Mucin Biosynthesis and Transport Mechanisms in Respiratory Health and Disease

项目摘要 每天，呼吸道表面都暴露在数十亿颗粒物中，如果不这样做，这些颗粒物的积累可能是有害的。 通过粘膜纤毛清除迅速消除。在健康状态下，粘液很薄，很容易运输。在粘膜- 阻塞性疾病，如哮喘，粘液丰富，厚，运输不畅。粘液功能障碍是 现有的治疗方法无法充分治疗。为了开发更有效的治疗方法， 我们对粘蛋白生物学的机械理解。两种聚合粘蛋白MUC 5AC/Muc 5ac和MUC 5 B/Muc 5 b 是气道粘液中的主要大分子。它们是非常大的糖蛋白，形成更大的多- 单元聚合物在合成过程中，聚合粘蛋白通过保守的羧基和氨基末端组装， 在血管性血友病因子（VWF）中也发现了这些结构域。已发表的关于VWF的报告已经确定了三个C-末端 在内质网（ER）中链间二聚化所需的半胱氨酸，然后转运到高尔基体。 这些半胱氨酸也存在于MUC 5AC/Muc 5ac和MUC 5 B/Muc 5 b中。在急诊室组装后，大多数 蛋白质在90 nm直径的COPII包被的囊泡中运输，所述囊泡从ER出芽并被运输到高尔基体。 然而，单个粘蛋白单体的长度>500 nm，因此将组装的粘蛋白二聚体包装成囊泡 因为运输需要替代机制。本提案的目的是了解 粘蛋白从内质网到高尔基体的聚合和运输机制。本提案的目标1（K99）将 使用在假设的半胱氨酸中具有点突变的小鼠来测试它们在粘蛋白组装、ER 高尔基体的运输和分泌。在目标2（K99/R 00）中，非常规ER向高尔基体运输的作用 将使用敲低方法在人细胞中测定运输粘蛋白二聚体中的组分MIA 3 线和主要文化在健康和疾病。目标3（R 00）将确定MIA 3的功能， 在人类细胞中，通过询问其亚型、特异性结构域和结合伴侣将粘蛋白ER转运到高尔基体 线和主要文化..总的来说，这项工作有可能确定粘蛋白的新机制， 聚合和粘蛋白运输途径在健康和疾病。该提案利用了 申请人在粘蛋白生物学和囊泡运输方面的专业知识。额外的培训将有助于领导 候选人的目标是建立一个独立的实验室，研究粘蛋白生物学在健康和疾病。

PROJECT SUMMARY Each day, respiratory surfaces are exposed to billions of particles whose accumulation could be harmful if not rapidly eliminated by mucociliary clearance. In health, mucus is thin and easily transported. In muco- obstructive diseases such as asthma, mucus is abundant, thick, and poorly transported. Mucus dysfunction is not adequately treated with existing therapies. To develop more effective therapies, it is essential to improve our mechanistic understanding of mucin biology. Two polymeric mucins MUC5AC/Muc5ac and MUC5B/Muc5b are the major macromolecules in airway mucus. They are very large glycoproteins that form even larger multi- unit polymers. During synthesis, polymeric mucins assemble via conserved carboxy and amino terminal domains also found in von Willebrand factor (VWF). Published reports on VWF have identified three C-terminal cysteines required for inter-chain dimerization in the endoplasmic reticulum (ER) and then transit into the Golgi. These cysteines are also present in MUC5AC/Muc5ac and MUC5B/Muc5b. After assembly in the ER, most proteins traffic in 90 nm diameter COPII coated vesicles that bud from the ER and get transported to the Golgi. However, individual mucin monomers are >500 nm long, so packaging assembled mucin dimers into vesicles for transport requires alternatives mechanisms. The objective of this proposal is to understand the polymerization and trafficking mechanisms of mucin from the ER to the Golgi. Aim 1 of this proposal (K99) will employ mice with point mutations in hypothesized cysteines to test their requirements in mucin assembly, ER to Golgi transport and secretion. In Aim 2 (K99/R00), the role of a non-conventional ER to Golgi trafficking component, MIA3, in trafficking mucin dimers will be determined using knock down approaches in human cell lines and primary cultures in health and disease. Aim 3 (R00) will determine the functions of MIA3 required for mucin ER to Golgi transport by interrogating its isoforms, specific domains, and binding partners, in human cell lines and primary cultures.. Overall, this work has the potential to identify novel mechanisms of mucin polymerization and mucin trafficking pathways in health and disease. This proposal takes advantage of the applicant’s expertise in mucin biology and vesicle trafficking. The additional training will help lead the candidate’s goal of establishing an independent lab studying mucin biology in health and disease.