A New Platform for Studying Protein Folding in Live Cells

研究活细胞中蛋白质折叠的新平台

• 批准号: 10337041
• 负责人: Dante Tenielle Johnson
• 金额: $ 2.85万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-01 至 2022-10-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10337041
• 关键词: Address; Amino Acids; Amyloidosis; Biological Assay; Biological Models; Black American; Cell Culture Techniques; Cells; Chemicals; Complex; Computer software; Coupled; Couples; Crowding; Development; Disease; Environment; Gel; Genes; Goals; Health; Humidity; Hydrogen Peroxide; Hydroxyl Radical; In Vitro; Incubators; Inherited; Knowledge; Lasers; Life; Luciferases; Manuals; Mass Spectrum Analysis; Methods; Molecular Chaperones; Mutation; Optics; Organism; Pathway interactions; Physiologic pulse; Positioning Attribute; Prealbumin; Protein Engineering; Protein Footprinting; Proteins; Pump; Resolution; Role; Serpins; Solvents; Sterility; System; Techniques; Technology; Temperature; Testing; Time; Transfection; Variant; alpha 1-Antitrypsin; base; cell growth; crosslink; design; efficacy evaluation; experimental study; gel electrophoresis; mutant; oxidation; photolysis; protein folding; protein function; protein misfolding; tool

Project Summary Understanding protein folding is critical for understanding proper protein function. It is well known that various diseases are associated with misfolding or folding deficiencies. Protein folding has been studied in vitro with residue resolution. However, it is well established that folding in the cell can be significantly different from folding studies in an isolated environment, notably due to the interactions of chaperones. Studying protein folding in a cellular context is limited with few approaches being able to tease out high resolution structural detail from a complex cellular environment. To overcome these limitations, we aim to develop a new method to study protein folding. This method, pulse chase in-cell fast photochemical oxidation of proteins (pcIC-FPOP), couples pulse- chase technology with mass spectrometry-based in cell footprinting which will allow for quick analysis of short- lived protein folding intermediates. This method aims to fill gaps in knowledge of protein folding and its role in various diseases. We designed a new platform for in cell hydroxyl radical protein footprinting. The system called Platform Incubator with an XY movable stage (PIXY) includes a sterile incubation system, XY movable stage with controllers, peristaltic pumps, a 248 nm KrF excimer laser, and optic mirrors. The system has been tested and optimized as the new platform for cell growth and IC-FPOP studies, referred to as PIXY IC- FPOP. Transient transfections were performed to assess and compare cell culture quality under standard incubator and stage top incubator conditions. Furthermore, a luciferase assay was performed to quantitate transfection efficiency. PIXY IC-FPOP experiments were also carried out to access comparability with the standard IC-FPOP flow system technique and results. PIXY will be configured into an automated system through LABVIEW software. Once optimized, PIXY will be further validated as a protein folding tool by observing the folding dynamics of well- studied abundant serpin protein, alpha-1 antitrypsin as well as its chaperone interaction. The sensitivity of the PIXY platform to decipher between wild type protein folding dynamics and subsequent mutants will allow for the expansion of this method to other protein groups. pcIC-FPOP will be used to study transthyretin (TTR) misfolding and aggregation known to be associated with the amyloid disease, hereditary transthyretin amyloidosis (hATTR).

项目摘要 了解蛋白质折叠对于了解正确的蛋白质功能至关重要。众所周知， 各种疾病都与错误折叠或折叠缺陷有关。蛋白质折叠已经在体外进行了研究 具有残留物分辨率。然而，众所周知，细胞中的折叠可以显著不同于 在孤立的环境中折叠研究，特别是由于伴侣的相互作用。蛋白质折叠的研究 在细胞环境中是有限的，几乎没有方法能够梳理出高分辨率的结构细节 一个复杂的蜂窝环境。为了克服这些限制，我们的目标是开发一种新的方法来研究蛋白质 折叠。这种方法，脉冲追逐细胞内蛋白质的快速光化学氧化(PCIC-FPOP)，耦合脉冲. Chase技术和基于质谱学的细胞足迹，将允许快速分析短 活蛋白折叠中间体。该方法旨在填补蛋白质折叠及其作用方面的知识空白 各种疾病。我们设计了一个新的细胞内羟基自由基蛋白质足迹平台。该系统名为 带XY活动平台的孵化器(PIXY)包括无菌孵化系统、XY活动平台 配备控制器、蠕动泵、248 nm KrF准分子激光器和光学反射镜。该系统已经过测试 并优化为细胞生长和IC-FPOP研究的新平台，简称Pixy IC-FPOP。瞬变 在标准培养箱和舞台顶上进行细胞培养质量评估和比较。 孵化器条件。此外，荧光素酶活性测定也被用来定量检测转染率。Pixy 还进行了IC-FPOP流动实验，以获得与标准IC-FPOP流动系统的相似性 技术和结果。Pixy将通过LabVIEW软件配置成自动化系统。一次 经过优化，Pixy将通过观察Well-Well的折叠动力学进一步验证其作为蛋白质折叠工具的有效性。 研究了丰富的丝氨酸蛋白、α-1抗胰蛋白酶及其与伴侣的相互作用。它的敏感度 Pixy平台破译野生型蛋白质折叠动力学和后续突变体之间的关系将允许 将该方法扩展到其他蛋白质基团。PCIC-FPOP将用于研究转甲状腺素(TTR)的错误折叠 和聚集已知与淀粉样病，遗传性转甲状腺蛋白淀粉样变性(HATTR)有关。