Investigating the effect of lipid environment on ABC transporter structure and function

ABC(三磷酸腺苷结合盒)转运体超家族存在于从细菌到人类的所有生物体中，它们利用三磷酸腺苷水解产生的能量来推动分子的跨膜运输。人类转运蛋白参与多种功能，包括对毒素的保护、新陈代谢、控制药物在体内的分布、介导炎性反应和运输脂质。一些成员负责遗传性疾病，如囊性纤维化和肾上腺脑白质营养不良，而其他成员参与了癌症治疗过程中导致多药耐药的过程。已有的研究表明，许多膜蛋白的功能受其脂双层环境的影响。特定的脂类可以直接与转运体相互作用并调节功能，或者简单地影响双层的一般性质(厚度、流动性)。本项目的目的是详细研究这一蛋白：两个模型ABC转运蛋白的脂质关系，首先是细菌转运蛋白Atm1，其次是人类转运蛋白MRP4/ABCC4(多药耐药蛋白4)。ATM1可以在大肠杆菌中表达，用苯乙烯马来酸聚合物(SMA)提取和纯化，形成SMA脂质颗粒(SMALPs)。我们之前已经证明，Atm1可以从SMALP重组为脂质体。在这个项目中，Atm1将被重组成具有特定脂组成/性质的脂质体，并监测其对蛋白质功能的影响。这将与使用电子显微镜的结构研究相结合。MRP4可在Sf9昆虫细胞和Freestyle HEK哺乳动物细胞中表达。将测量这些不同表达系统对蛋白质产量和功能的影响，并将使用SMA聚合物从每个表达系统中提取和纯化MRP4，以便对共提取的脂类进行质谱分析。

The ABC (ATP Binding Cassette) transporter superfamily is found in all organisms from bacteria to humans, and they utilise energy from ATP hydrolysis to power the transport of molecules across a membrane. The human transporters are involved in a wide range of functions including protection from toxins, metabolism, controlling drug distribution in the body, mediating inflammatory responses and transporting lipids. Several members are responsible for genetic diseases, such as cystic fibrosis and adrenoleukodystrophy, whilst others are involved in causing multi-drug resistance during cancer treatment. It is well established that the function of many membrane proteins is affected by their lipid bilayer environment. Specific lipids may interact directly with a transporter and modulate function, or simply affect the general properties (thickness, fluidity) of the bilayer. The aim of this project is to investigate this protein:lipid relationship in detail for two model ABC transporter proteins, firstly a bacterial transporter, Atm1, and secondly the human transporter MRP4/ABCC4 (multidrug resistance protein 4). Atm1 can be easily expressed in E.coli, extracted and purified using styrene maleic acid polymer (SMA) to form SMA lipid particles (SMALPs). We have previously demonstrated Atm1 can be reconstituted from SMALPs into liposomes. In this project Atm1 will be reconstituted into liposomes of defined lipid composition/properties and the affect on protein function monitored. This will be combined with structural studies using electron microscopy. MRP4 can be expressed in either Sf9 insect cells or Freestyle HEK mammalian cells. The effects of these different expression systems on protein yield and function will be measured, and SMA polymer will be used to extract and purify MRP4 from each expression system to enable analysis of the co-extracted lipids by mass spectrometry.