Unveiling the Proteostasis Network of Normal and Disease_Causing Collagen_I

 描述（由申请人提供）：常染色体显性成骨不全症 (OI) 通常是由 I 型胶原蛋白基因突变引起，导致骨质脆化和其他病理表型。严重的 OI 病理可能与畸形的、含有突变链的 I 型胶原蛋白三螺旋的分泌有关，或者与由于错误折叠的胶原蛋白链在细胞内积聚并最终导致细胞凋亡而产生的细胞应激有关。由于 I 型胶原蛋白分泌减少导致的单倍体不足也可能导致具有中度病理表型的 OI。 以细胞的蛋白质稳态（或蛋白质稳态）网络为目标来解决 I 型胶原蛋白折叠和质量控制方面的失败问题，有一天可能会带来一种新的成骨不全治疗范例。这种针对系统的治疗策略也可能对其他胶原蛋白病（例如埃勒斯-当洛斯综合征）有价值。然而，我们首先必须更多地了解细胞如何解决 I 型胶原蛋白折叠问题以及质量控制机制如何处理 I 型胶原蛋白错误折叠。在这里，我们部署基于定量质谱的蛋白质组学来识别负责（1）折叠和分泌野生型 I 型胶原蛋白链，（2）折叠和分泌导致 OI 的错误折叠胶原蛋白 -α1(I) Gly247Ser 和 Cys1299Trp 变体，以及（3）识别和处理错误折叠的 I 型胶原蛋白链的蛋白质稳态网络机制。 由于缺乏合适的 I 型胶原蛋白表达细胞模型系统，以前尚未对 I 型胶原蛋白进行过相互作用组学研究。最近，我们通过生成永生化纤维肉瘤细胞克服了这一关键障碍，这些细胞可诱导表达带有不同抗体表位标记的野生型和引起成骨不全的 I 型胶原蛋白。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及其相互作用的伙伴，从而首次使比较相互作用组学研究成为可能。 我们将根据多个参数仔细确定我们确定的 I 型胶原蛋白相互作用伙伴的优先顺序。我们将使用我们实验室已建立的 RNAi 耗竭和测定法来验证热门命中，以阐明这些相互作用的伙伴如何影响 I 型胶原蛋白稳态。我们最重要的发现最终将在通过 Coriell 细胞存储库从 OI 患者获得的突变匹配原代细胞系中得到验证。从长远来看，我们将把这些研究扩展到其他 I 型胶原蛋白变体，研究细胞解决 I 型胶原蛋白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，并建立新的策略，调整蛋白质稳态网络以增强 I 型胶原蛋白稳态和/或防止错误折叠的 I 型胶原蛋白三螺旋的分泌。

 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.