Chemical Biology Approaches to Studying Collagen IV Stability

研究胶原蛋白 IV 稳定性的化学生物学方法

• 批准号: 10723042
• 负责人: Jason E Gestwicki
• 金额: $ 43.57万
• 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: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10723042
• 关键词: Affinity; Anabolism; Animals; Automobile Driving; Basement membrane; Binding; Biochemical; Biogenesis; Biological Assay; Biology; Blindness; C-terminal; Cartilage; Cataract; Cell Line; Cell secretion; Cells; Characteristics; Chemicals; Collagen; Collagen Gene; Collagen Type IV; Corneal dystrophy; Defect; Diagnosis; Disease; Drug Combinations; Ehlers-Danlos Syndrome; Endoplasmic Reticulum; Extracellular Matrix; Extracellular Space; Family; Future; Genetic; Genetic Diseases; Glaucoma; Glues; Goals; Gould Syndrome; Hearing; Hereditary nephritis; Individual; Kidney; Laboratories; Length; Methods; Microphthalmos; Missense Mutation; Modeling; Molecular; Muscle Weakness; Mutagenesis; Mutation; N-terminal; Nonsense Mutation; Organ; Osteogenesis Imperfecta; Pathway interactions; Penetrance; Persons; Pharmaceutical Preparations; Phenotype; Physiologic Intraocular Pressure; Play; Post-Translational Protein Processing; Process; Protein Engineering; Proteins; Protomer; Recombinants; Role; Signaling Molecule; Site; Skin; Symptoms; System; Technology; Testing; Tissues; Variant; Work; autosome; biochemical model; bone; cell motility; design; drug discovery; drug-like compound; experience; eye dryness; gene therapy; high throughput screening; improved; innovation; mechanical properties; mouse model; preclinical study; rare genetic disorder; self assembly; skeletal; small molecule; tool; triple helix

PROJECT SUMMARY/ABSTRACT Collagens are essential components of the extra-cellular matrix and basement membranes, where they serve to maintain tissue integrity, facilitate cell migration, and organize signaling molecules. To achieve these activities, mature collagens are secreted from cells as stable trimers. Each collagen protomer is composed of three domains: a C-terminal, non-collagenous (NC1) domain, a collagen domain and an N-terminal domain (NTD). Newly synthesized collagen proteins undergo a unique “C-to-N” folding pathway in the endoplasmic reticulum (ER), in which individual, C-terminal NC1 domains first assemble into trimers prior to collagen triple helix formation. Collagens are also heavily modified by post-translational modifications (PTMs) prior to secretion of mature collagen trimers to the extracellular space. Collagen-associated disorders (CADs), such as Osteogenesis Imperfecta, Ehlers-Danlos syndrome (EDS) and Alport’s syndrome, are relatively common (1:5,000 to 1:10,000) genetic diseases that most commonly arise from mutations in collagen genes. Because of the key roles played by collagens in ocular tissues, vision loss is a common feature of CADs. For example, ocular dysgenesis is a feature of Gould Syndrome, a rare genetic disorder associated with mutations in type IV collagen a1 (COL4A1) and type IV collagen a2 (COL4A2). We propose that stabilizing NC1 interactions with genetic (e.g., gene therapy) or chemical (e.g., molecular glues) approaches is a potential, new way to treat Gould Syndrome. Specifically, we envision that stabilization of COL4A1-COL4A2 contacts will partially overcome the impact of missense or nonsense mutations by driving assembly of type IV collagen [a1a2a2(IV)] heterotrimers. To explore this idea, we used computational protein design to create a “stabilizer” variant, COL4A2S150W, in which residue packing at the COL4A2-COL4A1 interface is optimized. Indeed, we found that expression of COL4A2S150W enhanced biogenesis of collagen [a1a2a2(IV)] by 7-fold in a quantitative, cell-based model. Now, we are poised to (Specific Aim 1) screen Gould Syndrome- associated mutations to reveal which ones can be partially corrected in cell-based and biochemical models. Then, we will (Specific Aim 2) conduct high throughput chemical screens, which leverage our innovative, cell- based assays and “stabilizer” COL4A2S150W tool, to identify drug-like molecules that promote collagen [a1a2a2(IV)] biogenesis. Together, we expect these studies to reveal the relationship between NC1 affinity and collagen secretion in models of Gould Syndrome. We also hope to produce starting points for new Gould Syndrome treatments and provide a framework for potentially treating other CADs.

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 维持组织完整性、促进细胞迁移并组织信号分子以实现这些活动， 成熟的胶原蛋白作为稳定的三聚体从细胞中分泌出来，每个胶原蛋白原聚体由三个组成。 结构域：C 端非胶原 (NC1) 结构域、胶原蛋白结构域和 N 端结构域 (NTD)。 新合成的胶原蛋白在内质网中经历独特的“C-N”折叠途径 (ER)，其中各个 C 端 NC1 结构域首先在胶原三螺旋之前组装成三聚体 胶原蛋白在分泌之前也会通过翻译后修饰 (PTM) 进行大量修饰。 成熟的胶原三聚体进入细胞外空间。 胶原相关疾病 (CAD)，例如成骨不全症、埃勒斯-当洛斯综合征 (EDS) 和 奥尔波特综合征是相对常见的（1:5,000 至 1:10,000）遗传性疾病，最常见于 由于胶原蛋白在眼组织中发挥着关键作用，因此视力丧失是一个原因。 例如，眼部发育不全是古尔德综合症的一个特征，这是一种罕见的遗传病。 与 IV 型胶原蛋白 a1 (COL4A1) 和 IV 型胶原蛋白 a2 (COL4A2) 突变相关的疾病。 提出稳定 NC1 与遗传（例如基因治疗）或化学（例如分子胶）的相互作用 方法是治疗古尔德综合症的一种潜在的新方法，具体来说，我们设想这种方法可以稳定病情。 COL4A1-COL4A2接触将通过驱动部分克服错义或无义突变的影响 IV 型胶原 [a1a2a2(IV)] 异源三聚体的组装为了探索这个想法，我们使用了计算蛋白。 设计创建“稳定器”变体 COL4A2S150W，其中残留物堆积在 COL4A2-COL4A1 界面上 事实上，我们发现 COL4A2S150W 的表达增强了胶原蛋白的生物合成 [a1a2a2(IV)]。 现在，我们准备（具体目标 1）筛查古尔德综合症 - 相关突变揭示哪些突变可以在基于细胞和生化模型中部分纠正。 然后，我们将（具体目标 2）进行高通量化学筛选，利用我们的创新、细胞- 基于测定和“稳定剂”COL4A2S150W 工具，识别促进胶原蛋白的药物样分子 我们期望这些研究能够共同揭示 NC1 亲和力与 [a1a2a2(IV)] 生物发生之间的关系。 古尔德综合症模型中的胶原蛋白分泌我们也希望为新的古尔德综合症提供起点。 综合征治疗并为潜在治疗其他 CAD 提供框架。