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理解适当的蛋白质功能至关重要。众所周知 各种疾病与错误折叠或折叠缺陷有关。蛋白质折叠已在体外研究 与残留分辨率。但是，众所周知，细胞中的折叠可能与 在孤立环境中的折叠研究，特别是由于伴侣的相互作用。研究蛋白质折叠 在蜂窝情况下，有限的方法很少能从 一个复杂的细胞环境。为了克服这些局限性，我们旨在开发一种研究蛋白质的新方法 折叠式的。这种方法是脉冲追逐蛋白质（PCIC-FPOP）的快速光化学氧化，夫妻脉冲 - 基于质谱的追逐技术在细胞足迹中，这将允许快速分析短期 生活的蛋白质折叠中间体。该方法旨在填补蛋白质折叠知识的空白及其在 各种疾病。我们为细胞羟基自由基蛋白足迹设计了一个新平台。系统调用 具有XY可移动阶段（Pixy）的平台孵化器包括无菌孵化系统，XY可移动阶段 带有控制器，蠕动泵，248 nm KRF准分子激光器和光学镜。该系统已进行了测试 并优化为细胞生长和IC-FPOP研究的新平台，称为Pixy IC-fpop。瞬态 进行转染以评估和比较标准孵化器和阶段顶部的细胞培养质量 孵化器条件。此外，进行了荧光素酶测定以定量转染效率。魉 还进行了IC-FPOP实验以与标准IC-FPOP流系统的可比性访问 技术和结果。 Pixy将通过LabView软件配置为自动系统。一次 通过观察良好的折叠动力学，优化的，小精灵将作为蛋白质折叠工具进一步验证 研究了丰富的SERPIN蛋白，Alpha-1抗胰蛋白酶及其伴侣相互作用。敏感性 在野生型蛋白质折叠动力学和随后的突变体之间解密的小精灵平台将允许 将此方法扩展到其他蛋白质基团。 PCIC-FPOP将用于研究甲状腺素蛋白（TTR）错误折叠 并且已知与淀粉样蛋白疾病，遗传性甲状腺素蛋白淀粉样变性（HATTR）相关的聚集。