MOLECULAR BASIS OF P53 INDUCTION TO DNA DAMAGE

Mdm 2在调节p53稳定性和响应DNA损伤的p53诱导中起着非常关键的作用。Mdm 2与p53的结合是靶向p53降解所必需的。然而，p73与Mdm 2结合，但对Mdm 2的降解和DNA损伤的诱导具有抗性，这表明与Mdm 2的结合不足以降解，并且必须存在p53特有的序列，该序列决定其对Mdm 2介导的降解和DNA损伤的响应。通过利用p53和p73之间的结构同源性，我们产生了p53-p73嵌合体，其中每个p53结构域被p73的相应区域所取代，以确定p53的序列元件对于调节其稳定性至关重要。我们发现，取代p53的氨基酸92-112废除其响应Mdm 2介导的降解和损伤诱导的能力。值得注意的是，交换该片段将p73从对Mdm 2介导的降解和DNA损伤诱导不敏感转变为对Mdm 2介导的降解和DNA损伤诱导敏感的发现支持了p53的氨基酸92-112是调节其稳定性和对DNA损伤应答的重要元件。初步研究还提供了令人信服的证据，表明存在与该元素相关的其他蛋白质，并且对Mdm 2介导的降解至关重要。该建议旨在鉴定蛋白质并研究其在Mdm 2介导的降解和DNA损伤诱导的p53积累中的作用。还将研究p53决定簇在调节p53核输入/输出中的潜在参与。从所提出的工作中获得的结果可能会提供深入了解p53稳定性如何调节以及p53如何响应DNA损伤而被诱导的机制。我们还建议使用我们的体外降解测定系统筛选Mdm 2介导的p53降解的抑制剂。所鉴定的蛋白质和抑制剂可以是用于开发治疗剂以重新激活癌细胞中的p53活性的潜在候选物。具体目标包括：1）鉴定Mdm 2介导的p53降解所必需的p53决定子相互作用蛋白，并检查p53决定子在调节p53核输入/输出中的作用：2）研究该蛋白在响应DNA损伤的Mdm 2靶向p53降解和p53诱导中的作用：3）利用体外降解实验系统筛选小分子化合物，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.