Vif-CBF-β-ElonginB-ElonginC-Cullin5 E3复合物结构及相互作用机制研究

发布时间：2018-04-23 11:49

本文选题：HIV-1 + 相互作用　；参考：《吉林大学》2013年博士论文

【摘要】：Vif（viral infectivity factor）基因编码的蛋白作为HIV-1（AIDS病原体）的六个辅助因子之一，是一个高碱性的分子量为23kDa的磷蛋白，它在逆转录病毒生命周期的早期步骤和晚期步骤中发挥功能。HIV-1对Vif的需求受产病毒细胞的类型限制，即Vif对于HIV-1在非允许型细胞系中产生有传染效力的子代病毒和有效感染其他细胞至关重要，缺失了Vif的HIV-1能在允许细胞中而不能在非允许细胞有效复制。而特异性表达于非允许细胞中的APOBEC3G（CEM15）提供了这个非允许特性。它在2002年被鉴定为抗HIV-1的宿主因子。它主要作用于病毒生命周期的早期，能够包装进入多种逆转录病毒（HIV-1/HIV-2，马传染性贫血病毒，多种猴免疫缺陷病毒等等），随即启动大规模的针对逆病毒新生负义链cDNA的脱氨基作用，，借此引发病毒基因组的高突变，导致病毒蛋白发生错义突变或提前终止病毒蛋白转录。 Vif能够对A3G的抗病毒作用进行中和，它通过将APOBEC3G募集到人体内的CBF-β-ElonginB-ElonginC-Cullin5-Rbx（CBF-b-EloB-EloC-Cul5-Rbx）复合物上形成E3泛素连接酶上介导其降解。Vif用于募集E3复合物的功能位点已经被研究得比较透彻，Vif的N端包含了与CBF-β和A3G相互作用的位点，Vif的C端包含一个用于结合EloB-EloC的SOCS-box基序，一个用于结合Cul5的HCCH锌指基序。E3复合物中的每个组分对于降解A3G都不可或缺。Vif在复合物中作为A3G底物的接头分子，新发现的CBF-β通过调节Vif折叠作为调节子，EloB-EloC也是调节亚基而Cul5为基架蛋白。尽管关于Vif各区域功能的报道与日俱增，Vif结构仍未能得到彻底解析，2008年Vif SOCS基序的结构得到解析然而更多的Vif结构信息仍有待探索。作为新发现的调节亚基，CBF-β乃至整个E3的功能机制仍有待完善。本论文针对Vif-EloB-EloC复合物结构以及该复合物各组分间的相互作用机制展开了研究。我们对该复合物的组成蛋白进行多种截短和组合，通过X-蛋白晶体衍射技术试图进一步解析Vif结构及其与EloB-EloC的相互作用界面。以期获得最直接的作用机制信息指导治疗药物设计。通过蛋白共折叠可溶性分析，RNA沉默，免疫共沉淀，体内稳定性，关键基序突变等多种分子生物学手段分析E3复合物各组分相互作用机制。在蛋白复合物结构研究部分，应用改良载体PMR1以及具有多个读码框，能够同时表达多种蛋白的原核表达载体pST39作为复合物表达载体，我们构建，表达并纯化了HIV-1Vif蛋白单体及其六种复合物：PMR1-Vif；pST-EloC-EloB-vif；pST-EloC-EloB-Vif-Cul511-191； pST-EloC-EloB-Vif-Cul51-159；pST-EC-EB-Vif-Cul51-230； pST-EloCΔN17-EloB-VifΔN97； pST-EloCΔN17-EloBΔC20-VifΔN97。均获得可供晶体生长的可溶蛋白，其中Vif单体，EloCΔN17-EloB-VifΔN97，EloCΔN17-EloBΔC20-VifΔN97获得了纯度在95%以上的寡聚蛋白，Vif单体蛋白获得衍射为1.6的晶体，然而由于缺乏结构模型，硒代衍生物结晶失败而无法解析。随后我们对纯化得到的EloCΔN17-EloB-VifΔN97蛋白复合物进行了温和的胰蛋白酶剪切处理，去掉折叠松散，肽链骨架灵活度过高的区段，使得该复合物结晶概率提高。得到分辨率2.4可供结构解析的晶体，然而由于该晶体为孪晶难以成功解析，现正对已获得的结晶条件进行结晶温度优化，沉淀剂浓度优化，PH值优化等一系列优化，以期提高晶体质量，得到成功解析。对酶切处理前后的复合物VifΔN97-EB-ECΔN17进行免疫印迹法分析，发现酶切后EloB，EloC分子量没有明显改变，而Vif的分子量显著减小到4-5kDa，通过液相串联质谱联用分析认为酶切后Vif片段约为残基123-168，说明该区段折叠紧实因而防止了胰蛋白酶的剪切，揭示了Vif与EloB-EloC共折叠时C端的折叠核心。而对酶切前后复合物进行浊度分析等物理化学性质分析发现酶切后的复合物性质与酶切前有所不同，酶切后的蛋白热稳定性更优于酶切前。通过该部分研究，我们对从一个新的角度分析了Vif C端的折叠。在复合物功能分析部分，为了深入研究Vif-EloB-EloC-CBF-β复合物成员的功能与组装，我们沉默了内源EloB的表达，发现以下结论：（1）和以前一些研究结果相同，沉默了内源的EloB后，EloC的内源表达量将随之下降，再次肯定了EloB和EloC是作为专性复合物存在的。（2）EloB表达下调后，影响了Vif对A3G的降解，使得Vif阻止A3G包装进病毒的功能下降，从而使Vif拮抗A3G抗病毒的能力下降。（3）研究这一现象的机理，我们发现EloB表达下调后，会显著削弱CBF-β, Cul5与Vif的结合而不影响A3G与Vif的结合。（4）我们使用了VifΔSLQ这一能够削弱EloB-EloC结合的Vif突变体，发现其结合CBF-β的能力显著弱于野生型Vif。（5）我们构建了删除Vif结合位点的EloBΔC34突变体，发现该突变体的过表达能够削弱Vif对A3G的降解能力且该削弱为剂量依赖性的，也能够削弱Vif对A3G抗病毒功能的拮抗。（6）使用删除了EB作用位点的VifΔPPL突变体，发现该突变能够削弱CBF-β与Vif的作用。（7）在大肠杆菌内通过共表达可溶性验证发现，只有EloB-EloC存在的情况下，才能够通过辅助Vif折叠使得Vif与CBF-β大量存在于上清中。我们由以上发现得到结论：Vif与EloB-EloC的相互作用对于Vif与CBF-β的相互作用十分重要。此外我们还发现EloB及其泛素样结构域的过表达能够显著稳定Vif在细胞内的蛋白水平，该发现丰富了泛素样分子的功能多样性。本研究为解析Vif-EloB-EloC复合物相互作用的结构界面提供了基础，延伸了我们对Vif-Cul5E3复合物功能机制的理解，为HIV的药物治疗提供了新的可能性。
[Abstract]:The protein encoded by the Vif (viral infectivity factor) gene is one of the six cofactors of the HIV-1 (AIDS pathogen), and is a highly alkaline phosphor with a molecular weight of 23kDa. It plays the function of.HIV-1 to Vif in the early steps of the retroviral life cycle and in the late steps, which is limited by the type of virus producing cells, that is, Vif pairs. HIV-1 is essential for the generation of infective offspring viruses in non permissible cells and the effective infection of other cells, and the missing Vif HIV-1 can not be effectively replicated in non permissible cells in permissible cells. APOBEC3G (CEM15) specifically expressed in non permissible cells provides this non permissible characteristic. In 2002, it was identified. It is defined as the host factor of anti HIV-1. It mainly acts on the early stage of the virus's life cycle, and can be packed into a variety of retroviruses (HIV-1/HIV-2, equine infectious anemia virus, multiple monkey immunodeficiency virus, etc.), and then initiates a large-scale deamination of cDNA for the negative sense chain of the reverse virus, thus triggering the high genome of the virus. Mutation leads to missense mutation or premature termination of viral protein transcription.
Vif can neutralize the antiviral effect of A3G, which has been well studied through the formation of a E3 ubiquitin ligase on the CBF- beta (CBF-b-EloB-EloC-Cul5-Rbx) complex of the human body to form a E3 ubiquitin ligase on the CBF- beta -ElonginB-ElonginC-Cullin5-Rbx (CBF-b-EloB-EloC-Cul5-Rbx) complex in the human body. The N end of the Vif is included with CB. The site of the interaction of F- beta and A3G, the C end of Vif contains a SOCS-box motif for the combination of EloB-EloC, and a HCCH zinc finger based.E3 complex for Cul5, which is indispensable for the degradation of A3G, which is indispensable for.Vif in the complex as a A3G substrate. EloB-EloC also regulates subunits and Cul5 as the base protein. Although reports on the functions of Vif are increasing, the structure of Vif has not been thoroughly resolved. In 2008, the structure of the Vif SOCS motif is resolved, but more Vif structure information remains to be explored. As a newly discovered subunit, the functional mechanism of CBF- beta and even the whole E3 is still available. Be perfect.
In this paper, the structure of Vif-EloB-EloC complex and the interaction mechanism between the components of the complex are studied. We make a variety of truncation and combination of the composition of the complex. Through the X- crystal diffraction technique, we try to further analyze the Vif structure and the interaction interface with EloB-EloC. The interaction mechanism of E3 complex was analyzed by a variety of molecular biological means, such as protein co folding soluble analysis, RNA silencing, immunoprecipitation, stability in the body, key sequence mutation and other molecular biological means.
In the protein complex structure research part, we constructed, expressed and purified the HIV-1Vif protein monomer and its six complexes, PMR1-Vif; pST-EloC-EloB-vif; pST-EloC-EloB-Vif-Cul5, with the modified carrier PMR1 and a number of reading frame, which can express a variety of proteins at the same time as a complex expression vector. 11-191; pST-EloC-EloB-Vif-Cul51-159; pST-EC-EB-Vif-Cul51-230; pST-EloC Delta N17-EloB-Vif Delta N97; pST-EloC Delta N17-EloB Delta C20-Vif Delta N97. all obtained soluble protein for crystal growth, including Vif monomer, EloC Delta N17-EloB-Vif Delta. White obtained a crystal of 1.6 diffraction, however, due to the lack of structural models, the crystallization of the selenide derivatives failed to be resolved. Then we treated the purified EloC Delta N17-EloB-Vif Delta N97 protein complex by mild trypsin treatment to remove the areas of folded looseness and high activity of the peptide chain framework, making the complex crystallization almost. The resolution 2.4 can be obtained for the crystal of structure resolution. However, because the crystal is difficult to analyze the twins, the crystallization temperature is optimized, the concentration of precipitant is optimized, the pH value is optimized, so as to improve the crystal quality and obtain the successful analysis.
The Vif Delta N97-EB-EC Delta N17 before and after the enzyme digestion was analyzed by immunoblotting, and the molecular weight of EloC was not significantly changed after the enzyme was cut, and the molecular weight of Vif decreased to 4-5kDa significantly, and the Vif fragment of the enzyme was about 123-168 after the digestion of the enzyme by the liquid phase tandem mass spectrometry. It showed that the section was tightly folded and thus prevented the pancreas and thus prevented the pancreas. The shear of protease reveals the folding core of the C end when Vif and EloB-EloC are folded. And the physicochemical properties of the turbidimetric analysis of the complex before and after the enzyme digestion show that the properties of the compound after the enzyme are different from that before the enzyme, and the thermostability of the protein after the enzyme is better than that before the enzyme. The angle of the Vif C end is analyzed.
In the complex function analysis part, in order to study the function and assembly of Vif-EloB-EloC-CBF- beta complex members, we silence the expression of endogenous EloB, and find the following conclusion: (1) after the same research results, after the silence of the endogenous EloB, the endogenous expression of EloC will decrease, and EloB and EloC are reaffirmed as special. The existence of sexual complex. (2) the downregulation of EloB affects the degradation of Vif to A3G, which prevents the Vif from decreasing the function of A3G packaging into the virus, thus reducing the ability of Vif to antagonize the antiviral ability of A3G. (3) the mechanism of this phenomenon is studied. We found that the downregulation of EloB expression will significantly weaken the combination of CBF- beta, Cul5 and Vif without affecting A3G and Combined. (4) we used Vif Delta SLQ, a Vif mutant that could weaken EloB-EloC binding, and found that its ability to bind CBF- beta was significantly weaker than that of wild type Vif. (5). We constructed a EloB Delta C34 mutant that deleted the Vif binding site, and found that the overexpression of the mutant could weaken the degradability of Vif to A3G and should be reduced to a dose-dependent manner. It can also weaken the antagonism of Vif to A3G anti-virus function. (6) using the Vif Delta PPL mutant that deletes the EB action site, it is found that the mutation can weaken the effect of CBF- beta and Vif. (7) in Escherichia coli, by co expression of soluble validation, it is found that only EloB-EloC exists in the presence of auxiliary Vif folding to make Vif and CBF- beta large. It is found in the supernatant. We have found that the interaction between Vif and EloB-EloC is very important for the interaction between Vif and CBF- beta. In addition, we also found that the overexpression of EloB and its ubiquitin like domain can significantly stabilize the protein level of Vif in the cell, and the discovery that the functional diversity of ubiquitin like molecules is rich.
This study provides a basis for the analysis of the structural interface of Vif-EloB-EloC complex interactions, and extends our understanding of the functional mechanisms of Vif-Cul5E3 complexes, providing new possibilities for the drug treatment of HIV.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R512.91

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