STRUCTURE-INFORMED STUDIES OF THE EXTRACELLULAR REGION OF THE HUMAN NOTCH RECEPTOR IN HEALTH & 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. Abnormal 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. Because it is such a large protein of unknown flexibility, with a fibrous rather than globular organisation, it is not technically feasible to study Notch in its intact, full-length state. Instead, due to its modular construction it can be divided into different regions based on functional importance. In previous work, we have established a structural model for the EGF4-13 polypeptide which contains a previously unrecognized region of flexibility between EGF9 and EGF10. We now wish to extend our structural and functional studies to EGF20-27 and the domains (EGF14-20) that link this to the ligand-binding region. We are keen to understand whether domains within EGF20-27 which incorporate a region known as Abruptex (Ax) can interact intra or intermolecularly with the ligand-binding region (LBR).This will enable us to understand the overall shape of the extracellular domain of Notch from EGF4 to EGF27, to identify regions that are rigid and those that are flexible, to understand the significance of the Ax/LBR interaction for structure, function and regulation of Notch activity at the cell surface. 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.