Structure/Function Implications of Phospholipid Recognition by Notch-Ligands

The Notch signalling pathway is an essential signal transducer which regulates many different decisions in cell biology. These are crucial for embryonic development and for the maintenance of adult tissue. In its simplest form an extracellular protein/ ligand activates the pathway by binding to the Notch receptor protein on the outside of the cell. This leads to a cleavage event such that the intracellular portion of the receptor is released, travels to the nucleus, where it forms a complex to activate specific genes responsible for regulating cell behaviour. Aberrant loss or gain of Notch activity is associated with many human developmental disorders, adult onset diseases and cancers, making it a key target for therapeutic intervention. We have recently identified a new property of one of the activators or ligands of Notch signalling, which indicates the ligand can bind to phospholipids. Phospholipids are molecules which form the major component of the limiting cell membrane and vesicles with various transport functions. This project aims to understand in great detail how the ligand binds to phospholipid and how this interaction affects Notch binding, signalling and regulation. This will give us new insights into how the Notch signalling pathway works, how various diseases linked to the pathway arise, and will give us new ways of designing drugs such as monoclonal antibodies or small molecule activators or inhibitors .These may prove invaluable in a clinical and/ or research setting to manipulate the Notch signal.

This grant aims to investigate the structural and functional significance of a novel property of the N-terminus of Jagged-1, identified by structure determination. We wish to establish the role of this novel property in the molecular mechanisms underlying Notch binding, trans-activation and cis-inhibition. Using the well-established collaboration between the Lea (biophysics) and Handford (biochemical) laboratories, we will investigate i) the structural basis for this novel property in other Notch ligands belonging to the Jagged/Serrate and Delta-like families (Drosophila Serrate, human J-2, human Dll-1,-4) using X-ray crystallography , NMR and SPR methods ii) the functional importance of this novel property for Notch binding, trans-activation and cis-inhibition using molecular and cellular assays including ELISA, flow cytometry and luciferase complementation reporter assays. These data will give new mechanistic insights into the Notch signalling pathway which may be tested in in vivo models, provide a rational basis for the design of novel modulating antagonists and agonists, and facilitate the understanding of a number of diseases caused by mutations in genes encoding Notch ligands. Results will be disseminated at international conferences on Notch function which are held annually, and any commercial exploitation handled by ISIS Innovation, university of Oxford.

The central role of the Notch signalling pathway in metazoan development and homeostasis is such that any new mechanistic insight should prove invaluable in understanding the normal functions of this pathway in different aspects of biology as well as giving new insight into a plethora of diseases associated with Notch signalling. The new data presented in this application have already significantly enhanced our understanding of the molecular basis of Alagille syndrome which may be associated with genetic mutations in human Jagged-1. It has also facilitated research in other fields namely complement regulation, since we have identified the N-terminal region of Jagged-1 to be a ligand for CD46, a complement regulator.
At this early stage the focus of the research is on mechanistic understanding which is valuable to other researchers, but there is clearly potential to develop reagents which may be clinically valuable to the public such as monoclonal antibodies which inhibit Notch signalling. Such reagents are likely to be valuable in the treatment of breast, melanoma, and T-cell acute lymphoblastic leukemia which are all associated with abberant Notch signalling. Both Professors Handford and Lea are part of a CRUK Programme grant which aims to develop monoclonal antibodies which can inhibit Notch signalling. Thus there is an established infrastructure and pipeline already in place to exploit the findings of this research. ISIS Innovation, the University of Oxford technology transfer company , according to the usual terms applied by the MRC will exploit any intellectual property arising from this grant.
Both Professors Handford and Lea believe that public understanding of science is an important aspect of scientific endeavour. We expect that our discoveries would be communicated to the public by the MRC for which we would willingly provide any support, such as oral or written presentations. We will also use other Public Understanding of Science events such as University of Science Open Days, School visits, Support Group visits, to publicise our work and its relevance to disease.