Structure determination of the closed dimeric conformation of TAp63α and investigation of its CK1 dependent activation

生殖细胞作为所有后代的体细胞和生殖细胞的来源的特殊作用需要特殊的质量控制机制。卵母细胞只在胚胎发生期间产生，雌性哺乳动物出生时有有限数量的卵母细胞在减数分裂前期被阻止。这种减数分裂停止是在通过一个检查点后开始的，检查点消除了所有没有成功修复在之前的同源重组过程中引入的DNA双链断裂的卵母细胞。这个检查点在卵母细胞中保持活跃，并负责消除遭受DNA损伤的卵母细胞，例如，作为癌症治疗的一部分，化疗或放射治疗。因此，女性癌症患者往往变得不育，并遭受过早导致更年期的痛苦。这个卵母细胞特异性质量控制系统的核心是P53同源TAp63α。在以往的研究中，我们发现TAp63α在静止的初级卵母细胞中以一种自抑制、致密且只有二聚体的构象存在。DNA损伤的检测触发了一系列的激酶级联反应，通过Chk2和CK1的磷酸化，破坏了自身抑制的结构，形成了开放的、活性的四聚体，从而诱导了促凋亡的转录程序。我们优化了一种生产自抑制二聚体的细菌表达系统。基于突变分析和SAXS测量，我们建立了自抑制状态的第一个模型，并表明C-末端抑制结构域与N-末端反式激活结构域一起形成了阻断四聚界面的六链β-SHAP。我们现在想用低温电子显微镜来确定TAp63α的高分辨结构。TAp63α在负染模式下的初步图像显示了良好的图像质量，表明用低温EM确定结构是可行的。此外，我们还想研究磷酸化依赖的激活机制。到目前为止，我们已经证明激活需要Chk2和CK1的磷酸化。Chk2将C端抑制结构域前面的环中的单个丝氨酸残基磷酸化，从而招募CK1，CK1又增加了四个磷酸基团。静电斥力导致抑制性六链β-折叠的破坏。我们想要用核磁共振波谱来确定磷酸化和激活动力学，并确定CK1是使用过程还是分布模式的磷酸化。我们也已经开始通过确定CK1与三个磷酸化的多肽的复合体的结构来研究P63多肽与激酶CK1的相互作用，并希望通过X射线结晶学和生物物理方法进一步研究不同磷酸化的多肽之间的相互作用。

The special role of germ cells as the source for both somatic and germ cells of all following generations requires special quality control mechanisms. Oocytes are only generated during embryogenesis and female mammals are born with a finite number of oocytes that are arrested in prophase of meiosis I. This dictyate arrest starts after passing a check point that eliminates all oocytes that have not managed to repair the DNA double strand breaks that were introduced during the preceding process of homologous recombination. This checkpoint stays active in oocytes and is responsible for the elimination of oocytes suffering from DNA damage caused for example by chemotherapeutics or irradiation as part of a cancer therapy. Consequently, female cancer patients often become infertile and suffer from premature induction of menopause. Central player of this oocyte specific quality control system is the p53-homolog TAp63 alpha. In previous research we could show that TAp63 alpha adopts an autoinhibited, compact and only dimeric conformation in resting primary oocytes. Detection of DNA damage triggers a kinase cascade that results through phosphorylation by Chk2 and CK1 in the disruption of the autoinhibited structure and the formation of open and active tetramers that induce a pro-apoptotic transcriptional program. We have optimized a bacterial expression system for the production of the autoinhibited dimer. Based on mutational analysis and SAXS Measurements we have created a first model of the autoinhibited state and have shown that the C-terminal inhibitory domain together with the N-terminal transactivation domain forms a six-stranded β-sheet that blocks the tetramerization interface. We now want to determine the high resolution structure of TAp63α using cryo electronmicroscopy. Preliminary images of TAp63 alpha in negative stain mode show images of good quality suggesting that determining the structure by cryo EM is feasible. In addition we want to investigate the mechanism of phosphorylation dependent activation. So far we have shown that activation requires phosphorylation both by Chk2 and by CK1. Phosphorylation of a single serine residue in a loop preceding the C-terminal inhibitory domain by Chk2 recruits CK1 which adds four more phosphate groups. Electrostatic repulsion leads to the disruption of the inhibitory six-stranded beta-sheet. We want to determine the phosphorylation and activation kinetics using NMR spectroscopy and identify if CK1 uses a processive or distributive mode of phosphorylation. We have also started to investigate the interaction of p63 peptides with the kinase CK1 by determining a structure of CK1 in complex with a triple phosphorylated peptide and want to further investigate the interaction of differently phosphorylated peptides by x-ray crystallography and biophysical methods.