Development of methods to photocontrol the degradation of a target protein in order to finely regulate in space and time cellular and physiological processes in multicellular organisms.
The understanding of the complex workings of living systems relies on the development of tools to study and control intrinsically complex cellular networks. To attain deeper knowledge of the spatial and temporal organization of such networks new perturbation tools are required. In this context, methods relying on light actuation are particularly promising due to their high spatiotemporal resolution. Chemists and biologists have designed optical methods to control protein function targeting numerous cellular mechanisms involved in gene regulation (e.g. transcription, RNA degradation, translation, cellular localization, post-translational modifications). However, surprisingly protein degradation, which is an important mechanism of regulation of gene expression, has been kept aside. The discovery of the complex cascade of the ubiquitin/proteasome pathway has revolutionnalized the way one thinks of protein degradation, and it is now well accepted that protein degradation is a highly complex, temporally controlled, highly specific and tightly regulated process. In this context, we propose that the spatiotemporal control of the degradation of a specific protein with light could be a general method to regulate locally protein activity on a post-translational timescale (from tens of seconds to tens of minutes). The main goal of this project is to evaluate this yet unexplored way of controlling networks by light actuation. We propose to design artificial light-regulated degradation pathways to regulate in space and time the level of a target protein. These artificial pathways have the potential to enable the degradation of a broad range of proteins, independently of their functions and structures. The long-term goal of this project is to provide optical methods to interrogate, dissect and understand cellular networks and physiological processes in multicellular organisms. <br />
To design artificial light-regulated degradation pathways, two complementary strategies are envisioned, both based on the idea of using light actuation rather than post-translational modification to conditionally control the interaction between a target protein and a ubiquitin ligase, photocontrolling hence ubiquitylation and downstream degradation by the proteasome.
The development of new ways to systematically perturb and interrogate biological processes is indispensable to accelerate our understanding of the workings of living systems. Light- controllable perturbative tools have recently emerged as particularly powerful technologies that enable to study biological systems with high spatiotemporal resolution at the cellular and subcellular level. In this project we aim at developing methods to photocontrol the function of a target protein in order to finely regulate in space and time cellular and physiological processes in multicellular organisms (e.g. zebrafish embryo). To do so, we propose to develop artificial light-regulated cell degradation pathways that can be actuated with light to specifically trigger the knockout of a protein of interest. Light-triggered protein depletion would allow one to rapidly and locally switch off the function of a protein, and therefore interrogate, dissect and understand a complex cellular network by specifically degrading an inhibitory subunit or a catalyst. Two strategies will be followed in parallel. The first one will consist in developing a chemical optogenetic tool using a photo-inducible degradation pathway composed of an artificial ubiquitin ligase that can interact and ubiquitinate a target protein tagged with a specific degron upon photoreleasing of a small inducer of degradation, promoting hence its degradation. The second approach will be a fully optogenetic method that relies on a light-inducible degron, which binds to an artificial ubiquitin ligase protein only in its photoexcited state, promoting subsequent ubiquitination and degradation by the proteasome. Both methods will be first developed in mammalian cells, in order to fully characterize the efficacy, and the spatiotemporal resolution of the method, and then implemented in zebrafish embryo to demonstrate the power of such techniques in developmental studies.
Monsieur Arnaud Gautier (Processus d’Activation Sélective par Transfert d’Energie Uni-électronique ou Radiatif) – Arnaud.Gautier@ens.fr
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
PASTEUR Processus d’Activation Sélective par Transfert d’Energie Uni-électronique ou Radiatif
CIRB Centre Interdisciplinaire de Recherche en Biologie
Help of the ANR 329,879 euros
Beginning and duration of the scientific project: December 2012 - 36 Months