The Use Of Novel Polymer-Lipid Nanoparticles To Study G-Protein-Coupled Receptor Activation And Dynamics

使用新型聚合物脂质纳米颗粒研究 G 蛋白偶联受体的激活和动力学

• 批准号: BB/I019960/1
• 负责人: David Poyner
• 金额: $ 38.65万
• 依托单位: Aston University
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FI019960%2F1
• 关键词: Use; Novel; Polymer; Lipid; Nanoparticles

Most chemical messengers in the body, work through proteins on the cell surface known as receptors. The biggest class of receptors is the G-protein-coupled receptor (GPCRs) family, which are important for cell signalling and as drug targets. GPCRs are thus of enormous interest, both in terms of basic biology and also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level is fundamentally important and is one of the 'big questions' in biology today. For a few receptors we have a crystal structure showing their 3-dimensional shape when bound to a 'blocking drug'. This has deepened our understanding of receptor architecture which should aid future drug discovery. However, it is clear that GPCRs are very flexible proteins that change their shape on binding hormones and drugs, whereas the crystal structures provide snap-shots of a single conformation, usually of the non-signalling receptor. To fully understand GPCRs, we need to understand how they change their shape during signalling. This is also important for 'allosteric' agents that bind to a different part of the receptor to the natural hormone to modify receptor activity. This opens the way to a whole new class of drugs. In this application we describe a new approach to understanding GPCRs. We have found a novel way to make large quantities of natural GPCRs 'solubilised' within very small particles of their membrane environment without the need for detergent. Previously, there has been an absolute requirement for detergent to solubilise receptors. This formed a major barrier to progress in this field as its presence disrupted the normal properties and shape of the receptor, de-stabilising the protein. For the first time anywhere, we can now circumvent the detergent barrier to progress. This important advance opens up the prospect of applying powerful biophysical techniques to provide detailed information on how GPCRs dynamically change their shape in response to drugs of different classes. The project brings together four scientists from the Universities of Birmingham and Aston, with complementary skills to exploit our new technologies. We will study the adenosine A2a receptor (A2aR) as an example of the GPCR family. This is a well-characterised receptor with a large literature, defined drugs, useful tools and importantly it is one of the few receptors for which there is an atomic level crystal structure. In addition, our industrial collaborators Heptares Therapeutics will provide A2aRs constrained into functionally-defined shapes (conformations) which will be a great help to us. Finally we have a formal collaboration with an experienced computational chemist at Essex University who will construct molecular models and dynamic simulations of different functional states of the receptor based on our experimental data. We are ahead of the field and our pilot data show that we have the capability at this very moment to produce very large amounts of A2aR by growing them in a yeast and to rapidly 'solubilise' active receptors in their natural state in a styrene maleic acid lipid particle (SMALP). We will engineer A2aR so that 'reporter groups', including fluorescent groups, can be introduced at defined locations in the receptor architecture. We can then study A2aR-SMALP by a range of techniques. For example, fluorescence can tell us about the changes in the environment around important parts of the receptor when drugs bind. A related technique can also be used to provide a molecular ruler that allows us to calculate the distance between two parts of the receptor to determine how it changes on binding drugs of different signalling capability or in conformationally-constrained A2aRs provided by Heptares Therapeutics. Overall, this project will provide insight into the changes of shape that underlie GPCR activation and may aid rational drug design in the future.

体内的大多数化学信使通过细胞表面的蛋白质（称为受体）发挥作用。最大的一类受体是G蛋白偶联受体（GPCR）家族，它对细胞信号传导和药物靶点都很重要。因此，GPCR在基础生物学方面以及在制药工业的商业开发方面都具有巨大的意义。了解GPCR如何在分子水平上发挥作用是非常重要的，也是当今生物学中的“大问题”之一。对于一些受体，我们有一个晶体结构，显示它们与“阻断药物”结合时的三维形状。这加深了我们对受体结构的理解，这将有助于未来的药物发现。然而，很明显，GPCR是非常灵活的蛋白质，在结合激素和药物时会改变它们的形状，而晶体结构提供了单一构象的快照，通常是非信号受体。为了充分理解GPCR，我们需要了解它们在信号传导过程中如何改变形状。这对于与天然激素受体的不同部分结合以改变受体活性的“变构”剂也很重要。这为一种全新的药物开辟了道路。在本申请中，我们描述了一种理解GPCR的新方法。我们已经发现了一种新的方法，使大量的天然GPCR“溶解”在其膜环境的非常小的颗粒中，而不需要洗涤剂。以前，绝对需要去污剂来溶解受体。这形成了该领域进展的主要障碍，因为它的存在破坏了受体的正常特性和形状，使蛋白质不稳定。这是第一次，我们现在可以绕过清洁剂的障碍。这一重要进展开辟了应用强大的生物物理技术提供GPCR如何动态改变其形状以响应不同类别药物的详细信息的前景。该项目汇集了来自伯明翰大学和阿斯顿大学的四位科学家，他们具有互补的技能，可以利用我们的新技术。我们将研究腺苷A2 a受体（A2 aR）作为GPCR家族的一个例子。这是一种具有大量文献、定义的药物、有用的工具的良好表征的受体，重要的是，它是存在原子水平晶体结构的少数受体之一。此外，我们的工业合作伙伴Heptares Therapeutics将提供限制为功能定义形状（构象）的A2 aR，这将对我们有很大帮助。最后，我们与埃塞克斯大学一位经验丰富的计算化学家进行了正式合作，他将根据我们的实验数据构建受体不同功能状态的分子模型和动态模拟。我们在该领域处于领先地位，我们的试验数据表明，我们有能力在这个非常时刻通过在酵母中培养它们来产生非常大量的A2 aR，并在苯乙烯马来酸脂质颗粒（SMALP）中以其天然状态快速“溶解”活性受体。我们将设计A2 aR，使“报告基团”，包括荧光基团，可以在受体结构中的定义位置引入。然后，我们可以通过一系列技术来研究A2 aR-SMALP。例如，当药物结合时，荧光可以告诉我们受体重要部分周围环境的变化。相关技术也可用于提供分子标尺，其允许我们计算受体的两个部分之间的距离，以确定其如何在不同信号传导能力的结合药物上或在由Heparin Therapeutics提供的构象约束的A2 aR中变化。总的来说，这个项目将提供深入了解形状的变化，GPCR激活的基础，并可能有助于合理的药物设计在未来。

项目成果

Ligand-induced conformational changes in a SMALP-encapsulated GPCR

• DOI: 10.1016/j.bbamem.2020.183235
• 发表时间: 2020-06-01
• 期刊: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
• 影响因子: 3.4
• 作者: Routledge, Sarah J.;Jamshad, Mohammed;Wheatley, Mark
• 通讯作者: Wheatley, Mark

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin.

• DOI: 10.1186/s12934-017-0656-2
• 发表时间: 2017-03-09
• 期刊: Microbial cell factories
• 影响因子: 6.4
• 作者: Cartwright SP;Darby RA;Sarkar D;Bonander N;Gross SR;Ashe MP;Bill RM
• 通讯作者: Bill RM

Structural Determinants of Oligomerization of the Aquaporin-4 Channel.

• DOI: 10.1074/jbc.m115.694729
• 发表时间: 2016-03-25
• 期刊: The Journal of biological chemistry
• 作者: Kitchen P;Conner MT;Bill RM;Conner AC
• 通讯作者: Conner AC

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks.

• DOI: 10.1186/s12934-014-0127-y
• 发表时间: 2014-09-04
• 期刊: Microbial cell factories
• 影响因子: 6.4
• 作者: Bawa Z;Routledge SJ;Jamshad M;Clare M;Sarkar D;Dickerson I;Ganzlin M;Poyner DR;Bill RM
• 通讯作者: Bill RM

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent.

• DOI: 10.1042/bsr20140171
• 发表时间: 2015-04-16
• 期刊: Bioscience reports
• 影响因子: 4
• 作者: Jamshad M;Charlton J;Lin YP;Routledge SJ;Bawa Z;Knowles TJ;Overduin M;Dekker N;Dafforn TR;Bill RM;Poyner DR;Wheatley M
• 通讯作者: Wheatley M