Unveiling the Proteostasis Network of Normal and Disease_Causing Collagen_I

揭示正常和疾病的蛋白质稳态网络_Causing Collagen_I

• 批准号: 8973926
• 负责人: Matthew Donald Shoulders
• 金额: $ 7.8万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8973926
• 关键词: Address; Amyloidosis; Antibodies; Apoptosis; Biogenesis; Biological Assay; Biological Models; Cell Line; Cell model; Cells; Cellular Stress; Collagen; Collagen Type I; Data; Defect; Disease; Ehlers-Danlos Syndrome; Ensure; Epitopes; Extracellular Matrix Proteins; Face; Failure; Foundations; Future; Genes; Goals; Grant; Homeostasis; Huntington Disease; Lead; Learning; Link; Literature; Mass Spectrum Analysis; Methods; Modeling; Molecular; Molecular Chaperones; Mutation; Osteogenesis Imperfecta; Outcome; Pathologic; Pathology; Patients; Phenotype; Play; Prealbumin; Production; Proteins; Proteomics; Quality Control; RNA Interference; Research; Role; Stem cells; Structure; System; Testing; Therapeutic; Time; Validation; Variant; Work; base; bone; bone turnover; comparative; effective therapy; fibrillogenesis; fibrosarcoma; gene therapy; high throughput screening; improved; insight; killings; mutant; new therapeutic target; novel therapeutics; prevent; protein misfolding; protein protein interaction; protein transport; public health relevance; repository; skeletal; therapeutic target; trafficking; triple helix

 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.

 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，这种突变会导致脆性骨骼和其他病理表型。严重的OI病理可能与分泌畸形的、含有突变的I型胶原链的三螺旋有关，也可能与细胞应激有关，因为错误折叠的胶原链在细胞内堆积，最终导致细胞凋亡。由于I型胶原分泌减少而导致的单倍性功能不全也可导致中度病理表型的OI。以细胞的蛋白质稳态(或蛋白质稳态)网络为靶点来解决I型胶原折叠和质量控制的故障，有朝一日可能会导致OI的新治疗范例。这种系统靶向的治疗策略也可能被证明对其他胶原病有价值，例如埃勒斯-丹洛斯综合征。然而，我们首先必须更多地了解细胞如何解决I型胶原折叠问题，以及质量控制机制如何处理I型胶原的错误折叠。在这里，我们采用基于定量质谱学的蛋白质组学来确定负责(1)折叠和分泌野生型胶原-I链，(2)折叠和分泌导致OI的错误折叠的胶原-α1(I)Gly247Ser和Cys1299Trp变体的蛋白质平衡网络机制，以及(3)识别和处理错误折叠的I型胶原链。由于缺乏合适的表达I型胶原的细胞模型系统，以前还没有对I型胶原进行交互作用的研究。我们最近克服了这一关键障碍，通过产生永生化的纤维肉瘤细胞，诱导表达野生型和引起OI的胶原蛋白-I，并标记了不同的抗体表位。我们现在可以有选择地从这些细胞中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，使比较相互作用研究第一次成为可能。我们将在多个参数的基础上仔细确定I型胶原相互作用伙伴的优先顺序。最高的命中率将通过RNAi耗尽和我们实验室已经建立的分析来验证，以阐明那些相互作用的伙伴如何影响I型胶原的动态平衡。我们最重要的发现最终将在通过科里尔细胞库从OI患者那里获得的突变匹配的原代细胞系中得到验证。从长远来看，我们将把这些研究扩展到其他I型胶原变体，研究细胞解决I型胶原折叠和错误折叠问题的分子机制，开发高通量的胶原折叠和分泌方法，并建立新的策略，使蛋白质平衡网络适应于增强I型胶原的动态平衡和/或防止I型胶原错折叠的三螺旋的分泌。