Chemical probes to decode the subcellular redox-regulated proteome

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.

Who will benefit from this research?

The general public and society as a whole, and the pharmaceutical/biotechnology industries, will benefit from this research in the long term. In the shorter term, this research will benefit fundamental and applied scientists in academia and industry beyond the field of chemical biology, including drug discovery, cell biology and plant/crop science. The people involved in this research - the PDRA, myself, collaborators Prof. Christine Foyer and Prof. Alison Baker - will benefit directly from the opportunities this project will afford.

How will they benefit from this research?

1. Creation of tools and methodology to enable interrogation of biology.

Chemical proteomics approaches to understand native protein biology are well-recognised as contributing to pre-clinical drug discovery and there are calls to move towards clinical settings (Sci. Trans. Med. 2017, 9, 386). This move will require the further development of technologies to label proteins in live cells and tissues (not just in cell lysates) - one of the outcomes of this project. Furthermore, there is reviving interest in targeted covalent inhibitors as drugs, many of which react with cysteine residues in proteins, and in efforts to understand the chemically reactive proteome (Mol. Biosyst. 2016, 12, 1728). New tools to map accessible cysteines in cells are of significant interest in this endeavour. Methods developed during this proposal will therefore be of relevance to pharmaceutical companies.
Similarly, by addressing a methodology gap, this research will be of value to cell biologists seeking to understand molecular mechanisms with spatial resolution in cells. Small molecule tools are generally straightforward to apply across different biological systems, thus providing a complementary approach to molecular biology approaches.

2. Insights into redox biology.

By taking a tool- and data-driven approach, chemical tools can reveal currently unstudied and untapped biology, such as novel protein targets for drug discovery, or previously unknown signalling pathways and mechanisms. Redox regulation and signalling are of fundamental interest in a broad range of fields, for example being both implicated in diseases of human aging, including cancer, and in how plants respond to changing environments such as drought. The molecular mechanisms involved in most of these processes are not yet understood. For example, compared to healthy cells, cancer cells both produce increased levels of reactive oxygen species and have increased expression of antioxidant response systems. What this balance means for cellular physiology is not understood. Could a drug be used to specifically interfere with redox regulation in cancer cells to "tip the balance" towards cell death? Only an understanding of the underlying biology will enable such intervention. Whilst the focus of this grant is methodology development, tools can be readily translated for application to specific questions in collaboration with relevant experts.

3. Training of skilled people.

The training provided in interdisciplinary research to the PDRA employed will contribute to the workforce of skilled people. As a new PI, I will gain scientific and administrative skills and demonstrate my ability to successfully apply for and manage a grant from a UK agency, an important step in my academic career.