RESUMO
Trypanosoma equiperdum belongs to the subgenus Trypanozoon, which has a significant socio-economic impact by limiting animal protein productivity worldwide. Proteins involved in the intracellular Ca2+ regulation are prospective chemotherapeutic targets since several drugs used in experimental treatment against trypanosomatids exert their action through the disruption of the parasite intracellular Ca2+ homeostasis. Therefore, the plasma membrane Ca2+-ATPase (PMCA) is considered as a potential drug target. This is the first study revealing the presence of a PMCA in T. equiperdum (TePMCA) showing that it is calmodulin (CaM) sensitive, revealed by ATPase activity, western-blot analysis and immuno-absorption assays. The cloning sequence for TePMCA encodes a 1080 amino acid protein which contains domains conserved in all PMCAs so far studied. Molecular modeling predicted that the protein has 10 transmembrane and three cytoplasmic loops which include the ATP-binding site, the phosphorylation domain and Ca2+ translocation site. Like all PMCAs reported in other trypanosomatids, TePMCA lacks a classic CaM binding domain. Nevertheless, this enzyme presents in the C-terminal tail a region of 28 amino acids (TeC28), which most likely adopts a helical conformation within a 1-18 CaM binding motif. Molecular docking between Trypanosoma cruzi CaM (TcCaM) and TeC28 shows a significant similarity with the CaM-C28PMCA4b reference structure (2kne). TcCaM-TeC28 shows an anti-parallel interaction, the peptide wrapped by CaM and the anchor buried in the hydrophobic pocket, structural characteristic described for similar complexes. Our results allows to conclude that T. equiperdum possess a CaM-sensitive PMCA, which presents a non-canonical CaM binding domain that host a 1-18 motif.
Assuntos
ATPases Transportadoras de Cálcio/análise , Calmodulina/metabolismo , Membrana Celular/enzimologia , Trypanosoma/enzimologia , Sequência de Aminoácidos , Western Blotting , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Clonagem Molecular , Imunoensaio , Modelos Moleculares , Estudos Prospectivos , Conformação Proteica , Domínios Proteicos , Alinhamento de Sequência , Trypanosoma/genéticaRESUMO
The plasma membrane Ca(2+)-ATPase (PMCA) plays a key role in the regulation of the intracellular Ca(2+) concentration. Ethanol stimulates this Ca(2+) pump in an isoform-specific manner. On search for a physiological molecule that could mimic the effect of ethanol, we have previously demonstrated that some sphingolipids containing free "hydroxyl" groups, like ceramide, are able to stimulate the PMCA. Since diacylglycerol (DAG) structurally shares some characteristics with ceramide, we evaluate its effect on the PMCA. We demonstrated that DAG is a potent stimulator of this enzyme. The activation induced is additive to that produced by calmodulin, protein-kinase C and ethanol, which implies that DAG interacts with the PMCA through a different mechanism. Additionally, by different fluorescent approaches, we demonstrated a direct binding between PMCA and DAG. The results obtained in this work strongly suggest that DAG is a novel effector of the PMCA, acting by a direct interaction.
Assuntos
Cálcio/química , Diglicerídeos/química , Membrana Eritrocítica/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Cálcio/metabolismo , Calmodulina/química , Calmodulina/metabolismo , Ceramidas/química , Ceramidas/metabolismo , Diglicerídeos/metabolismo , Ativação Enzimática/fisiologia , Etanol/química , Etanol/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ligação Proteica/fisiologia , Proteína Quinase C/química , Proteína Quinase C/metabolismoRESUMO
Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Membrana Eritrocítica/enzimologia , Pré-Eclâmpsia/enzimologia , Adolescente , Adulto , Anticorpos/imunologia , Pressão Sanguínea/fisiologia , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/imunologia , Calmodulina/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/imunologia , Etanol/metabolismo , Feminino , Humanos , Peroxidação de Lipídeos , Peso Molecular , Placenta/metabolismo , Placenta/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática , Pré-Eclâmpsia/sangue , Gravidez , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
The plasma-membrane Ca(2+)-ATPase is a key enzyme in the regulation of the intracellular Ca(2+) concentration. On the other hand, sphingolipids have been recognized recently as important second messengers, acting in many systems in combination with Ca(2+). In view of the fact that the Ca(2+)-ATPase is stimulated by ethanol, and since sphingolipids possess free hydroxy groups, we decided to study the possible effect of ceramide and sphingosine on this calcium pump. Here we show that ceramide stimulates the Ca(2+)-ATPase in a dose-dependent manner and additively to the activation observed in the presence of calmodulin or ethanol, when compared with any of these effectors added alone. Ceramide affects both the affinity for Ca(2+) and the V(max) of the enzyme. Furthermore, this second messenger also stimulates Ca(2+) transport in inside-out plasma-membrane vesicles from erythro cytes. Conversely, sphingosine, which is reported to act in many systems antagonistically with ceramide, showed an inhibitory effect on Ca(2+)-ATPase activity. This inhibition was also observed on the calmodulin-stimulated enzyme. These results, taken together, suggest that ceramide and sphingosine act antagonistically on the plasma-membrane Ca(2+)-ATPase. This is in accordance with the frequently reported opposite effect of these sphingolipids on intracellular Ca(2+) concentration.
Assuntos
ATPases Transportadoras de Cálcio/antagonistas & inibidores , Ceramidas/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Esfingosina/farmacologia , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Ativação Enzimática , Membrana Eritrocítica/enzimologia , HumanosRESUMO
The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...