Structural and functional studies of an insect membrane transporter

昆虫对杀虫剂不敏感的靶蛋白的选择降低了现有害虫防治对粮食作物的效率。因此，杀虫剂研究的新型蛋白质靶点对未来的粮食安全至关重要（HEMINGWAY et al, 2002）。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。然后通过囊泡乙酰胆碱转运体（VAChT）将其泵入存储囊泡，并在神经元去极化时释放到突触间隙中（PRADO等，2002）。在昆虫中，乙酰胆碱的作用仅限于中枢神经系统。含有螺吲哚碱支架的VAChT抑制化合物已被证明对几种昆虫具有杀虫活性，如黑腹大蠊（SLUDER等，2012）。VAChT是一种反向转运蛋白，它利用atp酶产生的质子梯度，用两个质子运输一个乙酰胆碱分子（图1）（NGUYEN et al, 1998）。亲水性研究预测，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 et al, 1994）。目前还没有晶体结构和功能分析可用于VAChT。图1所示。VAChT的乙酰胆碱积累项目的目的是用已知的抑制剂来解决VAChT的晶体结构，并开发一种功能测定方法。确定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。生化特性首先在酿酒酵母中进行表达和优化。如果不能从酵母中获得稳定的VAChT，则将研究范围扩大到昆虫细胞培养。一旦优化的表达条件建立，VAChT将通过与vesamicol（已知抑制剂）的结合试验进行表征。将设计一个功能分析，通过将转运体和atp酶重组为蛋白质脂质体来进一步表征VAChT的活性。由于乙酰胆碱通过VAChT转运不涉及酶促反应，因此需要一种间接检测底物的方法。生物物理表征VAChT的物理结构将通过x射线晶体学在钻石光源下进行数据收集来解决。纯化的VAChT将进行结晶试验，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，与洗涤剂筛选（即DDM， LMNG等）一起，脂质立方相（LCP）的结晶也将被考虑。将尝试在有或没有抑制剂（维萨米醇）的情况下结晶蛋白质。理想情况下，载脂蛋白和维萨姆醇结合结构可以被分解。新型抑制剂的检测也将与先正达合作进行。erickson， J. D.等。囊泡乙酰胆碱转运蛋白的功能鉴定及其“胆碱能”基因位点的表达。生物化学学报，vol . 26, n. 35, p. 21929- 32,1994。ISSN 0021 - 9258。海明威,j .;场,l;陈晓明，王晓明。我国杀虫剂抗性研究进展。《科学》，2002年10月，第298期，第5591期，第96-7页。ISSN 1095 - 9203。阮，m.l；考克斯博士；囊泡型乙酰胆碱转运体的动力学参数：两个质子交换一个乙酰胆碱。生物化学，vol . 37, n. 38, p. 13400- 10,1998年9月。ISSN 0006 - 2960。普拉多，m.a.等。乙酰胆碱合成和储存的调控。神经化学杂志，vol . 41, n. 5, p. 291- 9,2002年11月。ISSN 0197 - 0186。斯莱德，A.等人。螺络啉类药物发现囊状乙酰胆碱转运体是一种新的杀虫剂作用靶点。科学通报，第7期，第5期，p. 374 - 374, 2012。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.