The long-term goal of the studies proposed here is to understand the structural basis for normal activation of Notch receptors, to define the biochemical mechanism underlying pathophysiologic activation of Notch by mutations found in T cell acute lymphocytic leukemia/lymphoma (T-ALL), and ultimately to harness this knowledge for the treatment of T-ALL. To understand how the non-covalently associated subunits of Notch proteins are normally held together, find out how cancer-associated mutations increase Notch signaling, and elucidate how the Notch transcriptional activation complex is assembled, we propose the following specific aims:
Aim 1. Determine the structure, dynamics, and interactions of a Notch heterodimerization domain.
Aim 2. Determine how tumor-derived heterodimerization domain mutations activate Notch.
Aim 3. Determine the structural basis underlying activation of transcription by Notch receptors. 3A. Solve the structure of a Notch transcriptional activation complex or a key subcomplex containing the Notch ankyrin repeat domain. 3B. Identify small molecules that prevent assembly of the Notch transcriptional activation complex. The structural studies proposed in this proposal will fill a major gap in our current understanding of Notch signaling. The proposed NMR studies of a Notch heterodimerization domain will reveal the nature of an intramolecular interface that is crucial for maintaining Notch in its resting conformation, and that is now known to harbor activating mutations found frequently in Notch-dependent human T-ALLs. The proposed Xray crystallographic studies of Notch transcriptional activation complexes will uncover how the ankyrin repeat domain of Notch cooperates with the CSL transcription factor to recruit the mastermind co-activator. These advances will identify candidate residues for mutational studies designed to probe the importance of specific ankyrin repeat contacts in coordinating mastermind recruitment, and in binding to CSL. Finally, the availability of purified ternary complexes of Notch, Mastermind-like polypeptides and CSL bound to cognate DNA creates an opportunity for us to identify small molecules that disrupt the complex, with potential future utility in probing Notch signaling in vivo, and as compound leads for targeted therapy in T-ALL. Because of the broad importance of NOTCH in differentiation and proliferation, interventions that prevent NOTCH signaling may not only lead to new forms of treatment for T-ALL, but manipulation of NOTCH activity may also be of general value in management of breast and other cancers as well.
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