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体细胞和生殖细胞来源的特殊作用需要特殊的质量控制机制。卵母细胞仅在胚胎发生过程中产生，雌性哺乳动物出生时具有有限数量的卵母细胞，这些卵母细胞在减数分裂I前期被阻止。在通过一个检查点后，这种网状细胞停滞开始，该检查点消除了所有未设法修复在先前同源重组过程中引入的DNA双链断裂的卵母细胞。该检查点在卵母细胞中保持活性，并负责消除遭受DNA损伤的卵母细胞，所述DNA损伤例如由化疗或作为癌症治疗的一部分的辐射引起。因此，女性癌症患者往往变得不孕，并遭受过早诱导绝经。该卵母细胞特异性质量控制系统的中心参与者是p53同源物TAp 63 α。在以前的研究中，我们可以表明TAp 63 α在静息的初级卵母细胞中采用自抑制的、紧凑的和仅二聚体的构象。DNA损伤的检测触发激酶级联反应，其通过Chk 2和CK 1的磷酸化导致自抑制结构的破坏和诱导促凋亡转录程序的开放和活性四聚体的形成。我们已经优化了一个细菌表达系统的自动抑制二聚体的生产。基于突变分析和SAXS测量，我们已经创建了自抑制状态的第一个模型，并且已经表明C-末端抑制结构域与N-末端反式激活结构域一起形成阻断四聚化界面的六链β-折叠。我们现在想用低温电子显微镜确定TAp 63 α的高分辨率结构。TAp 63 α在负染色模式下的初步图像显示了良好质量的图像，表明通过冷冻EM确定结构是可行的。此外，我们还想研究磷酸化依赖性激活的机制。到目前为止，我们已经表明，激活需要磷酸化的Chk 2和CK 1。Chk 2在C-末端抑制结构域之前的环中的单个丝氨酸残基的磷酸化募集CK 1，其增加了四个磷酸基团。静电排斥导致抑制性六链β折叠的破坏。我们想用核磁共振光谱来确定磷酸化和活化动力学，并确定CK 1是否使用磷酸化的进行性或分布模式。我们还开始研究p63肽与激酶CK 1的相互作用，通过确定CK 1与三磷酸化肽复合的结构，并希望通过X射线晶体学和生物物理学方法进一步研究不同磷酸化肽的相互作用。

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.