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.

项目摘要/摘要 胶原是细胞外基质和地下膜的重要组成部分，它们用于 增强组织完整性，介导细胞迁移并组织信号传导。胶原蛋白具有三个域： C末端三聚结构域（CTD/NC1），胶原蛋白结构域和7S N末端结构域。胶原蛋白 在分泌为 细胞外环境。 NC1结构域的修剪化是胶原蛋白折叠的第一步，需要 用于形成胶原蛋白三重螺旋。胶原蛋白的突变会导致严重疾病，例如成骨 Imperfecta，Alport的疾病和脑小血管疾病会损害骨骼的完整性，肾脏的完整性 和大脑和眼睛中的血管。尽管胶原蛋白中许多引起疾病的突变 已经鉴定出来，仍然对发病机理的机制仍然存在不完全的理解。目标 该建议的是了解最初的修剪化如何影响成熟和 胶原蛋白IV的生物合成。在我的第一个目的中，我将开发新的基于细胞的方法来量化NC1的效果 胶原蛋白组件上的域扰动。具体来说，我开发了一种分裂的荧光素酶测定法，以测量 NC1结构域的相互作用分离，并已开始量化致病性突变的作用。此外， 我将开发出表达全长胶原蛋白IV的完整细胞系，包括最有趣的突变体 在分裂荧光素酶测定中确定，以更好地了解这些扰动的功能后果。 在第二个目标中，我在NC1接口上设计了点突变，并确定了那些增加或 触发稳定性降低。令人兴奋的是，其中一个突变大大增加了NC1关联和 分泌。这些研究将共同​​揭示与胶原蛋白相关疾病的机制，并提出方法。 绕过它们。主要的创新是应用尖端计算方法的应用 设计点突变，以探测有关胶原蛋白组装和稳定性的特定问题。我们期望在一起 这些研究提出了治疗胶原蛋白相关疾病的新途径。最后，这些 实验是一个令人兴奋的培训机会，为我提供了研究经验的问题 纪律，翻译研究。