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.

项目概要 每天，呼吸表面都会暴露于数十亿颗粒，如果不进行这些颗粒的积累，可能会造成危害。 通过粘液纤毛清除迅速消除。健康时，粘液稀薄且易于运输。在粘液中- 哮喘等阻塞性疾病时，粘液丰富、粘稠，且输送不良。粘液功能障碍是 现有疗法未得到充分治疗。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。两种聚合粘蛋白 MUC5AC/Muc5ac 和 MUC5B/Muc5b 是气道粘液中的主要大分子。它们是非常大的糖蛋白，可以形成更大的多糖蛋白 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 血管性血友病因子 (VWF) 中也发现了结构域。已发表的有关 VWF 的报告已确定了三个 C 端 内质网 (ER) 中链间二聚化所需的半胱氨酸，然后转运至高尔基体。 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 包被的囊泡中运输，这些囊泡从内质网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，因此将组装好的粘蛋白二聚体包装到囊泡中 运输需要替代机制。该提案的目的是了解 粘蛋白从内质网到高尔基体的聚合和运输机制。该提案的目标 1 (K99) 将 使用假设的半胱氨酸发生点突变的小鼠来测试它们对粘蛋白组装、ER 的需求 高尔基体的运输和分泌。在目标 2 (K99/R00) 中，非常规 ER 对高尔基体贩运的作用 运输粘蛋白二聚体中的成分 MIA3 将使用人类细胞中的敲除方法来确定 健康和疾病的品系和原代培养物。目标 3 (R00) 将确定 MIA3 所需的功能 通过询问人类细胞中的同种型、特定结构域和结合伴侣，将粘蛋白 ER 转运至高尔基体 总体而言，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。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，研究健康和疾病中的粘蛋白生物学。