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拟和加速自然进化，以创造有用的 新的生物分子和研究进化过程。尽管定向进化的方法论很好- 在试管中以及在大肠杆菌和酵母菌等简单生物体中建立，仍然是一个重大挑战。具体来说- 通常，从这些平台的定向进化活动中衍生出来的新生物分子常常无法发挥作用 当转移到更复杂的细胞环境时，比如人类细胞。要解决这一关键问题 问题，我们的实验室最近开创了一个定向进化平台，可以用来重复生成 大量的突变生物分子文库，同时不断地选择和丰富最具功能的变体。 蚂蚁直接进入人体细胞环境。从化学生物学的角度来看，我们也深入参与了 在研究蛋白质平衡网络的功能--人类细胞环境的一个重要和独特的方面 这确保了蛋白质被正确折叠、加工和交易。我们已经开发出一系列化学物质 基因工具来调节蛋白平衡，我们现在已经准备好将这些工具与我们的定向进化- TION平台既进化了以前无法实现的生物分子功能，又加深了对 细胞如何解决蛋白质折叠问题。 总之，这个NIGMS Mira应用程序试图结合我的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产生生物分子- (2)将进化与蛋白质的化学调控相结合。 停滞，以获得对蛋白质平衡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可靠和可靠地执行有价值的生物分子 在复杂的人类细胞环境中的新功能。例子包括受控的G蛋白偶联受体 由神经科学应用的合成调节剂，将非天然氨基酸加入到 蛋白质，以及与疾病相关的重要信号通路的抑制物。所有这些目标都被证明是前- 在低等生物体或试管中很难可靠地进化。除了这些实际进展外，我们还将 还将人类基于细胞的定向进化与蛋白调控结合起来，以深入了解 网络解决了蛋白质折叠问题。例如，我们将利用我们的能力来调节蛋白质平衡来测试 这一假说认为，伴侣分子可以通过提供Ac-Ac来“涡轮增压”定向进化运动。 进入突变景观中其他生物物理上不可接受的区域。此外，我们会继续推行 了解伴侣在人类蛋白质进化中的作用，这一过程在 肿瘤发生的背景和癌基因中耐药的发展。总而言之，我们的内容- TIONS将影响从生物技术和药物开发到蛋白质折叠生物物理、进化和 词典生物学和癌症研究。

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.