The long-term goal of the studies proposed here is to understand the structural basis for normal activationof Notch receptors, to define the biochemical mechanism underlying pathophysiologic activation of Notch bymutations found in T cell acute lymphocytic leukemia/lymphoma (T-ALL), and ultimately to harness thisknowledge 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 Notchtranscriptional 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 theNotch 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 Notchsignaling. The proposed NMR studies of a Notch heterodimerization domain will reveal the nature of anintramolecular interface that is crucial for maintaining Notch in its resting conformation, and that is nowknown to harbor activating mutations found frequently in Notch-dependent human T-ALLs. The proposed Xraycrystallographic studies of Notch transcriptional activation complexes will uncover how the ankyrin repeatdomain of Notch cooperates with the CSL transcription factor to recruit the mastermind co-activator. Theseadvances will identify candidate residues for mutational studies designed to probe the importance of specificankyrin repeat contacts in coordinating mastermind recruitment, and in binding to CSL.Finally, the availability of purified ternary complexes of Notch, Mastermind-like polypeptides and CSLbound 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 inT-ALL. Because of the broad importance of NOTCH in differentiation and proliferation, interventions thatprevent NOTCH signaling may not only lead to new forms of treatment for T-ALL, but manipulation ofNOTCH activity may also be of general value in management of breast and other cancers as well.
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