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

研究卵母细胞中 p63 与 p300 和 iASPP 的相互作用。

基本信息

批准号：
319849750
负责人：
Professor Dr. Volker Dötsch
金额：
--
依托单位：
Institut für Biophysikalische Chemie
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2018-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/319849750?language=en
关键词：
Investigation interaction p63 p300 iASPP

项目摘要

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.

P63属于肿瘤抑制基因P53家族。尽管这两种蛋白的序列高度同源，但p63并不是一个典型的肿瘤抑制因子。到目前为止，p63有两种不同的功能：1)它在复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能力；2)它在生殖细胞中作为一个质量控制因子。我们主要研究最长的异构体TAp63a如何参与卵母细胞的质量控制。在哺乳动物中，卵母细胞在完成同源重组后进入二叠体停滞期。在这个阶段，卵母细胞被长期保存，在人类体内持续几十年，直到被招募排卵。在发育停滞的过程中，卵母细胞表达高水平的TAp63a。我们已经证明，TAp63a在静止的卵母细胞中保持着一种不活跃的、封闭的、只有二聚体的构象。检测到DNA双链断裂导致TAp63a的磷酸化，导致开放的、活性的和四聚体状态的形成。基于突变分析、SAXS测量和单个结构域的结构确定，我们建立了闭合状态的结构模型，表明该状态是一个动力学陷阱状态。活化遵循弹簧加载机制，产生热力学上更稳定的四聚体形式。我们已经证明，闭合和二聚体的DNA结合亲和力比开放四聚体的DNA亲和力低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受到抑制。我们想要研究p63和p300中与反式激活结构域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，我们还证明了TAp63a在低剂量照射后的激活明显慢于高剂量照射后的激活。由于大多数卵母细胞在低辐射剂量下存活，细胞必须有灭活激活的TAp63a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个可能的机制。在线虫中，生殖细胞只表达P53家族中的一个成员(CEP-1)和ASPP家族中的一个成员(Ce-iASPP)。Ce-iASPP被RNAi耗尽可促进生殖细胞的凋亡，提示Ce-iASPP是Cep-1的负调控因子，两种蛋白之间的这种抑制相互作用在维持生殖细胞活性方面发挥着重要作用。在这项应用中，我们建议通过相互作用和结构分析来研究iASPP的调节功能，以及与牛津大学新路实验室合作在小鼠卵母细胞中进行功能研究，该实验室已经创造了iASPP基因敲除小鼠。