Mucin Biosynthesis and Transport Mechanisms in Respiratory Health and Disease

呼吸系统健康和疾病中的粘蛋白生物合成和运输机制

• 批准号: 10664598
• 负责人: Ana Maria Jaramillo Hernandez
• 金额: $ 10.97万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10664598
• 关键词: Affect; Allergic; Anabolism; Asthma; Binding; Biology; Breathing; COPII-Coated Vesicles; Cell Line; Cells; Collagen; Cysteine; Cytoplasmic Tail; Data; Diameter; Dimerization; Disease; Disulfides; Drosophila genus; Economics; Endoplasmic Reticulum; Exposure to; Fibrinogen; Foundations; Functional disorder; Glycoproteins; Goals; Golgi Apparatus; Health; Health Care Costs; Homeostasis; Homologous Gene; Human; Human Cell Line; Impairment; In Vitro; Individual; Inflammatory; Intervention; Knock-in Mouse; Length; Link; Lung; MUC5AC gene; MUC5B gene; Mediating; Membrane; Molecular; Molecular Chaperones; Mucins; Mucociliary Clearance; Mucous body substance; Mus; Pathologic; Pathway interactions; Personal Satisfaction; Point Mutation; Polymers; Procollagen; Productivity; Protein Isoforms; Proteins; Publishing; Reporting; Research; Research Personnel; Respiratory Disease; Risk; Role; SH3 Domains; Site; Surface; Testing; Thick; Thinness; Training; Translations; Vesicle; Work; airway obstruction; asthma model; dimer; disulfide bond; effective therapy; improved; knock-down; lead candidate; macromolecule; monomer; mouse model; novel; particle; polymerization; protein complex; protein folding; public health relevance; respiratory; respiratory health; salivary mucins; secretory protein; trafficking; vesicle transport; von Willebrand Factor

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.

项目摘要 每天，呼吸表面暴露于数十亿个颗粒，如果不积累，它们的积累可能是有害的 在健康中，粘液很薄且容易运输。在粘液中 哮喘，粘液等阻塞性疾病丰富，浓密且运输良好。粘液功能障碍是 现有疗法没有充分治疗。要开发更有效的疗法，必须改善 我们对粘蛋白生物学的机械理解。两个聚合物粘蛋白MUC5AC/MUC5AC和MUC5B/MUC5B 它们是气道粘液中的主要大分子。它们是非常大的糖蛋白，形成更大的多糖蛋白 单位聚合物。在合成过程中，聚合物粘蛋白通过配置的羧基和氨基终端组装 在von Willebrand因子（VWF）中也发现了域。关于VWF的已发表报告已经确定了三个C末端 内质网中（ER）中链二聚化所需的半胱氨酸，然后转移到高尔基体中。 这些半胱氨酸也存在于MUC5AC/MUC5AC和MUC5B/MUC5B中。在ER中组装后，大多数 蛋白质直径为90 nm的copii涂层蔬菜，这些蔬菜从急诊室芽并运输到高尔基体。 但是，单个粘蛋白单体长度> 500 nm，因此包装成粘蛋白二聚体成蔬菜 对于运输需要替代机制。该提议的目的是了解 从ER到高尔基体的聚合和运输机制。本提案的目标1（K99）将 在假设半胱氨酸中具有点突变的员工小鼠，以测试其在粘蛋白组装中的需求，ER 向高尔基运输和分泌。在AIM 2（K99/R00）中，非常规ER对高尔基人贩运的作用 分量MIA3在运输粘蛋白二聚体中将使用人类细胞中的敲除方法确定 健康和疾病的线条和主要文化。 AIM 3（R00）将确定MIA3的功能 通过询问其同工型，特定领域和结合伙伴，在人类细胞中粘膜转运的粘蛋白传输 线条和初级文化。总的来说，这项工作有可能识别粘蛋白的新机制 健康和疾病中的聚合和粘蛋白运输途径。该建议利用了 申请人在粘蛋白生物学和囊泡贩运方面的专业知识。额外的培训将有助于领导 候选人的目标是建立一个独立研究健康和疾病中粘蛋白生物学的实验室。