MOLECULAR BASIS OF P53 INDUCTION TO DNA DAMAGE

MDM2在调节P53的稳定性和诱导P53对DNA损伤的反应中起着非常关键的作用。MDM2与P53的结合是靶向P53降解所必需的。然而，P73与MDM2结合，但对MDM2的降解和DNA损伤的诱导具有抵抗力，这表明与MDM2的结合不足以进行降解，必须有p53特有的序列来决定其对MDM2介导的降解和DNA损伤的反应。利用P53和p73的结构同源性，我们构建了P53-P73嵌合体，其中每个P53结构域都被相应的P73区域所取代，以确定对其稳定性调节所必需的P53序列元件。我们发现，替换P53的92-112位氨基酸会取消其对MDM2介导的降解和损伤诱导的反应能力。值得注意的是，交换这个片段可以将p73从难治性转变为对MDM2介导的降解和DNA损伤诱导的敏感性，这一发现支持了P53的92-112氨基酸是调节其稳定性和对DNA损伤反应的关键元件。初步研究也提供了令人信服的证据，表明存在与该元件相关的额外蛋白质(S)，并且对mdm2介导的降解至关重要。本研究旨在鉴定该蛋白(S)，并研究其在MDM2介导的降解和DNA损伤诱导的P53积聚中的作用。还将研究P53决定子在调节P53核进出口中的潜在作用。从拟议的工作中获得的结果可能会为如何调节P53稳定性以及如何诱导P53响应DNA损伤的机制提供深入的见解。我们还建议使用我们的体外降解试验系统来筛选MDM2介导的P53降解的抑制剂。确定的蛋白(S)和抑制物可能是潜在的候选开发治疗药物，以恢复癌细胞中的P53活性。其具体目的包括：1)鉴定MDM2介导的P53降解所必需的P53决定簇相互作用蛋白，并研究P53决定簇在调控P53核输入/输出中的作用；2)研究该蛋白在MDM2靶向的P53降解和DNA损伤诱导中的作用：3)利用我们的体外降解检测系统筛选小分子来寻找P53降解的抑制剂。

Mdm2 plays a very crucial role in the regulation of p53 stability and p53-induction in response to DNA damage. The binding of Mdm2 to p53 is required for targeting p53 for degradation. p73, however, binds to Mdm2 but is resistant to degradation by Mdm2 and to the induction by DNA damage, indicating that binding to Mdm2 is not sufficient for degradation and there must be a sequence unique to p53 that determines its response to Mdm2-mediated degradation and to DNA damage. By utilizing the structural homology between p53 and p73, we generated p53-p73 chimeras, where each of p53 domains was replaced by the corresponding region of p73, to determine the sequence element of p53 essential for regulation of its stability. We found that replacing the amino acid 92-112 of p53 abolishes its ability to respond to Mdm2-mediated degradation and to the induction by damage. Significantly, the finding that swapping this fragment converts p73 from refractory into sensitive to Mdm2-mediated degradation and induction by DNA damage supports that the amino acid 92-112 of p53 is an essential element in the regulation of its stability and response to DNA damage. Preliminary studies also provide compelling evidences to indicate a presence of additional protein(s) that associates with the element and is essential for Mdm2-mediated degradation. This proposal aims at identifying the protein(s) and investigating its role in Mdm2-mediated degradation and in DNA damage-induced p53 accumulation. Potential involvement of the p53 determinant in the regulation of p53 nuclear import/export will also be examined. Results obtained from the proposed work will likely provide insights into mechanism of how p53 stability is regulated and how p53 is induced in response to DNA damage. We also propose to screen for inhibitors of Mdm2-mediated p53 degradation using our in vitro degradation assay system. The identified protein(s) and the inhibitors can be potential candidate for the development of therapeutic agent to reactivate p53 activity in cancer cells. The Specific Aims include: 1) to identify the p53 determinant-interacting protein essential for Mdm2-mediated p53 degradation and to examine role of the p53 determinant in regulation of p53 nuclear import/export: 2) To investigate role of the protein in Mdm2-targeted p53 degradation and p53-induction in response to DNA damage: 3) to search for inhibitors of p53 degradation by screening small compounds using our in vitro degradation assay system.