Functional roles of the C2 phospholipid-binding domain in Notch ligands

The organs and tissues in our bodies are built from millions of cells that have to be organized with great precision to generate the appropriately sized and shaped structures. To achieve this, the cells communicate with one another to ensure that the right number and type of cells are made in the correct places. These cell communication pathways are therefore very important in ensuring that tissues are built correctly and are properly maintained throughout life. Their communication relies on so-called receptors, proteins that are present at the surface of the cell, interacting correctly with a signalling protein --a ligand-- produced by another cell. Notch is one such essential receptor, whose normal activity is critical for tissue development and maintenance and whose inappropriate activity is associated with many diseases including cancers. Understanding how its ligands operate is therefore very important, as this could have implications for disease treatments.

Our research investigates a novel hypothesis that has emerged from studies of the shape adopted by the Notch ligands. Their shapes suggest that these signalling proteins may contact the cell surface, the membrane, to help them find or stick to the Notch receptor. Because we can engineer subtle changes in the ligand shapes, we can directly test this hypothesis and distinguish the specific contribution that membrane binding makes to their ability to signal effectively. We will do this by engineering the changes in the genome of the fruit fly and looking at how this impacts on the way its' tissues are organized as a consequence. The fly is a powerful system for these studies as it contains all of the key players, indeed over 75% of human disease-causing genes are present in flies, yet it is easily and rapidly manipulated. Our results will have the potential to transform current understanding of how ligands interact with the Notch receptor-bearing cells. They will thus have wide impact and could suggest novel strategies to aid treatment of Notch-related diseases.

The Notch pathway is a conserved metazoan cell-cell signalling system, whose normal function is essential both during development and throughout life. Aberrations in Notch signalling underpin many diseases and are causal in some cancers, hence understanding the mechanisms of activity is of widespread importance. Notch ligands are highly conserved transmembrane proteins, which interact with the Notch receptor via a modified EGF-repeat-like domain (the DSL domain). Recent structural data revealed that their conserved N-terminal region forms a C2 domain, which is a phospholipid-binding module that is usually involved in targeting proteins to cell membranes. Although the C2 domain is essential for ligand activity, as illustrated by disease-causing missense mutations in human ligands, the contribution made by phospholipid-binding to ligand function remains enigmatic. Furthermore, sequence variations between the ligands' C2 domains imply they could preferentially interact with different lipids in the cell membrane. Building on this recent breakthrough in ligand structure, the aims of this project will be to determine the significance of the C2-domain phospholipid-binding activity for ligand function in vivo and to investigate the potential for different ligands to exhibit phospholipid selectivity. To achieve this we will (1) engineer targeted mutations into the C2 domain of Drosophila ligands, using CRISPR gene editing strategies; (2) analyze the consequences of C2 domain mutations on well characterized Notch dependent processes; (3) probe in vivo binding-preferences of the C2 domains; (4) test the consequences of manipulating membrane phospholipid composition on Notch pathway activity. Deciphering the fundamentals of ligand function in vivo has wide significance, given that optimal Notch pathway activity is important for healthy tissues and that it could provide new directions to bias cell selectivity of Notch signalling in disease and regenerative contexts.

IMPACT SUMMARY
Beneficiaries:
-Industry involved in pharmaceutical research and drug development.
-Business, industrial and public sectors recruiting graduate level staff.
-The general public and schools, through our involvement in public engagement.

Benefits to industry will come from the scientific results and the methodologies we develop:
1) Notch pathway is a major target for cancer and other therapeutics. Increased knowledge about the mechanisms regulating ligand-mediated activation can lead to novel approaches for targeting the pathway and can be important in informing about unforeseen side effects. Benefit is likely to be realized in the longer term 5-10 years and it would impact especially on enhancing quality of health.
2) Considerable interest is focussed on the concept of phospholipid rafts and on other localized differences in phospholipids that could modulate membrane protein activities. Our strategy to develop a novel C2-domain phospholipids sniffer has potential for wide applications, including uses of benefit to pharmaceutical research.

Benefits to business, industrial and public sector recruiting graduate level staff will come from the development of relevant research skills and professional skills:
The project's interdisciplinary nature ensures that the staff will acquire a broad range of technical skills (sophisticated imaging approaches, high-end molecular biology techniques), which will be applicable in wide range of life sciences, pharmaceutical, employment. We note that our staff already contributes to training courses. Alongside technical skills, staff will at the same time develop generic professional skills e.g. presentational skills; writing skills; data handling, including statistics; generic computational skills; project management. Evidence of our track record in this aspect comes from subsequent employment of some staff from our groups (e.g. investment banking, parliamentary advisor, publishing, venture capital advisor).

Benefits to the general public and schools, through our involvement in public engagement:
We actively participate in communicating modern scientific methodologies and approaches to the wider community and in extending the concepts from our research into other fields. The University of Cambridge encourages and provides excellent support for public dissemination of research through the Office of Community Affairs, including the Cambridge Science Festival. We actively participate in the Festival each year, and will continue to do so, several PDRAs are part of the Cambridge Pint of Science Festival.