The Ral-RLIP76 (RalBP1) signalling axis: plugging the GAP

Our work focuses on the relationship between structure and function in proteins involved in signalling within the cell. These proteins control many signal transmission pathways that can become deregulated in cancer and other diseases. The proteins that we study are important in the progression of a cell from being normal to being cancerous, a process that requires a combination of abnormal cell growth and resistance to normal cell death. The proteins that we concentrate on are collectively called small G proteins. These fascinating proteins act as molecular switches in the cell, turning signalling pathways on and off and thus regulating a large number of cellular functions. One pair of small G proteins are called the Ral proteins. These Ral proteins are important for both of the processes involved in turning a normal cell into a rogue cancer cell: RalA increases cell growth while RalB suppresses cell death. The focus of this project is a protein called RLIP76, a binding partner for the Ral proteins that is also involved in pathways that are important in cancer progression. For example, RLIP76 can act as a pump that removes chemotherapy drugs from cells, making them less effective. RLIP76 also increases the radiation resistance of cancer cells making them more difficult to kill using radiation therapies.

We have already worked out how RalB and RLIP76 bind to each other by studying their structures, both individually and together in a complex, in three dimensions. This is crucial, because once we know exactly how proteins bind to each other and which particular regions of the proteins are important for the interaction, it will be possible to design drugs that either prevent or enhance this binding, This is one of the avenues that we are already pursuing with Ral and RLIP76.

Additionally, our previous work with RLIP76 also revealed new properties of RLIP76 that we now intend to characterize, as we predict that there will be other ways to exploit these traits and therefore attack this protein in disease.

RLIP76 can itself control another family of small G proteins, the Rho family. RLIP76 has the ability to turn off the Rho family of molecular switches. As the Rho family proteins are themselves heavily implicated in disease progression, the ability to alter RLIP76 activity would therefore be therapeutically beneficial. We now intend to determine which of the Rho family proteins is the true target of RLIP76: this knowledge will be crucial for understanding which cellular processes RLIP76 modifies, by flicking the switch on Rho family proteins. We will also determine the minimal region of RLIP76 required to affect Rho family proteins and will go on to investigate how other proteins modulate this effect. Taken together, this work will further our knowledge of small G proteins and RLIP76 and permit the development of molecules that will guide future drug design projects.

Ral and RLIP76 have been shown to signal aberrantly in a variety of different cancers e.g. pancreatic, colorectal, bladder, prostate and melanoma, so therapies directed towards these proteins will be potentially helpful to a wide variety of cancer patients.

It is hypothesized that the minimal platform for oncogenic transformation involves abnormal proliferation and suppression of apoptosis. The Ral GTPases are involved in both processes: RalA is required for anchorage-independent proliferation, while RalB is involved in suppression of apoptosis. Thus, a therapeutic agent directed against Ral-controlled pathways would target both supports of this platform and be a powerful treatment to fight cancer. Recently, we have built up a wealth of knowledge on one RalA/B effector protein, RLIP76 (RalBP1), which is known to function aberrantly in multiple cancers. We have solved the structure of RalB in complex with the GTPase binding domain (GBD) of RLIP76 and used RalB mutants to reveal the thermodynamic hot spots in the interaction surface. We have also characterized the ability of RLIP76 to behave as a GTPase activating protein (GAP) for the Rho family of small G proteins. We have solved the structure of the GAP domain of RLIP76 and revealed why this isolated domain has rather poor catalytic activity towards Cdc42 and Rac1. In this new project we will fully characterize the RLIP76 GAP properties: we will identify exactly which regions of RLIP76 are necessary for full, functional GAP activity and we will determine which of the Rho family proteins are true substrates for this GAP. We will investigate the interaction between epsin and RLIP76, which together regulate cell migration and invasion. We will use our structures to design mutants in RLIP76 and epsin1 that disrupt their interaction and will test them in cell-based assays for their ability to suppress RLIP76 mediated migration and invasion. We will also design peptide-based inhibitors of the interaction and test them in the same assays.
This multidisciplinary, innovative and strategically important research will reveal the cellular ramifications of these specific protein interactions for RLIP76 function and may also identify new therapeutic targets and/or early drug-leads

The proteins we study are crucial to cancer progression. Ral proteins are required for both tumour cell proliferation and decreased apoptosis and are implicated in a growing roster of cancers, including pancreatic, colorectal, bladder, prostate and melanoma: these are in the top ten cancers diagnosed in the UK. RLIP76 is overexpressed in several cancers and has been shown to pump chemotherapeutics out of the cell. We will use our expertise to characterize the GAP activity of RLIP76 and define its true cellular targets. We will use these data in conjunction with our structures to understand the interactions vital for its regulation. This knowledge will be used to design peptide inhibitors and test them in vivo. Peptides identified will form the basis for future therapies directed towards RLIP76 inhibition. This work will be immediately beneficial to academic/industrial labs, both nationally and internationally, as it will provide proof of principle for the use of peptides to inhibit small G protein controlled pathways. It will also significantly add to the knowledge base of the action of small G proteins in signalling pathways and disease progression. Thus this work will also benefit researchers working in the wider fields of cell signalling and in drug design.
During this project, we will generate a powerful resource comprising a suite of Rho family expression constructs, including wild type and activated forms for bacterial and mammalian expression. Importantly, we will also have the purified proteins themselves ready to be tested for their ability to interact with a range of other molecules. Furthermore we will have a panel of Rho family effectors capable of identifying the activated forms of all Rho family members in vitro and in vivo. This resource is partially available in labs in the US/Europe but not in the UK. This will be an essential asset for the UK signalling community who can work with us by accessing our collection, allowing the community to move beyond the traditional approach of testing proteins for interactions with a limited set of Rho family proteins.
The staff working on this project will immediately and directly benefit, as they will develop skills in structure/function analysis of protein interactions, enzyme analysis and in vivo assays leading to therapeutic target validation. This will improve their employment prospects in both academic and industrial arenas. Training expert post-doctoral scientists in these skills will also be highly beneficial to future employers. By adding to the trained pool of researchers in the UK we should increase the economic competitiveness of the country by strengthening its position in the global academic/biotech/pharmaceutical market.
This work will benefit the pharmaceutical industry in the longer term (>10 yrs), by providing early lead molecules for drug design. Only in collaboration with the biotech and pharmaceutical industries can such leads realistically be developed into drugs.
In the long term, this work will also benefit patients who suffer from a wide variety of cancers. 1 in 3 of the UK population will suffer from cancer in their lifetime and cancer was responsible for 27% of all deaths in the UK in 2008 (CR-UK). Aside from the incalculable human cost, the economic burden on our society is huge and growing annually: cancer cost the UK economy £12 billion in 2008 (Health Economics Research Centre, University of Oxford). There is significant potential for enhancing quality of life and health of the population once tractable lead therapeutic molecules have been identified. Alongside this, the economic burden should decrease concomitantly. Effective targetted therapies can only be developed from an understanding of the underlying molecular principles of the biology involved. This proposal is based on research funded by MRC and therefore involves strengthening our knowledge base and understanding of signalling pathways, and building drug leads on basic research.