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
基本信息
- 批准号:10192108
- 负责人:
- 金额:$ 10万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2021
- 资助国家:美国
- 起止时间:2021-04-01 至 2023-03-31
- 项目状态:已结题
- 来源:
- 关键词:AddressAlzheimer&aposs DiseaseBacteriophage M13BindingBiological AssayCell LineCellsColorCommunitiesCoupledCystic FibrosisCystic Fibrosis Transmembrane Conductance RegulatorDelta F508 mutationDevelopmentDiagnosticDiseaseEngineeringFab ImmunoglobulinsFluorescence MicroscopyGenetic TranslationHandHealthHeterogeneityHumanHuntington DiseaseImageIn VitroIntrabodyKineticsLabelLeadLeftLengthLightLocationMeasuresMessenger RNAMethodsModelingNucleotidesParkinson DiseasePhage DisplayPhasePositioning AttributePostdoctoral FellowProteinsReagentReporterResearchResearch PersonnelResolutionRibosomesSignal TransductionSiteSpottingsSystemTechnologyTestingTimeTranslationsTubeWorkbasecombatdensityexperiencegraduate studentimaging modalityimaging platformin vivomethod developmentmutantnanobodiesnew therapeutic targetnovelprotein foldingprotein misfoldingreal-time imagessingle moleculespatiotemporalthermostability
项目摘要
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.
项目概述: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
人体健康虽然蛋白质折叠已经被广泛研究了很多年,但实时折叠的蛋白质已经被广泛研究。
由于缺乏足够的实验时空,蛋白质尚未在活细胞中被捕获和定量
分辨率为了填补差距,我将联合收割机结合单分子荧光显微镜和新的胞内抗体,
区分未折叠和折叠的蛋白质,以成像活细胞中的共翻译蛋白质折叠动力学。与
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,因为它具有
已经证明在体外主要是聚集性折叠。为了表征生活中的CFTR折叠
细胞,我将开发基因编码的胞内抗体,结合折叠和未折叠的CFTR胞质结构域
(Aim 1)。同时,我将建立方法来捕获和量化共翻译蛋白质折叠动力学,
基于GFP及其预先存在的胞内抗体的模型蛋白质折叠系统(Aim 2)。随着技术从
目的1和2在手(K99期),我将图像CFTR共翻译折叠动力学在单个mRNA水平
在活细胞中(目标3; R 00阶段)。这将精确地揭示何时、何地以及共翻译到何种程度
CFTR折叠在完全自然的环境中调节。总的来说,这项工作不仅将揭示新的光
共翻译蛋白质折叠动力学,但也将导致新的战略,以打击囊性纤维化和其他
蛋白质错误折叠疾病
项目成果
期刊论文数量(0)
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{{ truncateString('Ning Zhao', 18)}}的其他基金
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:
10759518 - 财政年份:2021
- 资助金额:
$ 10万 - 项目类别:
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 - 财政年份:2021
- 资助金额:
$ 10万 - 项目类别: