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

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.

Inflammation plays a crucial role in multiple aetiologies. In the respiratory tract, exacerbated inflammation strongly contributes to non-communicable diseases such as asthma or COPD, and is a hallmark of respiratory virus infections causing severe tissue damage and pulmonary obstruction. How to therapeutically manipulate inflammation in the airway remains an unmet target that can benefit multiple patient populations. We have recently screened a large number of cellular Cullin-RING ubiquitin ligases (CRLs) for their role in regulating inflammatory responses driven by the master transcription factor NF-kappaB. This screen has identified up to 11 CRLs with no previous known roles in NF-kappaB activation, some of which we have characterised at the molecular level. Some of these acted as negative regulators limiting the extent of NF-kappaB responses, whereas others acted as positive regulators needed for NF-kappaB to become active. This project aims to determine the extent to which these molecules modulate respiratory inflammation and how they operate. To establish the impact of these CRLs, we will conduct loss-of-function and gain-of-function assays in A549 and BEAS-2B cells and record NF-kappaB activity and cytokine production in response to cytokines and respiratory virus infection. To establish mechanism of action, we will conduct targeted and untargeted assays including quantitative proteomics to map the activity of these CRLs and identify their physiological cellular partners. These will be followed by detailed mutagenesis and protein-protein interaction assays offering invaluable mechanistic insights. Collectively, this project will uncover several novel regulatory mechanisms affecting the biology of CRLs and inflammatory responses and offer exciting novel avenues into therapeutic manipulation of inflammation in the airway.