结肠癌相关SAMHD1突变体的功能研究

发布时间：2018-03-02 00:20

本文关键词： SAMHD1 结肠癌 dNTPs 酶活性 LINE-1　出处：《吉林大学》2017年硕士论文　论文类型：学位论文

【摘要】：SAMHD1是真核生物基因编码的一种蛋白质。它是一种天然的抗病毒因子,具有d NTPase活性,能够降低细胞内的d NTPs水平,从而能够抑制病毒的复制。并且SAMHD1在生物体内含量丰富,在人体内的很多组织中均有表达,是一种丰富度较高的蛋白。SAMHD1不仅能够抑制病毒的复制,而且在很多肿瘤细胞中表达的SAMHD1也会消耗细胞内的d NTPs,从而起到抑制肿瘤细胞增殖的作用。但在肿瘤细胞中,SAMHD1通常会发生突变,这一突变将会影响它的活性,进而影响它的抗肿瘤作用,而且它的表达量也会有不同程度的降低。通过比较结肠癌中的五种单一位点突变体与野生型(109aa-626aa),我们发现,发生突变的SAMHD1依然能够在原核生物中表达,只是可溶性发生了不同程度的变化,虽然以包涵体形式表达的蛋白占大部分,但通过扩大培养,仍然可以得到足够量的可溶性蛋白用于纯化和后续的结构方面的研究。在一定条件下,在适量的纯化后的SAMHD1及其突变体中加入底物d GTP后,反应一段时间,通过高效液相色谱法测定SAMHD1及其突变体的活力,我们发现,结肠癌中SAMHD1相关突变体其d NTPase活性大部分几乎完全丧失,这极有可能是造成它不能抑制肿瘤细胞增殖的原因。相关报道表明,SAMHD1可以抑制逆转录元件LINE-1的转座,而这一功能与肿瘤的发生也密切相关。通过在细胞中共转染LINE-1及SAMHD1及其突变体质粒,我们发现,突变后的SAMHD1对LINE-1的抑制能力降低,这可能是导致肿瘤发生的一个因素。之前对SAMHD1的研究结果还表明,SAMHD1的抗病毒作用可以被HIV-2/SIV的附属蛋白Vpx抑制,Vpx通过招募E3泛素复合物,使SAMHD1发生泛素化,从而被蛋白酶体降解。在SAMHD1及其相关结肠癌突变体的研究中,我们发现SAMHD1及其突变体在与Vpx互作方面,除了D497Y,和野生型一样,依然可以被Vpx降解。这可能是由于D497Y距Vpx互作区比较近,其突变引发的构象改变导致了其不能被Vpx降解。本文通过对结肠癌中的SAMHD1五种突变体进行研究,主要研究突变后的SAMHD1和野生型的SAMHD1相比在消耗细胞内d NTPs的差异,也就是SAMHD1的酶活性的差异,以及被Vpx降解的差异及对逆转录转座子LINE-1的影响。最终我们得到的一些数据,对于SAMHD1及其相关疾病突变体的功能深入研究提供了一些参考。
[Abstract]:SAMHD1 is a protein encoded by eukaryotic genes. It is a natural antiviral factor with d NTPase activity, which can reduce the level of d NTPs in cells and thus inhibit the replication of virus. Expressed in many tissues of the human body, it is a rich protein. SAMHD1 can not only inhibit the replication of the virus, Moreover, SAMHD1 expressed in many tumor cells also consumes d NTPsin the cell, which inhibits the proliferation of tumor cells. But in tumor cells, SAMHD1 usually mutates, and this mutation will affect its activity. By comparing the five single-locus mutants in colon cancer with wild type 109aa-626a, we found that the mutated SAMHD1 could still be expressed in prokaryotes. But the solubility changed in different degrees, although the protein expressed in the form of inclusion body accounted for the majority, but through the expansion of culture, A sufficient amount of soluble protein can still be obtained for purification and subsequent structural studies. Under certain conditions, the substrates d GTP were added to the purified SAMHD1 and its mutants for a period of time. The activity of SAMHD1 and its mutants was determined by HPLC. We found that the d NTPase activity of SAMHD1 related mutants in colon cancer was almost completely lost. This is probably the reason why it can not inhibit the proliferation of tumor cells. It has been reported that SAMHD1 can inhibit the transposition of the reverse transcription element LINE-1. By co-transfection of LINE-1 and SAMHD1 and their mutant particles, we found that the inhibition of LINE-1 by mutated SAMHD1 was decreased. This may be a factor leading to tumorigenesis. Previous studies of SAMHD1 have also shown that the antiviral effect of SAMHD1 can be inhibited by Vpx, a subsidiary protein of HIV-2/SIV, which induces ubiquitin in SAMHD1 by recruiting the E3 ubiquitin complex. In the study of SAMHD1 and its associated colon cancer mutants, we found that SAMHD1 and its mutants interact with Vpx, except for D497Y, which is similar to the wild type. It is possible that D497Y can not be degraded by Vpx due to the conformational change caused by the conformation of D497Y, which is close to the interaction region between Vpx and D497Y. In this paper, we studied five SAMHD1 mutants in colon cancer. The difference of d NTPs in consuming cells between mutant SAMHD1 and wild type SAMHD1, that is, the difference of enzyme activity of SAMHD1, the difference of degradation by Vpx and the effect on retrotransposon LINE-1 were studied. Some references are provided for the further study of the function of SAMHD1 and its related disease mutants.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35

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