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 保持组织完整性，促进细胞迁移并组织信号分子。为了实现这些活动， 成熟的胶原蛋白从细胞中分泌为稳定的三聚体。每个胶原蛋白原始物由三个 域：C末端，非胶原（NC1）结构域，胶原蛋白结构域和N末端结构域（NTD）。 新合成的胶原蛋白会在内质网中经历独特的“ C-t-n”折叠途径 （ER），其中单个，C末端NC1结构域在胶原蛋白三重螺旋之前首先组装成三聚体 形成。在分泌之前，胶原蛋白还通过翻译后修饰（PTM）进行了大量修饰 成熟的胶原蛋白三聚体到细胞外空间。 胶原蛋白相关性疾病（CADS），例如成骨剂，Ehlers-Danlos综合征（EDS）和 Alport的综合征是相对常见的（1：5,000至1：10,000），最常见的是遗传疾病 胶原基因的突变。由于胶原蛋白在眼组织中扮演的关键作用，因此视力丧失是一个 CAD的共同特征。例如，眼部发病障碍是古尔德综合征的特征，这是一种罕见的遗传 与IV型胶原蛋白A1（COL4A1）和IV型胶原蛋白A2（COL4A2）中的突变相关的疾病。我们 提议稳定NC1与遗传（例如基因治疗）或化学（例如，分子胶）相互作用的建议 方法是治疗古尔德综合症的一种潜在的新方法。具体来说，我们设想了 COL4A1-COL4A2触点将通过驾驶部分克服错义或废话突变的影响 IV型胶原蛋白[A1A2A2（IV）]异三聚体的组装。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，其中保留在COL4A2-COL4A1接口处填充 已优化。确实，我们发现COL4A2S150W的表达增强了胶原蛋白的生物发生[A1A2A2（iv）] 基于定量的基于细胞的模型在7倍中。现在，我们被中毒为（特定目标1）筛查古尔德综合征 - 相关的突变以揭示哪些突变可以在基于细胞的和生化模型中部分校正。 然后，我们（特定的目标2）进行高通量化学筛选，这利用了我们创新的细胞 - 基于基于的测定和“稳定剂” Col4a2S150W工具，以识别促进胶原蛋白的药物样分子 [A1A2A2（IV）]生物发生。我们共同希望这些研究揭示了NC1亲和力与 古尔德综合征模型中的胶原蛋白分泌。我们还希望为新古尔德创造起点 综合征治疗，并为可能治疗其他CAD提供框架。