Optogenetic control of site-specific proteolysis and protein stability

点击翻译按钮获取中文摘要

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.