Towards a GTPase/GEF assay. Background.


My name is Carmen, and I a postdoc at the SGC in Oxford. I work in multidisciplinary projects in Paul Brennan and Opher Gileadi groups.

Today I am going to introduce one of the projects I have been working for the last year which is part of the group’s endeavour to target small GTPases through their GEF regulating proteins. If you are interested in this topic, read this awesome review, published earlier this year in Angewante Chemie by Janine Gray, one of our PhD students.

Small GTPases are a super family of enzymes working as biological switches. They take part in cell signalling and regulate many processes inside the cell. There are 5 families: Ras, Rho, Rab, Ran, and Arf. Our efforts have been focussed mainly on Rac1, a member of the Rho family.

GTPases are switched on for signal transduction when they are bound to GTP, and they are switched off when they are bound to GDP. However, the intrinsic activity to cycle from active to inactive is very low, and therefore they require two other players: GAPs and GEFs.

GAPs or GTPase activating proteins, help the GTPase hydrolysing GTP into GDP. GEFs or Guanine nucleotide exchange factors facilitate the exchange of GDP in the GTPase with a new molecule of GTP Figure 1. GDIs or Guanine nucleotide dissociation inhibitors also play an important role in Rho and Rab GTPases, but I will not talk about them for now.

Figure 1 Small GTPase cycle, from active to inactive. Adapted from Gray, J. L. et al. Angew. Chem. Int. Ed. 2019. Accepted article.

Because of the many processes they are involved, GTPases are interesting molecules to target, and a lot of effort was invested through the years. The main challenge we face when targeting GTPases is that active site will be occupied by GTP or GDP which bind to the GTPase with picomolar affinity. This, together with the high intracellular concentration of GTP and GDP makes it challenging to create a competitor.

However, few years ago it was observed that the affinity of the GTPase for GTP/GDP decreases when the GTPase is in complex with a GEF protein. Figure 2.

Figure 2  Mechanism of GEF-induced nucleotide exchange. Bos, J. L. et al. Cell 2007, 129 (5), 865–877.

Within our group we are exploring this opportunity and trying to find modulators of the GTPases when in complex with their GEFs. My input in this project is to develop an enzymatic assays that we can use in most of the GTPase-GEF complexes to measure their exchange activity and how this changes when we add a modulator.


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