Mucin Biosynthesis and Transport Mechanisms in Respiratory Health and Disease

项目总结 每天，呼吸道表面暴露在数十亿个颗粒中，如果不积累，这些颗粒可能是有害的。 通过粘液纤毛清除迅速消除。在健康方面，粘液很薄，很容易运输。在粘液中- 阻塞性疾病，如哮喘，粘液丰富、稠密，运输不便。粘液功能障碍 没有用现有的疗法进行充分的治疗。为了开发更有效的治疗方法，必须改进 我们对粘蛋白生物学的机械理解。两种聚合物粘蛋白MUC5AC/Muc5Ac和MUC5B/MUC5B 是呼吸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，聚合物粘蛋白通过保守的羧基和氨基末端组装。 在von Willebrand因子(VWF)中也发现了结构域。已发表的关于VWF的报告确定了三个C-末端 内质网(ER)中链间二聚所需的半胱氨酸，然后进入高尔基体。 这些半胱氨酸也存在于MUC5AC/Muc5ac和MUC5B/MUC5B中。在急诊室组装后，大多数 蛋白质在直径90 nm的COPII包被囊泡中运输，这些囊泡从内质网萌发并被运输到高尔基体。 然而，单个粘蛋白单体的长度为500纳米，因此包装将粘蛋白二聚体组装成小泡 因为运输需要替代机制。这项建议的目的是了解 粘蛋白从内质网到高尔基体的聚合和运输机制。本建议书的目标1(K99)将 利用假设半胱氨酸点突变的小鼠来测试它们对粘蛋白组装的需求，ER 促进高尔基体的运输和分泌。在目标2(K99/R00)中，非常规ER对高尔基人贩运的作用 人类细胞中运输粘蛋白二聚体的成分MIA3将通过击倒方法确定 健康和疾病方面的线条和原代培养。目标3(R00)将确定MIA3所需的功能 粘蛋白ER通过询问其异构体、特定结构域和结合伙伴在人类细胞中向高尔基体转运 线条和原始文化..总的来说，这项工作有可能确定粘蛋白的新机制 健康和疾病中的聚合和粘蛋白运输途径。这项建议充分利用了 申请人在粘蛋白生物学和水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，研究健康和疾病中的粘蛋白生物学。

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.