The Structure and Function of S100 Proteins

描述(申请人提供)：在过去的四年里，我们的实验室已经证明：(I)。在原发恶性黑色素瘤中，S100B蛋白与野生型P53结合；(Ii)S100B通过促进Hdm2依赖的P53泛素化/降解来下调P53蛋白水平及其肿瘤抑制功能；(Iii)与Hdm2一样，S100B启动子在肿瘤抑制因子水平过高时作为反馈环的一部分由P53激活；(Iv)针对S100B的siRNA恢复P53蛋白水平及其转录激活活性，这是细胞周期停滞和细胞凋亡所必需的，与我们先前的S100B下调P53的结果一致；(V)S100B在有P53的情况下与钙的结合比在没有P53的情况下更紧密，这表明S100B在有靶蛋白存在的情况下最容易截留钙；(Vi)锌与S100B的结合改变了S100B的高分辨结构，增强了钙和P53的结合；(Vii)核磁共振确定apo-mts1(亚家族1)的高分辨结构更像S100A6(亚家族1)，而不是S100B(亚家族2)，这使我们能够区分两个S100蛋白亚家族之间的结构差异；CA2与S100A1(亚家族2)的结合引起S100A1的构象变化，与S100B相似，但“铰链”和C-末端环中两个蛋白质结构的差异解释了为什么这两个S100蛋白质与不同的蛋白质靶点紧密结合；(Ix)结合这些数据，我们能够开始开发特异性阻断S100B-P53相互作用的抑制剂，并在恶性黑色素瘤中恢复野生型P53的活性。在下一个授权期，我们将扩大这些研究的具体目标：(1)我们将利用核磁共振松弛和停流方法确定依赖钙的S100B-P53相互作用所涉及的分子决定因素和蛋白质动力学，并进一步表征锌与S100B和钙S100B结合的作用；(2)我们将展示其他S100蛋白是否与P53结合并下调其功能，其程度与P53相同；这些研究与其他S100-靶蛋白相互作用(即S100A1、S100A2和S100A4)的比较将确定在S100-靶蛋白复合体中提供特异性的分子决定因素；以及(3)我们将确定S100B如何在体外和癌细胞中促进P53的降解。在已建立的合作中，我们还将研究S100A1与ryanodine受体的结构/功能关系，以及mtsl与非肌肉肌球蛋白IIA的结构/功能关系。

DESCRIPTION (provided by applicant): 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.