RESUMO
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.
Assuntos
Entamoeba histolytica/patogenicidade , Proteínas Ativadoras de GTPase/fisiologia , Proteínas de Protozoários/fisiologia , Proteínas rac de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Núcleo Celular/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Entamoeba histolytica/enzimologia , Eritrócitos/parasitologia , Proteínas Ativadoras de GTPase/química , Humanos , Simulação de Dinâmica Molecular , Fagocitose , Transporte Proteico , Proteínas de Protozoários/químicaRESUMO
Rho GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to cytoskeleton. The Rho guanine nucleotide exchange factors (RhoGEFs) have been implicated in direct activation of these GTPases. Here, we describe a novel RhoGEF, denominated EhGEF3 from the parasite Entamoeba histolytica, which encodes a 110 kDa protein containing the domain arrangement of a Dbl homology domain in tandem with a pleckstrin homology domain, the DH domain of EhGEF3 is closely related with the one of the Vav3 protein. Biochemical analysis revealed that EhGEF3 is capable of stimulating nucleotide exchange on the E. histolytica EhRacA and EhRho1 GTPases in vitro, however only a partial GEF activity toward Cdc42 was observed. Conserved residue analysis showed that the N816 and L817 residues are critical for EhGEF3 activity. Cellular studies revealed that EhGEF3 colocalises with EhRacA in the rear of migrating cells, probably regulating the retraction of the uroid and promoting the activation of these GTPase during the chemotactic response toward fibronectin, and that EhGEF3 also regulates EhRacA activation during the capping of cell receptors. These results suggest that EhGEF3 should have a direct role in activating EhRacA, and in bringing the activated GTPase to specific target sites such as the uroid.
Assuntos
Quimiotaxia , Entamoeba histolytica/enzimologia , Entamoeba histolytica/fisiologia , Regulação da Expressão Gênica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Entamoeba histolytica/genética , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/análise , Fatores de Troca do Nucleotídeo Guanina/química , Técnicas In Vitro , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Proteínas rac de Ligação ao GTP/genéticaRESUMO
EhABP-120 is the first filamin identified in the parasitic protozoan Entamoeba histolytica. Filamins are a family of cross-linking actin-binding proteins that promote a dynamic orthogonal web. They have been reported to interact directly with more than 30 cellular proteins and some phosphoinositides. The biochemical consequences of these interactions may have either positive or negative effects on the cross-linking function and also form a link between the cytoskeleton and plasma membrane. In this study, the EhABP-120 carboxy-terminal domain (END) was biochemically characterized. This domain was able to associate to 3-sulfate galactosyl ceramide, a new lipid target for a member of the filamin family. Also, the END domain was able to dimerize "in vitro." Molecular modeling analysis showed that the dimeric region is stabilized by a disulfide bond. Electrostatic and docking studies suggest that an electropositive concave pocket at the dimeric END domain interacts simultaneously with several sulfogalactose moieties of the sulfatide.
Assuntos
Proteínas Contráteis/química , Proteínas Contráteis/metabolismo , Entamoeba histolytica/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas Contráteis/genética , Entamoeba histolytica/genética , Filaminas , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , TransfecçãoRESUMO
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 significantly 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.
Assuntos
Entamoeba histolytica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Concanavalina A/farmacologia , Sequência Conservada , DNA Complementar/química , DNA Complementar/isolamento & purificação , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Entamoeba histolytica/genética , Expressão Gênica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/química , Fatores de Troca do Nucleotídeo Guanina/genética , Microscopia Confocal , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Reação em Cadeia da Polimerase , Transfecção , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/fisiologia , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/fisiologiaRESUMO
In the present work, the complete amino acid sequence of the Entamoeba histolytica ribosomal protein L10 (EhL10) is reported. cDNA of 630bp revealed an open reading frame that encodes a protein of 210 amino acids. Analysis of EhL10 ribosomal protein revealed 75% similarity and 57% identity with QM protein from Homo sapiens and 78 and 60%, respectively, with Arabidopsis thaliana. Western blot analysis of ribosomal proteins from E. histolytica showed that EhL10 protein is part of the ribosomal complex. Immunofluorescence analysis of EhL10 distribution in a transfected E. histolytica strain showed that EhL10 protein was mainly localized in the nucleus of trophozoites. Overexpression of EhL10 ribosomal protein in trophozoites transfected with the pExEhNeo/EhL10 vector exhibited a 60% reduction in cellular growth. DNA mobility-shift assays demonstrated that EhL10 ribosomal protein was able to destabilize the activating protein 1 (AP-1) complex binding specifically to the c-Jun-like protein. It is proposed in this study that the complex formation of EhL10 with c-Jun-like protein interferes with transcriptional activation of genes controlled by Jun (i.e. gene involved in cell growth). It is also being reported identification of a member of the AP-1 complex, the c-Jun-like protein, in nuclear extracts of E. histolytica using human-specific antibodies against this protein. The observations suggest that EhL10 may have an extraribosomal function in E. histolytica involved in suppression of cell proliferation in E. histolytica similar to the QM protein.