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。

• 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; Investigation; interaction; p63; p300; iASPP

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p53的家族。尽管两种蛋白质之间的序列同源性很高，但p63并不是典型的肿瘤抑制剂。到目前为止，已经确定了P63：1）在分层上皮组织的基础室中高度表达了它，在这些基础室中，它调节了这些细胞的增殖潜力，2）它是生殖细胞中质量控制因子。我们专注于表征最长的同工型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dict骨停滞阶段。在此阶段，卵母细胞长时间保存，持续数十年，直到被招募进行排卵。在Dictyate停滞期间，卵母细胞表达高水平的TAP63A。我们已经证明，静息卵母细胞中的TAP63A保持在不活跃，封闭和仅二聚体构象中。 DNA双链断裂的检测导致TAP63A的磷酸化，从而形成开放，活跃和四聚体状态。基于突变分析，SAXS的测量和单个领域的结构确定我们创建了封闭状态的结构模型，并表明该状态是动力学捕获的状态。激活遵循弹簧加载机制，该机制产生了热力学更稳定的四聚体形式。我们已经证明，闭合和二聚体形式的DNA结合亲和力比开放四聚体的DNA结合亲和力小于20倍。但是，DNA结合亲和力的这种降低只是抑制机制的一部分。此外，我们的初步数据表明，与转录机械的相互作用受到抑制。我们想研究与反式激活结构域相互作用的p63与p300域之间的相互作用，并开发了整个抑制机制的定量模型。此外，我们已经证明，用低辐射剂量的辐照后TAP63A的激活比较高剂量的辐射后的激活明显慢。由于大多数卵母细胞在低辐照剂量细胞中存活，必须具有灭活活化的TAP63A的机制。除了泛素化和蛋白酶体降解外，与IASPP相互作用抑制是一种可能的机制。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。 RNAi对CE-ISPP的耗竭增强了生殖细胞凋亡，表明CE-iSPP是CEP-1的负调节剂，并且两种蛋白质之间的这种抑制作用在维持可行的生殖细胞种群中起着重要作用。在此应用中，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Xin Lu实验室合作的小鼠卵母细胞的功能研究，该实验室创造了IASPP敲除小鼠。