Structure/function analysis of the Ral/RLIP signalling pathway as a therapeutic target
Lead Research Organisation:
University of Cambridge
Department Name: Biochemistry
Abstract
Our work addresses the relationship between structure and function in proteins involved in signalling within the cell. These proteins control many signal transmission pathways that 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 increased growth and resistance to normal cell death. The protein that we are proposing to concentrate on here, RLIP-76, is involved in many processes. It is important for stress responses and also for bringing molecules into the cell, a process that is strictly controlled. Both of these pathways are important in cancer progression. RLIP-76 can also act as a pump that removes chemotherapy drugs from cells, making them less effective. If we can understand more about how RLIP-76 is activated in all of these different processes, we might be able to find ways to inhibit it; so one of the things we will look at is the mechanism of RLIP-76 activation. One of the most important things that proteins do in cells is to bind to other proteins. Although we know which proteins RLIP-76 binds to, we do not know how it binds to them. So one of the other major aims of our research is to understand how RLIP-76 binds to these other proteins. We do this by working out what the complex of RLIP-76 and its partner looks like in three dimenensions. This is crucial, because if we know 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,
Technical Summary
It has been hypothesized that in non-cancerous cells proliferation and apoptosis are tightly associated and that the minimal platform to drive oncogenic transformation involves enhanced proliferation in conjunction with abrogation of apoptosis. The small GTPase RalA is required for anchorage-independent proliferation, while its close relative, RalB, is involved in the suppression of apoptosis, implying that Ral GTPases contribute to both critical aspects of carcinogenesis. This, coupled with the fact that loss of RalB function induces apoptosis in cancer cell lines but not normal cell lines, indicates that a potential therapeutic avenue directed against Ral-controlled signal transduction pathways would target both supports of the minimal platform simultaneously.
We will use a combination of structural biology, in vitro and in vivo analyses to investigate the interaction of the Ral effector RLIP-76 with RalB and with other signalling molecules. We have already solved the structures of the RLIP-76 GTPase binding domain (GBD) and RalB by NMR methods. We will now go on to solve the structure of the RLIP-76 GBD/RalB complex using NMR; initial spectra of the complex are of a high quality. We will use the structure of this complex to design Ral mutants that specifically abrogate binding to RLIP-76, while maintaining binding to other effector proteins (Exo84, Sec5, ZONAB and PLCdelta). These mutants will then be validated in vivo. We will go on to investigate the RhoGAP activity of RLIP-76 and its regulation by Ral and R-Ras GTPases. We will solve structures by NMR or X-ray crystallography of the RLIP-76 RhoGAP-GBD di-domain and of the RhoGAP/R-Ras complex. These analyses will shed light on the regulation of RLIP-76 by GTPases. We will also investigate the binding of the RLIP-76 C- and N-terminal regions to molecules involved in endocytosis (POB1 and AP2) and heat-shock (HSF1). We will use in vitro biochemical techniques to study these interactions and whether they bind independently or enhance or inhibit each other. We will also study the dependence of POB1, HSF1 and AP2 binding on Ral binding to RLIP-76. We will take these complexes forward to structural studies if possible.
The data that we generate will allow us to validate specific signalling pathways as therapeutic targets. The combination of structural information and thermodynamic parameters for these complexes could be used to aid design of small molecule inhibitors (or stabilisers as appropriate) of these interactions for therapeutic purposes.
We will use a combination of structural biology, in vitro and in vivo analyses to investigate the interaction of the Ral effector RLIP-76 with RalB and with other signalling molecules. We have already solved the structures of the RLIP-76 GTPase binding domain (GBD) and RalB by NMR methods. We will now go on to solve the structure of the RLIP-76 GBD/RalB complex using NMR; initial spectra of the complex are of a high quality. We will use the structure of this complex to design Ral mutants that specifically abrogate binding to RLIP-76, while maintaining binding to other effector proteins (Exo84, Sec5, ZONAB and PLCdelta). These mutants will then be validated in vivo. We will go on to investigate the RhoGAP activity of RLIP-76 and its regulation by Ral and R-Ras GTPases. We will solve structures by NMR or X-ray crystallography of the RLIP-76 RhoGAP-GBD di-domain and of the RhoGAP/R-Ras complex. These analyses will shed light on the regulation of RLIP-76 by GTPases. We will also investigate the binding of the RLIP-76 C- and N-terminal regions to molecules involved in endocytosis (POB1 and AP2) and heat-shock (HSF1). We will use in vitro biochemical techniques to study these interactions and whether they bind independently or enhance or inhibit each other. We will also study the dependence of POB1, HSF1 and AP2 binding on Ral binding to RLIP-76. We will take these complexes forward to structural studies if possible.
The data that we generate will allow us to validate specific signalling pathways as therapeutic targets. The combination of structural information and thermodynamic parameters for these complexes could be used to aid design of small molecule inhibitors (or stabilisers as appropriate) of these interactions for therapeutic purposes.
