Channelling a path for substrates through a multidrug transporter

了解自然界的“保镖”：多药泵的机制所有细胞都被称为细胞膜的保护屏障所包围。细胞控制它们通过这层膜运输的东西，以便能够吸收（输入）或去除（输出）特定的化学物质。重要转运的例子包括从饮食中摄取营养物质进入肠道细胞，将毒素和废弃化学品输出到尿液和胆汁中，以及将维生素和营养物质分泌到乳腺组织的乳汁中。这些转运过程是通过细胞膜中的“泵蛋白”进行的。泵蛋白通常以设定的方向（即进入细胞或离开细胞）运输特定分子。有些泵蛋白质是不寻常的，因为它们从细胞中输出的不仅仅是一种化学物质，而是数百种不同且不相关的化学物质。这些蛋白质被称为“多药（MDR）泵”，它们相当于细胞的夜总会保镖，其作用可以产生很大的影响。例如，细菌中的抗生素耐药性-一个巨大的医疗挑战-可能会发生，因为多药泵从其靶细胞中去除抗生素。在人类中，MDR泵保护癌细胞免受化疗，因为泵在肿瘤细胞中表达更多。它们将旨在杀死肿瘤的抗癌药物从细胞中踢出来，导致化疗失败。这些MDR泵还可以影响我们对各种疾病的药物反应，包括心脏病的他汀类药物和癫痫的抗惊厥药。肠道、肝脏和肾脏中的MDR泵控制这些药物如何被吸收和从体内清除。MDR泵并不总是“坏消息”--化学工业希望利用细菌从单糖中制造化学品（如燃料），而不是使用日益减少的化石燃料。MDR泵在这种“绿色化学”中是重要的，因为它们可以用于从细菌中输出有用的化学产物以供收获。因此，深入了解MDR泵的工作原理以及我们如何劫持或阻止这一过程非常重要。在其他只运输一种化学物质的泵蛋白中，这种化学物质在被“泵”时通过蛋白质的路线可以很好地定义。然而，了解MDR泵的主要挑战之一是，这些泵可以处理如此多的不同药物和化学品，因此绘制它们通过泵蛋白的路线非常困难。我们将在我们的提案中解决一种MDR泵ABCG 2的这个大问题。使我们的研究与众不同的是，我们已经开发出一种新的方法来“观察”ABCG 2如何在微观水平上识别化学底物。我们将使用分子“饼干切割机”来制造细胞膜的微小环，每个环只包含一个泵蛋白。然后，我们观察一种荧光药物（当我们照射激光时会发光）结合我们含有ABCG 2的膜“饼干”。这告诉我们，我们的底物在泵工作时识别泵的能力如何，以及其他药物如何阻止这一过程。我们可以通过改变泵蛋白的部分来测试我们的底物路线图，看看它的功能会发生什么变化，并使用计算机建模来模拟相互作用。我们还将寻找结合ABCG 2并调节功能的新型药物类型，期望这些将成为有用的实验工具和未来药物的起点。这些技术将适用于观察任何泵蛋白与其底物的相互作用，这将激发人们对泵蛋白在生理学各个方面的基本作用的兴趣。我们已经在与制药和化学品生产公司讨论如何更好地了解泵：基质相互作用对药物设计过程和化学品制造有益。因此，该研究项目将促进对重要的人类MDR泵的基本生物化学的理解，并对膜泵蛋白的大家族进行更广泛的了解。

Understanding nature's "bouncers": the mechanism of multidrug pumpsAll cells are surrounded by a protective barrier known as the cell membrane. Cells control what they transport across this membrane, in order to be able to take up (import) or remove (export) specific chemicals. Examples of important transport include taking up nutrients from the diet into the cells lining the gut, export of toxins and waste chemicals into urine and bile, and secretion of vitamins and nutrients into milk in mammary tissue. These transport processes are carried out by "pump proteins" in the cell membrane. Pump proteins usually transport a specific molecule in a set direction (i.e. either into the cell or out of the cell). Some pump proteins are unusual in that they export from cells not just one type of chemical, but hundreds of different and unrelated chemicals. These proteins are called "multidrug (MDR) pumps" and they are the cell's equivalent of a nightclub bouncer, with actions that can have a big impact. For example, antibiotic resistance in bacteria - a huge healthcare challenge - can occur because multidrug pumps remove antibiotics from their target cells. In humans, MDR pumps protect cancer cells from chemotherapy because the pumps are expressed more in tumour cells. They kick the anticancer drugs designed to kill tumours back out of cells and cause chemotherapy to fail. These MDR pumps can also affect how we all respond to medication for a wide range of conditions including statins for heart disease and anticonvulsants for epilepsy. MDR pumps in the gut, liver and kidney control how these medicines are absorbed and removed from the body. MDR pumps are not always "bad news" - the chemicals industry wants to use bacteria to make chemicals (such as fuels) from simple sugars, rather than using dwindling fossil fuels. MDR pumps are important in this "green chemistry", because they can be used to export the useful chemical products from bacteria for harvesting. A deep understanding of how MDR pumps work, and how we can hijack or block this process, is therefore really important. In other pump proteins that transport only one chemical, the route this chemical takes through the protein as it is 'pumped' can be well defined. However, one of the main challenges in understanding MDR pumps is that these can deal with so many different drugs and chemicals so mapping their route through the pump protein is very difficult. We are going to tackle this big question for one MDR pump, ABCG2, in our proposal. What makes our research unique is that we have developed a new way to "see" how ABCG2 recognises chemical substrates at a microscopic level. We will use molecular "cookie cutters" to make tiny rings of cell membrane, each containing just one pump protein. We then watch a fluorescent drug (that glows when we shine laser light on it) bind our membrane "cookies" containing ABCG2. This tells us how well our substrates recognise the pump as it works, and how other drugs might stop this process. We can test our substrate route map by changing parts of the pump protein involved to see what happens to its function, and using computer modelling to simulate the interactions. We will also look for novel drug types that bind ABCG2 and regulate function, expecting that these will be useful experimental tools and starting points for future medicines.The techniques will be adapted to look at any pump protein interacting with its substrate, which will excite people interested in the fundamental roles of pump proteins in all aspects of physiology. We are already discussing with pharmaceutical and chemical production companies how a better understanding of pump:substrate interactions benefits the drug design process and chemicals manufacturing. This research project will therefore advance understanding of the basic biochemistry of an important human MDR pump, and cast a broader light on the large family of membrane pump proteins.