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

P2X7 Interactome：P2X7 C 末端的蛋白质相互作用及其在炎症信号传导中的作用

• 批准号: BB/J017345/1
• 负责人: Mark Young
• 金额: $ 42.1万
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2013
• 资助国家: 英国
• 起止时间: 2013 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FJ017345%2F1
• 关键词: P2X7; Interactome; Protein; interactions; terminus

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.

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，如英国，与年龄相关的慢性炎症的不良和痛苦的后果，如关节炎，心血管疾病和阿尔茨海默病，对健康和生活质量有显着的负面影响。例如，关节炎目前仅在英国就影响着200多万人。这就是为什么研究炎症和炎症信号的分子理解对全球人类健康产生重大影响的原因，并且符合BBSRC老龄化研究的战略重点：终身健康和福祉。这项研究的目的是通过发现和表征信号通路中的关键步骤，精确了解免疫细胞（如巨噬细胞）如何对炎症信号做出反应。促炎信号由巨噬细胞细胞表面上的受体识别。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，它表明细胞由于压力，感染或组织损伤而死亡。释放的ATP被P2 X7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（特征是受影响区域的肿胀和疼痛）。P2 X7受体包含3个大的结构域;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）。虽然我们知道ATP如何与受体结合，以及离子如何进入细胞，但我们不知道信号级联是如何启动的。我们假设P2 X7的胞内结构域必须具有明确的三维结构和组织，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，也不知道相互作用蛋白的身份是什么。为了了解炎症过程在健康人体中的功能，并设计治疗老年人群中不必要的炎症的策略，我们必须了解P2 X7细胞内结构域的结构，组织和相互作用。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备和纯化P2 X7胞内结构域的主要部分，即胞内C-末端。我们的目标是利用这一进步，并以两种主要方式使用我们的纯化蛋白。首先，我们想研究分离蛋白的结构和组织，看看哪些部分对炎症信号传导很重要，哪些部分对保持其结构很重要。第二，我们想用C末端作为诱饵，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，我们可以分离相互作用的蛋白质并鉴定它们，为P2 X7的C-末端发现新的相互作用蛋白质伴侣。一旦我们发现P2 X7 C-末端结构是如何组织的，并鉴定其相互作用伴侣，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P2 X7激活如何打开炎症信号通路，并显着推进我们对人类健康中炎症过程的理解。

项目成果

Nucleoside reverse transcriptase inhibitors possess intrinsic anti-inflammatory activity.

• DOI: 10.1126/science.1261754
• 发表时间: 2014-11-21
• 期刊: Science (New York, N.Y.)
• 作者: Fowler BJ;Gelfand BD;Kim Y;Kerur N;Tarallo V;Hirano Y;Amarnath S;Fowler DH;Radwan M;Young MT;Pittman K;Kubes P;Agarwal HK;Parang K;Hinton DR;Bastos-Carvalho A;Li S;Yasuma T;Mizutani T;Yasuma R;Wright C;Ambati J
• 通讯作者: Ambati J