CAREER: Integrating Chemical Biology Methods and RNA Virus Models to Elucidate How the Metazoan Proteostasis Network Modulates Protein Evolutionary Landscapes

蛋白质中可能发生哪些有害或有益的突变？这个基本问题的答案支撑着理解和预测进化的能力。当试图通过在实验室中引入突变来研究这个问题时，人们了解到突变通常会导致产生在空间中无法正确折叠的蛋白质变体。在活细胞中，有几个辅助蛋白质折叠过程的系统（称为细胞蛋白质稳态网络），它们帮助一些突变蛋白质正确折叠。通过扰乱蛋白质稳态系统，研究人员希望更多地了解蛋白质进化过程中实际发生的突变。该项目将使用可以在实验室中快速进化的流感病毒和可以打开和关闭蛋白质稳定机制各个部分的化学探针。该研究与开发和测试针对当地在家上学的学生社区的外展计划有关。提供的机会包括与研究相关的高中生实习、化学演示以及开发和测试用于带回家实验的生物分子模型，这些实验教给学生有关进化和蛋白质折叠的知识。这项研究揭示了 HSP90 伴侣之外的后生动物蛋白质稳态机制如何在分子水平上影响蛋白质进化。一系列定制设计的化学生物学工具被用来利用小分子高精度地扰乱后生动物蛋白质稳态网络关键成分的组成和活性。通过利用在后生动物细胞中繁殖的快速进化的RNA病毒（重点是甲型流感病毒）来研究这些不同的蛋白质稳态环境对蛋白质进化轨迹的影响。高度跨学科的实验方法包括对单个病毒蛋白质的深度突变扫描，以及优化的测序、建模和生物物理分析策略，以实现对结果的定量评估。结果将是对蛋白质稳态网络和客户蛋白进化之间复杂相互作用的新认识，对于在分子水平上理解和预测病毒进化具有重要意义。测试蛋白质稳态网络在缓冲蛋白质进化中的作用也将阐明特定蛋白质稳态网络组件以前未知的功能。因此，基础科学的贡献将影响从病毒学、进化生物学到生物物理学等领域。

What are the mutations, harmful or beneficial, that may occur in a protein? The answers to this fundamental question underpin the ability to understand and predict evolution. While trying to study this question by introducing mutations in the lab, it was learned that mutations very often result in the production of protein variants that do not fold correctly in space. In the living cell, there are several systems that assist the protein folding process (known as the cellular proteostasis network), and they help some of the mutant proteins fold correctly. By perturbing the proteostasis systems, the investigator hopes to learn more about the mutations that actually happen during protein evolution. This project will use the influenza viruses, which can evolve rapidly in the lab and chemical probes that can turn on and off various parts of the proteostasis machinery. The research is linked to developing and testing an outreach program targeted at the local homeschooled student community. Opportunities offered include high school student internships related to the research, chemistry demonstration shows, and the development and testing of biomolecule models for take-home experiments that teach students about evolution and protein folding.This research is uncovering how metazoan proteostasis mechanisms beyond the HSP90 chaperone influence protein evolution at the molecular level. A battery of custom-designed chemical biology tools is used to perturb the composition and activities of key components of the metazoan proteostasis network with high precision using small molecules. The effects of these different proteostasis environments on protein evolutionary trajectories are studied by leveraging rapidly evolving RNA viruses propagating in metazoan cells, with a focus on influenza A. The highly interdisciplinary experimental approach encompasses deep mutational scanning of individual viral proteins, and optimized sequencing, modeling, and biophysical analysis strategies to enable quantitative assessment of the results. The outcomes will be a new appreciation for the complex interplay between the proteostasis network and client protein evolution, with important implications for understanding and predicting viral evolution at the molecular level. Testing roles of the proteostasis network in buffering protein evolution will also elucidate previously unknown functions of specific proteostasis network components. Thus, fundamental science contributions will impact fields ranging from virology and evolutionary biology to biophysics.