Defining the role of NC1 domain assembly in collagen biosynthesis and collagen associated disorders

定义 NC1 结构域组装在胶原蛋白生物合成和胶原蛋白相关疾病中的作用

• 批准号: 10679523
• 负责人: Zachary Gale-Day
• 金额: $ 4.05万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-03 至 2025-04-02
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10679523
• 关键词: Affinity; Anabolism; Basement membrane; Biochemical; Biological Assay; Biology; Biotechnology; C-terminal; Categories; Cell Line; Cells; Cerebral small vessel disease; Cerebrovascular system; Chemicals; Clinical; Collagen; Collagen Diseases; Collagen Type IV; Collection; Defect; Disease; Environment; Extracellular Matrix; Extracellular Space; Eye; Foundations; Future; Genetic; Goals; Homo; Impairment; In Vitro; Kidney; Knowledge; Length; Luciferases; Measures; Mediating; Mentors; Methods; Missense Mutation; Modeling; Mutation; N-terminal; Osteogenesis Imperfecta; Pathogenesis; Pathogenicity; Pathway interactions; Phenotype; Point Mutation; Protein Biochemistry; Protein Engineering; Proteins; Protomer; Reporting; Research; Role; Signal Transduction; Structure; Testing; Therapeutic; Tissues; Work; bone; cell motility; design; disease-causing mutation; experience; experimental study; extracellular; innovation; mechanical properties; mutant; protein protein interaction; skills; tool; training opportunity; translational study; triple helix; white matter

PROJECT SUMMARY/ABSTRACT Collagens are essential components of the extra-cellular matrix and basement membranes, where they serve to bolster tissue integrity, mediate cell migration, and organize signaling. Collagen proteins have three domains: the C-terminal trimerization domain (CTD/NC1), the collagen domain and the 7S N-terminal domain. Collagens undergo folding followed by trimerization and collagen triple helix formation in the ER prior to secretion to the extra-cellular environment. Trimerization of the NC1 domain is the first step in collagen folding and is required for formation of the collagen triple helix. Mutations in collagens result in severe diseases, such as Osteogenesis Imperfecta, Alport’s Disease and Cerebral Small Vessel Disease which impair the integrity of bones, kidneys and blood vessels in the brain and eye respectively. Although many disease-causing mutations in collagens have been identified, there still is an incomplete understanding of the mechanisms of pathogenesis. The goal of this proposal is to understand how perturbing the initial trimerization effects the maturation and biosynthesis of collagen IV. In my first aim, I will develop new cell-based assays to quantify the effect of NC1 domain perturbation on collagen assembly. Specifically, I have developed a split luciferase assay that measures NC1 domain interactions in isolation and have begun to quantify the effect of pathogenic mutations. Additionally, I will develop complementary cell lines that express full-length collagen IV, including the most interesting mutants identified in the split luciferase assays, to better understand the functional consequences of these perturbations. In my second aim, I have designed point mutations at the NC1 interface, and identified those that increase or decrease trimer stability. Excitingly, one of these mutations significantly increased NC1 association and secretion. Together, these studies will reveal the mechanisms of collagen-associated disease and suggest ways of circumventing them. The major innovation is the application of cutting-edge computational approaches to design point mutations that probe specific questions about collagen assembly and stability. Together, we expect these studies to suggest new avenues for the treatment of collagen associated disorders. Finally, these experiments serve as an exciting training opportunity, providing me with a mentored research experience in inter- disciplinary, translational studies.

项目概要/摘要 胶原蛋白是细胞外基质和基底膜的重要组成部分，它们的作用是 增强组织完整性、介导细胞迁移和组织信号传导。胶原蛋白具有三个结构域： C 端三聚化结构域 (CTD/NC1)、胶原蛋白结构域和 7S N 端胶原蛋白结构域。 在内质网中进行折叠，然后三聚化和胶原三螺旋形成，然后分泌到 NC1 结构域的三聚化是胶原蛋白折叠的第一步，也是必需的。 胶原蛋白三螺旋的形成会导致严重的疾病，例如成骨。 损害骨骼、肾脏完整性的不完美症、奥尔波特病和脑小血管病 以及大脑和眼睛的血管，虽然胶原蛋白有许多致病突变。 已确定，但对发病机制的了解仍不完全。 该提案的目的是了解扰动初始三聚化如何影响成熟和 IV 型胶原蛋白的生物合成 在我的第一个目标中，我将开发新的基于细胞的检测方法来量化 NC1 的效果。 具体来说，我开发了一种测量裂解荧光素酶测定法。 NC1 结构域的相互作用是孤立的，并且已经开始量化致病突变的影响。 我将开发表达全长 IV 型胶原蛋白的互补细胞系，包括最有趣的突变体 在分裂荧光素酶测定中鉴定，以更好地了解这些扰动的功能后果。 在我的第二个目标中，我在 NC1 界面设计了点突变，并鉴定了那些增加或减少的点突变。 令人兴奋的是，其中一种突变显着增加了 NC1 关联性并降低了三聚体稳定性。 这些研究将共同​​揭示胶原蛋白相关疾病的机制并提出方法。 规避它们的主要创新是尖端计算方法的应用。 我们期望设计点突变来探讨有关胶原蛋白组装和稳定性的具体问题。 这些研究为治疗胶原蛋白相关疾病提出了新的途径。 实验是一个令人兴奋的培训机会，为我提供了在跨领域的指导性研究经验。 学科、转化研究。