Systematic identification and characterization of missing GEFs for GTPases
Cell Biology
Final Report Abstract
Eukaryotic cells are highly complex entities. In order to respond to changing conditions, these cells need to convert extracellular stimuli into adaptable intracellular signals. Here, the family of small GTPases play a crucial role as they integrate and propagate these signals. Therefore, small GTPases are under tight temporal and spatial regulation in order to control cellular signaling. The activation of these proteins is regulated by an important heterogeneous class of enzymes referred to as guanine nucleotide exchange factors (GEFs). Even though small GTPases can unambiguously identified on the basis of their amino acid sequences and structures, their GEFs cannot be easily deduced and assigned. Since the understanding of intracellular signaling networks requires the characterization of GTPase regulation, it is important to identify elusive GEFs of given GTPases. In this project, we established a general concept for the enrichment and identification of GEFs. The method is based on the biochemical feature of GTPase to strongly bind to their GEFs when they have been depleted from their co-factors (i.e. guanosine diphosphates (GDP) or guanosine triphosphates (GTP)). Immobilizing GDP/GTP depleted GTPases on solid support allowed us to selectively enrich and subsequently elute binding partners. With the help of high performance liquid chromatography (HPLC) mass spectrometry (MS) the enriched proteins could be identified. This method can be applied to mammalian lysates, but also to bacteria that manipulate mammalian GTPases via the secretion of GEFs during infection. Even though the experimental setup for GEF enrichment is easily realized, the access to mass spectrometry proteomics is absolutely crucial for the identification of putatively enriched proteins. Furthermore, the direct validation of GEF activity requires the purification of the protein in moderate quantities and purities. Clearly, the dependence on protein production limits the overall scientific output and has been the most challenging part of the project. Nevertheless, alternative complementary experimental setups such as yeast-two-hybrid gave first indications that several new GTPase binding partners could be identified that may possess GEF or GTPase-stabilizing activities.
Publications
- (2016) A pull-down procedure for the identification of unknown GEFs for small GTPases. Small GTPases 7, 93-106
Koch, D., Rai, A., Ali, I., Bleimling, N., Friese, T., Brockmeyer, A., Janning, P., Goud, B., Itzen, A., Mueller, M.P. & Goody, R.S.
(See online at https://doi.org/10.1080/21541248.2016.1156803) - (2018) Structure of the tandem PX-PH domains of Bem3 from Saccharomyces cerevisiae. Acta Cryst. F74, 315–321
Ali, I., Eu, S., Koch, D., Bleimling, N., Goody, R.S. & Müller, M.P.
(See online at https://doi.org/10.1107/S2053230X18005915)
