A new nucleocytoplasmic RhoGAP protein contributes to control the pathogenicity of Entamoeba histolytica by regulating EhRacC and EhRacD activity.
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. Immunofluorescence 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 3,5-bisphosphate. 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.
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Araceli
A
Hernandez-Flores
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Ma de Jesus
MJ
Almaraz-Barrera
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Daniela
D
Lozano-Amado
High School of Medicine of the I.P.N, Molecular Modeling Laboratory and Drug Design, Mexico City, D.F., Mexico.
Jose
J
Correa-Basurto
Cuajimalpa Unit., Department of Natural Sciences, Metropolitan Autonomous University, Mexico City, D.F., Mexico.
Arturo
A
Rojo-Dominguez
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Eva
E
Luna-Rivera
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Michael
M
Schnoor
Institut Pasteur, Department of Cell Biology and Infection, Paris, France.
Nancy
N
Guillen
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico.
Rosaura
R
Hernandez-Rivas
Department of Molecular Biomedicine, Center of Research and Advanced Studies of the I.P.N, Mexico City, D.F., Mexico. mavargas@cinvestav.mx.
Miguel
M
Vargas
D000595:Amino Acid Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000378:metabolism; D004748:Entamoeba histolytica / Q000201:enzymology / Q000472:pathogenicity*; D004912:Erythrocytes / Q000469:parasitology; D020690:GTPase-Activating Proteins / Q000737:chemistry / Q000502:physiology*; D006801:Humans; D056004:Molecular Dynamics Simulation; D010587:Phagocytosis; D021381:Protein Transport; D015800:Protozoan Proteins / Q000737:chemistry / Q000502:physiology*; D020744:rac GTP-Binding Proteins / Q000378:metabolism*
D020690:GTPase-Activating Proteins; D015800:Protozoan Proteins; C085379:rho GTPase-activating protein; D020744:rac GTP-Binding Proteins