Notch signaling is a general transmembrane signal transduction pathway that is essential for cell fate determination, development, homeostasis, and nervous system function in eukaryotes. Genetic lesions in the pathway are linked to human disease states such as cancer and neurological disorders. A central component of this pathway is the cytosolic -1000 residues of the Notch transmembrane receptor; upon activation of the pathway, this receptor fragment is released by a protease, and potentiates downstream signaling events. The activity of this soluble fragment, termed NIC, is modulated by at least nine intracellular effector proteins through what is thought to be direct interactions. Thus, NIC is the hub of a complex network of interactions.
The aim of the research proposed here is to elucidate and quantify the molecular mechanisms and structural details of how this network modulates signaling and determines cell fate. Dr. Barrick will identify and characterize the structural and functional domains along NIC through analysis of solution structure and stability of nested and overlapping polypeptides derived from NIC. Structural domain maps will also be determined for intracellular effectors. These domains will then be used to analyze pairwise and higher order interactions. A systematic high-throughput approach will screen qualitatively for all possible pairwise interactions; conventional solution thermodynamic methods will be used to quantify the strength of pairwise interactions. Dr. Barrick will study the effect of existing Notch pathway mutations on structural stability and pairwise interactions, to help connect in vitro results to the biology of the signaling pathway. Multi-protein allosteric interactions among our NIC and effector domains will be identified and quantified; elucidation of such interactions will help to clarify the molecular mechanisms that modulate Notch signaling. Finally, Dr. Barrick will crystallize structural domains and complexes to provide an atomic level description of domains and complexes by x-ray crystallography.
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