【作者】 陈明谅；

【导师】 林圣彩； 李勤喜；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 Pin1是一个新颖的磷酸化后修饰酶,它能特异性异构化磷酸化的Ser/Thr-Pro,从而改变底物蛋白的稳定性、细胞内定位以及与其它蛋白的相互作用等。Axin是一个含有多个功能域的构架蛋白,它执行很多重要的生物学功能。Axin作为负调控因子结合APC、β-catenin、GSK-3β及LRP5等Wnt信号通路中的蛋白并通过促进β-catenin的降解来调控Wnt信号通路。此外,Axin还参与了p53、JNK和TGF-β信号通路。但是到目前为止,Axin本身的调控以及Axin对这些复杂的信号通路进行调控的机制还不是很清楚。本论文通过体内和体外实验首次证实了Axin能与Pin1蛋白相互作用,Pin1与Axin的结合区域为WW结构域。随后我们发现虽然它们的相互作用并没有影响Axin对Wnt信号通路的抑制作用,但是Pin1和Axin对TGF-β信号通路的激活具有叠加效应,在293细胞中进行的荧光素酶报告基因实验表明Axin与Pin1能够协同激活p53蛋白的转录活性,为深入研究Axin激活p53的机制提供了新的思路。完成了上述研究后,本论文还对Daxx与Pin1之间的相互作用进行了初步研究。Daxx是一个高度保守的,多功能性的蛋白。大量的研究表明Daxx通过不同的信号途径参与细胞生长和凋亡的调节,但是Daxx在细胞凋亡中究竟是起促进还是抑制作用存在很大争议。本论文克隆了人类Daxx全长的cDNA,首次证实Daxx与肽基脯氨酰异构酶Pin1之间存在相互作用并初步确定了两个蛋白相互作用的位点。通过免疫荧光染色发现Daxx和Pin1共定位于细胞核内。该发现揭示Pin1可能在Daxx调节细胞凋亡的过程中发挥了重要作用,为进一步揭示Daxx的生物学功能提供了新的途径。更多还原

【Abstract】 Pin1 is a novel peptidyl-prolyl isomerase that can specifically isomerize the phosphorylated Ser/Thr-Pro (pSer/Thr-Pro) bond, and regulates the function of a defined subset of phosphoproteins. Axin, which plays a critical role in diverse biological processes, is a scaffold protein with multiple function domains. It binds to APC,β-catenin, GSK-3βand LRP5, serving as a negative regulator which promotes the degradation ofβ-catenin in Wnt signal pathway. In addition, Axin is also involved in the p53, JNK and TGF-βsignal pathways. However, it is still not well understood how Axin is regulated and how Axin is able to regulate such complex signal pathways. In this thesis we present the first evidence that Axin interacts with the peptidyl-prolyl isomerase Pin1 and the Axin-binding site of Pin1 locates at the WW domain. Though Pin1 does not influence the regulatory role of Axin in the Wnt signal pathway, Pin1 and Axin additively activate TGF-βsignaling. In addition, Pin1 and Axin synergically enhance the transcriptional activity of p53, indicating that Pin1 can selectively regulate the function of Axin. Our data shed new light on the mechanism of the Axin-mediated p53 activation.Futhermore, we studied the interaction between Pin1 and Daxx, a highly conserved, multi-functional protein. Despite a number of studies attempting to uncover the function of Daxx in apoptosis and growth, its precise role in these processes is still in controversy. Here we cloned human Daxx gene and showed that Daxx interacted and co-localized with Pin1. This founding implicated the Pin1’s role in the regulation of Daxx-modulated cell death and provided a potential approach to the understanding of the function of Daxx.更多还原