A Novel Approach to Osteogenesis Imperfecta_ The Collagen Protein Folding Problem

成骨不全症的新方法_胶原蛋白折叠问题

• 批准号: 9232077
• 负责人: Andrew Stephen DiChiara
• 金额: $ 4.05万
• 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: 2015-03-04 至 2018-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9232077
• 关键词: Address; Alleles; Alpha Cell; Amino Acids; Antibodies; Architecture; Area; Autophagosome; Biochemical; Biological Assay; Biological Models; Bone structure; Cell Culture Techniques; Cell Line; Cell model; Cells; Collagen; Collagen Gene; Collagen Type I; Data; Defect; Deformity; Disease; Endoplasmic Reticulum; Enzymes; Epitopes; Exhibits; Failure; Fracture; Genes; Goals; Hereditary Disease; Homeostasis; Human; Individual; Investigation; Lead; Learning; Length; Mass Spectrum Analysis; Methods; Missense Mutation; Molecular; Molecular Biology; Molecular Chaperones; Mutate; Mutation; Osteogenesis; Osteogenesis Imperfecta; Pain; Pathologic; Pathology; Pathway interactions; Patients; Phenotype; Play; Population; Preventive treatment; Process; Production; Proteins; Quality Control; Rare Diseases; Regulation; Research; Research Infrastructure; Role; Route; Stable Isotope Labeling; Stem cells; Symptoms; System; Terminator Codon; Testing; Therapeutic; Toxic effect; Variant; Work; base; biological adaptation to stress; bone; comparative; curative treatments; experimental study; gene therapy; healing; insight; mouse model; multicatalytic endopeptidase complex; mutant; new therapeutic target; novel; novel strategies; novel therapeutics; prevent; protein folding; protein misfolding; proteostasis; public health relevance; response; vector

 DESCRIPTION (provided by applicant): Osteogenesis imperfecta (OI) is a rare disease that causes patients to suffer from brittle or broken bones and varying degrees of deformities. Currently, there is no cure, and patients must rely on situational treatment as their symptoms arise, 'healing' them only until another painful break or fracture occurs. The most common cases of OI are caused by missense mutations in one of the two types I collagen genes, producing a mutant protein that compromises the integrity of bone structure. In recent years, studies show that protein misfolding stress responses are activated in a select number of OI mouse models. Additionally, in rare forms of OI, collagen modifying enzymes are rendered inactive and result in the same brittle bones and deformities. Taken together, these studies strongly suggest that OI represents a failure of the protein folding machinery to selectively identify and degrade poorly folded collagen. Unfortunately, a cell model system for studying collagen using the high throughput molecular biology and biochemical assays available today does not exist. Cells used to study collagen production and secretion are slow growing, and difficult to manipulate, while common cell lines used in many other applications express their own, endogenous collagen-I. In the absence of a proper, malleable cell model to probe different protein folding pathways with the ability to isolate each wild type and mutant strand individually, progress in this area remains slow. I have overcome the aforementioned difficulties by creating wild type and mutant collagen-I vectors that have genetically encoded, orthogonal antibody epitope tags for easily distinguishing between each collagen strand population. Additionally, I have used these vectors to create a cell model system that inducibly expresses my collagen constructs in the absence of endogenous collagen that would convolute any data obtained. With my cell model as the main route to discovery, I am investigating the interactions required for wild type collagen-I folding and secretion using state of the art mass spectrometry and biochemical assays that are accessible only because of our cell model system. I am also performing similar biochemical experiments on the OI-causing mutant collagen strands, and carefully examining differences that the mutation may induce in the collagen folding pathway. Finally, I am performing a SILAC-assisted (stable isotope labeling of amino acids in cell culture) mass spectrometry experiment comparing wild type and mutant collagen, providing the first quantitative, comparative interactomics study of full length human collagen-I. Our approach has dual benefits of both increasing our understanding of how wild type collagen-I folds and also delving into the specifics of the molecular causes of OI, by providing the first hypothesis-driven, directed investigation of how different OI-causing collagen strands misfold and diverge from the normal intracellular homeostatic pathways.

 描述（由申请人提供）：成骨不全（OI）是一种罕见的疾病，导致患者遭受脆性或骨折和不同程度的畸形。目前，还没有治愈的方法，病人必须在症状出现时依靠情景治疗，“治愈”他们，直到另一个痛苦的骨折或骨折发生。最常见的OI病例是由两种I型胶原基因之一的错义突变引起的，产生了一种损害骨结构完整性的突变蛋白。近年来，研究表明，蛋白质错误折叠应激反应在选择数量的OI小鼠模型中被激活。此外，在罕见形式的OI中，胶原蛋白修饰酶被灭活并导致相同的脆骨和畸形。总之，这些研究强烈表明，OI代表蛋白质折叠机制选择性识别和降解折叠不良的胶原蛋白的失败。不幸的是，目前还不存在使用高通量分子生物学和生物化学测定来研究胶原蛋白的细胞模型系统。用于研究胶原蛋白产生和分泌的细胞生长缓慢，难以操作，而用于许多其他应用的常见细胞系表达其自身的内源性胶原蛋白I。在缺乏适当的，可塑性的细胞模型，以探测不同的蛋白质折叠途径的能力，以单独分离每个野生型和突变链，在这一领域的进展仍然存在 慢了我已经通过创建野生型和突变型胶原蛋白-I载体克服了上述困难，所述载体具有遗传编码的正交抗体表位标签，用于容易地区分每个胶原蛋白链群体。此外，我已经使用这些载体来创建细胞模型系统，其在不存在内源性胶原的情况下诱导表达我的胶原构建体，所述内源性胶原将使所获得的任何数据重叠。以我的细胞模型为主要发现途径，我正在研究野生型胶原蛋白-I折叠和分泌所需的相互作用，使用最先进的质谱和生物化学测定法，这些方法只能通过我们的细胞模型系统才能获得。我也在对引起OI的突变胶原链进行类似的生化实验，并仔细检查突变可能在胶原折叠途径中诱导的差异。最后，我正在进行一项SILAC辅助（细胞培养中氨基酸的稳定同位素标记）质谱实验，比较野生型和突变型胶原蛋白，首次对全长人胶原蛋白I进行定量、比较性相互作用研究。我们的方法具有双重益处，既增加了我们对野生型胶原蛋白-I如何折叠的理解，又通过提供第一个假设驱动的、对不同的引起OI的胶原蛋白链如何错误折叠和偏离正常细胞内稳态途径的定向调查，深入研究了OI的分子原因的细节。