EXCEED THE SPACE PROVIDED. Higher than normal levels of S100 proteins are used to diagnose cancer in numerous tissues; however, the role of these tumor markers in cancer progression is not well understood. In the last granting period, we determined that S 100B binds to the tumor suppressor protein, p53, in primary malignant melanoma cells and that the S100B-p53 complex forms in a cell-cycle dependent manner (during G1). The S100B-p53 interaction was found to inhibit p53 function, which likely contributes to cancer progression when S100B is overproduced. We also found that p53 activates the transcription of S100B in a regulatory feedback loop (i.e. p53 activates S100B, and then S100B down- regulates p53). Similar feedback control occurs for the only other protein known to down-regulate p53, namely mdm2. To explore S100-p53 interactions in detail, NMR structures were determined for apo-S 100B, apo-S 100A1, apo-mts 1, CaZ+-s100B, and CaZ+-s100B bound to p53 (residues 367-388). The 3D structure of CaZ+-s100B bound to a high affinity target peptide (TRTK-12) was also determined and could be useful for drug-design. A comparison of the S 100B structures (apo-, holo-, p53-bound) illustrates why p53 binding to S100B is CaZ+-dependent in vitro. In this granting period, we will continue to characterize the CaZ+-dependent conformational change in S 100B that is required for p53 binding (Aim 1). Further, demonstrating that the S 100B-p53 interaction is CaZ+-dependent in vivo remains to be done (Aim 1). This goal is very important because if the S100-p53 interaction is dependent on Ca 2+in vivo, as expected, then a direct link will be established between p53 biology and CaZ+-mediated signal transduction pathways initiated outside the cell. It is also known that Zn 1+binds S 100B and increases the CaZ+-binding affinity to S 100B (>10- fold). Likewise, Zn 2+binding enhances the affinity of target proteins such as p53 to S 100B (>3-fold). Therefore, Zn 2+ complexes of S100B will be explored (Aim 2). Because p53 activity is u_E-regulated by covalent modifications in vivo (i.e. phosphorylation, acetylation, sumoylation), we will investigate how these modifications affect S100B-p53 binding. Based on our S100B-p53367-388 structure and preliminary data in vivo, it is hypothesized that covalent modifications of p53 will protect the tumor suppressor from S 100B binding. This hypothesis will be tested (Aim 3). Another potential mechanism for protecting p53 from S100B is heterodimer formation. In particular, S 100A2 is a tumor suppressor that localizes to the nucleus (the only $100 protein to do so) and does not inhibit p53 function in vivo. Therefore, the effects of heterodimer formation between S 100B and S100A2 will be explored in p53 assays (Aim 3). Again, the hypotheses for protecting p53 from S100B (in Aim 3) are similar to what is found for mdm2 (i.e. p53 phosphorylated at Thr-18 binds mdm2 an order of magnitude more weakly than unmodified p53, and mdm2 no longer down-regulates p53 when it forms heterodimers with a structural homologue, mdmX). Lastly, another dimeric S 100 protein, the metastasis protein (mtsl; S100A4), binds p53 and inhibits tumor suppression in vivo. Therefore, the structure and function of mts 1 will be examined and compared to S100B to determine if general relationships can be found in S 100-p53 interactions (Aim 4), as is necessary for developing useful S 100 inhibitors (i.e. for a cancer therapeutic). PERFORMANCE SITE ========================================Section End===========================================
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