Ric-8: un nuevo GEF con estructura tipo "armadillo"
RIC-8 is a highly conserved cytosolic protein (63 KDa) initially identified in C. elegans as an essential factor in neurotransmitter release and asymmetric cell division. Two different isoforms have been described in mammals, RIC-8A and RIC-8B; each possess guanine nucleotide exchange activity (GEF) on heterotrimeric G-proteins, but with different Gα subunits specificities. To gain insight on the mechanisms involved in RIC-8 cellular functions it is essential to obtain some information about its structure. Therefore, the aim of this thesis was to study the relationship between structure and function on RIC-8, using as model RIC-8 from X. laevis. Analysis in its primary structure did not give us information about the function of xRIC-8 and RIC-8 proteins were showed as a single family without similarity with others. For this reason, to obtain a 3D model of xRIC-8, different bioinformatics approaches that include protein folding and structure prediction were used. The RIC-8 structural model is composed of 10 armadillo folding motifs, organized in a right-twisted alpha-alpha super helix. In order to validate the structural model, a His-tag fusion construct of RIC-8 was expressed in E. coli, purified by affinity and anion exchange chromatography and subjected to circular dichroism analysis (CD) and thermostability studies. This model together with the comparison among RIC-8 proteins that shows a high conservation in the carboxy region, deletion mutants that remove the last three armadillo domain were created in order to search a loss of the GEF function. The mutants could not be expressed in bacteria for in vitro assays, but their expression in HEK293T culture showed that all of them preserved the GEF activity. Furthermore, confocal microscopy showed that all the mutants could translocate to plasmatic membrane under stimulation with isoproterenol, and have the capacity to interact with Gs. The results of this thesis has allowed present the first 3D model for a RIC-8 protein, as well as classify RIC-8 as a new member of the protein family with armadillo repeats. Functionally, the carboxi terminal region, the most conserved among RIC-8, did not show has the GEF activity, and did not alter its behavior in HEK293T cell cultures while was stimulated with isoproterenol.
Life sciences, Biochemistry, biophysics & molecular biology, Sciences du vivant, Biochimie, biophysique & biologie moléculaire
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