The P2X7 Interactome: Protein interactions in the P2X7 C-terminus and their role in inflammatory signalling

炎症是对感染或损伤的正常、健康的生理反应。然而，在人口老龄化的国家，如联合王国，与年龄有关的慢性炎症，如关节炎、心血管疾病和阿尔茨海默病，所带来的不受欢迎的痛苦后果，对健康和生活质量产生了重大的负面影响。例如，目前仅在英国就有超过200万人患有关节炎。这就是为什么对炎症和炎症信号的分子理解的研究对全世界的人类健康有重大影响，并且符合BBSRC老龄化研究的战略重点：终身健康和福祉。本研究的目的是通过发现和表征信号通路中的关键步骤，准确了解免疫细胞（如巨噬细胞）如何对炎症信号做出反应。促炎信号被巨噬细胞表面的受体识别。一些促炎信号是关键的细胞代谢产物，如ATP，因为当它们从细胞中释放出来时，表明细胞因压力、感染或组织损伤而死亡。释放的ATP被P2X7受体识别，这导致下游信号级联，放大促炎信号，并可能导致全面的炎症反应（以受影响区域肿胀和疼痛为特征）。P2X7受体包含3个大结构域；一个识别ATP（在细胞外部），一个允许离子在激活时进入细胞（嵌入细胞膜），一个在激活时打开信号级联（在细胞内部）。虽然我们对ATP如何与受体结合以及离子如何进入细胞有一些了解，但我们不了解信号级联是如何开启的。我们假设P2X7的胞内结构域必须具有明确的3D结构和组织，使其能够与其他信号蛋白相互作用，但我们不知道它是什么，或者相互作用蛋白的身份是什么。为了了解炎症过程如何在健康人体内发挥作用，并设计出治疗老龄化人群中不必要的炎症的策略，了解P2X7细胞内结构域的结构、组织和相互作用至关重要。了解蛋白质结构域的结构和功能的一个好方法是将其与蛋白质的其他部分分开研究。我们最近已经能够在酵母中制造和纯化P2X7胞内结构域的主要部分，胞内c端。我们的目标是利用这一进步，并以两种主要方式使用我们纯化的蛋白质。首先，我们想研究分离蛋白的结构和组织，通过观察哪些部分对炎症信号很重要，哪些部分对保持其结构在一起很重要。其次，我们想用c端作为诱饵，在巨噬细胞的细胞内提取物中寻找与之相互作用的蛋白质。然后我们可以分离出相互作用的蛋白并鉴定它们，发现P2X7 c端新的相互作用蛋白伙伴。一旦我们发现了P2X7 c端结构是如何组织的，并确定了它的相互作用伙伴，我们就可以测试破坏这些相互作用对巨噬细胞中促炎信号通路的影响。这将使我们能够准确地了解P2X7激活如何在炎症信号通路上切换，并显著推进我们对人类健康中炎症处理的理解。

Inflammation is a normal, healthy physiological response to infection or injury. However, in countries with ageing populations, such as the UK, the unwanted and painful consequences of age-related chronic inflammation, in conditions such as arthritis, cardiovascular disease and Alzheimer's disease, have a significant negative effect on health and quality of life. Arthritis, for example, currently affects greater than 2 million people in the UK alone. This is why research towards a molecular understanding of inflammation and inflammatory signalling is of great impact to human health worldwide, and fits within the BBSRC strategic priority of ageing research: lifelong health and wellbeing.The purpose of this research is to understand precisely how immune cells such as macrophages respond to inflammatory signals, by discovering and characterising a key step in the signalling pathway. Pro-inflammatory signals are recognised by receptors on the cell surface of macrophages. Some pro-inflammatory signals are key cellular metabolites such as ATP, because when they are released from cells, it indicates that cells are dying due to stress, infection or tissue injury. Released ATP is recognised by P2X7 receptors, and this leads to a downstream signalling cascade, that amplifies the pro-inflammatory signal and can lead to a full-scale inflammatory response (characterised by swelling and pain in the affected area). The P2X7 receptor contains 3 large domains; one which recognises ATP (on the outside of the cell), one which allows ions to enter the cell upon activation (embedded in the cell membrane), and one which switches on a signalling cascade upon activation (inside the cell). While we have some information about how ATP binds to the receptor, and how ions can enter the cell, we do not understand how the signalling cascade is switched on. We hypothesize that the intracellular domain of P2X7 must have a defined 3D structure and organisation that enables it to interact with other signalling proteins, but we do not know what it is, or what the identities of the interacting proteins are. To understand how the inflammatory process functions in healthy humans, and to devise strategies to treat unwanted inflammation in the ageing population, it is vital that we understand the structure, organisation and interactions of the intracellular domain of P2X7.A good way to understand the structure and functions of a protein domain is to study it in isolation from other parts of the protein. We have recently been able to make and purify a major portion of the P2X7 intracellular domain, the intracellular C-terminus, in yeast. We aim to exploit this advance, and use our purified protein in two major ways. First, we want to investigate the structure and organisation of the isolated protein, by looking at which parts are important for inflammatory signalling, and which parts are important for holding its structure together. Second, we want to use the C-terminus as bait, to fish for proteins which interact with it, in intracellular extracts from macrophages. We can then isolate the interacting proteins and identify them, discovering novel interacting protein partners for the C-terminus of P2X7.Once we have discovered how the P2X7 C-terminus structure is organised, and identified its interacting partners, we can test the effects of disrupting these interactions on pro-inflammatory signalling pathways in macrophages. This will enable us to understand precisely how P2X7 activation switches on inflammatory signalling pathways, and significantly advance our understanding of inflammatory processed in human health.