ABSTRACT
BACKGROUND: Inorganic PPases are essential metal-dependent enzymes that convert pyrophosphate into orthophosphate. This reaction is quite exergonic and provides a thermodynamic advantage for many ATP-driven biosynthetic reactions. We have previously demonstrated that cytosolic PPase from R. microplus embryos is an atypical Family I PPase. Here, we explored the functional role of the cysteine residues located at the homodimer interface, its redox sensitivity, as well as structural and kinetic parameters related to thiol redox status. METHODS: In this work, we used prokaryotic expression system for recombinant protein overexpression, biochemical approaches to assess kinetic parameters, ticks embryos and computational approaches to analyze and predict critical amino acids as well as physicochemical properties at the homodimer interface. RESULTS: Cysteine 339, located at the homodimer interface, was found to play an important role in stabilizing a functional cooperativity between the two catalytic sites, as indicated by kinetics and Hill coefficient analyses of the WT-rBmPPase. WT-rBmPPase activity was up-regulated by physiological antioxidant molecules such as reduced glutathione and ascorbic acid. On the other hand, hydrogen peroxide at physiological concentrations decreased the affinity of WT-rBmPPase for its substrate (PPi), probably by inducing disulfide bridge formation. CONCLUSIONS: Our results provide a new angle in understanding redox control by disulfide bonds formation in enzymes from hematophagous arthropods. The reversibility of the down-regulation is dependent on hydrophobic interactions at the dimer interface. GENERAL SIGNIFICANCE: This study is the first report on a soluble PPase where dimeric cooperativity is regulated by a redox mechanism, according to cysteine redox status.
Subject(s)
Inorganic Pyrophosphatase/metabolism , Protein Multimerization , Sulfhydryl Compounds/metabolism , Ticks/enzymology , Amino Acids/metabolism , Animals , Calcium/pharmacology , Disulfides/metabolism , Electrophoresis, Polyacrylamide Gel , Fluorides/pharmacology , Glutathione Disulfide/metabolism , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/metabolism , Oxidants/pharmacology , Oxidation-Reduction , Protein Multimerization/drug effects , Recombinant Proteins/metabolism , Reducing Agents/pharmacologyABSTRACT
Ehrlichia sp. e Anaplasma platys são micro-organismos Gram negativos, parasitos intracelulares obrigatórios, residindo em vacúolos citoplasmáticos de leucócitos e plaquetas, encontrados no sangue periférico ou em tecidos. Poucos relatos têm sido feitos sobre erliquiose e anaplasmose em gatos no Brasil, os quais são baseados na presença de mórulas em leucócitos e plaquetas, ou pela detecção de anticorpos. O objetivo deste trabalho foi investigar a infecção natural por Ehrlichia sp. e A.platys em gatos no Município de Campos dos Goytacazes-RJ, através da hematoscopia e pela detecção do DNA desses agentes. Foram utilizadas amostras de sangue total e de soro de 91 gatos, independente de raça, sexo e idade. Realizaram-se hemograma, bioquímica sérica e PCR, utilizando oligonucleotídes para Ehrlichia sp. e A.platys. Os dados de hematoscopia mostraram que 9,89 por cento dos gatos apresentaram mórulas em macroplaquetas. O DNA de A.platys foi detectado em 13,18 por cento dos 91 animais e em 44,44 por cento das amostras positivas à hematoscopia. O DNA de Ehrlichia sp. não foi detectado em nenhuma amostra. Nenhuma alteração foi observada nos sinais clínicos nem nos resultados laboratoriais nos animais estudados. Os dados sugerem que os felinos domésticos podem atuar como potenciais reservatórios para A. platys, como forma não sintomática das enfermidades relacionadas.
Ehrlichia sp. and Anaplasma platys are Gram-negative micro-organisms, obligate intracellular parasites, residing in cytoplasmic vacuoles of leukocytes and platelets, found in peripheral blood or tissue. Few reports have been made about ehrlichiosis and anaplasmosis in cats in Brazil, which are based on the presence of morulae in leukocytes and platelets, or by detecting antibodies. The objective of this study was to investigate the natural infection with Ehrlichia sp. and A. platys in cats in Campos dos Goytacazes-RJ, by hematoscopia and DNA detection of these agents. Samples of whole blood and serum from 91 cats, regardless of race, gender and age. Blood count, serum biochemistry and PCR using primers for Ehrlichia sp. and A. platys were perfomed. Data from hematoscopia showed 9.89 percent of morulae only in platelets. The DNA of A. platys was detected in 13.18 percent of the 91 samples and 44.44 percent of the positive at hematoscopia. The DNA of Ehrlichia sp. was not detected in any sample. All animals studied did not show clinical signs neither positive laboratory results. The data suggest that domestic cats can serve as potential reservoirs for A. platys as asymptomatic form of related diseases.
