Chondronoids for Studying Collagen-II Homeostasis and Diseases

项目摘要/摘要 II型胶原病是一种由II型胶原蛋白突变引起的疾病，最常见的 软骨中含有丰富的蛋白质。这些疾病表现出影响关节结构的各种症状， 软骨特性和骨骼发育。目前，他们还没有治愈方法。患者依赖姑息疗法 当他们的症状出现时，他们会接受治疗。两个例子是由基因突变引起的软骨发育不良 COL2A1导致II型胶原中的G1170S或R719C替换。在场的患者有一台 或更多下列症状：性早熟骨关节炎，股骨头缺血性坏死， 和莱格-卡尔韦-珀斯病。以前的细胞和小鼠模型阻碍了对这些的研究 由于对人类表型的概括不佳而引起的疾病，以及对 需要分子生物学和生化分析来了解疾病的分子起源。 在缺乏探索II型胶原错误折叠对细胞和组织影响的模型的情况下， 在确定治疗靶点和新的治疗选择方面的进展仍然缓慢。 这些挑战可以通过使用一组诱导的多潜能干细胞(IPSC)来克服 能够分化为软骨产生软骨细胞的细胞系以阐明潜在的 胶原蛋白的产生和错误折叠缺陷。利用之前生成的经过基因编辑的面板， 携带G1170S和R719C II型胶原突变和野生型的基因匹配的IPSCs 作为对照，这种疾病的基础可以在体外以前所未有的分辨率进行检查。这 高度相关的系统将有助于阐明致病胶原蛋白的生化表现- II组织、细胞和蛋白质水平的突变。多亏了麻省理工学院杰出的设施和专家， 培训将包括各种广泛有用的技术，如蛋白质组学、RNA测序、 干细胞培养和类器官生成，以检查诸如基质组成和 结构、胶原蛋白的稳定性和应激通路的激活。在分子水平上，最先进的 质谱学和生化分析将使研究所需的相互作用 野生型和易错折叠的II型胶原蛋白组装、折叠和分泌。此方法基于 一个高度相关的模型将阐明野生型蛋白是如何折叠的，同时还将探索特定的 通过利用假说驱动的直接研究突变如何 胶原蛋白-II错误折叠并偏离正常的蛋白平衡途径。总的来说，这项研究是 有望对我们理解II型胶原在健康中的动态平衡做出相当大的贡献 并为寻找解决胶原蛋白的新治疗靶点奠定了坚实的基础 这一错误折叠的问题，以及潜在的许多其他相关的胶原病。

PROJECT SUMMARY/ABSTRACT Type II collagenopathies are a class of disease caused by mutations in collagen-II, the most abundant protein in cartilage. These diseases present with various symptoms affecting joint structure, cartilage properties, and skeletal development. Currently, they have no cure. Patients rely on palliative treatments as their symptoms arise. Two examples are the chondrodysplasias caused by mutations in COL2A1 leading to either the G1170S or R719C substitutions in collagen-II. Patients present with one or more of the following symptoms: precocious osteoarthritis, avascular necrosis of the femoral head, and Legg-Calvé-Perthes disease. Previous cell and mouse models hindered research on these diseases owing to poor recapitulation of the human phenotype, and poor adaptability for the necessary molecular biology and biochemical assays required to understand molecular origins of the disorders. In the absence of amenable models to probe the effect of collagen-II misfolding on cells and tissues, progress on identifying therapeutic targets and novel treatment options remains slow. These challenges can be overcome by using a panel of induced pluripotent stem cell (iPSC) lines capable of being differentiated into cartilage-producing chondrocytes to elucidate the underlying collagen production and misfolding defects. Harnessing a panel of previously generated gene-edited, genetically matched iPSCs harboring the G1170S and R719C collagen-II mutations and wild-type control, the underpinnings of the disease can be examined in vitro with unprecedented resolution. This highly pertinent system will help illuminate the biochemical manifestations of disease-causing collagen- II mutations at tissue, cellular, and protein levels. Thanks to outstanding facilities and experts at MIT, training will include a wide variety of broadly useful techniques such as proteomics, RNA-sequencing, stem cell culture, and organoid generation to examine characteristics such as matrix composition and structure, collagen stability, and activation of stress pathways. At the molecular-level, state-of-the-art mass spectrometry and biochemical assays will enable investigation of the interactions required for wild-type and misfolding-prone collagen-II to assemble, fold, and be secreted. This approach based in a highly relevant model will elucidate how the wild-type protein folds, while also probing the specific molecular causes of pathology by harnessing a hypothesis-driven, direct investigation of how mutant collagen-II misfolds and diverges from the normal proteostasis pathways. Overall, this research is expected to make a considerable contribution to our understanding of collagen-II homeostasis in health and disease, and to lay a strong foundation for identifying novel therapeutic targets to solve the collagen misfolding problem in this and potentially the many other related collagenopathies.