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

定向进化在实验室中模仿和加速自然进化， 新的生物分子和研究进化过程。尽管定向进化的方法学很好- 在试管和简单的生物体中建立，如E.大肠杆菌和酵母菌，仍然是一个重大的挑战。具体- 最重要的是，在这些平台上，从定向进化运动中衍生出来的新生物分子通常不能发挥作用， 当转移到更复杂的细胞环境中时，例如人类细胞。处理这个关键 问题，我们的实验室最近开创了一个定向进化平台，可用于重复生成 大量的突变生物分子库，同时不断选择和富集最具功能的瓦里， 蚂蚁直接进入人类细胞环境。从化学生物学的角度来看， 在研究蛋白质稳态网络的功能-人类细胞环境的一个重要和独特的方面 确保蛋白质正确折叠、加工和运输。我们开发了一系列化学物质 基因工具来调节蛋白质稳态，我们现在准备将这些工具与我们的定向进化相结合， 一个新的平台，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应用程序旨在将我实验室的两个主要兴趣联合收割机结合起来：（1） 开发和应用下一代基于人类细胞的定向进化平台来产生生物分子， 在复杂细胞中优化功能的cules和（2）整合进化与蛋白质的化学调节， 以获得对蛋白质稳定网络功能的基本原理的新见解。在此，我们建议 整合这些研究领域，以提供一系列生物分子， 在复杂的人类细胞环境中的新功能。实例包括G蛋白偶联受体控制的 通过用于神经科学应用的合成调节剂， 蛋白质和与疾病相关的重要信号通路的抑制剂。所有这些目标都被证明是前- 在低等生物体或试管中可靠地进化非常困难。除了这些实际的进展，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 网络解决了蛋白质折叠问题。例如，我们将利用我们调节蛋白质稳态的能力来测试 这一假设，伴侣可以用来“涡轮增压”定向进化运动，提供交流， 放弃其他生物药理学上不可接受的突变区域。此外，我们将继续 了解分子伴侣在人类蛋白质进化中的作用，这一过程对人类蛋白质进化特别重要。 肿瘤发生的背景和癌基因耐药性的发展。总之，我们的贡献- 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， 生物学和癌症研究。

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.