In the last four years, our lab has demonstrated that: (i). S100B protein binds to wild-type p53 in primary malignant melanoma; (ii) S100B down-regulates p53 protein levels and its tumor suppression function via promoting hdm2-dependent ubiquitination/degradation of p53; (iii) like hdm2, the S100B promoter is activated by p53 as part of a feedback loop when levels of the tumor suppressor get too high; (iv) siRNA directed against S100B restores p53 protein levels and its transcription activation activity, as necessary for cell cycle arrest and apoptosis, and consistent with our previous results that S100B down-regulates p53; (v) S100B binds calcium more tightly in the presence of p53 than in its absence showing that S100B most readily sequesters calcium when its target protein is present; (vi) Zn2+binding to Ca2+S100B changes the high resolution structure of S100B and enhances Ca2+ and p53-binding; (vii) the high resolution structure of apo-mts1 (sub-family 1), as determined by NMR, is more like S100A6 (sub-family 1) than S100B (sub-family 2) and enabled us to distinguish structural differences between the two S100 protein sub-families; (viii); Ca2+binding to S100A1 (sub-family 2) induces a conformational change in S100A1 similar to that of S100B, but differences in the structures of two proteins in the """"""""hinge"""""""" and C-terminal loop explains why these two S100 proteins bind tightly to different protein targets; (ix) together these data enabled us to start developing inhibitors that specifically block the S100B-p53 interaction and restore wild-type p53 activity in malignant melenoma. In the next granting period, we will extend these studies with the following specific Aims: (1) We will identify the molecular determinants and protein dynamics involved in the Ca2+dependent S100B-p53 interaction using NMR relaxation and stopped-flow methods as well as further characterize the role of Zn2+ binding to S100B and Ca2+S100B; (2) We will show whether other S100 proteins bind p53 and down- regulate its function to the same extent as p53; these studies together with comparisons to other S100-target protein interactions (i.e. for S100A1, S100A2, and S100A4) will identify the molecular determinants that provide specificity in S100-target protein complexes; and (3) we will determine how S100B contributes to p53 degradation in vitro and in cancer cells. In established collaborations, we will also study the structure/function relationships of S100A1 with the ryanodine receptor and mtsl with non-muscle myosin IIA. ? ? ?

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Macromolecular Structure and Function B Study Section (MSFB)
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Wehrle, Janna P
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University of Maryland Baltimore
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