ABSTRACT
The Dbl family of guanine nucleotide exchange factors (GEFs) is made up of a vast array of members that participate in the activation of the Rho family of small GTPases. Dbl-family proteins promote the exchange of guanosine diphosphate/guanosine triphosphate (GDP/GTP) in their target molecules, resulting in the activation of a variety of signaling pathways involved in diverse cellular events, such as actin-cytoskeleton remodeling, cellular invasion, cell movement, and other functions. It has been reported that members of the Dbl family have important roles in several cellular events in Entamoeba histolytica. These include activation of the actin cytoskeleton, cytokinesis, capping, uroid formation, cellular proliferation, erythrophagocytosis, cell migration, and chemotaxis. Here, we report the identification and testing of inhibitors of the E. histolytica guanine nucleotide exchange factor 1 (EhGEF1) protein (the research compounds 2BYRF, 2BY05, 2BYT6, 2BYLX, and 2BYPD), which decreased the in vitro ability of the protein to exchange GDP/GTP at its target GTPases, EhRacG and EhRho1, by 14.9-85.2%. Interestingly, the drug 1,1'-(1,2-phenylene)-bis-(1H-pyrrole-2,5-dione), which completely inhibits the GEF activity of the Trio protein in human cells, decreases the GEF activity of the EhGEF1 protein on the EhRacG and EhRho1 GTPases by 55.7% and 3.2%, respectively. The identification and evaluation of such inhibitors opens up the possibility of obtaining a new pharmacological tool to study the function of amoeba GEF proteins, their roles in various Rho GTPase-mediated signaling pathways, and the repercussions of modulating their activities with respect to several mechanisms related to E. histolytica pathogenesis.
Subject(s)
Entamoeba histolytica/metabolism , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/chemistry , Cloning, Molecular , Gene Expression , Glutathione Transferase/chemistry , Ligands , Models, Molecular , Phosphoproteins/chemistry , Protein Conformation , Signal Transduction , rho GTP-Binding Proteins/antagonists & inhibitors , rho GTP-Binding Proteins/chemistryABSTRACT
The actin cytoskeleton consists of multiple actin binding proteins (ABPs) that participate cooperatively in different cellular functions such as the maintenance of polarity and cell motility as well as the invasion of target cells and regulation of gene expression, among others. Due to the important role of ABPs in the pathogenesis of Entamoeba histolytica, the role of a new nucleocytoplasmic ABP from E. histolytica named EhNCABP166 was investigated. The EhNCABP166 gene encodes a protein with an estimated molecular weight of 166kDa. Structurally, this peptide is composed of two CH domains arranged in tandem at the N-terminus of the protein, followed by an alpha-helical region containing a number of different domains with a low level of homology. Two (Bin1/Amphiphysin/Rvs167) (BAR) domains, one GTPase-binding/formin 3 homology (GBD/FH3) domain, three Bcl2-associated athanogene (BAG) domains, one basic-leucine zipper (bZIP) domain and one poly(A)-binding protein C-terminal (PABC) domain were also present. Molecular and biochemical studies showed that the EhNCABP166 protein is transcribed and translated in trophozoites of E. histolytica. It was also shown that the CH domains are functional and bind to F-actin, whereas the BAR and GBD/FH3 domains interact in vitro and in vivo with different families of GTPases such as Rho and Ras, and with different phosphoinositides. These findings suggest that these domains have the conserved functional properties described in other eukaryotic systems. These domains also interacted with additional GTPase and lipid targets that have not been previously described. Finally, cellular studies showed that EhNCABP166 is localized to the cytoplasm and nucleus of E. histolytica and that it has an important role in phagocytosis, proliferation, and motility of E. histolytica.
Subject(s)
Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Actins/metabolism , Amino Acid Sequence , Cell Nucleus/chemistry , Cytoplasm/chemistry , Entamoeba histolytica/physiology , GTP Phosphohydrolases/metabolism , Gene Expression Profiling , Lipid Metabolism , Microfilament Proteins/biosynthesis , Microfilament Proteins/chemistry , Molecular Sequence Data , Molecular Weight , Phagocytosis , Protein Binding , Protein Biosynthesis , Protein Conformation , Protein Interaction Domains and Motifs , Protein Structure, Tertiary , Protozoan Proteins/biosynthesis , Protozoan Proteins/chemistry , Sequence Homology, Amino Acid , Transcription, GeneticABSTRACT
This paper reports the EhGEF1-EhRacG and EhGEF1-EhRho1 molecular complexes from Entamoeba histolytica. The not conserved amino acids Gln201,Tyr299, Gln302, Lys312, Asn313, Phe314 and Ile324 were localized, by means of an in silico computational analysis, at the interface of the exposed face from the DH domain of EhGEF1, which are important to establish the contact with its target GTPases EhRacG and EhRho1. Functional studies of nucleotide exchange of Phe314Ala mutant showed a decrement of 80% on EhRacG GTPase; in contrast the Ile324Ala mutant exhibited a reduction of 77%, specifically on EhRho1; meanwhile the Gln302Ala mutant showed a reduction of approximately 50% on the exchange activity for both GTPases. Moreover, the functional studies of the protein EhGEF1 mutants in the conserved residues Thr194Ala, Asn366Ala and Glu367Ala indicated that contrary to what has been reported for other systems, the mutation of these residues did not alter considerably its catalytic activity.
