Optogenetic control of site-specific proteolysis and protein stability

利用光遗传工具调控细胞行为是生物医学研究和代谢工程的有力手段。设计这种通用工具的一种策略是通过调节降解来控制蛋白质的丰度，这一过程在真核生物中通常由泛素-蛋白酶体系统实施。以前，我们通过将LOV光感受器结构域与降解序列相结合来构建这样的工具，以诱导所选蛋白质的光降解。在此基础上，我们希望开发出用于光控制蛋白(De)稳定的改进和新型工具。我们的目标受益于基于结构的蛋白质工程方法和在酵母细胞中进行有效筛选的组合，以获得高效、功能强大的工具。从我们以前的光敏降解模块出发，我们想要构建蛋白质稳定模块，它(1)在光照条件下发挥蛋白质的稳定而不是失稳，(2)对不同的光照条件(发光状态的波长、强度、温度相关的稳定性)敏感。此外，我们将利用我们之前在基于TEV蛋白酶的调控系统方面的专业知识来构建(3)蛋白质完整性模块，其中定点蛋白分解受到光的严格控制。最后，我们有兴趣产生(4)异源二聚LOV结构域对，因为这将允许以模块化的方式对蛋白质-蛋白质相互作用进行光控制。我们计划设计的光遗传工具对于控制酵母菌的生物技术过程和在高等真核生物中的广泛应用特别感兴趣。该项目将结合我们在光生物化学、蛋白质工程和酵母遗传学方面的专业知识，构建新的光遗传蛋白质稳定性模块，从而为优先计划SPP1926--下一代光遗传学：工具开发和应用--做出贡献。由于结构/功能关系的并行研究，光感受器工程和结构表征将产生超越工具生成的影响，这是进一步合理开发光遗传工具所需的基础知识。

Regulation of cellular behavior by optogenetic tools is a powerful approach for biomedical research and metabolic engineering. One strategy for engineering such generic tools is to control protein abundance by regulated degradation, a process commonly exerted in eukaryotes by the ubiquitin-proteasome system. Previously, we constructed such a tool by coupling a LOV photoreceptor domain with a degradation sequence to induce degradation of a chosen protein by light. Based on this, we want to generate improved and novel tools for light-controlled protein (de)stabilization. Our objectives benefit from a combination of structure-based protein-engineering approaches and efficient screening in yeast cells to obtain highly efficient, functional tools. Starting from our previous photosensitive degron module we want to construct protein stability modules, which (1) exert protein stabilization instead of destabilization under illuminating conditions and (2) are susceptible to different illumination conditions for activity (wavelength, intensity, temperature-dependent stability of the lit state). Furthermore, we will use our previous expertise on TEV protease-based regulatory systems to construct (3) protein integrity modules, where site-directed proteolysis is tightly controlled by light. Finally, we are interested to generate (4) heterodimerizing LOV domain pairs, as these will allow light-control of protein-protein interactions in a modular way. The optogenetic tools, which we plan to engineer, are of particular interest for the control of biotechnological processes in yeasts and for a wide range of applications in higher eukaryotes. This project will contribute to the Priority Programme SPP1926 -Next Generation Optogenetics: Tool Development & Application- by joining our expertise in photobiochemistry, protein engineering and yeast genetics for constructing novel optogenetic protein stability modules. The photoreceptor engineering and construct characterization will have impact beyond tool generation due to the parallel investigation of structure/function relationships, which is basic knowledge required for further rational development of optogenetic tools.