结核分枝杆菌疫苗候选物蛋白UgpB结构与功能研究
发布时间：2018-12-08 20:38
【摘要】：结核病仍是目前威胁人类健康的重大传染病之一,而TB/HIV双重感染和结核耐药菌株的出现,使得全球范围内结核病防控工作更加困难。卡介苗BCG是当前唯一的结核疫苗,但仅对新生儿效果良好。目前结核病标准疗程需要3到4种药混合使用,治疗周期长达6个月,治疗过程中患者依从度较差。同时,抗药结核患者数量持续增加,甚至已有完全抗药结核病例报道。因此,亟待新的抗结核药物和疫苗来控制结核病的全球肆虐。针对结核分枝杆菌蛋白质组中药物靶点蛋白和疫苗候选物的三维结构研究将推动新型抗结核药物的发现与开发,以及促进抗结核药理学和生物学行为背后的生化机制描述。 ABC转运体超家族是膜整合蛋白,大部分家族成员是耦合ATP水解能量来跨膜转运溶质的转运蛋白。根据溶质运输方向,分为内向转运体和外向转运体。原核生物内向转运蛋白介导了细胞生存和生长相关营养物质的跨膜摄取。目前结核分枝杆菌在人宿主体内的关键营养物质和相应的转运体仍未知。UgpABCE是结核分枝杆菌中5个可辨别碳水化合物输入转运体之一,基因组分析认为它是sn-G3P转运体。相关研究表明其周质结合蛋白UgpB对于结核分枝杆菌体外最优生长非常关键,也被认为是一个潜在的抗结核疫苗候选物。 本研究中,我们解析了结核分枝杆菌UgpB的三维高分辨率结构(1.5A),并基于结构进行了相关生化实验分析。有趣的是,ITC实验结果显示,结核UgpB并不结合sn-G3P,而是结合GPC,暗示了GPC是结核分枝杆菌在宿主体内利用的磷源和碳源之一。结构比对分析表明,大肠杆菌UgpB中对于底物G3P结合至关重要的Trp169在结核UgpB中被Leu205代替。此外,大肠UgpB中其它一些与G3P相互作用的氨基酸残基在结核UgpB中也不保守。因此结核UgpB丧失了对G3P的亲和力。针对Leu205的突变实验也证实了它对于UgpB底物结合的重要性。ITC实验还表明,G2P和麦芽糖不是结核UgpB结合的底物。本课题研究不仅有助于理解结核分枝杆菌在宿主体内对碳源和磷源的利用,也将为抗结核药物或疫苗的开发提供结构基础,进而促进结核病化学疗法的改进。
[Abstract]:Tuberculosis is still one of the major infectious diseases threatening human health at present, and the emergence of TB/HIV double infection and TB resistant strains make it more difficult to prevent and control tuberculosis worldwide. BCG is the only TB vaccine at present, but it only works well in newborns. The current standard course of treatment for tuberculosis requires a combination of 3 to 4 drugs for a period of 6 months with poor compliance. At the same time, the number of drug-resistant TB patients continues to increase, even drug-resistant TB cases have been reported. Therefore, new anti-TB drugs and vaccines are urgently needed to control the global spread of tuberculosis. The research on the three-dimensional structure of drug target protein and vaccine candidate in proteome of Mycobacterium tuberculosis will promote the discovery and development of new anti-tuberculosis drugs and the description of biochemical mechanism behind the pharmacological and biological behaviors of anti-tuberculosis drugs. The ABC transporter superfamily is a membrane integrin, and most of the family members are transporters coupled with ATP hydrolysis energy to transport solute across the membrane. According to the transport direction of solute, it can be divided into inward transporter and extroverted transporter. Prokaryotic inward transporter mediates the transmembrane uptake of nutrients associated with cell survival and growth. At present, the key nutrients and corresponding transporters of Mycobacterium tuberculosis in human host are unknown. UgpABCE is one of the five identifiable carbohydrate transporters in Mycobacterium tuberculosis, which is considered to be a sn-G3P transporter by genomic analysis. Related studies have shown that its periplasmic binding protein UgpB is critical to the optimal growth of Mycobacterium tuberculosis in vitro and is also considered as a potential candidate for anti-tuberculosis vaccine. In this study, we analyzed the three-dimensional high-resolution structure (1.5A) of Mycobacterium tuberculosis UgpB, and analyzed the related biochemical experiments based on the structure. Interestingly, ITC results show that TB UgpB does not bind to sn-G3P, but GPC, suggests that GPC is one of the phosphorus and carbon sources used by Mycobacterium tuberculosis in the host. Structural comparison analysis showed that Trp169, which is important for substrate G3P binding in Escherichia coli UgpB, was replaced by Leu205 in tuberculous UgpB. In addition, some other amino acid residues interacting with G3P in colorectal UgpB are not conserved in tuberculous UgpB. Therefore, TB UgpB lost its affinity to G 3 P. The mutation test for Leu205 also confirmed the importance of G2P and maltose for the binding of UgpB substrates. The ITC experiment also showed that G2P and maltose were not the substrates of UgpB binding of tuberculosis. This study will not only help to understand the utilization of carbon and phosphorus sources by Mycobacterium tuberculosis in the host, but also provide a structural basis for the development of anti-tuberculosis drugs or vaccines, and promote the improvement of TB chemotherapy.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52
，
本文编号：2368966
[Abstract]:Tuberculosis is still one of the major infectious diseases threatening human health at present, and the emergence of TB/HIV double infection and TB resistant strains make it more difficult to prevent and control tuberculosis worldwide. BCG is the only TB vaccine at present, but it only works well in newborns. The current standard course of treatment for tuberculosis requires a combination of 3 to 4 drugs for a period of 6 months with poor compliance. At the same time, the number of drug-resistant TB patients continues to increase, even drug-resistant TB cases have been reported. Therefore, new anti-TB drugs and vaccines are urgently needed to control the global spread of tuberculosis. The research on the three-dimensional structure of drug target protein and vaccine candidate in proteome of Mycobacterium tuberculosis will promote the discovery and development of new anti-tuberculosis drugs and the description of biochemical mechanism behind the pharmacological and biological behaviors of anti-tuberculosis drugs. The ABC transporter superfamily is a membrane integrin, and most of the family members are transporters coupled with ATP hydrolysis energy to transport solute across the membrane. According to the transport direction of solute, it can be divided into inward transporter and extroverted transporter. Prokaryotic inward transporter mediates the transmembrane uptake of nutrients associated with cell survival and growth. At present, the key nutrients and corresponding transporters of Mycobacterium tuberculosis in human host are unknown. UgpABCE is one of the five identifiable carbohydrate transporters in Mycobacterium tuberculosis, which is considered to be a sn-G3P transporter by genomic analysis. Related studies have shown that its periplasmic binding protein UgpB is critical to the optimal growth of Mycobacterium tuberculosis in vitro and is also considered as a potential candidate for anti-tuberculosis vaccine. In this study, we analyzed the three-dimensional high-resolution structure (1.5A) of Mycobacterium tuberculosis UgpB, and analyzed the related biochemical experiments based on the structure. Interestingly, ITC results show that TB UgpB does not bind to sn-G3P, but GPC, suggests that GPC is one of the phosphorus and carbon sources used by Mycobacterium tuberculosis in the host. Structural comparison analysis showed that Trp169, which is important for substrate G3P binding in Escherichia coli UgpB, was replaced by Leu205 in tuberculous UgpB. In addition, some other amino acid residues interacting with G3P in colorectal UgpB are not conserved in tuberculous UgpB. Therefore, TB UgpB lost its affinity to G 3 P. The mutation test for Leu205 also confirmed the importance of G2P and maltose for the binding of UgpB substrates. The ITC experiment also showed that G2P and maltose were not the substrates of UgpB binding of tuberculosis. This study will not only help to understand the utilization of carbon and phosphorus sources by Mycobacterium tuberculosis in the host, but also provide a structural basis for the development of anti-tuberculosis drugs or vaccines, and promote the improvement of TB chemotherapy.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R52
，
本文编号：2368966
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