Pin1结构、稳定性及与磷酸化蛋白相互作用的核磁共振研究

发布时间：2018-05-05 09:37

  本文选题：Pin1 + Pin1　； 参考：《湘潭大学》2017年硕士论文

【摘要】：Pin1是一个含有163个氨基酸的肽基脯氨酰同分异构酶,它由一个N端的WW结构域和一个C端的PPIase结构域组成。Pin1的WW结构域特异性识别含pSer/Thr-Pro基序的蛋白质,而它的PPIase结构域则催化相应肽键的顺反异构化。Pin1通过对磷酸化蛋白的结合与催化异构化调控一系列细胞过程,如细胞周期和细胞成长等。Pin1能够与链状的pSmad 2/3、pTau结合,从而影响神经组织中酶的形成,对于治疗神经性疾病有很重要的意义。如在老年痴呆症缠结的神经元纤维中,Pin1蛋白通过调节激酶、磷酸化酶的活性进一步调控与微管相关的Tau蛋白。Pin1蛋白能够识别并结合Smad3中的PEpTPPP结构,进而通过诱导泛素蛋白酶的降解来抑制TGF-β信号传递。在乳腺癌组织中,Pin1与蛋白激酶JNK的协同作用增强了磷酸化c-Jun调控cyclin D1启动子的转录活性,导致癌症基因cyclin D1的过表达。本论文主要采用配备了超低温探头的700 MHz高场液体核磁共振谱仪来研究Pin1 WW WT(野生株型)及单点突变体Pin1 WW S16R(G20D)的结构、稳定性及与人类疾病密切相关的几个磷酸化蛋白如pTau和pSmad3的相互作用。首先通过二维的1H-15N异核单量子相关谱(HSQC)和三维的HNCO、HN(CO)CA、HNCA、HNCACB及CBCA(CO)NH谱确定骨架和支链的信号,然后通过5-25℃的核磁变温实验获得Pin1 WW单点突变结构域氢键的变化信息,根据核磁共振滴定实验获得Pin1 WW结构域与磷酸化多肽的相互作用位点信息。首先根据自然演变挑选出带不同电荷的Pin1 WW结构域S16R和G20D突变体,采用核磁的方法与野生株型的Pin1 WW结构域比较,研究它们的结构、稳定性及与磷酸化Tau的相互作用。采用牛血清蛋白(BSA)来模拟细胞环境,研究类细胞环境对于Pin1 WW结构域蛋白质结构、稳定性及与pSmad3相互作用的影响,首次反映了类细胞环境下原子分辨率水平的其它大分子蛋白对Pin1与磷酸化底物结合的影响。虽然BSA只是单一的大分子,不能模拟细胞内复杂的拥挤环境,但是可以初步的获得类细胞环境下的相关信息,为将来采用细胞裂解液或者真实细胞的相关实验提供参照。通过比较c-Jun中两个磷酸化位点与全长Pin1蛋白质的作用提出合理的多位点磷酸化c-Jun与Pin1的动态结合模型,阐明了Pin1特异性结合多位点磷酸化c-Jun的结构基础,为后期进一步细致研究Pin1蛋白质在乳腺癌等疾病中的作用机理打下基础。
[Abstract]:Pin1 is a peptidyl prolyl isoisomerase containing 163 amino acids. It is composed of a N-terminal WW domain and a C-terminal PPIase domain. The WW domain of Pin1 specifically recognizes proteins containing pSer/Thr-Pro motifs. Its PPIase domain catalyzes the cis-transisomerization of the corresponding peptide bonds. Pin1 regulates a series of cellular processes by binding and catalyzing isomerization of phosphorylated proteins, such as cell cycle and cell growth. Pin1 can bind to the chained pSmad 2 / 3 ptau. Thus affecting the formation of enzymes in nerve tissue, for the treatment of neurological diseases have a very important significance. For example, in Alzheimer's tangle neurons, Pin1 protein further regulates the microtubule-related Tau protein. Pin1 protein can recognize and bind to the PEpTPPP structure in Smad3 by regulating kinase, phosphorylase activity. Furthermore, TGF- 尾 signal transduction was inhibited by inducing the degradation of ubiquitin protease. In breast cancer tissues, the synergistic effect of Pin1 and protein kinase JNK enhanced the transcriptional activity of cyclin D1 promoter regulated by phosphorylated c-Jun, resulting in the overexpression of the cancer gene cyclin D1. In this paper, 700 MHz high field liquid nuclear magnetic resonance spectrometer equipped with ultra-low temperature probe was used to study the structure of Pin1 WW WTand single point mutant Pin1 WW S16 RG20D. Stability and interaction of several phosphorylated proteins closely related to human disease such as pTau and pSmad3. Firstly, the signals of skeleton and branched chain were determined by two-dimensional 1H-15N heteronuclear single quantum correlation spectroscopy (HSQC) and three-dimensional HNCO-HNCO-HNCACB and CBCA(CO)NH spectra, and then the variation information of hydrogen bond in Pin1 WW single-point mutation domain was obtained by nuclear magnetic field temperature variation experiments at 5-25 鈩，

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