The long-term goal of the proposed research is to understand the molecular mechanisms by which the nuclear factor of activated T cells (NFAT) transmits the common calcium signal to elicit diverse transcriptional responses. To address this question, a combination of structural, biochemical and cell-based studies will be used to analyze the interactions between NFAT and distinct transcription factor partners. For well-characterized NFAT complexes, small peptide inhibitors will be developed to further analyze the functions of these complexes in vivo. The main goal of Aim 1 is to establish the high-resolution structural model of the NFAT/GATA interaction. This interaction has been implicated in many important physiological processes, such as heart hypertrophy, IGF-induced myocyte hypertrophy and T cell development. This structural information will provide a foundation for further analyzing the mechanism and function of NFAT/GATA synergy in vivo. The focus of Aim 2 is to characterize the detailed protein-protein interactions between NFAT and MEF2 in an effort to understand the mechanism and functions of NFAT/MEF2 synergy in a variety of calcium-mediated biological responses, such as cardiac hypertrophy, skeletal muscle fiber switching T cell differentiation and proliferation. These studies will also allow us to characterize the structure and function of NFAT1 beyond the DNA binding domain. Finally, the goal of Aim 3 will be to develop small molecule inhibitors to disrupt the protein-protein interactions between NFAT and Fos-Jun. These studies will test whether relatively small molecules can effectively inhibit the large protein interface between NFAT and Fos-Jun. These peptides will be useful tools for analyzing the function of the NFAT/Fos-Jun interaction inside cells. Overall, the three Specific Aims address the structural and functional versatility of NFAT from different perspectives. These studies will establish an integrated molecular model for understanding how the calcineurin/NFAT pathway branches downstream from the calcineurin nodal point and whether a specific branch can be selectively targeted by mutations or peptide inhibitors. These studies will further dissect the function of the calcineurin/NFAT pathway and serve as a basis for developing compounds for therapeutic applications.
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