A structure-function analysis of USP7 and NF-kB interaction.

Inflammation is the complex biological response of the body's immune system to harmful stimuli, such as microbes, tissue damage or irritants. It is a protective attempt to remove the cause of injury as well as initiate the healing process for the affected tissue or organ. In the absence of inflammation, wounds and infections would never heal and progressive destruction of the affected tissue would eventually lead to serious illness or death. However, inflammation which runs unchecked can also lead to a host of diseases including inflammatory bowel disease, rheumatoid arthritis, autoimmunity, sepsis and atherosclerosis. Recent research has also revealed a role for unregulated or dysregulated inflammatory processes in cancer and neurodegeneration.

The proposed research will focus on a family of proteins collectively known as Nuclear Factor kappaB (NF-kB). NF-kB is a critical regulator of gene expression and plays key roles in the immune response to infection and inflammation. The triggers of such responses may include microbes or their associated products as well as signals from neighbouring cells or tissues. These stimuli induce NF-kB to increase the expression of genes that encode for pro-inflammatory factors such as cytokines and chemokines which orchestrate the movement and activation of immune cells to sites of infection or injury. While these processes are essential for the clearance of infection and the repair of damaged tissue, they pose serious threats to healthy tissue and can cause severe damage in areas of inflammation. For this reason the expression of these pro-inflammatory mediators by NF-kB is normally tightly controlled. However in certain diseases, including those listed above, NF-kB is inadequately regulated and sustained expression of these potentially harmful mediators of inflammation occurs. As a result, NF-kB is considered an important target for the development of new therapies to prevent inflammation in the treatment of inflammatory diseases.

In recent years it has emerged that the controlled destruction of NF-kB proteins is an important way used by the immune system to reduce the production of pro-inflammatory factors. Our previous studies discovered a protein called USP7 that prevents the destruction of NF-kB by interacting directly with it. We hypothesise that interfering with the interaction of these two proteins will reduce NF-kB protein levels and thereby reduce inflammation. In this proposal we aim to understand in detail how USP7 and NF-kB interact and to investigate the consequences of interfering with this interaction on the function of immune cells. The information provided from these studies could be used to develop new drugs in the future that inhibit NF-kB activity to reduce inflammation in the treatment of inflammatory disease.

The NF-kB transcription factor is a master regulator of the immune response and plays a critical role in inflammatory disease by mediating the expression of pro-inflammatory factors. The NF-kB-directed transcription of genes that promote cell survival and proliferation also implicates it as an important factor in cancers and neurodegenerative disorders. The key roles for NF-kB in the pathogenesis of these and other diseases have established it as an important therapeutic target, which to date remains unharnessed. Previous strategies focussed on inhibiting the IKK kinases, critical activators of NF-kB, have failed to make clinical impact due to severe side-effects, and so new approaches to targeting NF-kB for therapeutic benefit are required. This project aims to exploit the regulation of NF-kB by the ubiquitin proteasome system in order to inhibit NF-kB mediated inflammatory responses. The ubiquitin-triggered proteasomal degradation of NF-kB is a major limiting factor in the expression of pro-inflammatory genes. We have previously identified the deubiquitinase USP7 as a key regulator of NF-kB transcriptional activity by reversing NF-kB ubiquitination and preventing its proteasomal degradation. We have extended these initial findings to identify a distinct NF-kB binding site in USP7 that selectively mediates the interaction of USP7 with NF-kB. We hypothesise that this binding site could be targeted to selectively inhibit NF-kB-directed inflammatory responses by promoting its ubiquitination and degradation. We propose a structure-function based study of the USP7 and NF-kB interface that will define the NF-kB binding site and the functional impact of its disruption. We will use structural biology, transcriptomic and proteomic techniques to test this hypothesis. The results will facilitate the rational structure-led design of substrate-selective inhibitors of USP7 to inhibit NF-kB mediated inflammatory responses.

In this proposal we will perform a structure-function based analysis of the USP7 and NF-kB interface in order to define the NF-kB binding site and the functional impact of its disruption. The data generated will be used to test our hypothesis that USP7 possesses a distinct NF-kB binding site that cold be targeted to selectively inhibit NF-kB directed inflammatory responses.

The impact of the proposed research can therefore be identified as follows:

1) Academic
The data from the proposed research will be of importance to academic and private sector researchers both in the UK and internationally. The focus of this proposal, NF-kB, is an important factor in human health and disease and so is relevant to a wide range of disciplines including, but not limited to, immunology, cancer biology, cardiovascular biology and neurobiology. Furthermore, USP7 is regarded as an important therapeutic target for the modulation of numerous cellular processes implicated in cancer. Therefore, the impact of this proposal on UK based researchers will be to advance the knowledge economy. In addition, by communicating the research through our continued teaching and outreach activities we also hope to inform and educate, with impact, other beneficiaries in the University of Glasgow and local communities.

2) Private sector/biotech industry
The proposed research will experimentally define the structure of the USP7 and NF-kB interface and elucidate the functional consequences of disrupting it in the context of resting and activated macrophages. These data will facilitate the rational structure-led development of novel, substrate selective inhibitors of USP7 activity in order to inhibit NF-kB-mediated inflammation. This will have impact in the biotech sector since this represents a novel strategy of targeting NF-kB activity for therapeutic benefit. The data from our proposal will be of benefit in assessing the anti-inflammatory potential of this approach with potential for attracting R&D investment and developing intellectual property in this area. The approach adopted in this proposal may also be applied to other USP7 - substrate interactions in order to develop further substrate-selective inhibitors.

3) Economic and societal impact
NF-kB is intimately linked to the pathology of a wide range of diseases with profound social and economic consequences. These include chronic inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease and autoimmunity, as well as cancer, atherosclerosis and neurodegenerative disease. This proposal focuses on a fundamental aspect of NF-kB biology which is relevant to the future development of therapies in these areas which will benefit the quality of life, health and well-being of UK citizens.

4) Training of researchers at an advanced level
This proposal exploits state of the art techniques in the structure - function analysis of USP7 and NF-kB interaction incorporating structural biology, molecular biology and proteomic techniques. The multidisciplinary approach to addressing our research questions offers exceptional opportunities for the training of researchers at an advanced level.