本文選題：Pin1 + Pin1　； 參考：《湘潭大學(xué)》2017年碩士論文

【摘要】：Pin1是一個(gè)含有163個(gè)氨基酸的肽基脯氨酰同分異構(gòu)酶,它由一個(gè)N端的WW結(jié)構(gòu)域和一個(gè)C端的PPIase結(jié)構(gòu)域組成。Pin1的WW結(jié)構(gòu)域特異性識(shí)別含pSer/Thr-Pro基序的蛋白質(zhì),而它的PPIase結(jié)構(gòu)域則催化相應(yīng)肽鍵的順?lè)串悩?gòu)化。Pin1通過(guò)對(duì)磷酸化蛋白的結(jié)合與催化異構(gòu)化調(diào)控一系列細(xì)胞過(guò)程,如細(xì)胞周期和細(xì)胞成長(zhǎng)等。Pin1能夠與鏈狀的pSmad 2/3、pTau結(jié)合,從而影響神經(jīng)組織中酶的形成,對(duì)于治療神經(jīng)性疾病有很重要的意義。如在老年癡呆癥纏結(jié)的神經(jīng)元纖維中,Pin1蛋白通過(guò)調(diào)節(jié)激酶、磷酸化酶的活性進(jìn)一步調(diào)控與微管相關(guān)的Tau蛋白。Pin1蛋白能夠識(shí)別并結(jié)合Smad3中的PEpTPPP結(jié)構(gòu),進(jìn)而通過(guò)誘導(dǎo)泛素蛋白酶的降解來(lái)抑制TGF-β信號(hào)傳遞。在乳腺癌組織中,Pin1與蛋白激酶JNK的協(xié)同作用增強(qiáng)了磷酸化c-Jun調(diào)控cyclin D1啟動(dòng)子的轉(zhuǎn)錄活性,導(dǎo)致癌癥基因cyclin D1的過(guò)表達(dá)。本論文主要采用配備了超低溫探頭的700 MHz高場(chǎng)液體核磁共振譜儀來(lái)研究Pin1 WW WT(野生株型)及單點(diǎn)突變體Pin1 WW S16R(G20D)的結(jié)構(gòu)、穩(wěn)定性及與人類疾病密切相關(guān)的幾個(gè)磷酸化蛋白如pTau和pSmad3的相互作用。首先通過(guò)二維的1H-15N異核單量子相關(guān)譜(HSQC)和三維的HNCO、HN(CO)CA、HNCA、HNCACB及CBCA(CO)NH譜確定骨架和支鏈的信號(hào),然后通過(guò)5-25℃的核磁變溫實(shí)驗(yàn)獲得Pin1 WW單點(diǎn)突變結(jié)構(gòu)域氫鍵的變化信息,根據(jù)核磁共振滴定實(shí)驗(yàn)獲得Pin1 WW結(jié)構(gòu)域與磷酸化多肽的相互作用位點(diǎn)信息。首先根據(jù)自然演變挑選出帶不同電荷的Pin1 WW結(jié)構(gòu)域S16R和G20D突變體,采用核磁的方法與野生株型的Pin1 WW結(jié)構(gòu)域比較,研究它們的結(jié)構(gòu)、穩(wěn)定性及與磷酸化Tau的相互作用。采用牛血清蛋白(BSA)來(lái)模擬細(xì)胞環(huán)境,研究類細(xì)胞環(huán)境對(duì)于Pin1 WW結(jié)構(gòu)域蛋白質(zhì)結(jié)構(gòu)、穩(wěn)定性及與pSmad3相互作用的影響,首次反映了類細(xì)胞環(huán)境下原子分辨率水平的其它大分子蛋白對(duì)Pin1與磷酸化底物結(jié)合的影響。雖然BSA只是單一的大分子,不能模擬細(xì)胞內(nèi)復(fù)雜的擁擠環(huán)境,但是可以初步的獲得類細(xì)胞環(huán)境下的相關(guān)信息,為將來(lái)采用細(xì)胞裂解液或者真實(shí)細(xì)胞的相關(guān)實(shí)驗(yàn)提供參照。通過(guò)比較c-Jun中兩個(gè)磷酸化位點(diǎn)與全長(zhǎng)Pin1蛋白質(zhì)的作用提出合理的多位點(diǎn)磷酸化c-Jun與Pin1的動(dòng)態(tài)結(jié)合模型,闡明了Pin1特異性結(jié)合多位點(diǎn)磷酸化c-Jun的結(jié)構(gòu)基礎(chǔ),為后期進(jìn)一步細(xì)致研究Pin1蛋白質(zhì)在乳腺癌等疾病中的作用機(jī)理打下基礎(chǔ)。
[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，

本文編號(hào)：1847180