Optogenetic control of site-specific proteolysis and protein stability
Participants:
1) Lars-Oliver Essen (PI; University of Marburg), coworker: Sebastian Hepp
2) Christof Taxis (PI; University of Marburg), coworker: Jonathan Trauth
Abstract:
Optogenetic control of site-specific proteolysis and protein stability.
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 approach and efficient screening in yeast cells to obtain highly efficient, functional tools. Starting from our previous photosensitive degron (psd) 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.
Relevant Publications:
Usherenko S, Stibbe H, Muscò M, Essen LO, Kostina EA, and Taxis C (2014). Increasing the light-sensitivity of Lov2 domain-based optogenetic tools. BMC Syst Biol 8: 128.
Renicke C, Schuster D, Usherenko S, Essen LO, and Taxis C (2013). An optogenetic tool to control protein degradation and cellular function. Chemistry & Biology 20: 619-626.
Renicke C, Spadaccini R, and Taxis C (2013). A tobacco etch virus protease with increased substrate tolerance at P1' position. PLOS One 8: e67915.
Jungbluth M, Renicke C, and Taxis C (2010). Targeted protein depletion in Saccharomyces cerevisiae by activation of a bidirectional degron. BMC Syst Biol 4: 176.
Taxis C, Stier G, Spadaccini R, Knop M (2009) Efficient protein depletion by genetically controlled deprotection of a dormant N-degron. Mol Syst Biol 5: 267.
Geisselbrecht Y, Frühwirth S, Schroeder C, Pierik AJ, Klug G, Essen LO (2012). CryB from Rhodobacter sphaeroides: a unique class of cryptochromes with new cofactors. EMBO Rep. 13:223-229.
Schroeder C, Werner K, Otten H, Krätzig S, Schwalbe H, Essen LO (2008). Influence of a joining helix on the BLUF domain of the YcgF photoreceptor from Escherichia coli. Chembiochem. 9:2463-2473.
Banerjee A, Herman E, Kottke T, and Essen LO (2015). Structure of a native-like aureochrome 1a LOV domain dimer from Phaeodactylum tricornutum. Structure, in press.
Lutz AP, Renicke C, and Taxis C (2016). Controlling protein activity and degradation using blue-light. In: Methods in Molecular Biology: Optogenetics - Methods and Protocols. Kianianmomeni A (ed.). Springer, Heidelberg, in press.
Taxis C, and Knop M (2012). TIPI: TEV protease-mediated induction of protein instability. In: Methods in Molecular Biology: Ubiquitin family modifiers and the proteasome: Reviews and protocols. Dohmen/Scheffner (eds.). 832: 611-626. Springer, Heidelberg.