黄芩苷对Ⅱ型核糖体失活蛋白活性的抑制作用和机制研究
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摘要
核糖体失活蛋白家族是一类广泛存在于植物和细菌中的蛋白毒素，从蓖麻中提取的蓖麻毒素是一种蛋白类毒素，与大肠杆菌分泌的志贺样毒素都隶属于核糖体失活蛋白（RIPs）家族，是存在于自然界中的最强的毒素之一。它们都由两个亚基组成，A亚基具有N-糖苷酶活性，能从第4324位腺嘌呤处切断RNA，导致蛋白质翻译的停止从而引起细胞死亡；B亚基参与蛋白与细胞受体的结合。蓖麻毒素提取工艺简单、中毒剂量低，是一种潜在的生物恐怖武器，对公众的安全存在潜在的威胁。
产志贺毒素大肠杆菌（STEC）是一种常见的革兰氏阴性致病菌，是一种重要的人兽共患致病菌，可以导致动物和人的多种疾病。肠出血性大肠杆菌（EHEC）O157:H7是典型代表菌株，可以引起多种严重疾病，如出血性肠炎（HC）、急性溶血性尿毒综合征（HUS）等，严重威胁人类健康。尽管O157:H7感染不推荐用抗生素进行治疗，但临床中分离的菌株对多种抗生素有耐药性，尤其对磺胺类和四环素类抗菌药物的耐药较为常见。由于受到耐药性的限制和抗生素治疗的局限性导致临床中对O157:H7感染的治疗面临无药可用的局面，因此，临床急需开发新的抗O157:H7感染的药物。志贺样毒素（Stxs）是STEC分泌的主要毒力因子，其中II型志贺毒素（Stx2）毒性较强，是引起人HC和HUS的物质基础。传统抗生素以杀菌或抑菌为目的，作用后导致菌体裂解大量的毒素释放到体内，不仅对疾病没有治疗作用还会加重患者的症状，提高死亡的风险。因此，近年来对抗STEC感染的药物研发从抑菌转变为抑制菌体主要毒力因子表达和活性，Stx2作为STEC最重要的毒力因子，有望成为抗大肠杆菌感染药物开发的靶标。
由于Stxs和蓖麻毒素的强毒性，因此研究中和Stxs和蓖麻毒素活性的化合物或抗体成为目前的研究热点。目前抗体研究已经取得了显著的进展，已经研发出有20余种可以中和志贺毒素和蓖麻毒素活性的抗体，然而小分子抑制剂却没有太大进展，多数小分子对动物模型没有保护作用。中草药的使用在我国有悠久的历史，在治疗感染性疾病和中毒性疾病中也有广泛的应用。尤其在近年来细菌耐药性高发的后抗生素时代，中草药治疗感染性疾病的应用将丰富感染性疾病治疗的内容。我国中草药资源丰富，所含化学成分复杂多样，是未来药物开发的天然宝库。
本研究以金葡菌α-溶血素为靶标，筛选能抑制其活性的小分子，发现千层纸素A能通过抑制寡聚化而抑制α-溶血素的活性，进一步应用分子对接技术发现千层纸素A和类似物黄芩苷能与EHEC O157:H7分泌的Stx2结合。因此以Stx2和蓖麻种子中分离的蓖麻毒素为靶标，从清热解毒类中药主要化学成分中筛选能抑制两种毒素的天然化合物。首先通过基因克隆、原核表达和纯化得到了纯度高的重组II型志贺毒素，通过硫酸铵沉淀和亲和层析从蓖麻种子中提取到了蓖麻毒素蛋白；将得到的蛋白分别通过对Hela细胞的毒性作用、对体外蛋白质翻译的抑制作用和对小鼠的致死作用评价了其生物学活性。从细胞水平研究了9种受试化合物对Stx2和蓖麻毒素引起的Hela细胞的细胞毒性的保护作用，结果发现从中药黄芩中提取的黄芩苷对Stx2和蓖麻毒素引起的Hela细胞死亡有保护作用。进一步通过无细胞蛋白质翻译系统研究发现，黄芩苷对由两种毒素引起的萤火虫荧光素酶合成的抑制有恢复作用，且呈现剂量依赖性，该结果提示黄芩苷可能直接抑制了毒素的活性而发挥作用。
通过腹腔注射纯化的重组Stx2和蓖麻毒素建立了小鼠急性溶血性尿毒综合征模型和小鼠蓖麻毒素中毒模型，考察给予黄芩苷后对小鼠模型的治疗作用。结果显示，黄芩苷治疗后两个小鼠模型的存活率显著提高；对由Stx2引起的小鼠体重下降有显著的恢复作用；小鼠血清中尿素和肌酐的含量显著下降，提示对肾功能下降有改善作用；组织病理学研究发现小鼠给予黄芩苷后肾小球肿胀减轻，没有管型出现，肾小管上皮细胞脱落减轻；肾组织细胞因子分析发现，黄芩苷治疗后小鼠肾组织中的主要炎性因子IL-1β、IL-4、IL-6、TNF-α和IFN-γ的含量显著降低。
为了阐明黄芩苷抑制Stx2和蓖麻毒素活性的机制，我们应用结构生物学方法得到了蓖麻毒素A亚基和黄芩苷的复合物晶体，通过X射线衍射技术解析了分辨率为2.2复合物的晶体结构。根据结构信息分析发现黄芩苷促进蓖麻毒素A亚基形成聚合而失去功能，主要结合位点为R189, T190, R193, Y194, R235和R258，通过比对蓖麻毒素A亚基和II型志贺毒素A亚基的蛋白序列发现其可能的结合位点为R179, Q180, S183, E184和V218。
为了验证结构生物学得到的结合位点，我们应用QuikChange点突变方法将结合位点做了相应的点突变，应用分析超速离心技术和体外无细胞蛋白翻译系统研究了突变体蛋白与黄芩苷的作用。结果发现，野生型蓖麻毒素A亚基加入黄芩苷后可形成大量多聚体，而突变体蛋白形成多具体显著减少。结合体外蛋白质翻译试验发现蓖麻毒素与黄芩苷的主要结合位点为R189、T190和R193；Stx2突变体与黄芩苷的主要结合位点为R179、E184和V218。
综上所述，黄芩苷可以直接作用于Stx2和蓖麻毒素A亚基，诱导其形成寡聚体，从而抑制其活性的发挥，对Stx2引起的小鼠急性溶血性尿毒综合征和蓖麻毒素引起的中毒有治疗作用。本研究通过结构生物学阐明了黄芩苷抗Stx23和蓖麻毒素活性的作用机制，为制备抗EHEC感染和蓖麻毒素中毒药物的研发提供依据。
Ricin extracted from Ricinus communis and Stx2belong to type II ribosomeinactivating proteins (RIPs II), which is one of the most toxic toxins in the world.Both toxins are composed of A and B subunits, A subunit has N-glycosidase activitythat can cut off ribosome on the28S rRNA at the position of4324, which leads to celldeath. Ricin is easy to extract and has high toxicity, making it a potential bioterroristweapon, which can cause severe threat to the public.
Shiga toxin-producing Escherichia Coli (STEC) is a gram-negative bacterium,which also is an important zoonotic pathogen that can cause a variety of diseases bothin animals and human. Enterohemorrhage E.Coli (EHEC) O157:H7is a typical strainof STEC that can cause severe diseases, such as hemorrhagic colitis (HC) andhemolytic uremic syndrome (HUS). EHEC O157:H7infections is becoming a seriousthreat to human health, which can cause public health problems in the world.Antibiotics are not recommended for the treatment of O157:H7infections, butantibiotics resistance was observed from strains isolated from clinic, particularresistance to tetracyclines and sulfonamides are more common. Due to the limit oftreatment with antibiotics and antibiotics resistance, there is an urgent need to identifynew antimicrobial agents to combat with these lethal infections associated withO157:H7. Shiga like toxins (Stxs) are the major virulence factors secreted by STEC.Furthermore, shiga like toxin2(Stx2) is about1000times more toxic than shiga liketoxin1(Stx1), which is considered that associated with HC and HUS. Purpose oftraditional antibiotics is bactericidal or bacteriostatic, however treatment with theseagents can increase symptoms and raise the risk of death. The strategy of developingnovel drugs against STEC infections is tuned to inhibit the expression or activity ofvirulence factors. Stx2, the major virulence factor produced by STEC, is expected tobe target of anti-STEC infections.
Because of the high toxicity of ricin and Stxs, there is an urgent need for the development of small-molecule inhibitors or antibodies against these lethal toxins.There are more than20antibodies have been reported that can neutralize the toxicityof ricin and Stxs. Nevertheless, there is only few small-molecular inhibitors that wasdiscovered, and most of these inhibitors showed little activity in animal models.Chinese traditional herbs have been applied for thousands of years in China, whichalso play an important role in the treatment of infectious diseases. Particularly, in theage of post-antibiotic, usage of Chinese herbs can abundant the treatment of infectiousdiseases. There are thousands of herbs in our country which contain multi naturalcompounds, and therefore can be a natural source for the drug discovery in the future.
In this study, we screened oroxylin A that can inhibit the activity of α-hemolysinsecreted by S. aureusvia inhibiting formation of oligomers. Furthermore, moleculardocking was performed and identified that oroxylin A could bind on the surface ofStx2. Then Stx2and ricin were used as target of novel drug discovery from Chineseherbs. We amplified stx2gene from EHEC O157:H7and overexpressed recombinantStx2(rStx2) using prokaryotic expression system in E. coli and isolated ricin fromcastor beans. Firstly, the biological activity of the purified toxins was evaluated byHela cells, cell-free translation assays and lethal effect to mice both in vitro and invivo.9compounds were tested for the protection of Hela cells induced by rStx2andricin, the results showed that baicalin, a flavonoid compound extracted fromScutellaria baicalensis Georgi, could protect Hela cells against rStx2and ricin,oroxylin A and baicalein only showed the activity on inhibiting the activity ofhemolysin secreted by Staphylococcus aureus. Furthermore, we found that baicalincould recover suppress of luciferase expression in a dose-dependent manner. Theseresults indicated that baicalin could inhibit the activity of thses toxins directly.
Mice model of HUS and ricin toxication was established by injection of purifiedtoxins intraperitoneally to evaluate the therapeutical effect to these toxins. The resultsshowed that treatment with baicalin could significantly increase the survival rate ofmice in both model. Baicalin could recover the body weight loss caused by theinjection of rStx2; it could also reduce the level of blood urea nitrogen and creatininewhich indicate that can improve the renal function. Histopathological study found thatbaicalin could decrease the damage in tubules and glomerulus. Analysis of cytokines in renal tissue showed that mice treated with baicalin could significantly reduce theexpression of IL-1β, IL-4, IL-6, TNF-α and IFN-γ.
We obtained the crystal of ricin A chain (RTA) and baicalin complex to clarify themechanism of baicalin inhibiting the activity of ricin and Stx2. The structure of thecomplex was solved by X-ray crystallography to2.2, and according to the structurebaicalin could bind to RTA and promote oligomerization of RTA which led to loss ofactivity. The main binding site was R189, T190, R193, Y194, R235and R258.Furthermore, we superimposed the structure of RTA and Stx2, and found that thepotential binding site of baicailin and Stx2which is composed of R179, Q180, S183,E184and V218.
We performed QuikChange site Mutagenesis to obtain mutant proteins of the toxinsto verify the binding site. Then analytical ultracentrifugation and cell free translationassays were performed to investigate the effect of baicalin on mutant toxins. Theresults showed that wild type RTA could form large amount of oligomer in thepresence of baicalin compared to those mutations. Furthermore, the cell freetranslation assay showed that mutations of R189A, T190A and R193A with baicalincould still inhibit the expression of luciferase. And Stx2revealed the same tendency,R179, E184, V218served as the main binding site.
In summary, baicalin can inhibit the activity of RTA and Stx2A by formingoligomer, baicalin can protect mice against HUS induced by Stx2and ricin toxication.Furthermore, we solved the structure of RTA-baicalin complex and clarified themechanism of baicalin. Our study will lay the development of innovative drugsofanti-EHEC infections and ricin.
产志贺毒素大肠杆菌（STEC）是一种常见的革兰氏阴性致病菌，是一种重要的人兽共患致病菌，可以导致动物和人的多种疾病。肠出血性大肠杆菌（EHEC）O157:H7是典型代表菌株，可以引起多种严重疾病，如出血性肠炎（HC）、急性溶血性尿毒综合征（HUS）等，严重威胁人类健康。尽管O157:H7感染不推荐用抗生素进行治疗，但临床中分离的菌株对多种抗生素有耐药性，尤其对磺胺类和四环素类抗菌药物的耐药较为常见。由于受到耐药性的限制和抗生素治疗的局限性导致临床中对O157:H7感染的治疗面临无药可用的局面，因此，临床急需开发新的抗O157:H7感染的药物。志贺样毒素（Stxs）是STEC分泌的主要毒力因子，其中II型志贺毒素（Stx2）毒性较强，是引起人HC和HUS的物质基础。传统抗生素以杀菌或抑菌为目的，作用后导致菌体裂解大量的毒素释放到体内，不仅对疾病没有治疗作用还会加重患者的症状，提高死亡的风险。因此，近年来对抗STEC感染的药物研发从抑菌转变为抑制菌体主要毒力因子表达和活性，Stx2作为STEC最重要的毒力因子，有望成为抗大肠杆菌感染药物开发的靶标。
由于Stxs和蓖麻毒素的强毒性，因此研究中和Stxs和蓖麻毒素活性的化合物或抗体成为目前的研究热点。目前抗体研究已经取得了显著的进展，已经研发出有20余种可以中和志贺毒素和蓖麻毒素活性的抗体，然而小分子抑制剂却没有太大进展，多数小分子对动物模型没有保护作用。中草药的使用在我国有悠久的历史，在治疗感染性疾病和中毒性疾病中也有广泛的应用。尤其在近年来细菌耐药性高发的后抗生素时代，中草药治疗感染性疾病的应用将丰富感染性疾病治疗的内容。我国中草药资源丰富，所含化学成分复杂多样，是未来药物开发的天然宝库。
本研究以金葡菌α-溶血素为靶标，筛选能抑制其活性的小分子，发现千层纸素A能通过抑制寡聚化而抑制α-溶血素的活性，进一步应用分子对接技术发现千层纸素A和类似物黄芩苷能与EHEC O157:H7分泌的Stx2结合。因此以Stx2和蓖麻种子中分离的蓖麻毒素为靶标，从清热解毒类中药主要化学成分中筛选能抑制两种毒素的天然化合物。首先通过基因克隆、原核表达和纯化得到了纯度高的重组II型志贺毒素，通过硫酸铵沉淀和亲和层析从蓖麻种子中提取到了蓖麻毒素蛋白；将得到的蛋白分别通过对Hela细胞的毒性作用、对体外蛋白质翻译的抑制作用和对小鼠的致死作用评价了其生物学活性。从细胞水平研究了9种受试化合物对Stx2和蓖麻毒素引起的Hela细胞的细胞毒性的保护作用，结果发现从中药黄芩中提取的黄芩苷对Stx2和蓖麻毒素引起的Hela细胞死亡有保护作用。进一步通过无细胞蛋白质翻译系统研究发现，黄芩苷对由两种毒素引起的萤火虫荧光素酶合成的抑制有恢复作用，且呈现剂量依赖性，该结果提示黄芩苷可能直接抑制了毒素的活性而发挥作用。
通过腹腔注射纯化的重组Stx2和蓖麻毒素建立了小鼠急性溶血性尿毒综合征模型和小鼠蓖麻毒素中毒模型，考察给予黄芩苷后对小鼠模型的治疗作用。结果显示，黄芩苷治疗后两个小鼠模型的存活率显著提高；对由Stx2引起的小鼠体重下降有显著的恢复作用；小鼠血清中尿素和肌酐的含量显著下降，提示对肾功能下降有改善作用；组织病理学研究发现小鼠给予黄芩苷后肾小球肿胀减轻，没有管型出现，肾小管上皮细胞脱落减轻；肾组织细胞因子分析发现，黄芩苷治疗后小鼠肾组织中的主要炎性因子IL-1β、IL-4、IL-6、TNF-α和IFN-γ的含量显著降低。
为了阐明黄芩苷抑制Stx2和蓖麻毒素活性的机制，我们应用结构生物学方法得到了蓖麻毒素A亚基和黄芩苷的复合物晶体，通过X射线衍射技术解析了分辨率为2.2复合物的晶体结构。根据结构信息分析发现黄芩苷促进蓖麻毒素A亚基形成聚合而失去功能，主要结合位点为R189, T190, R193, Y194, R235和R258，通过比对蓖麻毒素A亚基和II型志贺毒素A亚基的蛋白序列发现其可能的结合位点为R179, Q180, S183, E184和V218。
为了验证结构生物学得到的结合位点，我们应用QuikChange点突变方法将结合位点做了相应的点突变，应用分析超速离心技术和体外无细胞蛋白翻译系统研究了突变体蛋白与黄芩苷的作用。结果发现，野生型蓖麻毒素A亚基加入黄芩苷后可形成大量多聚体，而突变体蛋白形成多具体显著减少。结合体外蛋白质翻译试验发现蓖麻毒素与黄芩苷的主要结合位点为R189、T190和R193；Stx2突变体与黄芩苷的主要结合位点为R179、E184和V218。
综上所述，黄芩苷可以直接作用于Stx2和蓖麻毒素A亚基，诱导其形成寡聚体，从而抑制其活性的发挥，对Stx2引起的小鼠急性溶血性尿毒综合征和蓖麻毒素引起的中毒有治疗作用。本研究通过结构生物学阐明了黄芩苷抗Stx23和蓖麻毒素活性的作用机制，为制备抗EHEC感染和蓖麻毒素中毒药物的研发提供依据。
Ricin extracted from Ricinus communis and Stx2belong to type II ribosomeinactivating proteins (RIPs II), which is one of the most toxic toxins in the world.Both toxins are composed of A and B subunits, A subunit has N-glycosidase activitythat can cut off ribosome on the28S rRNA at the position of4324, which leads to celldeath. Ricin is easy to extract and has high toxicity, making it a potential bioterroristweapon, which can cause severe threat to the public.
Shiga toxin-producing Escherichia Coli (STEC) is a gram-negative bacterium,which also is an important zoonotic pathogen that can cause a variety of diseases bothin animals and human. Enterohemorrhage E.Coli (EHEC) O157:H7is a typical strainof STEC that can cause severe diseases, such as hemorrhagic colitis (HC) andhemolytic uremic syndrome (HUS). EHEC O157:H7infections is becoming a seriousthreat to human health, which can cause public health problems in the world.Antibiotics are not recommended for the treatment of O157:H7infections, butantibiotics resistance was observed from strains isolated from clinic, particularresistance to tetracyclines and sulfonamides are more common. Due to the limit oftreatment with antibiotics and antibiotics resistance, there is an urgent need to identifynew antimicrobial agents to combat with these lethal infections associated withO157:H7. Shiga like toxins (Stxs) are the major virulence factors secreted by STEC.Furthermore, shiga like toxin2(Stx2) is about1000times more toxic than shiga liketoxin1(Stx1), which is considered that associated with HC and HUS. Purpose oftraditional antibiotics is bactericidal or bacteriostatic, however treatment with theseagents can increase symptoms and raise the risk of death. The strategy of developingnovel drugs against STEC infections is tuned to inhibit the expression or activity ofvirulence factors. Stx2, the major virulence factor produced by STEC, is expected tobe target of anti-STEC infections.
Because of the high toxicity of ricin and Stxs, there is an urgent need for the development of small-molecule inhibitors or antibodies against these lethal toxins.There are more than20antibodies have been reported that can neutralize the toxicityof ricin and Stxs. Nevertheless, there is only few small-molecular inhibitors that wasdiscovered, and most of these inhibitors showed little activity in animal models.Chinese traditional herbs have been applied for thousands of years in China, whichalso play an important role in the treatment of infectious diseases. Particularly, in theage of post-antibiotic, usage of Chinese herbs can abundant the treatment of infectiousdiseases. There are thousands of herbs in our country which contain multi naturalcompounds, and therefore can be a natural source for the drug discovery in the future.
In this study, we screened oroxylin A that can inhibit the activity of α-hemolysinsecreted by S. aureusvia inhibiting formation of oligomers. Furthermore, moleculardocking was performed and identified that oroxylin A could bind on the surface ofStx2. Then Stx2and ricin were used as target of novel drug discovery from Chineseherbs. We amplified stx2gene from EHEC O157:H7and overexpressed recombinantStx2(rStx2) using prokaryotic expression system in E. coli and isolated ricin fromcastor beans. Firstly, the biological activity of the purified toxins was evaluated byHela cells, cell-free translation assays and lethal effect to mice both in vitro and invivo.9compounds were tested for the protection of Hela cells induced by rStx2andricin, the results showed that baicalin, a flavonoid compound extracted fromScutellaria baicalensis Georgi, could protect Hela cells against rStx2and ricin,oroxylin A and baicalein only showed the activity on inhibiting the activity ofhemolysin secreted by Staphylococcus aureus. Furthermore, we found that baicalincould recover suppress of luciferase expression in a dose-dependent manner. Theseresults indicated that baicalin could inhibit the activity of thses toxins directly.
Mice model of HUS and ricin toxication was established by injection of purifiedtoxins intraperitoneally to evaluate the therapeutical effect to these toxins. The resultsshowed that treatment with baicalin could significantly increase the survival rate ofmice in both model. Baicalin could recover the body weight loss caused by theinjection of rStx2; it could also reduce the level of blood urea nitrogen and creatininewhich indicate that can improve the renal function. Histopathological study found thatbaicalin could decrease the damage in tubules and glomerulus. Analysis of cytokines in renal tissue showed that mice treated with baicalin could significantly reduce theexpression of IL-1β, IL-4, IL-6, TNF-α and IFN-γ.
We obtained the crystal of ricin A chain (RTA) and baicalin complex to clarify themechanism of baicalin inhibiting the activity of ricin and Stx2. The structure of thecomplex was solved by X-ray crystallography to2.2, and according to the structurebaicalin could bind to RTA and promote oligomerization of RTA which led to loss ofactivity. The main binding site was R189, T190, R193, Y194, R235and R258.Furthermore, we superimposed the structure of RTA and Stx2, and found that thepotential binding site of baicailin and Stx2which is composed of R179, Q180, S183,E184and V218.
We performed QuikChange site Mutagenesis to obtain mutant proteins of the toxinsto verify the binding site. Then analytical ultracentrifugation and cell free translationassays were performed to investigate the effect of baicalin on mutant toxins. Theresults showed that wild type RTA could form large amount of oligomer in thepresence of baicalin compared to those mutations. Furthermore, the cell freetranslation assay showed that mutations of R189A, T190A and R193A with baicalincould still inhibit the expression of luciferase. And Stx2revealed the same tendency,R179, E184, V218served as the main binding site.
In summary, baicalin can inhibit the activity of RTA and Stx2A by formingoligomer, baicalin can protect mice against HUS induced by Stx2and ricin toxication.Furthermore, we solved the structure of RTA-baicalin complex and clarified themechanism of baicalin. Our study will lay the development of innovative drugsofanti-EHEC infections and ricin.
引文
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