RESUMO
Entamoeba histolytica is the causative agent of amoebiasis, and Entamoeba dispar is its noninvasive morphological twin. Entamoeba invadens is a reptilian parasite. In the present study, Western blot, phosphatase activity, immunofluorescence, and bioinformatic analyses were used to identify PP2C phosphatases of E. histolytica, E. dispar, and E. invadens. PP2C was identified in trophozoites of all Entamoeba species and cysts of E. invadens. Immunoblotting using a Leishmania mexicana anti-PP2C antibody recognized a 45.2 kDa PP2C in all species. In E. histolytica and E. invadens, a high molecular weight element PP2C at 75 kDa was recognized, mainly in cysts of E. invadens. Immunofluorescence demonstrated the presence of PP2C in membrane and vesicular structures in the cytosol of all species analyzed. The ~75 kDa PP2C of Entamoeba spp. shows the conserved domain characteristic of phosphatase enzymes (according to in silico analysis). Possible PP2C participation in the encystation process was discussed.
Assuntos
Entamoeba/enzimologia , Proteína Fosfatase 2C/metabolismo , Proteínas de Protozoários/metabolismo , Trofozoítos/enzimologia , Sequência de Aminoácidos , Animais , Entamoeba/isolamento & purificação , Entamebíase/parasitologia , Entamebíase/patologia , Humanos , Filogenia , Proteína Fosfatase 2C/química , Proteína Fosfatase 2C/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Homologia de Sequência de Aminoácidos , Trofozoítos/isolamento & purificaçãoRESUMO
Protein phosphatases are enzymes that dephosphorylate tyrosine and serine/threonine amino acid residues. Although their role in cellular processes has been best characterized in higher eukaryotes, they have also been identified and studied in different pathogenic microorganisms (e.g., parasites) in the last two decades. Whereas some parasite protein phosphatases carry out functions similar to those of their homologs in yeast and mammalian cells, others have unique structural and/or functional characteristics. Thus, the latter unique phosphatases may be instrumental as targets for drug therapy or as markers for diagnosis. It is important to better understand the involvement of protein phosphatases in parasites in relation to their cell cycle, metabolism, virulence, and evasion of the host immune response. The up-to-date information about parasite phosphatases of medical and veterinarian relevance is herein reviewed.
Assuntos
Evasão da Resposta Imune , Parasitos , Fosfoproteínas Fosfatases , Animais , Fosfoproteínas Fosfatases/genética , VirulênciaRESUMO
Protein phosphorylation and dephosphorylation are increasingly recognized as important processes for regulating multiple physiological mechanisms. Phosphorylation is carried out by protein kinases and dephosphorylation by protein phosphatases. Phosphoprotein phosphatases (PPPs), one of three families of protein serine/threonine phosphatases, have great structural diversity and are involved in regulating many cell functions. PP2C, a type of PPP, is found in Leishmania, a dimorphic protozoan parasite and the causal agent of leishmaniasis. The aim of this study was to clone, purify, biochemically characterize and quantify the expression of PP2C in Leishmania mexicana (LmxPP2C). Recombinant LmxPP2C dephosphorylated a specific threonine (with optimal activity at pH 8) in the presence of the manganese divalent cation (Mn+2). LmxPP2C activity was inhibited by sanguinarine (a specific inhibitor) but was unaffected by protein tyrosine phosphatase inhibitors. Western blot analysis indicated that anti-LmxPP2C antibodies recognized a molecule of 45.2 kDa. Transmission electron microscopy with immunodetection localized LmxPP2C in the flagellar pocket and flagellum of promastigotes but showed poor staining in amastigotes. Interestingly, LmxPP2C belongs to the ortholog group OG6_142542, which contains only protozoa of the family Trypanosomatidae. This suggests a specific function of the enzyme in the flagellar pocket of these microorganisms.
