Role of RhoGDI in RhoA prenylation and membrane targeting
RhoGTPases are key regulators of cell cytoskeleton remodelling, cell polarity and division. Most of the known Rho proteins are subjected to prenylation allowing them to function at the plasma membrane. The RhoGTPases operate via a simple switch mechanism. They are active and can recruit downstream effectors when bound to GTP and inactive when associated with GDP nucleotide. The activity of Rho GTPases is therefore under strict control from the GTP exchange factors (RhoGEFs) that load GTPases with GTP nucleotide, GTPase Activating Proteins (RhoGAPs) that trigger intrinsic GTP-hydrolysis reaction and Rho GDP Dissociation Inhibitors (RhoGDIs). The latter molecules solubilize Rhos from the membrane, thus preventing their interaction with RhoGEFs, RhoGAPs and the downstream effectors. RhoGDIs have been seen as key but, nevertheless, passive regulators of Rho proteins. Therefore functionality of RhoGDIs has been underestimated. Recently, the range of the regulatory activities of RhoGDIs has significantly expanded. They have been found to be chaperones of prenylated Rho proteins in the cell, regulate Rho proteins cross talk and are directly involved in the control of spatial Rho localization.
Despite the importance of RhoGDI in the regulation of Rho, the key molecular details of RhoGDI:Rho interaction and factors affecting it are still disputed. The inherent hydrophobicity of prenylated Rho GTPases prevents their direct biophysical characterization in vitro, whereas the data obtained in in vivo experiments is often speculative, due to the unavoidable restricted level of control of experimental conditions. Therefore a main goal in the field is to obtain unambiguous experimental quantification of Rho:RhoGDI interactions, essential for building models with high predictive power.
The goal of this project is to understand what factors drive membrane extraction/insertion of prenylated RhoGTPase by their key regulator RhoGDI. To achieve this, we developed a number of RhoGDI:Rho interaction sensors. We showed that Rho activation state is important, but probably not sufficient, in RhoGDI-mediated membrane extraction. Based on these findings we suggest that in the cell RhoGDI forms two different complexes with GTP- and GDP-bound Rho proteins which are characterised by different lifetimes and affinities. More importantly, the very low picomolar affinity of RhoGDI interaction with GDP-bound RhoA suggests that RhoGDI might operate in a coincidence detection mode in the cell. The concurrent presence of several critical factors triggers Rho membrane insertion by accelerating dissociation of the Rho:RhoGDI complex.
A number of positive feedback loop mechanisms that help localise activity of Rho proteins in the cell have been proposed. We demonstrated that RhoGDI that is tightly associated with prenylated RhoA protein can bind another molecule of unprenylated RhoA in a non-competitive manner. Using a series of biophysical protein:protein interaction techniques (fluorescence titration, fluorescence anisotropy titrations and microscale thermophoresis) we demonstrated that this interaction has a 5 to 10 micromolar affinity. More importantly, we have analyzed the solution structure of the crosslinked RhoA-GG:RhoGDI:RhoA complex by SAXS. The obtained scattering data was very well correlated with part of the RhoA-GG:RhoGDI complex that formed the asymmetric unit of our RhoAGMPPNP-GG:RhoGDI crystals. We propose a new mechanism of Rho membrane targeting that could involve a novel positive feedback loop based on the additional Rho-binding site on the RhoGDI.
Also, the highly hydrophobic Rho proteins are required to be either associated with membranes or RhoGDIs in the cell. We conjectured that RhoGDI could be involved in prenylation of the Rho proteins in the cell. Using newly developed prenylation assay we demonstrated that RhoGDI stimulates the geranylgeranylation reaction most likely via accelerating the dissociation of the Enzyme:Product complex.
Novel approaches for understanding protein prenylation
Protein prenylation is a widespread posttranslational modification among signalling proteins in eukaryotic cells. Although the mechanics of the enzymatic prenylation reaction are well understood, little is known about regulation of this reaction in the cell. The main approach for analysis of protein prenylation in vitro and in vivo is functionalization of isoprenoids with reporter groups. We developed a generic strategy for the synthesis of modified phosphoisoprenoids using amine-derivatized prenyl as a scaffold for the synthesis of phosphoisoprenoid compound libraries. This allowed us to avoid multistep organic synthesis that previously prevented production of isoprenoids with bright fluorescent groups. Using the described approach we synthesised a range of new compounds, including two novel fluorescent isoprenoids brighter than all previously generated fluorescent prenyl analogs.
Furthermore, we have shown that isoprenoid derivatization can be coupled to enzymatic prenylation in a one-pot reaction. This format is suitable for high-throughput construction of libraries of proteins prenylated with a randomized set of isoprenoid analogs. This may allow discovery of isoprenoids with desired properties much more efficiently than using conventional synthesis and testing approaches.