Investigation of the interaction of p63 with p300 and iASPP in oocytes.

P63属于肿瘤抑制因子p53家族。尽管两种蛋白之间的序列高度同源，但p63不是典型的肿瘤抑制因子。到目前为止，p63已经确定了两种不同的功能：1)它在分层上皮组织的基底室中高度表达，在那里它调节这些细胞的增殖潜能；2)它在生殖细胞中作为质量控制因子。我们重点描述了最长的同工异构体TAp63a如何参与卵母细胞的质量控制。在哺乳动物中，卵母细胞在同源重组完成后进入指令停止期。在这个阶段，卵母细胞被保存了很长一段时间，在人类中可以持续几十年，直到被招募用于排卵。在指令性阻滞期间，卵母细胞表达高水平的TAp63a。我们已经证明，在静止的卵母细胞中，TAp63a保持在一个不活跃的、封闭的、只有二聚体的构象中。检测到DNA双链断裂导致TAp63a磷酸化，形成开放、活性和四聚体状态。基于突变分析、SAXS测量和单个结构域的结构确定，我们建立了封闭态的结构模型，并表明该状态是一个动力学捕获态。激活遵循弹簧加载机制，产生热力学上更稳定的四聚体形式。我们已经证明，封闭和二聚体形式的DNA结合亲和力比开放的四聚体低20倍。然而，这种DNA结合亲和力的降低只是抑制机制的一部分。此外，我们的初步数据显示，与转录机制的相互作用被抑制。我们想要研究p63和p300中与transactivation domain相互作用的结构域之间的相互作用，并建立整个抑制机制的定量模型。此外，我们已经证明，低剂量辐照后TAp63a的活化明显慢于高剂量辐照后的活化。由于大多数卵母细胞在低照射剂量下存活，细胞必须具有使活化的TAp63a失活的机制。除了泛素化和蛋白酶体降解外，与iASPP相互作用的抑制也是一种可能的机制。在秀丽隐杆线虫中，只有p53家族的一个成员（CEP-1）和ASPP家族的一个成员（Ce-iASPP）在生殖细胞中表达。通过RNAi去除Ce-iASPP可增强生殖细胞凋亡，这表明Ce-iASPP是Cep-1的负调控因子，这两种蛋白之间的抑制相互作用在维持生殖细胞群的存活中起着重要作用。在本次申请中，我们提议通过相互作用和结构分析以及小鼠卵母细胞的功能研究来研究iASPP的调控功能，并与牛津大学Xin Lu实验室合作创建了iASPP敲除小鼠。

p63 belongs to the family of the tumor suppressor p53. Despite high sequence homology between both proteins, p63 is not a typical tumor suppressor. So far two different functions have been identified for p63: 1) It is highly expressed in the basal compartment of stratified epithelial tissues where it regulates the proliferative potential of these cells and 2) it serves as a quality control factor in germ cells. We have focused on characterizing how the longest isoform, TAp63a, is involved in the quality control of oocytes. In mammals oocytes enter the dictyate arrest phase after completion of homologous recombination. In this stage oocytes are kept for extended periods of time, lasting in humans several decades, until being recruited for ovulation. During the dictyate arrest oocytes express high levels of TAp63a. We have shown that TAp63a in resting oocytes is kept in an inactive, closed and only dimeric conformation. Detection of DNA double strand breaks leads to phosphorylation of TAp63a, resulting in the formation of an open, active and tetrameric state. Based on mutational analysis, SAXS measurements and structure determination of individual domains we have created a structural model of the closed state and shown that this state is a kinetically trapped state. Activation follows a spring loaded mechanism that creates the thermodynamically more stable tetrameric form. We have demonstrated that the DNA binding affinity of the closed and dimeric form is 20 fold less than that of the open tetramer. However, this reduction of the DNA binding affinity is only part of the inhibitory mechanism. In addition, our preliminary data show that the interaction with the transcriptional machinery is inhibited. We want to study the interaction between p63 and the domains of p300 that interact with transactivation domains and develop a quantitative model of the entire inhibitory mechanism. In addition, we have demonstrated that the activation of TAp63a after irradiation with a low dose of irradiation is significantly slower than the activation after irradiation with a higher dose. Since most oocytes survive a low irradiation dose cells must have a mechanism to inactivate activated TAp63a. In addition to ubiquitination and proteasomal degradation, inhibition by interaction with iASPP is a likely mechanism. In C. elegans only one member of the p53 family (CEP-1) and one member of the ASPP family (Ce-iASPP) are expressed in germ cells. Depletion of Ce-iASPP by RNAi enhances germ cell apoptosis, suggesting that Ce-iASPP is a negative regulator of Cep-1 and that this inhibitory interaction between both proteins plays an important role in maintaining a viable germ cell population. In this application we propose to study the regulatory function of iASPP by interaction and structural analysis as well as functional studies in mouse oocytes in collaboration with the laboratory of Xin Lu, Oxford, that has created an iASPP knock out mouse.