Comparative characterisation of an ecto-5'-nucleotidase (CD73) in non-tumoral MCF10-A breast cells and triple-negative MDA-MB-231 breast cancer cells.

Ecto-5'-nucleotidase (CD73) hydrolyses 5'AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA-MB-231) express high CD73 levels, and this enzyme has been found to play a tumour-promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto-5'-nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF-10A cells, triple-negative breast cancer MDA-MB-231 cells had a higher ecto-5'-nucleotidase expression level and enzymatic activity. Although ecto-5'-nucleotidase activity in the MDA-MB-231 cell line showed no selectivity among monophosphorylated substrates, 5'AMP was preferred by the MCF-10A cell line. Compared to the MCF-10A cell line, the MDA-MB-231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,ß­methylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto-5-nucleotidase significantly reduced the migratory and invasive capacity of MDA-MB-231 cells, suggesting that ecto-5-nucleotidase activity might play an important role in metastatic progression.

ATP synthase affects lipid metabolism in the kissing bug Rhodnius prolixus beyond its role in energy metabolism.

ATP synthase plays an essential role in mitochondrial metabolism, being responsible for the production of ATP in oxidative phosphorylation. However, recent results have shown that it may also be present in the cell membrane, involved in lipophorin binding to its receptors. Here, we used a functional genetics approach to investigate the roles of ATP synthase in lipid metabolism in the kissing bug Rhodnius prolixus. The genome of R. prolixus encodes five nucleotide-binding domain genes of the ATP synthase α and ß family, including the α and ß subunits of ATP synthase (RpATPSynα and RpATPSynß), and the catalytic and non-catalytic subunits of the vacuolar ATPase (RpVha68 and RpVha55). These genes were expressed in all analyzed organsn highest in the ovaries, fat body and flight muscle. Feeding did not regulate the expression of ATP synthases in the posterior midgut or fat body. Furthermore, ATP synthase is present in the fat body's mitochondrial and membrane fractions. RpATPSynß knockdown by RNAi impaired ovarian development and reduced egg-laying by approximately 85%. Furthermore, the lack of RpATPSynß increased the amount of triacylglycerol in the fat body due to increased de novo fatty acid synthesis and reduced transfer of lipids to lipophorin. RpATPSynα knockdown had similar effects, with altered ovarian development, reduced oviposition, and triacylglycerol accumulation in the fat body. However, ATP synthases knockdown had only a slight effect on the amount of ATP in the fat body. These results support the hypothesis that ATP synthase has a direct role in lipid metabolism and lipophorin physiology, which are not directly due to changes in energy metabolism.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.

Effect of starvation on lipophorin density in fifth instar larval Manduca sexta.

Lipophorin (Lp) is a major insect lipoprotein and is responsible for lipid transport between organs. In this study, the effect of starvation on Lp properties was analyzed in larval Manduca sexta during the fifth instar. Lp hemolymph concentrations in larvae at days 1 and 2 were around 2-3 mg/ml and at day 3 it increased to 8 mg/ml. When larvae were starved for 24 h, they did not grow, but their body mass and hemolymph volume did not decrease significantly. Differences in Lp densities were observed. In fed larvae, from days 1 to 4, two major Lp populations were found with densities of 1.124 ± 0.002 (high density Lp-larval1 , HDLp-L1 ) and 1.141 ± 0.002 g/ml (HDLp-L2 ). When larvae were starved for 24 h, only one Lp population was present, with density 1.114 ± 0.001 g/ml (HDLp-Ls ). When larvae were abdominally ligated at day 1 or 2 of fifth instar, only HDLp-Ls was found after 24 h, indicating that the formation of this HDLp population was not dependent on any factor released by head. On the other hand, larvae that were ligated at day 3 showed the same Lp populations as the fed ones. In 24-h starved larvae, lipid load in Lp was higher as compared to the fed controls. In 24-h ligated larvae Lp lipid content increased when ligation was performed on day 1 or 2, but not on day 3. So, different responses to starvation can be observed depending on the developmental phase of the same larval instar.

Trypanosoma cruzi: effects of heat shock on ecto-ATPase activity.

In this work, we demonstrate that Trypanosoma cruzi Y strain epimastigotes exhibit Mg2+-dependent ecto-ATPase activity that is stimulated by heat shock. When the epimastigotes were incubated at 37°C for 2h, the ecto-ATPase activity of the cells was 43.95±0.97 nmol Pi/h×10(7) cells, whereas the ecto-ATPase activity of cells that were not exposed to heat shock stress was 16.97±0.30 nmol Pi/h×10(7) cells. The ecto-ATPase activities of cells, that were exposed or not exposed to heat shock stress had approximately the same Km values (2.25±0.26 mM ATP and 1.55±0.23 mM ATP, respectively) and different Vmax values. The heat-shocked cells had higher Vmax values (54.38±3.07 nmol Pi/h×10(7) cells) than the cells that were not exposed to heat shock (19.38±1.76 nmol Pi/h×10(7) cells). We also observed that the ecto-phosphatase and ecto-5'nucleotidase activities of cells that had been incubated at 28°C or 37°C were the same. Interestingly, cycloheximide, an inhibitor of protein synthesis, suppressed the heat shock effect of ecto-ATPase activity on T. cruzi. The Mg2+-dependent ecto-ATPase activity from the Y strain (high virulence) was approximately 2-fold higher than that of Dm28c (a clone with low virulence). In addition, these two strains presented different responses to heat shock with regard to their ecto-ATPase activities; Y strain epimastigotes had a stimulation of 2.52-fold while the Dm28c strain had a 1.71-fold stimulation. In this context, the virulent trypomastigote form of T. cruzi, Dm28c, had an ecto-ATPase activity that was more than 7-fold higher (66.67±5.98 nmol Pi/h×10(7) cells) than that of the insect epimastigote forms (8.91±0.76 nmol Pi/h×10(7) cells). This difference increased to approximately 10-fold when both forms were subjected to heat shock stress (181.14±16.48 nmol Pi/h×10(7) cells for trypomastigotes and 16.71±1.17 nmol Pi/h×10(7) cells for epimastigotes at 37°C). The ecto-ATPase activity of a plasma membrane-enriched fraction obtained from T. cruzi epimastigotes was not increased by heat treatment, which suggested that cytoplasmic components had an influence on enzyme activation by heat shock stress.

Leishmania amazonensis: characterization of an ecto-3'-nucleotidase activity and its possible role in virulence.

Ecto-3'-nucleotidase/nuclease (3'NT/NU) is a membrane-bound enzyme that plays a key role in the nutrition of Leishmania sp. protozoan parasites. This enzyme generates nucleosides via hydrolyzes of 3'mononucleotides and nucleic acids, which enter the cell by specific transporters. In this work, we identify and characterize Leishmania amazonensis ecto-3'-nucleotidase activity (La3'-nucleotidase), report ammonium tetrathiomolybdate (TTM) as a novel La3'-nucleotidase inhibitor and approach the possible involvement of ecto-3'-nucleotidase in cellular adhesion. La3'-nucleotidase presented characteristics similar to those reported for the class I single-strand nuclease family; a molecular weight of approximately 40 kDa and optimum activity in an alkaline pH range were observed. Although it is conserved among the genus, La3'-nucleotidase displays different kinetic properties; it can be inhibited by vanadate, molybdate and Cu(2+) ions. Interestingly, ecto-3'-nucleotidase activity is 60-fold higher than that of ecto-5'-nucleotidase in L. amazonensis. Additionally, ecto-3'-nucleotidase activity is two-fold higher in virulent L. amazonensis cells than in avirulent ones. Notably, macrophage-parasite attachment/invasion was increased by 400% in the presence of adenosine 3'-monophosphate (3'AMP); however, this effect was reverted by TTM treatment. We believe that La3'-nucleotidase may play a significant role in the generation of adenosine, which may contribute to mammalian host immune response impairment and establishment of infection.

Localization and function of Rhipicephalus (Boophilus) microplus vitellin-degrading cysteine endopeptidase.

The tick Rhipicephalus (Boophilus) microplus is an important parasite of cattle in many areas of the tropics. Characterization of molecules involved in mechanisms such as vitellogenesis and embryo development may contribute to a better understanding of this parasite's physiology. The vitellin-degrading cysteine endopeptidase (VTDCE) is the most active enzyme involved in vitellin hydrolysis in R. microplus eggs. Here we show an association between VTDCE and vitellin in an additional site, apart from the active site. Our data also demonstrate cysteine endopeptidase activity in different tissues such as ovary, gut, fat body, salivary gland and female haemolymph, where it is controlled by a physiological inhibitor. In R. microplus female gut, VTDCE is localized in areas of protein synthesis and trafficking with the underlying haemolymph. VTDCE is also localized in the ovary basal region, in vesicle membranes of ovary pedicel cells and in oocyte cytosol. These results suggest that VTDCE plays a role in vitellin digestion during tick development.

Ecto-nucleotidase activities in the fat body of Rhodnius prolixus.

In this study, we describe the ability of intact fat body of an insect, Rhodnius prolixus, to hydrolyze extracellular ATP. In these fat bodies, the ATP hydrolysis was low in the absence of any divalent metal, and was stimulated by MgCl(2). Both activities (in the absence or presence of MgCl(2)) were linear with time for at least 30 min. In order to confirm the observed nucleotidase activities as ecto-nucleotidases, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid). This reagent inhibited both nucleotidase activities and its inhibitory effect was suppressed by ATP. Both ecto-nucleotidase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin, ouabain, vanadate, molybdate, sodium fluoride, levamizole, tartrate, p-NPP, sodium phosphate, and suramin. Concanavalin A, activator of some ecto-ATPases, was able to stimulate the Mg(2+)-independent nucleotidase activity, but not the Mg(2+)-dependent one. The Mg(2+)-independent nucleotidase activity was enhanced with increases in the pH in the range between 6.4-8.0, but the Mg(2+)-dependent nucleotidase activity was not affected. Besides MgCl(2) , the ecto-ATPase activity was also stimulated by CaCl(2),() MnCl(2), and SrCl(2), but not by ZnCl(2). ATP, ADP, and AMP were the best substrates for the Mg(2+)-dependent ecto-nucleotidase activity, and CTP, GTP, and UTP produced very low reaction rates. However, the Mg(2+)-independent nucleotidase activity recognized all these nucleotides producing similar reaction rates, but GTP was a less efficient substrate. The possible role of the two ecto-nucleotidase activities present on the cell surface of fat body of Rhodnius prolixus, which are distinguished by their substrate specificity and their response to Mg(2+), is discussed.

Trypanosoma rangeli: characterization of a Mg-dependent ecto ATP-diphosphohydrolase activity.

In this work we describe the ability of living Trypanosoma rangeli to hydrolyze extracellular ATP. In these intact parasites whose viability was assessed before and after the reactions by motility and by Trypan blue dye exclusion, there was a low level of ATP hydrolysis in the absence of any divalent metal (1.53+/-0.12 nmol P(i)/h x 10(7) cells). The ATP hydrolysis was stimulated by MgCl(2) and the Mg-dependent ecto-ATPase activity was 5.24+/-0.64 nmol P(i)/h x 10(7) cells. The Mg-dependent ecto-ATPase activity was linear with cell density and with time for at least 60 min. This stimulatory effect on the ATP hydrolysis was also observed when MgCl(2) was replaced by MnCl(2), but not by CaCl(2), SrCl(2), and ZnCl(2). The apparent K(m) for Mg-ATP2- was 0.53+/-0.11 mM. The optimum pH for the T. rangeli Mg-dependent ecto-ATPase activity lies in the alkaline range. This ecto-ATPase activity was insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, molybdate, sodium fluoride, tartrate, and levamizole. To confirm that this Mg-dependent ATPase was an ecto-ATPase, we used an impermeant inhibitor, DIDS (4, 4'-diisothiocyanostylbene 2'-2'-disulfonic acid) as well as suramin, an antagonist of P2 purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg(2+)-dependent ATPase activity in a dose-dependent manner. This ecto-ATPase activity was stimulated by carbohydrates involved in the attachment/invasion of salivary glands of Rhodnius prolixus and by lipophorin, an insect lipoprotein circulating in the hemolymph.

Oogenesis and egg development in triatomines: a biochemical approach

Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo.