Role of mucin in lung homeostasis and pathophysiology

项目摘要 在许多肺部疾病中--包括哮喘--过多的粘液会扰乱清除并阻碍气流，但粘液 现有疗法不能有效治疗功能障碍。气道粘液中的主要大分子是 MUC 5AC和MUC 5 B。为了研究它们的因果关系，我们制作了Muc 5ac和Muc 5 b敲除小鼠。我们发现 Muc 5 b是健康状态下粘膜纤毛清除（MCC）所必需的，而Muc 5ac则是必需的。Muc5ac 导致粘液堵塞，并且是哮喘模型中气道高反应性（AHR）所必需的。尽管如此，尽管 在小鼠和人类中的稳态需求，我们现在知道过量的MUC 5 B/Muc 5 b本身可以是 肺纤维化（PF）。这些发现显示了重要性，但它们也强调了找到方法 防止粘液功能障碍，同时还保持防御。我们假设，这可以部分实现， 通过对MUC 5AC和MUC 5 B组装机制的更好理解。 MUC 5AC/Muc 5ac和MUC 5 B/Muc 5 b是非常大的蛋白质，通过连接形成更大的聚合物 在它们的羧基（C-）和氨基（N-）末端之间。它们的病理性质依赖于共价二硫键 其减少逆转AHR并改善MCC，这表明治疗干预目标可能是 通过精确地确定粘蛋白如何组装来揭示。粘蛋白C-和N-末端聚合结构域是 与蛋白质血管性血友病因子（VWF）同源。VWF组装所需的半胱氨酸在VWF中是保守的。 粘蛋白。我们已经确定了气道粘蛋白的组装和功能所需的。在这里，我们寻求继续 确定粘蛋白聚合和功能的结构要求和细胞机制。 新出现的数据表明，被称为“半胱氨酸三联体”的三个半胱氨酸的簇是至关重要的。之一 三联体的成员是形成分子间二硫化物的半胱氨酸。这需要酸性环境 在高尔基体中提供，以促进二硫键交换，释放分子间二硫键所需的半胱氨酸。 低pH值也质子化组氨酸在域周围的三元组。半胱氨酸三联体和pH敏感组氨酸 在粘蛋白中是保守的我们假设气道多聚粘蛋白的生物合成受高尔基体的调控 调节粘液功能和功能障碍的特定机制。我们将通过1）检查组装如何 由高尔基体定位的半胱氨酸三联体依赖机制调节，2）决定保守的组氨酸 残基是pH依赖性N-末端组装所必需的，以及3）证明这些残基如何影响粘蛋白 聚合气道粘液的功能。这里的研究将推进粘蛋白生物学领域，同时奠定基础 在保持防御的同时防止粘液功能障碍的方法。虽然这里的研究重点是哮喘， 结果将对慢性阻塞性肺病（COPD）、CF等疾病产生影响， 粘液功能障碍普遍存在的病症。

PROJECT SUMMARY In many lung diseases--including asthma--excessive mucus disrupts clearance and obstructs airflow, but mucus dysfunction is not effectively treated by existing therapies. The chief macromolecules in airway mucus are MUC5AC and MUC5B. To study them causatively, we made Muc5ac and Muc5b knockout mice. We discovered that Muc5b is required for mucociliary clearance (MCC) in health, but Muc5ac is dispensable. Instead, Muc5ac causes mucus plugging and is required for airway hyperreactivity (AHR) in models of asthma. Nonetheless, despite its homeostatic requirements in mice and humans, we now know that excessive MUC5B/Muc5b can itself be detrimental in lung fibrosis (PF). These findings show significance, but they also highlight the need to find ways to prevent mucus dysfunction while also preserving defense. We postulate that this can be accomplished in part through an improved understanding of MUC5AC and MUC5B assembly mechanisms. MUC5AC/Muc5ac and MUC5B/Muc5b are very large proteins that form even larger polymers via linkages between their carboxyl (C-) and amino (N-) termini. Their pathologic properties depend on covalent disulfide bonds whose reduction reverses AHR and improves MCC, suggesting that therapeutic intervention targets could be revealed by determining precisely how mucins assemble. Mucin C- and N-terminal polymerization domains are homologous with the protein von Willebrand Factor (VWF). Cysteines required for VWF assembly are conserved in mucins. We have identified which are required for airway mucin assembly and function. Here we seek to continue determine structural requirements and cellular mechanisms for mucin polymerization and function. Emerging data suggest that a cluster of three cysteines referred to as a “cysteine triad” is crucial. One of member of the triad is the cysteine that forms an inter-molecular disulfide. This requires the acidic environment provided in the Golgi to facilitate disulfide exchanges that liberate cysteines needed for intermolecular disulfides. Low pH also protonates histidines in domains that surround the triad. The cysteine triads and pH-sensing histidines in VWF are conserved in mucins. We hypothesize that airway polymeric mucin biosynthesis is regulated by Golgi specific mechanisms that mediate mucus function and dysfunction. We will test this by 1) examining how assembly is regulated by Golgi-localized cysteine-triad dependent mechanisms, 2) determining the conserved histidine residues are required for pH-dependent N-terminal assembly, and 3) demonstrating how these affect mucin polymerization airway mucus functions. Studies here will advance the mucin biology field while laying groundwork for approaches to prevent mucus dysfunction while preserving defense. While research here focuses on asthma, results will have implications for diseases such as chronic obstructive pulmonary disease (COPD), CF, and other conditions where mucus dysfunction is prevalent.