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

研究脂质环境对ABC转运蛋白结构和功能的影响

• 批准号: 2883785
• 金额: --
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2883785
• 关键词: Investigating; effect; lipid; environment; ABC

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.

在从细菌到人类的所有生物体中都发现了ABC（ATP结合盒）转运蛋白超家族，它们利用来自ATP水解的能量来为分子跨膜的运输提供动力。人类转运蛋白参与了广泛的功能，包括保护毒素，代谢，控制体内药物分布，介导炎症反应和运输脂质。几个成员负责遗传疾病，例如囊性纤维化和肾上腺素肌营养不良，而另一些成员则参与癌症治疗期间引起多药耐药性。众所周知，许多膜蛋白的功能受其脂质双层环境的影响。特定的脂质可能直接与转运蛋白相互作用并调节功能，或者简单地影响双层的一般特性（厚度，流动性）。该项目的目的是研究这种蛋白质：两种模型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可以在大肠杆菌中轻松表达，使用苯乙烯马来酸聚合物（SMA）提取和纯化以形成SMA脂质颗粒（SMALPS）。我们以前已经证明，可以将ATM1从SMALP重组为脂质体。在该项目中，ATM1将被重构为定义的脂质组成/特性的脂质体，并对监测的蛋白质功能的影响。这将与使用电子显微镜的结构研究结合使用。 MRP4可以在SF9昆虫细胞或自由泳HEK哺乳动物细胞中表达。这些不同表达系统对蛋白质产量和功能的影响将被测量，SMA聚合物将用于从每个表达系统中提取和纯化MRP4，以通过质谱法分析共同提取的脂质。