Leveraging Next-Generation Directed Evolution Platforms and Chemical Control of Proteostasis to Deliver Robust Biotechnologies and Illuminate Roles of Chaperone Networks in Protein Evolution

利用下一代定向进化平台和蛋白质稳态的化学控制来提供强大的生物技术并阐明伴侣网络在蛋白质进化中的作用

• 批准号: 10387843
• 负责人: Matthew Donald Shoulders
• 金额: $ 8.52万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10387843
• 关键词: Address; Area; Bacteria; Biological; Biology; Biophysics; Biotechnology; Cells; Charge; Chemicals; Complex; Directed Molecular Evolution; Disease; Drug resistance; Ensure; Environment; Escherichia coli; Evolution; G-Protein-Coupled Receptors; Human; Laboratories; Libraries; Lower Organism; Malignant Neoplasms; Methodology; Molecular Chaperones; Mutation; National Institute of General Medical Sciences; Neurosciences; Oncogenes; Organism; Process; Proteins; Research; Role; Signal Pathway; System; Testing; Tube; Variant; Yeasts; anticancer research; base; chemical genetics; drug development; inhibitor/antagonist; insight; interest; mutant; next generation; novel; protein folding; proteostasis; technique development; tool; tumorigenesis; unnatural amino acids

Directed evolution mimics and accelerates natural evolution in the laboratory in order to create useful new biomolecules and to study evolutionary processes. Although methodologies for directed evolution are well- established in test tubes and in simple organisms like E. coli and yeast, there is still a major challenge. Specifi- cally, novel biomolecules derived from directed evolution campaigns in these platforms often fail to function when transferred to more complex cellular environments, such as that of human cells. To address this critical issue, our laboratory recently pioneered a directed evolution platform that can be used to repeatedly generate massive libraries of mutant biomolecules while continuously selecting and enriching the most functional vari- ants directly in the human cell environment. From a chemical biology perspective, we are also deeply engaged in studying functions of the proteostasis network – a vital and unique aspect of the human cellular environment that ensures proteins are correctly folded, processed and trafficked. We have developed an array of chemical genetic tools to modulate proteostasis, and we are now primed to integrate these tools with our directed evolu- tion platform to both evolve previously inaccessible biomolecule functions and gain a deeper understanding of how cells solve protein folding problems. Altogether, this NIGMS MIRA application seeks to combine two of my laboratory's primary interests: (1) Developing and applying next-generation, human cell-based directed evolution platforms to generate biomole- cules optimized for function in complex cells and (2) Integrating evolution with chemical modulation of proteo- stasis to gain new insights into fundamental principles of proteostasis network function. Here, we propose to integrate these research areas to deliver an array of biomolecules that reliably and robustly perform valuable new functions in the complex human cellular milieu. Examples include G-protein coupled receptors controlled by synthetic regulators for neuroscience applications, systems for incorporation of unnatural amino acids in proteins, and inhibitors of important signaling pathways related to disease. All of these targets have proven ex- ceedingly difficult to reliably evolve in lower organisms or test tubes. Beyond these practical advances, we will also integrate human cell-based directed evolution with proteostasis modulation to gain insights into how the network solves protein folding problems. For example, we will use our capacity to modulate proteostasis to test the hypothesis that chaperones can be used to “turbo-charge” directed evolution campaigns by providing ac- cess to otherwise biophysically unacceptable regions of the mutational landscape. Further, we will pursue an understanding of the roles of chaperones in human protein evolution, a process that is particularly important in the setting of tumorigenesis and in the development of drug resistance in oncogenes. Altogether, our contribu- tions will impact fields ranging from biotechnology and drug development to protein folding biophysics, evolu- tionary biology, and cancer research.

定向进化在实验室中模仿并加速自然进化，以创造有用的 新的生物分子并研究进化过程。 在试管以及大肠杆菌和酵母等简单生物体中建立的模型仍然存在重大挑战。 特别是，源自这些平台中的定向进化活动的新型生物分子通常无法发挥作用 当转移到更复杂的细胞环境（例如人类细胞）时，可以解决这一关键问题。 问题，我们实验室最近开创了一个定向进化平台，可以用来重复生成 海量突变生物分子库，同时不断选择和丰富最具功能性的变异体 从化学生物学的角度来看，我们也深入参与了蚂蚁直接在人类细胞环境中的研究。 研究蛋白质稳态网络的功能——人类细胞环境的一个重要而独特的方面 确保蛋白质正确折叠、加工和运输 我们开发了一系列化学品。 调节蛋白质稳态的遗传工具，我们现在准备将这些工具与我们的定向进化相结合 化平台，既可以进化以前无法实现的生物分子功能，又可以更深入地了解 细胞如何解决蛋白质折叠问题。 总而言之，这个 NIGMS MIRA 应用程序旨在结合我实验室的两个主要兴趣：(1) 开发和应用下一代基于人类细胞的定向进化平台来生成生物分子 针对复杂细胞中的功能进行优化的细胞和（2）将进化与蛋白质的化学调节相结合 在这里，我们建议对蛋白质稳态网络功能的基本原理有新的认识。 整合这些研究领域以提供一系列可靠且稳健地执行有价值的生物分子 复杂的人类细胞环境中的新功能包括受控的 G 蛋白偶联受体。 通过神经科学应用的合成调节剂，将非天然氨基酸掺入的系统 蛋白质以及与疾病相关的重要信号通路的抑制剂已被证明是有效的。 在低等生物体或试管中可靠地进化是非常困难的，除了这些实际进展之外，我们将。 还将基于人类细胞的定向进化与蛋白质稳态调节相结合，以深入了解 例如，我们将利用我们调节蛋白质稳态的能力来测试网络解决蛋白质折叠问题。 假设伴侣可以通过提供ac-来“加速”定向进化活动 此外，我们将寻求一种生物物理学上不可接受的突变景观。 了解伴侣在人类蛋白质进化中的作用，这一过程在 总而言之，我们的贡献是肿瘤发生的背景和癌基因耐药性的发展。 这些变化将影响从生物技术和药物开发到蛋白质折叠生物物理学、进化等领域 生物学和癌症研究。