Structural and functional studies of an insect membrane transporter

背景昆虫选择对杀虫剂不敏感的靶标蛋白，降低了现有害虫防治粮食作物的效率。因此，杀虫剂研究的新蛋白质靶标对未来的粮食安全至关重要(海明威等人，2002年)。乙酰胆碱(ACh)是由胆碱乙酰转移酶(ChAT)在神经末梢细胞质中合成的。然后，它被囊泡性乙酰胆碱转运体(VAChT)泵入储存囊，在神经元去极化时释放到突触间隙(Prado等人，2002年)。乙酰胆碱的作用仅限于昆虫的中枢神经系统。含有螺吲哚支架的VACHT抑制化合物已被证明在几种昆虫中具有杀虫活性(Sluder等人，2012年)。VAChT是一种逆向转运蛋白，它利用ATPase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年)。水病研究预测Vacht有12个跨膜结构域，在结构域I和结构域II之间有一个大的囊内环(Erickson等人，1994)。目前还没有Vacht的晶体结构和功能分析方法。图1.VAChTAim的乙酰胆碱积累该项目的目的是用已知的抑制剂解析Vacht的晶体结构，并开发一种功能性分析方法。识别对底物结合重要的氨基酸残基将是推动该蛋白杀虫化合物设计的动力。生物化学特征最初将在酿酒酵母中进行表达和优化。如果不能从酵母中获得令人满意的稳定的Vacht产量，搜索将扩展到昆虫细胞培养。一旦建立了优化的表达条件，Vacht将通过与维沙米考(已知的抑制剂)结合实验来鉴定。通过将转运蛋白和ATPase重组为蛋白脂质体，将设计一种功能分析来进一步表征Vacht的活性。由于ACh通过Vacht的运输不涉及酶反应，因此需要一种间接检测方法来检测底物。生物物理特征Vacht的物理结构将通过X射线结晶学和在钻石光源进行的数据收集来解析。提纯的Vacht将接受结晶试验，以确定适合蛋白质结晶的条件。MemGold稀疏矩阵筛选将是研究的起点，结合洗涤剂筛选(如DDM、LMNG等)，还将考虑在脂质立方相(LCP)中结晶。将尝试在使用和不使用抑制剂(维沙米考)的情况下使蛋白质结晶。理想情况下，可以拆分Apo和维沙米考的结合结构。使用新型抑制剂的试验也将与先正达合作进行。囊泡乙酰胆碱转运蛋白的功能鉴定及其在胆碱能基因座上的表达。《生物化学》，第269卷，第35号，21929-32页，1994年9月。ISSN 0021-9258。《杀虫剂抗药性概述》。《科学》，第298卷，第5591号，第96-7页，2002年10月。ISSN 1095-9203。NGUYEN，M.L.；COX，G.D.；Parsons，S.M.囊泡乙酰胆碱转运体的动力学参数：两个质子交换为一个乙酰胆碱。《生物化学》，第37卷，第38期，13400-10页，1998年9月。ISSN 0006-2960.PRADO，M.A.等人乙酰胆碱的合成和储存的调控。《神经化学国际》，第41卷，第5期，第291-9页，2002年11月。ISSN 0197-0186.SLUDER，A.等人。螺吲哚类化合物将囊泡型乙酰胆碱转运蛋白确定为杀虫剂作用的新靶点。《公共科学图书馆·综合》，第7卷，第5号，e34712页，2012年。ISSN 1932-6203。

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.