Chemical Biology Approaches to Studying Collagen IV Stability

项目摘要/摘要 胶原蛋白是细胞外基质和基底膜的基本成分，在那里它们起到 维持组织完整性，促进细胞迁移，并组织信号分子。为了实现这些活动， 成熟的胶原蛋白以稳定的三聚体的形式从细胞中分泌出来。每个胶原蛋白原由三个 结构域：C-末端非胶原区(NC1)、胶原区和N-末端结构域(NTD)。 新合成的胶原蛋白在内质网上经历独特的“C-to-N”折叠途径 (Er)，其中单个的C-末端NC1结构域首先组装成三聚体，然后再组装成胶原三螺旋 队形。胶原蛋白在分泌之前也被翻译后修饰(PTM)严重修饰 成熟的胶原蛋白三聚体进入细胞外空间。 胶原相关疾病(CADS)，如成骨不全、Ehler-Danlos综合征(EDS)和 阿尔波特综合征是相对常见的(1：5,000至1：10,000)遗传性疾病，最常见的原因是 胶原蛋白基因的突变。由于胶原蛋白在眼组织中扮演的关键角色，视力丧失是一种 CADS的共同特征。例如，眼睛发育不全是古尔德综合征的一个特征，这是一种罕见的遗传性疾病 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。我们 建议稳定NC1与遗传(如基因治疗)或化学(如分子胶)的相互作用 方法是治疗古尔德综合征的一种潜在的新方法。具体地说，我们设想稳定 COL4A1-COL4A2接触将部分克服错义或无义突变的影响 IV型胶原[a1a2a2(IV)]杂三聚体的组装。为了探索这个想法，我们使用了计算蛋白质 设计以创建一种“稳定剂”变体COL4A2S150W，其中残留物堆积在COL4A2-COL4A1界面 是经过优化的。事实上，我们发现COL4A2S150W的表达促进了胶原的生物生成[a1a2a2(IV)]。 在基于细胞的定量模型中增加7倍。现在，我们准备(具体目标1)筛查古尔德综合症- 相关突变，以揭示哪些突变可以在基于细胞的和生物化学模型中部分纠正。 然后，我们将(特定目标2)进行高通量化学筛选，这利用了我们创新的细胞- 基于分析和稳定剂COL4A2S150W工具，以识别促进胶原蛋白的类药物分子 [a1a2a2(IV)]生物发生。总之，我们希望这些研究能够揭示NC1亲和力和 古尔德综合征模型中的胶原分泌。我们也希望为新的古尔德创造起点 并为其他冠心病的潜在治疗提供了一个框架。

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.