Investigating the functional significance of lipid-binding properties of human Notch ligands in health and disease

The Notch pathway is an essential signal transduction system which regulates crucial decisions in cell biology. These are vital for embryonic development and for the maintenance of adult tissue. In its simplest form, a cell-surface expressed ligand activates the pathway by binding to the Notch receptor protein presented on the outside of a neighbouring cell. This leads to cleavage events such that the intracellular portion of the receptor is released and travels to the nucleus where it forms a complex with other proteins 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. Despite extensive study of the downstream consequences of activation, many aspects of the early events leading to Notch cleavage and generation of the signal remain unclear. This is because we lack understanding of the overall shape of Notch and the way in which it can interact with its ligands.
We have recently established a novel property of Notch ligands: the ability to bind to various lipids found in the cell membrane. Binding of lipid to ligand appears to be coupled to Notch binding, suggesting a 3-component complex forms dependent on the type of lipid and ligand available. This will be investigated further using an engineered form of ligand which should form more stable lipid/protein complexes. Furthermore the identity of the actual lipids which interact with ligand will be probed using high-throughput lipid binding assays. Lastly the importance of the lipid binding property for genetic and acquired disease will be investigated by testing the ability of various mutant ligands to activate a Notch reporter cell line , and cell type specific insights gained from the study of Notch signaling in hepatic cells.
Fundamental understanding of how the Notch signal is generated should allow us to develop reagents to manipulate Notch activity and facilitate advances in many aspects of cell biology, including stem cell biology. In addition, since dysfunction of the pathway results in many inherited and acquired forms of disease, such as cancer, these reagents are likely to have therapeutic potential.

Understanding the molecular basis of Notch signaling is of significant biomedical importance. We will investigate the role of lipid as a novel modulator of Notch signaling using the following methods:
i) Create affinity-matured forms of ligand receptor complexes using established expression systems for use in lipid-binding assays which utilize liposomes or Nanodiscs. This will provide biochemical (plate assays, lipid specificity) and biophysical information (eg SPR, NMR, mass spec) about the lipid/ligand interaction.
ii) Different high-throughput screening methods including commercial lipid arrays, LiMA, native mass spectrometry and lipid pathway inhibitors will be used to identify candidate lipid ligands for each ligand family.
iii) The effects of somatic mutations associated with various cancers found in the lipid binding region of C2 domains of Notch ligands on lipid/Notch binding and Notch activity will be tested, and why hepatic cells appear sensitive to the effects of lipid binding mutations giving rise to disease will be investigated.

These data will give new insights into the modulatory role of lipid binding to mammalian C2 domains in Notch signaling which can be tested in animal models. It will facilitate the understanding of disease-causing mutations affecting this domain. Results will be disseminated at international conferences on Notch function which are held annually. Any commercial exploitation will be handled by Oxford University Innovation.

The central role of the Notch signaling 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 providing new insight into a plethora of diseases associated with Notch signaling.

At this early stage the focus of the research is on mechanistic understanding of how the lipid/ligand/receptor complex forms and is modulated at the cell membrane which is valuable to other researchers, but there is clearly potential to develop reagents which may be of industrial value, facilitating growth of specialist cells for a variety of biomedical applications, and clinically valuable to the public such as monoclonal antibodies which inhibit Notch signaling. Such reagents are likely to be valuable in the treatment of breast cancer, melanoma, and T-cell acute lymphoblastic leukemia which are all associated with aberrant Notch signaling. Professors Handford and Lea have been part of a CRUK Programme grant which aims to develop monoclonal antibodies which can inhibit Notch signaling. Thus there is an established infrastructure and pipeline already in place to exploit the findings of this research, should regions of the ligand prove to be valid targets for antibodies. Oxford University Innovation, our technology transfer company, according to the usual terms applied by the MRC will help to exploit any intellectual property arising from this grant if deemed appropriate.

Public understanding of science is an important aspect of scientific endeavour. Any discoveries will be communicated to the public by the University of Oxford Press Office and the MRC for which the PI and PDRA would willingly provide any support, such as oral or written presentations. We will also use other Public Understanding of Science events such as University Science Open Days, Oxford Science Events, School visits, Support Group visits, our web pages and our graduate publication Phenotype to publicise our work and its relevance to disease.