The Structure and Function of S100 Proteins

描述（由申请人提供）：在过去的四年里，我们的实验室已经证明：（i）。S100 B蛋白与原发性恶性黑色素瘤中的野生型p53结合;（ii）S100 B通过促进hdm 2依赖的p53泛素化/降解下调p53蛋白水平及其肿瘤抑制功能;（iii）与hdm 2一样，当肿瘤抑制因子水平过高时，S100 B启动子被p53激活，作为反馈环的一部分;（iv）针对S100 B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凋亡所必需的，并且与我们先前的S100 B下调p53的结果一致;（v）S100 B在p53存在下比在其不存在下更紧密地结合钙，这表明当其靶蛋白存在时，S100 B最容易螯合钙;（vi）Zn ~（2+）与Ca ~（2+）S100 B的结合改变了S100 B的高分辨结构，增强了Ca ~（2+）和p53 ~-的结合;（vii）apo-mts 1的高分辨率结构（亚族1），如通过NMR测定的，更像是S100 A6（子系列1）比S100 B（亚家族2），并使我们能够区分两个S100蛋白亚家族之间的结构差异;（viii）; Ca 2+与S100 A1结合（亚家族2）诱导S100 A1中类似于S100 B的构象变化，但两种蛋白质在“铰链”和C-末端环中的结构差异解释了为什么这两种S100蛋白质与不同的蛋白质靶标紧密结合;（ix）这些数据使我们能够开始开发特异性阻断S100 B-p53相互作用并恢复恶性黑色素瘤中野生型p53活性的抑制剂。在下一个资助期内，我们将继续开展这些研究，具体目标如下：（1）利用NMR弛豫和停流方法鉴定Ca ~（2+）依赖的S100 B-p53相互作用的分子决定簇和蛋白动力学，并进一步表征Zn ~（2+）与S100 B和Ca ~（2+）S100 B结合的作用;（2）我们将展示其他S100蛋白是否与p53结合并下调其功能至与p53相同的程度;这些研究以及与其他S100-靶蛋白相互作用的比较（即S100 A1、S100 A2和S100 A4）将鉴定在S100-靶蛋白复合物中提供特异性的分子决定簇;以及（3）我们将确定S100 B如何在体外和癌细胞中促进p53降解。在已建立的合作中，我们还将研究S100 A1与ryanodine受体和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.