Chondronoids for Studying Collagen-II Homeostasis and Diseases

项目总结/摘要 II型胶原蛋白病是一类由II型胶原蛋白突变引起的疾病，其中最常见的是II型胶原蛋白突变。 软骨中含有丰富的蛋白质。这些疾病表现为影响关节结构的各种症状， 软骨特性和骨骼发育。目前，他们没有治愈。患者依赖姑息疗法 随着症状的出现而治疗。两个例子是由突变引起的软骨发育不良， COL 2A 1导致II型胶原中G1170 S或R719 C置换。患者出现1例 以下症状中的一种或多种：早发性骨关节炎，股骨头缺血性坏死， 和Legg-Calvé-Perthes病以前的细胞和小鼠模型阻碍了这些研究 疾病由于人类表型的重演性差，以及对必要的适应性差， 分子生物学和生物化学分析，以了解疾病的分子起源。 在缺乏适合的模型来探测胶原蛋白II错误折叠对细胞和组织的影响的情况下， 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多能干细胞（iPSC）来克服。 能够分化成软骨产生软骨细胞的细胞系，以阐明 胶原蛋白产生和错误折叠缺陷。利用一组先前生成的基因编辑， 携带G1170 S和R719 C胶原蛋白-II突变的基因匹配的iPSC和野生型 控制，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引起疾病的胶原蛋白的生化表现- 在组织、细胞和蛋白质水平的II突变。感谢麻省理工学院出色的设施和专家， 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、胶原稳定性和应激途径的激活。在分子水平上， 质谱和生物化学分析将能够调查所需的相互作用， 野生型和易错误折叠的II型胶原蛋白组装、折叠和分泌。这种方法基于 一个高度相关的模型将阐明野生型蛋白质如何折叠，同时也探测特定的 病理学的分子原因，利用假设驱动，直接调查突变如何 胶原-II错误折叠并偏离正常的蛋白质稳态途径。总的来说，这项研究是 预计将作出相当大的贡献，我们的理解胶原蛋白-II稳态在健康 和疾病，并为确定新的治疗靶点奠定坚实的基础，以解决胶原蛋白 在这方面的错误折叠问题和潜在的许多其他相关的胶原蛋白病。

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.