Imaging cotranslational protein folding with high spatiotemporal resolution in living cells

以高时空分辨率对活细胞中的共翻译蛋白折叠进行成像

• 批准号: 10377445
• 负责人: Ning Zhao
• 金额: $ 10万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10377445
• 关键词: Address; Alzheimer' s Disease; Bacteriophage M13; Binding; Biological Assay; Cell Line; Cells; Color; Communities; Coupled; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Delta F508 mutation; Development; Diagnostic; Disease; Engineering; Fab Immunoglobulins; Fluorescence Microscopy; Genetic Translation; Hand; Health; Heterogeneity; Human; Huntington Disease; Image; In Vitro; Intrabody; Kinetics; Label; Lead; Left; Length; Light; Location; Measures; Messenger RNA; Methods; Modeling; Nucleotides; Parkinson Disease; Persons; Phage Display; Phase; Positioning Attribute; Postdoctoral Fellow; Proteins; Reagent; Reporter; Research; Research Personnel; Resolution; Ribosomes; Signal Transduction; Site; Spottings; System; Technology; Testing; Time; Translations; Tube; Work; base; combat; density; experience; graduate student; imaging modality; imaging platform; in vivo; method development; mutant; nanobodies; new therapeutic target; novel; protein folding; protein misfolding; real-time images; single molecule; spatiotemporal; thermostability

Project Summary: Protein misfolding is implicated in many diseases, including cystic fibrosis, Alzheimer’s, Parkinson’s, and Huntington’s disease. Understanding protein folding mechanisms is therefore important for human health. While protein folding has been extensively studied for many years, the real-time folding of a protein has yet to be captured and quantified in a living cell due to a lack of adequate experimental spatiotemporal resolution. To fill the gap, I will combine single-molecule fluorescence microscopy and novel intrabodies that can distinguish unfolded and folded proteins to image cotranslational protein folding dynamics in living cells. With this technology, I propose to investigate the folding dynamics of the cystic fibrosis transmembrane conductance regulator (CFTR), the misfolding of which causes cystic fibrosis. CFTR is a good first application because it has already been demonstrated to predominantly fold cotranslationally in vitro. To characterize CFTR folding in living cells, I will develop genetically encodable intrabodies that bind folded and unfolded CFTR cytosolic domains (Aim 1). In parallel, I will establish methods to capture and quantify cotranslational protein folding dynamics using a model protein folding system based on GFP and its pre-existing intrabodies (Aim 2). With the technology from Aims 1 and 2 in hand (K99 phase), I will image CFTR cotranslational folding dynamics at the single mRNA level in living cells (Aim 3; R00 phase). This will reveal precisely when, where, and to what degree cotranslational CFTR folding is regulated within a fully natural context. Collectively, this work will not only shed new light on cotranslational protein folding dynamics, but will also lead to new strategies to combat cystic fibrosis and other protein misfolding diseases.

项目摘要：蛋白质错误折叠与许多疾病有关，包括囊性纤维化、阿尔茨海默氏症、 帕金森氏症和亨廷顿氏症。因此，了解蛋白质折叠机制对于 人类健康。虽然蛋白质折叠已经被广泛研究了很多年，但蛋白质的实时折叠 由于缺乏足够的时空实验，蛋白质还没有在活细胞中被捕获和量化。 决议。为了填补这一空白，我将结合单分子荧光显微镜和新的体内可以 区分未折叠和折叠的蛋白质以成像活细胞中共翻译的蛋白质折叠动力学。使用 利用这项技术，我建议研究囊性纤维化跨膜电导的折叠动力学 调节因子(CFTR)，其错误折叠导致囊性纤维化。CFTR是一个很好的第一个应用程序，因为它有 已经证明在体外主要以共翻译的方式折叠。活体中CFTRs折叠的特征 细胞，我将开发可遗传编码的体内，结合折叠和未折叠的CFTR胞浆结构域 (目标1)。同时，我将建立捕获和量化共翻译蛋白质折叠动力学的方法。 基于绿色荧光蛋白及其预先存在的体内的蛋白质折叠系统模型(目标2)。有了来自 目标1和2在手头(K99阶段)，我将在单个mRNA水平上成像CFTR共翻译折叠动力学 在活细胞中(目标3；R00期)。这将准确地揭示合作翻译的时间、地点和程度 CFTR折叠是在完全自然的背景下调节的。总体而言，这项工作不仅将为 共翻译蛋白质折叠动力学，但也将导致抗击囊性纤维化和其他 蛋白质错误折叠疾病。