Identification and characterization of novel Ras plasma membrane signalling complex modulators - the RAS-NANOME. (FNR co-application)

Ras proteins are important members of the communication systems (signalling networks) within cells. They sit on the inner surface of cell membrane from where they help to relay information that controls whether cells will proliferate, change or die. Cell signalling networks are often pre-grouped together to ensure that information transfer within them is efficient. Ras proteins are organised into small clusters (nanoclusters) at the inside of the cell membrane and this organisation is an essential requirement for proper Ras function. Several partner proteins have been identified that are capable of changing the nanoclustering specifically for one or a few of the four different Ras proteins. We define the RAS-NANOcluster modulating proteOME as the RAS-NANOME. They represent important targets for influencing the normal and disease-associated functions of Ras.
Our project aims to identify new members of the RAS-NANOME. We will label all proteins that sit next to Ras in the cell to identify likely candidates and then screen them for their ability to regulate Ras nanoclustering. We will pick examples that show the best evidence for specifically regulating nanoclustering and characterise how they do this. This will include identifying how they interact with Ras and how the cell activity is modulated by them. Thus, we will assess the extent to which these new RAS-NANOME members are able to influence Ras signalling networks. Finally, we will take a combined look at all of the new and previously identified RAS-NANOME members to determine whether they co-operate or compete to regulate Ras nanoclustering and signalling and how the conditions that cells typically experience might influence this.

Ras proteins operate from the plasma membrane to control cell proliferation, differentiation and survival pathways. Three Ras genes encode 4 protein isoforms (H-Ras, N-Ras, K-Ras4A, K-Ras4B) that are organised into distinct signalling complexes (nanoclusters) in the plasma membrane that integrate effector engagement and outputs. Ras nanoclustering is regulated by lipid and protein interactions. Evidence suggests that nanocluster modulatory proteins direct Ras isoform specific activities and thus fine-tune biological networks in a context dependent manner. However, so far only a handful of Ras nanocluster modulator proteins have been identified and their spectra of activity suggest further nanocluster modulatory proteins remain to be discovered.
Here we aim at systematically identifying the RAS-NANOcluster modulating proteOME, that we term the RAS-NANOME. We will start by proteomics-screening for all proteins proximal to individual Ras isoforms. Subsequently, a series of sensitive assays (nanoclustering-BRET and -EM coupled with high content confocal microscopy) will isolate novel, isoform-specific Ras nanocluster modulators, which will help us to define the candidate RAS-NANOME. These assays will furthermore help us to assign unknown isoform-specificities to the few currently known Ras-nanocluster modulators. Finally, we will select two new Ras nanocluster modulators and determine in detail the mechanistic basis of how they regulate Ras nanoclustering, signalling and biological activity in an isoform-specific manner. Our proteome-wide analysis will provide the first comprehensive characterisation of the RAS-NANOME and thus allow for a new perspective on the context-dependence of isoform-specific Ras biology.

The outcome of this research will not have immediate economic impact for the UK and EU but will contribute to the knowledge about an important class of proteins that will be relevant for the development of drugs to inhibit them as well as developing methodology to study them. The impact of the project can be divided into the following areas:
1. Development of the Institution and International Collaborative Networks.
This is a multidisciplinary project involving research groups at the Universities of Liverpool and Luxembourg. Our departments will benefit from exchanges of expertise and active interaction between these international groups. Whilst there are immediate benefits for the project, in the longer term we anticipate contributing to EU-funded international networks focussing on Ras biology that are currently being planned.
2. People and Skills Development.
An important aspect of this project will involve the training of the PDRA, preparing them for a future in the pharmaceutical or biotechnology industry or in academia. The development of the UK and EU economy critically relies on a technically skilled workforce. The PDRA will be trained in a variety of techniques, including proteomics, genetic screening, assay development, microscopic imaging (including EM), cell signalling and cell phenotypic analysis. Adding to the pool of highly trained, highly skilled researchers in the UK/EU will increase our economic competitiveness in the global marketplace.
3. Societal and Health Impact of the Research.
Small G proteins and their partners in signalling complexes impact on a number of diseases, and more information about them that helps them to be targeted more effectively will impact in the long term on the UK/EU health care development. Whilst this is a basic science proposal, there are translational implications because mutant Ras is found in ~17% of all human cancers and in developmental syndromes (RASopathies) affecting ~1 in 1000 people. Whilst directly drugging Ras has been realized in one case (K-RasG12C), significant attention must still be placed on targeting isoform specific regulators of Ras membrane association and nanoclustering since these are essential for normal and disease-associated Ras function. Therefore, the wider public could benefit from the novel RAS-targeting strategies derived from our work.
4. Pharmaceutical and Biotechnology Industry
Our work will provide expertise and methodologies for working with a large class of peripheral membrane proteins that are new and emerging drug targets. This will be of interest to pharmaceutical and biotechnology companies, many of whom are attempting to target these proteins directly. The Ras field has been re-energised in recent years due to significant advances in direct and indirect Ras inhibitor development. This has led to the significant new investment and interest in finding alternative inhibitory strategies. The identification of novel regulators of Ras nanoclustering will also suggest new possibilities for anti-Ras targeting strategies.