高15lo1依赖性细胞内氧化物应激，并且在气道AEC中也可以通过恶化 - 容易发生哮喘。在“ T2/IL-4/-13第一击中”的情况下，促肌to虫15lo1-PEBP途径被激活， 但有可能仅限于局部破坏性线粒体过程 自噬/线粒体（无细胞死亡）。这种依赖GSH的过程会产生氧化脆弱的细胞 随着秘密标记表达的增加和与细胞感应一致的较低的增殖。与 “氧化第二命中”，GSH跌落，降低了GPX4活性并引发了广义的铁毒性，破坏了上皮 障碍，增加促炎性因子释放并促进加剧。那我们假设 具有GPX4和LC3的15LO1和PEBP1从根本上调节了逆转和 线粒体，影响细胞功能，哮喘控制和恶化。使用体外和体内人类细胞 在体内动物模型我们将：1）确定T2相关的第一击的机制。 哮喘气道细胞中的稳态”，及其对哮喘严重程度和控制的影响，2）定义 “氧化第二击中”破坏“压力体内平衡”以诱导宽度肿瘤的铁肉芽作用的机制 并促进哮喘恶化。这，我们将研究基本的死亡和生存途径 哮喘并确定15lo1-PEBP活性和铁铁作用是否是哮喘和 它加重了。