Unveiling the Proteostasis Network of Normal and Disease_Causing Collagen_I

 描述（由申请人提供）：常染色体显性遗传性成骨不全（OI）通常是由I型胶原基因突变引起的，该基因突变可导致脆骨和其他病理表型。严重的OI病理可能与分泌畸形的、含有突变链的胶原蛋白-I三螺旋或由于错误折叠胶原蛋白链在细胞内积累并最终引起细胞凋亡而引起的细胞应激有关。由于I型胶原分泌减少导致的单倍不足也可导致中度病理表型的OI。 靶向细胞的蛋白质稳态（或蛋白质稳态）网络，以解决胶原蛋白I折叠和质量控制的失败可能有一天会导致一个新的治疗模式的OI。这种系统靶向的治疗策略也可以证明对其他胶原蛋白病有价值，如Ehlers-Danlos综合征。然而，我们必须首先了解更多关于细胞如何解决胶原-I折叠问题以及质量控制机制如何处理错误折叠的胶原-I。在这里，我们部署定量质谱为基础的蛋白质组学，以确定蛋白质稳态网络机制负责（1）折叠和分泌野生型胶原蛋白-I链，（2）折叠和分泌OI引起的，错误折叠胶原蛋白-α1（I）Gly 247 Ser和Cys 1299 Trp变体，（3）识别和处置错误折叠胶原蛋白-I链。 由于缺乏合适的表达I型胶原的细胞模型系统，先前未使用I型胶原进行Interactomics研究。我们最近克服了这一关键的障碍，通过产生永生化纤维肉瘤细胞，诱导表达野生型和OI引起的胶原蛋白-I标记不同的抗体表位。我们现在可以从这些细胞中选择性地免疫沉淀野生型和错误折叠的胶原蛋白，沿着它们的相互作用伙伴，这使得比较相互作用组学研究首次成为可能。 我们将根据多个参数仔细确定I型胶原相互作用伙伴的优先级。将使用我们实验室中已经建立的RNAi消除和测定来验证热门命中，以阐明胶原蛋白-I稳态如何受到这些相互作用伴侣的影响。我们最重要的发现最终将在通过Coriell Cell Repository从OI患者获得的突变匹配的原代细胞系中得到验证。从长远来看，我们将这些研究扩展到其他胶原蛋白-I变体，研究细胞解决胶原蛋白-I折叠和错误折叠问题的分子机制，开发胶原蛋白折叠和分泌的高通量检测方法，并建立适应蛋白质稳态网络的新策略，以增强胶原蛋白-I稳态和/或防止错误折叠的胶原蛋白-I三螺旋的分泌。

 DESCRIPTION (provided by applicant): Autosomal dominant osteogenesis imperfecta (OI) is typically caused by mutations in collagen-I genes that engender brittle bones and other pathologic phenotypes. Severe OI pathology may be linked to the secretion of malformed, mutant strand-containing collagen-I triple helices or to cellular stress owing to misfolding collagen strands accumulating inside cells and ultimately causing apoptosis. Haploinsufficiency owing to reduced collagen-I secretion can also cause OI with moderate pathologic phenotypes. Targeting the cell's protein homeostasis (or proteostasis) network to resolve failures in collagen-I folding and quality control could one day lead to a new therapeutic paradigm for OI. Such a system-targeted therapeutic strategy could also prove valuable for other collagenopathies, such as Ehlers-Danlos Syndrome. However, we must first learn much more about how the cell solves the collagen-I folding problem and how the quality control machinery handles misfolding collagen-I. Here, we deploy quantitative mass spectrometry-based proteomics to identify the proteostasis network machinery responsible for (1) folding and secreting wild-type collagen-I strands, (2) folding and secreting the OI-causing, misfolding collagen-α1(I) Gly247Ser and Cys1299Trp variants, and (3) identifying and disposing of misfolding collagen-I strands. Interactomics studies have not been previously performed with collagen-I owing to the absence of a suitable collagen-I expressing cell model system. We recently overcame this critical roadblock by generating immortalized fibrosarcoma cells that inducibly express wild-type and OI-causing collagen-I tagged with distinct antibody epitopes. We can now selectively immunoprecipitate wild-type and misfolding collagens, along with their interacting partners, from these cells, making comparative interactomics studies possible for the first time. We shall carefully prioritize collagen-I interacting partners we identify on the basis of multiple parameter. Top hits will be validated using RNAi depletion and assays already established in our lab to elucidate how collagen-I homeostasis is influenced by those interacting partners. Our most important findings will eventually be validated in mutation-matched primary cell lines obtained from OI patients via the Coriell Cell Repository. In the longer term, we will extend these studies to other collagen-I variants, study the molecular mechanisms by which the cell solves the collagen-I folding and misfolding problem, develop high throughput assays for collagen folding and secretion, and establish new strategies that adapt the proteostasis network to enhance collagen-I homeostasis and/or prevent the secretion of misfolded collagen-I triple helices.