Chemical probes to decode the subcellular redox-regulated proteome

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This proposal aims to create new small molecule tools to map molecular processes in cells. Proteins inside cells perform the fundamental functions of life, impacting on all aspects of health and disease in organisms such as ourselves. A cell is not a bag of molecules, however, but intricately divided into compartments possessing different chemical environments. The same protein may be present in different compartments but modified (decorated) in distinct ways, interacting with different groups of proteins, or performing different catalytic functions. Tools and methods that enable us to map these 'moonlighting' proteins - where they are, how their modifications differ depending on which compartment they are in, and what their function is in different compartments - contribute to our understanding of fundamental cell biology.One way in which cellular compartments differ is in their level of reactive oxygen species (ROS) and redox (reduction-oxidation) state. ROS are produced as a consequence of cell metabolism, but are also crucial signalling molecules that enable biological systems to respond rapidly to changing environments. ROS are detected via post-translational modifications on the cysteine residues of redox-sensitive proteins. This chemical signalling mechanism is thought to be deregulated in many diseases, such as cancer, diabetes and inflammation, and a lack of plasticity in the ability of a cell to maintain redox steady state may be an integral part of ageing. Plants sense redox changes to regulate their growth and determine cell fate, for example in times of drought and stress. However, current methods cannot capture a global and molecular picture of redox-related modifications on proteins in specific compartments of live cells, as this information is lost or distorted when cells are broken open for analysis.In this project we will develop new chemical tools that can be directed to different cellular compartments. Here they will release a reactive 'warhead' to capture cysteine residues in different redox states. We will design, synthesise and test tools, exploring two new approaches to mask the reactive warhead so that it can be released on demand. We will then showcase our new approach by designing tools to target a compartment called the peroxisome, which is involved in redox signalling via mechanisms that are not yet fully understood. We will use our tools to study how perturbed redox status in peroxisomes in plant cells influences cysteine modifications in this compartment.Therapeutic approaches based on the premise that boosting cellular antioxidants should have beneficial effects have largely failed to materialise. This is likely due to a lack of fundamental knowledge on how redox processes regulate cell biology and how they change over the lifetime of an organism. Tools to study redox signalling at the level of individual cellular compartments will enable our understanding of this biology.