Discovery and characterisation of novel immunomodulatory Cullin-RING ubiquitin ligases (CRLs) in the airway

我们的免疫系统通过我们细胞中复杂的分子网络对感染发出警报，这些分子网络感知感染并随后激活被称为细胞因子的小的可溶性信使的产生。细胞因子到达血流并触发细胞中的多种应激反应，目的是限制和对抗感染。缺乏细胞因子的产生会导致不受控制的感染;但过度的细胞因子产生可能会导致慢性炎症和严重的组织损伤。因此，细胞因子是极其重要的分子，理想情况下，它们的产生应受到仔细调控。由于大多数尚不清楚的原因，某些病原体过度刺激免疫系统，可能是一种不希望的结果或分散免疫系统的一种方式。这在呼吸道病原体中特别常见，如引起流感的病毒或引起COVID-19的新型冠状病毒，但也在非感染性炎症性疾病中，如哮喘，COPD或过敏。在所有这些情况下，过度的免疫反应和细胞因子的过度产生是有毒的，并导致呼吸道阻塞、损伤和最终死亡。该项目旨在了解细胞因子的产生和炎症是如何在气道中调节的，目的是提供新的策略来控制感染和其他疾病期间的这些反应。一种称为NF-κ B的关键细胞因子是控制气道炎症的关键。NF-κ B在正常细胞中受到很好的调节，其激活和失活由几个“激活”和“制动”点控制。然而，当这些控制点失败时，个体无法对感染作出反应或以不受控制的细胞因子产生作出反应。因此，了解NF-kappaB在呼吸组织中的功能对于了解炎症是如何控制的至关重要。为了解决这一知识缺口，并发现可能成为未来调节气道炎症的新分子，我们最近筛选了超过100种分子对NF-κ B反应的作用。这些分子属于一个被称为Cullins的蛋白质家族，我们战略性地选择了它，因为药物可以针对它们进行设计。我们的研究结果已经可靠地鉴定了多达11种新分子，这些分子强烈影响NF-κ B的激活程度，其中4种我们已经广泛表征为概念验证。基于这些强大的初步数据，我们现在建议研究剩余的分子，并确定它们如何影响细胞因子或病毒引发的炎症。为了确定这些分子在多大程度上控制NF-κ B炎症反应，我们将进行实验，其中这些分子将从（功能丧失）细胞中去除，或引入（功能获得）细胞中。然后将这些细胞暴露于细胞因子或呼吸道病毒，并通过一系列方法记录随后产生的炎症反应。为了确定这些分子如何控制炎症反应，我们将进行一系列有针对性的生化实验，其中我们将仔细确定每个Cullin分子如何促进或限制NF-κ B活化。这些实验将与Cullin相互作用组的全球分析相结合;也就是说，在给定时间与给定Cullin相关的每个单个分子的鉴定。这些有针对性的研究的结论将提供关于炎症生物学的新知识，而且也是医学上增加或减少气道炎症反应的新方法。因此，该项目将有可能影响我们目前对慢性炎症性疾病以及病毒引起的急性炎症过程的治疗。目前这些疾病的治疗方案的进步是一个迫切的需要，将显着提高患者的生活质量，并减轻在英国和超越医疗系统的压力。

Our immune system is alerted of an infection by a sophisticated network of molecules in our cells that sense the infection and subsequently activate the production of small soluble messengers known as cytokines. Cytokines reach the bloodstream and trigger multiple stress responses in cells with the objective of limiting and combating the infection. Lack of cytokine production results in uncontrolled infections; but excessive cytokine production may result in chronic inflammation and severe tissue damage. Cytokines are therefore extremely important molecules and ideally their production should be carefully regulated. For reasons that remain mostly unclear, certain pathogens overstimulate the immune system, possibly as an undesired outcome or a way to distract the immune system. This is particularly common in respiratory pathogens such as the virus causing flu or the new coronavirus causing COVID-19, but also in non-infectious inflammatory diseases such as asthma, COPD or allergies. In all these cases, the excessive immune response and over-production of cytokines is toxic and results in the obstruction of the respiratory tract, damage and eventually death. This project aims to understand how cytokine production and inflammation is regulated in the airway, with the aim of providing novel strategies to control these responses during infection and other diseases. A critical cellular factor termed NF-kappaB is key in controlling inflammation in the airway. NF-kappaB is very well regulated in normal cells and its activation and deactivation is controlled by several 'activation' and 'brake' points. However, when these control points fail, individuals fail to respond to infection or respond with an uncontrolled production of cytokines. Understanding how NF-kappaB functions in respiratory tissues is therefore crucial to understand how inflammation is controlled. In order to address this knowledge gap and to discover novel molecules that could become future targets to regulate inflammation in the airway, we recently screened >100 molecules for their role on NF-kappaB responses. These molecules belong to a family of proteins known as Cullins, which we strategically selected because drugs can be designed against them. Our results have reliably identified up to 11 novel molecules that strongly impact the extent to which NF-kappaB is activated, 4 of which we have extensively characterised as a proof of concept. Building on these robust preliminary data we now propose to study the remaining molecules and determine how they impact on inflammation triggered by cytokines or viruses. To establish to what extent these molecules control NF-kappaB inflammatory responses, we will conduct experiments in which these molecules will be removed from (loss-of-function), or introduced into (gain-of-function), cells. These cells will then be exposed to cytokines or respiratory viruses and the subsequent inflammatory response generated will be recorded by a range of methods. To establish how these molecules control the inflammatory response, we will conduct a series of targeted biochemical experiments in which we will meticulously determine how each Cullin molecule boosts or limits NF-kappaB activation. These experiments will be combined with global analyses of the Cullin interactome; that is, the identification of each single molecule that associates with a given Cullin at a given time. The conclusions from these targeted studies will provide novel knowledge on the biology of inflammation, but also novel ways to medically increase or reduce airway inflammatory responses. This project will therefore have potential to impact on our current treatments against chronic inflammatory diseases as well as acute inflammatory processes caused by viruses. Advancement of the current treatment options for these diseases is an urgent need that will significantly improve patient life quality and alleviate pressures on medical systems in the UK and beyond.