EhGEF3, a novel Dbl family member, regulates EhRacA activation during chemotaxis and capping inEntamoeba histolytica (original) (raw)
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Experimental Parasitology, 2005
The molecular, biochemical, and cellular characterization of EhGEF1 protein is described. Complete cDNA sequence of 1890 bp revealed an open reading frame that encodes a protein of 69 kDa. EhGEF1 is constituted of Dbl homology domain, pleckstrin homology domain, and several putative regulation sites. Studies of guanine nucleotide exchange activity of EhGEF1 on several GTPases from Entamoeba histolytica and Homo sapiens showed preferential activation on EhRacG, suggesting that EhGEF1 protein could be involved in mechanisms related to actin cytoskeleton activation, cytokinesis, capping, and uroid formation in trophozoite. Confocal microscopy studies of pExEhNeo/HSV-tagged-EhGEF1-transfected cells showed that trophozoites stimulated with ConA, EhGEF1, and EhRacG were localized at plasma membrane. Cellular studies showed that F-actin content of pExEhNeo/ HSV-tagged-EhGEF1-transfected trophozoites as well as cellular migration and cell damage capacity were signiWcantly altered. The observations suggest that EhRacG was the principal target of EhGEF1 and that EhGEF1 may provide a link between F-actin dynamics and EhRacG signaling. 2004 Elsevier Inc. All rights reserved.
Molecular and Biochemical Parasitology, 2007
Dbl proteins are a family of factors that exchange the guanine nucleotide which promote the activation of Rho small GTPases. This paper reports the molecular, structural, biochemical and functional characterization of EhGEF2, a new member of the Dbl family. EhGEF2 is the second GEF studied in parasites and in the protozoan Entamoeba histolytica, and it is also the first member of the Dbl family that was found to have Arm repeats. The catalytic domain (DH) of EhGEF2 has the conserved residues T421, N590 and E591, which are important for the activation of the GTPases. Biochemical studies on EhGEF2 showed that it could activate in vitro the amoebic GTPases EhRacA, EhRacB, EhRacC, EhRacD, EhRacG, EhRacH and EhCdc42, being EhRacG its main target. It was found that the DH domain binds specifically phosphatidic acid (PA); docking and lipid dot blot studies indicated that this binding does not interfere with the contact surface of EhRacG. Functional studies showed that both the Arm repeats and the catalytic domain of EhGEF2 participate in its localization at the amoebic membrane. Expression of a negative dominant version of EhGEF2 protein in E. histolytica provoked a 30% decrease in its ability to phagocyte human erythrocytes as well as severe effects on both the proliferation and the cellular chemotaxis which suggest that EhGEF2 participates in these cellular processes.
A unique Rab GTPase, EhRabA, is involved in motility and polarization of Entamoeba histolytica cells
Molecular and Biochemical Parasitology, 2005
Entamoeba histolytica, an enteric protozoan parasite, infects 10% of the world's population leading to 50 million cases of invasive amoebiasis annually. Motility, which requires cell polarization, is important to the virulence of this pathogen, as it may result in destruction of host tissues and invasion. To gain insight into these processes in Entamoeba, a unique Rab GTPase, EhRabA, which localizes to the leading edge of cells, was characterized. Cell lines expressing a dominant negative version of EhRabA (EhRabA-DN) were generated. These mutant cells exhibited alterations in cell shape, polarity, and motility, supporting a role for this Rab in the regulation of these processes. Consistent with the notion that a dynamic actin cytoskeleton is crucial to cell polarity and motility, these mutants also exhibited alterations in the actin cytoskeleton. Cells expressing EhRabA-DN also displayed defects in several virulence functions including the ability to adhere to host cells, destroy host cells, and release cysteine proteases. Mislocalization of a prominent adhesion molecule, the galactose/N-acetylgalactosamine (Gal/GalNAc) adherence lectin and reorganization of ordered lipid domains, known as lipid rafts, also accompanied expression of EhRabA-DN. Interestingly, several endocytic processes were unaffected by expression of EhRabA-DN. Together, these data suggest that EhRabA may be involved in the regulation of polarization, motility and actin cytoskeletal dynamics: functions that participate in the pathogenicity of Entamoeba.
G protein signaling in the parasite Entamoeba histolytica
Experimental & molecular medicine, 2013
The parasite Entamoeba histolytica causes amebic colitis and systemic amebiasis. Among the known amebic factors contributing to pathogenesis are signaling pathways involving heterotrimeric and Ras superfamily G proteins. Here, we review the current knowledge of the roles of heterotrimeric G protein subunits, Ras, Rho and Rab GTPase families in E. histolytica pathogenesis, as well as of their downstream signaling effectors and nucleotide cycle regulators. Heterotrimeric G protein signaling likely modulates amebic motility and attachment to and killing of host cells, in part through activation of an RGS-RhoGEF (regulator of G protein signaling-Rho guanine nucleotide exchange factor) effector. Rho family GTPases, as well as RhoGEFs and Rho effectors (formins and p21-activated kinases) regulate the dynamic actin cytoskeleton of E. histolytica and associated pathogenesis-related cellular processes, such as migration, invasion, phagocytosis and evasion of the host immune response by surfa...
Cellular Microbiology, 2016
Small GTPases are signalling molecules that regulate important cellular processes. GTPases are deactivated by GTPase-activating proteins (GAPs). While human GAPs have been intensively studied, no GAP has yet been characterized in Entamoeba histolytica. In this study, we identified and characterized a novel nucleocytoplasmic RhoGAP in E. histolytica termed EhRhoGAPnc. In silico analyses of the domain structure revealed a previously undescribed peptide region within the carboxy-terminal region of EhRhoGAPnc capable of interacting with phosphatidic acid and phosphatidylinositol 3,5-bisphosphate. The full structural GAP domain showed increase GAP activity compared with the minimum region able to display GAP activity, as analysed both by experimental assays and molecular dynamics simulations. Furthermore, we identified amino acid residues that promote interactions between EhRhoGAPnc and its target GTPases EhRacC and EhRacD. Immuno-fluorescence studies revealed that EhRhoGAPnc colocalized with EhRacC and EhRacD during uroid formation but not during erythrophagocytosis. Interestingly, during erythrophagocytosis of red blood cells, EhRhoGAPnc colocalized with phosphatidic acid and phosphatidylinositol 3,5bisphosphate. Overexpression of EhRhoGAPnc in E. histolytica led to inhibition of actin adhesion plate formation, migration, adhesion of E. histolytica to MDCK cells and consequently to an impairment of the cytopathic activity.
Unique structural and nucleotide exchange features of the Rho1 GTPase of Entamoeba histolytica
2011
Background: Rho family GTPases regulate Entamoeba histolytica pathogenesis. Results: Despite Ras-like structural features and fast intrinsic nucleotide exchange, EhRho1 engages classical Rho effectors and regulates actin. Conclusion: EhRho1 is a true Rho family GTPase with a unique mode of nucleotide interaction. Significance: Possibly representing an early Rho subfamily divergence from the Ras superfamily, EhRho1 likely regulates actin polymerization in E. histolytica. ) contains supplemental Figs. S1 and S2. The atomic coordinates and structure factors (codes 3REF and 3REG) have been deposited in the Protein . 5 The abbreviations used are: GEF, guanine nucleotide exchange factors; GDI, guanine nucleotide dissociation inhibitors; GBD, GTPase-binding domains; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; EhRho1, E. histolytica Rho1; Hs, Homo sapiens; FH3, formin homology 3 domain.
Structural determinants of RGS-RhoGEF signaling critical to Entamoeba histolytica pathogenesis
Structure (London, England : 1993), 2013
G protein signaling pathways, as key components of physiologic responsiveness and timing, are frequent targets for pharmacologic intervention. Here, we identify an effector for heterotrimeric G protein α subunit (EhGα1) signaling from Entamoeba histolytica, the causative agent of amoebic colitis. EhGα1 interacts with this effector and guanosine triphosphatase-accelerating protein, EhRGS-RhoGEF, in a nucleotide state-selective fashion. Coexpression of EhRGS-RhoGEF with constitutively active EhGα1 and EhRacC leads to Rac-dependent spreading in Drosophila S2 cells. EhRGS-RhoGEF overexpression in E. histolytica trophozoites leads to reduced migration toward serum and lower cysteine protease activity, as well as reduced attachment to, and killing of, host cells. A 2.3 Å crystal structure of the full-length EhRGS-RhoGEF reveals a putative inhibitory helix engaging the Dbl homology domain Rho-binding surface and the pleckstrin homology domain. Mutational analysis of the EhGα1/EhRGS-RhoGEF ...
BioMed Research International
Entamoeba histolytica (Eh) is a pathogenic eukaryote that often resides silently in humans under asymptomatic stages. Upon indeterminate stimulus, it develops into fulminant amoebiasis that causes severe hepatic abscesses with 50% mortality. This neglected tropical pathogen relies massively on membrane modulation to flourish and cause disease; these modulations range from the phagocytic mode for food acquisition to a complex trogocytosis mechanism for tissue invasion. Rab GTPases form the largest branch of the Ras-like small GTPases, with a diverse set of roles across the eukaryotic kingdom. Rab GTPases are vital for the orchestration of membrane transport and the secretory pathway responsible for transporting the pathogenic effectors, such as cysteine proteases (EhCPs) which help in tissue invasion. Rab GTPases thus play a crucial role in executing the cytolytic effect of E. histolytica. First, they interact with Gal/Nac lectins required for adhering to the host cells, and then, th...
Experimental Parasitology, 2005
EhRabB is an Entamoeba histolytica protein involved in phagocytosis. However, proteins that regulate the EhRabB activity are unknown. Here, we report the identiWcation of a putative G protein-coupled receptor of E. histolytica (EhGPCR-1) that binds to EhRabB. By two-hybrid screening, we found a 372-bp cDNA fragment that encodes the C-terminus of EhGPCR-1. The cloning and sequence of the full-length cDNA revealed that it predicts a polypeptide with two tyrosine-based sorting signals for endocytosis and seven transmembranal domains. These results suggest that EhGPCR-1 could be a GPCR involved in phagocytosis. EhGPCR-1 could be a member of the Rhodopsin family, characterized by a short N-terminus without cysteine residues.