The Structure and Function of S100 Proteins

描述（由申请人提供）：在过去的四年中，我们的实验室已经证明：(i)。S100B蛋白与野生型p53在原发性恶性黑色素瘤中的结合（ii） S100B通过促进hdm2依赖性的p53泛素化/降解，下调p53蛋白水平及其抑瘤功能；（iii）与hdm2一样，当肿瘤抑制因子水平过高时，S100B启动子作为反馈回路的一部分被p53激活；（iv）针对S100B的siRNA恢复p53蛋白水平及其转录激活活性，这是细胞周期阻滞和凋亡所必需的，与我们之前的结果一致，S100B下调p53；(v) p53存在时，S100B与钙的结合比p53不存在时更紧密，这表明当其靶蛋白存在时，S100B最容易将钙隔离；（vi） Zn2+与Ca2+S100B结合改变了S100B的高分辨率结构，增强了Ca2+与p53的结合；（vii）经核磁共振测定，apo-mts1（亚家族1）的高分辨率结构更像S100A6（亚家族1）而不是S100B（亚家族2），这使我们能够区分两个S100蛋白亚家族的结构差异；(八);Ca2+与S100A1（亚家族2）的结合诱导了S100A1类似于S100B的构象变化，但两种蛋白在“铰链”和c末端环中的结构差异解释了为什么这两种S100蛋白与不同的蛋白靶点紧密结合；（ix）这些数据使我们能够开始开发特异性阻断S100B-p53相互作用的抑制剂，并恢复恶性黑色素瘤中野生型p53的活性。在下一个授权期内，我们将以以下具体目标扩展这些研究：(1)我们将使用NMR松弛和停流方法确定Ca2+依赖性S100B-p53相互作用中涉及的分子决定因素和蛋白质动力学，并进一步表征Zn2+结合S100B和Ca2+S100B的作用；(2)我们将证明是否有其他S100蛋白与p53结合并下调其功能的程度与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靶蛋白复合物中提供特异性的分子决定因素；(3)我们将确定S100B在体外和癌细胞中如何促进p53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.