【作者】 王璐；

【导师】 范成鹏；

【作者基本信息】 武汉大学 ， 生物化学与分子生物学， 2020， 硕士

【摘要】 2016年10月至2017年5月爆发在我国华南地区的猪急性腹泻综合征冠状病毒(swine acute diarrhea syndrome coronavirus,SADS-CoV)曾造成20000多头仔猪死亡,并在后续2018年及2019年春季都曾再次爆发,造成大量仔猪死亡及严重的经济损失。2003年SARS冠状病毒爆发加快了科研工作者对冠状病毒研究,但是到目前为止,仍没有研制出能够有效抑制冠状病毒的抗病毒药物。针对冠状病毒刺突蛋白而研制抗体也因刺突蛋白的高度可突变性而应用受限。在该研究背景下,针对冠状病毒中高度保守的非结构蛋白研究冠状病毒抑制剂成为了抗病毒药物研制的新型靶标。冠状病毒编码的多蛋白在非结构蛋白PLPs及主蛋白酶作用下被切割成14-16种非结构蛋白而参与冠状病毒复制复合物的形成。PLP2是一种木瓜样蛋白酶,可以识别并特异性切割多蛋白N端序列,形成nsp1、nsp2和nsp3,对于SARSCoV PLpro的体外研究表明,PLpro具有去泛素化及去ISG化作用,推测其可以帮助冠状病毒在侵染宿主后逃逸机体的免疫应答。Ub和ISG15都是宿主针对病毒感染后先天免疫应答的重要信号传导元件,可被病毒DUB和de ISGylating酶负调控,因此可以作为新型抗病毒药物研制的靶标。本文以猪急性腹泻综合征冠状病毒(SADS-CoV)PLP2为研究对象,发现其与SARS-CoV或MERS-CoV的PLpro序列同源性较低,通过蛋白质晶体学及生物化学等方法,对SADS-CoV PLP2结构生物学研究,探讨其结构和功能的分子机制,并进行抑制剂筛选。我们通过原核表达系统表达和纯化了SADS-CoV PLP2,通过优化蛋白质晶体的生长条件,获得了具有高质量的蛋白质晶体,利用X-射线衍射法重构了PLP2蛋白的三维结构,分辨率为1.72(?)。PLP2的拓扑结构与SARS-CoV、MERS-CoV的PLpro类似,都由拇指-手掌-手指结构域组成。与SARS-CoV PLpro结构比对后,发现SADS-CoV PLP2在拇指域和手指域上存在较大差异,及通过与Ub建模分析发现,SADS-CoV PLP2活性位点为C101、H256以D269催化三联体以及周围其他氨基酸残基辅助Ub正确定位到活性位点。通过体外酶学实验方法对PLP2的去泛素化和去ISG化的能力进行研究,即通过PLP2蛋白与底物融合蛋白nsp1-e GFP、Ub-e GFP、ISG15-e GFP共孵育后检测PLP2对底物的特异性切割能力,发现SADS-CoV PLP2与SARS-CoV、MERS-CoV的PLpro蛋白具有相同的催化作用,都可以特异性识别并切割Ub和ISG15,使病毒具备了逃逸机体先天免疫的潜力。通过使用nsp1-AMC、Ub-AMC、ISG15-AMC和两种多肽-AMC底物,结合结构分析、定点突变和酶动力学研究方法,测定米氏常数Km和Kcat,进而确定了与底物结合和催化有关的关键活性位点氨基酸残基突变后对酶活性的影响,我们发现当活性位点及其周边的氨基酸残基突变后,PLP2的亲和力降低,酶活力也降低,但当缺失了N端的49个氨基酸后,PLP2的酶学活性增强。与SARS-CoV PLpro结构比对后,发现SADS-CoV PLP2在结构上存在较大差异,进一步通过与Ub建模分析发现,SADS-CoV PLP2活性位点为C101、H256以D269催化三联体以及周围其他氨基酸残基辅助Ub正确定位到活性位点。最后我们通过抑制剂筛选得到了三种具有显著抑制效果的抑制剂并测定IC50。发现化合物112与PLP2的催化三联体中H256的咪唑基形成氢键,化合物115和119与位于PLP2蛋白BL2环上的G255形成氢键,通过增大空间位阻或改变BL2环的构象从而抑制PLP2与底物的结合,抑制了PLP2蛋白酶的活性。对SADS-CoV PLP2详细描述对于理解该酶在冠状病毒复制酶复合体中的作用机制和对宿主免疫反应的影响是至关重要,也为后续研究及药物开发提供了重要的理论基础。更多还原

【Abstract】 From October 2016 to May 2017,the outbreak of swine acute diarrhea syndrome coronavirus(SADS-Co V)in southern China had caused the death of more than 20,000 piglets,and it had recurred in the subsequent 2018 and 2019 spring,resulted in a large number of piglets died and serious economic losses.The outbreak of SARS coronavirus accelerated the research of coronavirus by scientists,but so far,no antiviral drugs that can effectively inhibit coronavirus have been developed successfully.Antibodies developed against the spike protein of coronavirus are also limited in application due to the high mutability of the spike protein.Under the background of this research,the investigation of inhibitors against highly conserved non-structural proteins in coronaviruses has become a new target for the development of antiviral drugs.Coronavirus encodes polyprotein pp1 a and pp1 ab,which are cleaved into 14-16 nonstructural proteins under the action of non-structural protein PLPs and main protease to participate in the formation of coronavirus replication complex.PLP2 is a papain-like protease that will recognize and cleave specifically the N-terminal sequence of polyprotein pp1 a to form mature nsp1,nsp2,and nsp3.In vitro studies on SARS-Co V PLpro have shown that PLpro has deubiquitination and de ISGylating activity,which is speculated to help coronavirus to escape the immune response of the host after the infection.Both Ub and ISG15 are important signaling components of the host’s innate immune response to viral infections,which can be negatively regulated by the viral DUB and de ISGylating enzymes,so they can be used as targets for the development of new antiviral drugs.In this paper,SADS-Co V PLP2 was selected as the research object,and it was found that it has low sequence homology with PLpro of SARS-Co V or MERS-Co V.The structural biology of SADS-Co V PLP2 was studied by methods of protein crystallography and biochemistry,to explore the molecular mechanism of its structure and function,and to screen the inhibitors based on the structural and biochemical data.We expressed and purified the SADS-Co V PLP2 through the prokaryotic expression system,and obtained the protein crystals with high quality.The three-dimensional structure of the PLP2 protein was determined by X-ray diffraction with the resolution of 1.72 (?).After comparison with the structure of SARS-Co V PLpro,it is found that there are some significant differences in the structure of SADS-Co V PLP2 and SARSCo V PLpro.Based on the analysis of modeling with Ubiquitin-PLP2 complex,it was found that the active sites of SADS-Co V PLP2 were C101,H256 and D269 catalytic triad and other surrounding amino acid residues to help Ub to enter the active site correctly.Enzymatic characterization of PLP2 was carried out by in vitro enzymatic experiments,that is,PLP2 can effectively cleave nsp1-AMC,ubiquitin-AMC,ISG15-AMC and peptide-AMC.The key active site residues related to substrate binding and catalysis were determined by structural analysis,site-directed mutagenesis and enzyme kinetics,and the Michaelis constant Km and kcat and the activity changes after mutation of the amino acid residues at the active site were determined,to verify the results of structural analysis.Finally,we obtained three inhibitors with significant inhibitory effects through inhibitor screening and measured IC50.The detailed description of SADS-Co V PLP2 is crucial for understanding the mechanism of function of the enzyme in the coronavirus replicase complex and its impact on the host immune response,and also provides an important theoretical basis for subsequent research and drug development.更多还原