Structural and functional studies of an insect membrane transporter

昆虫膜转运蛋白的结构和功能研究

• 批准号: 2368379
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2368379
• 关键词: Structural; functional; studies; insect; membrane

BackgroundThe selection of target protein insensitive to insecticide by insect is reducing the efficiency of existing pest control toward food crops. Therefore, novel protein target for insecticide research is paramount for future food security (HEMINGWAY et al, 2002). Acetylcholine (ACh) is synthesised in the nerve terminal cytoplasm by choline acetyltransferase (ChAT). It is then pumped into storage vesicles by vesicular acetylcholine transporter (VAChT) to be released into the synaptic clefts upon neuronal depolarisation (PRADO et al, 2002). The role of acetylcholine is restricted to the CNS in insect. VAChT inhibiting compounds containing the spiroindoline scaffold have shown to possess insecticidal activities in several insect species such as D. melanogaster (SLUDER et al, 2012).VAChT is an antiporter, which utilises the proton gradient generated by ATPase, transporting one acetylcholine molecule for two proton (figure 1) (NGUYEN et al, 1998). Hydropathy studies predicts that VAChT have 12 transmembrane domains and a large intravesicular loop between domain I and II (ERICKSON et al, 1994). There is currently no crystal structural and functional assay available for VAChT. Figure 1. Acetylcholine accumulation by VAChTAimThe aim of the project is to resolve the crystal structure of VAChT with known inhibitors and develop a functional assay. Identifying the amino acid residues important for substrate binding would be an impetus in driving the design of insecticidal compound for this protein.Biochemical CharacterisationThe expression and optimisation will be performed in S. cerevisiae initially. If no satisfactory yield of stable VAChT is obtained from yeast, the search will be extended to insect cell culture. Once optimised expression condition have been established, VAChT will be characterised by binding assay with vesamicol (known inhibitor). A functional assay will be designed to further characterise VAChT activities by reconstituting the transporter and ATPase into a proteoliposome. Since the transport of ACh via VAChT involves no enzymatic reaction, an indirect detection method to detect the substrate will be required.Biophysical CharacterisationThe physical structure of VAChT will be resolved by X-ray crystallography with data collection conducted at the Diamond Light Source. Purified VAChT will be subjected to crystal trials to ascertain conditions suitable for protein crystallisation. MemGold sparse matrix screen will be the starting point of the investigation, in conjunction with detergents screening (i.e. DDM, LMNG etc.), crystallisation in lipid cubic phase (LCP) will also be considered. Crystallisation of the protein with and without inhibitor (vesamicol) will be attempted. Ideally the Apo and vesamicol bound structure can be resolved. Assay with novel inhibitors will also be performed in collaboration with Syngenta.ReferencesERICKSON, J. D. et al. Functional identification of a vesicular acetylcholine transporter and its expression from a "cholinergic" gene locus. J Biol Chem, v. 269, n. 35, p. 21929-32, Sep 1994. ISSN 0021-9258. HEMINGWAY, J.; FIELD, L.; VONTAS, J. An overview of insecticide resistance. Science, v. 298, n. 5591, p. 96-7, Oct 2002. ISSN 1095-9203.NGUYEN, M. L.; COX, G. D.; PARSONS, S. M. Kinetic parameters for the vesicular acetylcholine transporter: two protons are exchanged for one acetylcholine. Biochemistry, v. 37, n. 38, p. 13400-10, Sep 1998. ISSN 0006-2960.PRADO, M. A. et al. Regulation of acetylcholine synthesis and storage. Neurochem Int, v. 41, n. 5, p. 291-9, Nov 2002. ISSN 0197-0186.SLUDER, A. et al. Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action. PLoS One, v. 7, n. 5, p. e34712, 2012. ISSN 1932-6203.

背景昆虫对杀虫剂不敏感的靶蛋白的选择正在降低现有对粮食作物害虫防治的效率。因此，杀虫剂研究的新蛋白质靶标对于未来的粮食安全至关重要（HEMINGWAY 等，2002）。乙酰胆碱（ACh）由胆碱乙酰转移酶（ChAT）在神经末梢细胞质中合成。然后通过囊泡乙酰胆碱转运蛋白（VAChT）将其泵入储存囊泡，并在神经元去极化时释放到突触间隙（PRADO等人，2002）。乙酰胆碱的作用仅限于昆虫的中枢神经系统。含有螺吲哚啉支架的 VAChT 抑制化合物已显示出对多种昆虫物种（例如黑腹果蝇）具有杀虫活性（SLUDER 等人，2012）。VAChT 是一种反向转运蛋白，它利用 ATP 酶产生的质子梯度，将一个乙酰胆碱分子转运为两个质子（图 1）（NGUYEN 等人，1998）。水疗法研究预测 VAChT 具有 12 个跨膜结构域以及结构域 I 和 II 之间的大囊泡内环（ERICKSON 等人，1994）。目前还没有可用于 VAChT 的晶体结构和功能测定。 图 1. VAChTAim 积累乙酰胆碱该项目的目的是利用已知抑制剂解析 VAChT 的晶体结构并开发功能测定法。鉴定对底物结合重要的氨基酸残基将成为推动该蛋白质的杀虫化合物设计的动力。生化表征最初将在酿酒酵母中进行表达和优化。如果从酵母中没有获得令人满意的稳定 VAChT 产量，则研究将扩展到昆虫细胞培养。一旦建立了优化的表达条件，VAChT 将通过与维沙考（已知抑制剂）的结合测定来表征。将设计功能测定，通过将转运蛋白和 ATP 酶重建到蛋白脂质体中来进一步表征 VAChT 活性。由于通过 VAChT 传输 ACh 不涉及酶促反应，因此需要一种间接检测方法来检测底物。生物物理表征 VAChT 的物理结构将通过 X 射线晶体学和在钻石光源处进行的数据收集来解析。纯化的 VAChT 将进行晶体试验，以确定适合蛋白质结晶的条件。 MemGold 稀疏基质筛选将是研究的起点，结合洗涤剂筛选（即 DDM、LMNG 等），还将考虑脂质立方相 (LCP) 中的结晶。将尝试使用和不使用抑制剂（维沙考）的蛋白质结晶。理想情况下，可以解析 Apo 和维沙考结合的结构。新型抑制剂的检测也将与先正达合作进行。参考ERICKSON, J. D. 等人。囊泡乙酰胆碱转运蛋白的功能鉴定及其从“胆碱能”基因座的表达。 《生物化学杂志》，第 269 卷，n。 35，p。 21929-32，1994 年 9 月。ISSN 0021-9258。海明威，J.；菲尔德，L.； VONTAS, J. 杀虫剂抗性概述。 《科学》，第 298 卷，n。 5591，页。 96-7，2002 年 10 月。ISSN 1095-9203。NGUYEN，M.L.；考克斯，G.D.；帕森斯，S.M. 囊泡乙酰胆碱转运蛋白的动力学参数：两个质子交换为一个乙酰胆碱。生物化学，第 37 卷，n。 38，p。 13400-10，1998 年 9 月。ISSN 0006-2960。PRADO，M.A. 等人。乙酰胆碱合成和储存的调节。 Neurochem Int，第 41 卷，n。 5，p。 291-9，2002 年 11 月。ISSN 0197-0186。SLUDER，A. 等人。螺吲哚啉将囊泡乙酰胆碱转运蛋白识别为杀虫作用的新靶点。 《公共科学图书馆一》，第 7 卷，n。 5，p。 e34712，2012。ISSN 1932-6203。