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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058888-12
Application #
7758359
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Wehrle, Janna P
Project Start
1999-02-01
Project End
2011-07-31
Budget Start
2010-02-01
Budget End
2011-07-31
Support Year
12
Fiscal Year
2010
Total Cost
$433,361
Indirect Cost
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Melville, Zephan; Hernández-Ochoa, Erick O; Pratt, Stephen J P et al. (2017) The Activation of Protein Kinase A by the Calcium-Binding Protein S100A1 Is Independent of Cyclic AMP. Biochemistry 56:2328-2337
Cavalier, Michael C; Melville, Zephan; Aligholizadeh, Ehson et al. (2016) Novel protein-inhibitor interactions in site 3 of Ca(2+)-bound S100B as discovered by X-ray crystallography. Acta Crystallogr D Struct Biol 72:753-60
Roth, Braden M; Varney, Kristen M; Rustandi, Richard R et al. (2016) (1)H(N), (13)C, and (15)N resonance assignments of the CDTb-interacting domain (CDTaBID) from the Clostridium difficile binary toxin catalytic component (CDTa, residues 1-221). Biomol NMR Assign 10:335-9
Cavalier, Michael C; Ansari, Mohd Imran; Pierce, Adam D et al. (2016) Small Molecule Inhibitors of Ca(2+)-S100B Reveal Two Protein Conformations. J Med Chem 59:592-608
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Hartman, Kira G; Vitolo, Michele I; Pierce, Adam D et al. (2014) Complex formation between S100B protein and the p90 ribosomal S6 kinase (RSK) in malignant melanoma is calcium-dependent and inhibits extracellular signal-regulated kinase (ERK)-mediated phosphorylation of RSK. J Biol Chem 289:12886-95
Dhar, Amlanjyoti; Mallick, Shampa; Ghosh, Piya et al. (2014) Simultaneous inhibition of key growth pathways in melanoma cells and tumor regression by a designed bidentate constrained helical peptide. Biopolymers 102:344-58
Cavalier, Michael C; Pierce, Adam D; Wilder, Paul T et al. (2014) Covalent small molecule inhibitors of Ca(2+)-bound S100B. Biochemistry 53:6628-40
Ramagopal, Udupi A; Dulyaninova, Natalya G; Varney, Kristen M et al. (2013) Structure of the S100A4/myosin-IIA complex. BMC Struct Biol 13:31
Hartman, Kira G; McKnight, Laura E; Liriano, Melissa A et al. (2013) The evolution of S100B inhibitors for the treatment of malignant melanoma. Future Med Chem 5:97-109

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