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

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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.