RESUMO
Hyperglycemia is a common feature of diabetes mellitus, considered as a risk factor for cancer. However, its direct effects in cancer cell behavior are relatively unexplored. Herein we show that high glucose concentration induces aberrant glycosylation, increased cell proliferation, invasion and tumor progression of colon cancer. By modulating the activity of the rate-limiting enzyme, glutamine-fructose-6-phosphate amidotransferase (GFAT), we demonstrate that hexosamine biosynthetic pathway (HBP) is involved in those processes. Biopsies from patients with colon carcinoma show increased levels of GFAT and consequently aberrant glycans' expression suggesting an increase of HBP flow in human colon cancer. All together, our results open the possibility that HBP links hyperglycemia, aberrant glycosylation and tumor malignancy, and suggest this pathway as a potential therapeutic target for colorectal cancer.
RESUMO
BACKGROUND: Proliferation of Leishmania infantum depends on exogenous inorganic phosphate (P(i)) but little is known about energy metabolism and transport of P(i) across the plasma membrane in Leishmania sp. METHODS: We investigated the kinetics of 32P(i) transport, the influence of H+ and K+ ionophores and inhibitors, and expression of the genes for the Na+:P(i) and H+:P(i) cotransporters. RESULTS: The proton ionophore FCCP, bafilomycin A1 (vacuolar ATPase inhibitor), nigericin (K+ ionophore) and SCH28080 (an inhibitor of H+, K(+)-ATPase) all inhibited the transport of P(i). This transport showed Michaelis-Menten kinetics with K0.5 and V(max) values of 0.016 +/- 0.002 mM and 564.9 +/- 18.06 pmol x h(-1) x 10(-7) cells, respectively. These values classify the P(i) transporter of L. infantum among the high-affinity transporters, a group that includes Pho84 of Saccharomyces cerevisiae. Two sequences were identified in the L. infantum genome that code for phosphate transporters. However, transcription of the PHO84 transporter was 10-fold higher than the PHO89 transporter in this parasite. Accordingly, P(i) transport and LiPho84 gene expression were modulated by environmental P(i) variations. CONCLUSIONS: These findings confirm the presence of a P(i) transporter in L. infantum, similar to PHO84 in S. cerevisiae, that contributes to the acquisition of inorganic phosphate and could be involved in growth and survival of the promastigote forms of L. infantum. GENERAL SIGNIFICANCE: This work provides the first description of a PHO84-like P(i) transporter in a Trypanosomatide parasite of the genus Leishmania, responsible for many infections worldwide.
Assuntos
Leishmania infantum/enzimologia , Fosfatos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Meios de Cultura , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Cinética , Leishmania infantum/genética , Macrolídeos/farmacologia , Dados de Sequência Molecular , Nigericina/farmacologia , Fosfatos/farmacologia , Radioisótopos de Fósforo , Filogenia , Ionóforos de Próton/farmacologia , Simportadores de Próton-Fosfato/antagonistas & inibidores , Simportadores de Próton-Fosfato/genética , Simportadores de Próton-Fosfato/metabolismo , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato/antagonistas & inibidores , Proteínas Cotransportadoras de Sódio-Fosfato/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
BACKGROUND: Orthophosphate (Pi) is a central compound in the metabolism of all organisms, including parasites. There are no reports regarding the mechanisms of Pi acquisition by Trypanosoma cruzi. METHODS: (32)Pi influx was measured in T. cruzi epimastigotes. The expression of Pi transporter genes and the coupling of the uptake to Na(+), H(+) and K(+) fluxes were also investigated. The transport capacities of different evolutive forms were compared. RESULTS: Epimastigotes grew significantly more slowly in 2mM than in 50mM Pi. Influx of Pi into parasites grown under low Pi conditions took place in the absence and presence of Na(+). We found that the parasites express TcPho84, a H(+):Pi-symporter, and TcPho89, a Na(+):Pi-symporter. Both Pi influx mechanisms showed Michaelis-Menten kinetics, with a one-order of magnitude higher affinity for the Na(+)-dependent system. Collapsing the membrane potential with carbonylcyanide-p-trifluoromethoxyphenylhydrazone strongly impaired the influx of Pi. Valinomycin (K(+) ionophore) or SCH28028 (inhibitor of (H(+)+K(+))ATPase) significantly inhibited Pi uptake, indicating that an inwardly-directed H(+) gradient energizes uphill Pi entry and that K(+) recycling plays a key role in Pi influx. Furosemide, an inhibitor of the ouabain-insensitive Na(+)-ATPase, decreased only the Na(+)-dependent Pi uptake, indicating that this Na(+) pump generates the Na(+) gradient utilized by the symporter. Trypomastigote forms take up Pi inefficiently. CONCLUSIONS: Pi starvation stimulates membrane potential-sensitive Pi uptake through different pathways coupled to Na(+) or H(+)/K(+) fluxes. GENERAL SIGNIFICANCE: This study unravels the mechanisms of Pi acquisition by T. cruzi, a key process in epimastigote development and differentiation to trypomastigote forms.
Assuntos
Fosfatos/metabolismo , Potássio/metabolismo , Sódio/metabolismo , Trypanosoma cruzi/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Concentração de Íons de Hidrogênio , Imidazóis/farmacologia , Valinomicina/farmacologiaRESUMO
BACKGROUND: Trypanosoma rangeli is dependent on the presence of exogenous orthophosphate (Pi) for maximal growth and ecto-phosphatase activity is responsible for Pi supply under low Pi. Here we investigated the mechanisms of Pi uptake. METHODS: We investigated the kinetics of 32Pi transport, its Na+ and H+ dependence, its correlation with the Na+-ATPase and H+-ATPase, and gene expression of the Na+:Pi cotransporter and Na+-ATPase. RESULTS: T. rangeli grown under limiting Pi transports this anion to the cytosol in the absence and presence of Na+, suggesting that influx is mediated by both Na+-independent and Na+-dependent transporters. Cloning studies demonstrated that this parasite expresses a Pi transporter not previously studied in trypanosomatids. The H+ ionophore, carbonylcyanide-p-trifluoromethoxyphenylhydrazone, decreased both components of 32Pi influx by 80-95%. The H+-ATPase inhibitor, bafilomycin A1, inhibited the Na+-independent mechanism. Furosemide, an inhibitor of ouabain-insensitive Na+-ATPase, decreased both uptake mechanisms of 32Pi to the same extent, whereas ouabain had no effect, indicating that the former is the pump responsible for inwardly directed Na+ and the electric gradients required by the transporters. Parasite growth in high Pi had a lower Pi influx than that found in those grown in low Pi, without alteration in TrPho89 expression, showing that turnover of the transporters is stimulated by Pi starvation. CONCLUSIONS: Two modes of Pi transport, one coupled to Na+-ATPase and other coupled to H+-ATPase seem to be responsible for Pi acquisition during development of T. rangeli. GENERAL SIGNIFICANCE: This study provides the first description of the mechanism of Pi transport across the plasma membrane of trypanosomatids.
Assuntos
Fosfatos/metabolismo , Rhodnius/parasitologia , Sódio/metabolismo , Trypanosoma/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/metabolismo , Membrana Celular/metabolismo , Inibidores Enzimáticos/farmacologia , Macrolídeos/farmacologia , Ouabaína/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhodnius/metabolismo , Trypanosoma/crescimento & desenvolvimentoRESUMO
Quantitative real-time PCR (qPCR) has become one of the most used techniques to measure gene expression. However, normalization of gene expression data against reference genes is essential, although these are usually used without any kind of validation. The expression of seven genes was compared in organs of Rhodnius prolixus under diverse conditions, using published software to test gene expression stability. Rp18S and elongation factor 1 (RpEF -1) were the most reliable genes for normalization in qPCR when gene expression in different organs was compared. Moreover, both genes were found to be the best references when transcript levels were compared in the posterior midgut of insects infected with Trypanosoma cruzi. Rp18S was also the best reference gene in the fat bodies of unfed and fed insects. By contrast, RpEF-1 was found to be the best reference gene for comparison between posterior midguts, and RpMIP or RpActin should be used to compare gene expression in the ovaries. Although Rp18S is indicated here as the best reference in most cases, reports from the literature show that it is difficult to find an optimum reference gene. Nevertheless, validation of candidate genes to be taken as references is important when new experimental conditions are tested to avoid incorrect data interpretation.
Assuntos
Rhodnius/genética , Animais , Feminino , Expressão Gênica , Genes de Insetos , Genes de RNAr , Fator 1 de Elongação de Peptídeos/genética , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Padrões de ReferênciaRESUMO
[(14)C]Oleic acid injected into the hemocoel of Rhodnius prolixus females was shown to rapidly associate with lipophorin particles. Half of the lipophorin-associated [(14)C]oleic acid was transferred in about 5 min to different organs, but the midgut was the main organ to take it up on day 10 after a blood meal. The rate of [(14)C]oleic acid incorporation by the midgut was high up to 15 min after injection and then declined. The [(14)C]oleic acid incorporated by the midgut was found in phospholipids (58.6%) and neutral lipids (37.4%). The midgut capacity to incorporate [(14)C]oleic acid varied on different days after a meal: it increased up to day 10 and then decreased. The fate of the [(14)C]lipids synthesized by the midgut was followed and it was observed that 10 days after feeding diacylglycerol was the main lipid released to hemolymph and that most of phospholipids and triacylglycerols remained associated with the midgut. The metabolism of free fatty acids in Rhodnius prolixus females is discussed in the context of major biological events that follow a blood meal such as digestion and oogenesis.
Assuntos
Proteínas de Transporte/metabolismo , Hemolinfa/química , Lipoproteínas/metabolismo , Ácido Oleico/metabolismo , Rhodnius/metabolismo , Animais , Proteínas de Transporte/sangue , Cromatografia em Camada Fina/veterinária , Diglicerídeos/biossíntese , Diglicerídeos/sangue , Eletroforese em Gel de Poliacrilamida/veterinária , Feminino , Lipoproteínas/sangue , Ácido Oleico/sangue , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/sangue , Fosfatidiletanolaminas/biossíntese , Fosfatidiletanolaminas/sangue , Fosfatidilserinas/biossíntese , Fosfatidilserinas/sangue , Contagem de Cintilação/veterinária , Fatores de Tempo , Triglicerídeos/biossíntese , Triglicerídeos/sangueRESUMO
Lipophorin (Lp) is the major lipoprotein in insect hemolymph. The structural organization proposed for Lp is basically the same as that suggested for vertebrate lipoproteins, consisting of a hydrophobic core containing neutral lipids, stabilized in the aqueous environment by surrounding polar moieties of protein and phospholipids at the particle surface. After complete removal of phospholipids from Lp by phospholipase A2, the particle remains soluble [Gondim, K. C., Atella, G. C., Kawooya, J. K., & Masuda, H. (1992) Arch. Insect Biochem. Physiol. 20, 303-314]. However, studies on the roles of phospholipid on the structural stability of Lp are still lacking. In the present work, we have studied the structure and stability of dephospholipidated lipophorin (d-Lp). Trypsinolysis of d-Lp indicated no exposure of new cleavage sites on the protein when compared to Lp. However, an enhanced rate of proteolysis of the apoproteins (especially apolipophorin II) was observed in d-Lp. Circular dichroism analysis indicated that the secondary structure of Lp was not significantly affected by phospholipid removal. Furthermore, the exposure of tryptophan residues to the aqueous solvent in d-Lp was the same as in Lp, as indicated by intrinsic fluorescence emission spectra and fluorescence quenching experiments. Interestingly, d-Lp was more resistant to denaturation by guanidine hydrochloride than Lp. d-Lp was also found to be less sensitive than Lp to structural changes induced by hydrostatic pressure. Taken together, these results indicate that, although changes in its structural organization were subtle, dephospholipidated lipophorin may have additional protein-protein and/or protein-neutral lipid interactions that are responsible for the observed increase in stability. Therefore, phospholipids are not only not essential for Lp stability, but their presence in the particle seems to result in a less stable structure in the aqueous environment.
Assuntos
Proteínas de Transporte/química , Lipídeos/química , Lipoproteínas/química , Fosfolipídeos/química , Rhodnius/química , Animais , Dicroísmo Circular , Feminino , Guanidina , Guanidinas , Desnaturação Proteica , Espectrometria de Fluorescência , TriptofanoRESUMO
The density of lipophorin was determined in adult females of Rhodnius prolixus on different days after a meal. Several populations od lipoproteins, differing in density but always in the range of HDL, were found in the hemolymph. The density of the major population was analyzed and a complex profile of density variation was found associated with the principal metabolic events in these insects digestion and oogenesis. During the initial three days after the blood meal, with the onset of the digestive process, the density of lipophorin decreased from 1.1185 g/l to 1.1095 g/l, associated with the transfer of lipids from midgut to the lipophorin particles. During the period of intense vitellogenesis and lipid uptake by the ovary, the lipophorin density started to increase and reached the value, 1.1322 g/l, and remained stable up to the end of oogenesis. As soon as the requirement of lipids to build up the oocytes ceased, the density of lipophorin decreased to its initial value associated with the transfer of lipids from fat body to lipophorin. Soon after the blood meal the midgut was the main source of lipids capable of replenishing the lipophorin particles, while the fat body assumed this function during the succeeding days and reached its maximum capacity around day 10, as estimated by the rate of lipid transfer. The principal lipids transferred were phospholipids and diacylglycerols. Except in the protein/lipid ratio no major changes were observed among different lipids isolated from lipophorin of different densities.
Assuntos
Proteínas de Transporte/análise , Lipoproteínas/análise , Rhodnius/química , Animais , Feminino , Oogênese , Coelhos , Rhodnius/fisiologiaRESUMO
Purified lipophorin, metabolically labelled with 32P exclusively in the phospholipid moiety, was used to study the process of phospholipid delivery to the oocyte. The kinetics of phospholipid transfer "in vitro," from lipophorin to the oocytes, was linear at least up to 4 h and was impaired by low temperature. A net transfer of phospholipids from lipophorin particles to the oocytes was observed. The rate of phospholipid uptake was dependent on the concentration of lipophorin in the medium and was shown to be a saturable process. The addition of a molar excess of purified unlabelled lipophorin to the culture medium resulted in a substantial decrease in the transfer of [32P]phospholipids, but no reduction occurred in the presence of a molar excess of albumin. The lipophorin binding sites were localized in the oocytes by immunogold techniques using two different protocols for oocyte fixation. Strong labelling was observed especially at the microvilli. No labelling was detected in the yolk granules.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Insetos/imunologia , Lipoproteínas/metabolismo , Oócitos/metabolismo , Ovário/metabolismo , Fosfolipídeos/metabolismo , Rhodnius/metabolismo , Animais , Sangue , Proteínas de Transporte/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Feminino , Imuno-Histoquímica , Técnicas In Vitro , Proteínas de Insetos/metabolismo , Lipoproteínas/ultraestrutura , Microscopia Eletrônica , Oócitos/ultraestrutura , Oogênese , Radioisótopos de Fósforo , Coelhos , Rhodnius/química , Fatores de Tempo , Vitelogeninas/análiseRESUMO
The lipophorin of Rhodnius prolixus metabolically labelled with 32P exclusively in the phospholipid moiety was purified on a potassium bromide gradient and treated with phospholipase A2 in the presence of an excess of fatty acid-free albumin. The treatment completely removed the phospholipids from the particles and generated [32P]-lysophosphatidylcholine, [32P]-lysophosphatidylethanolamine, and free fatty acids that remained bound to albumin. The phospholipid-depleted lipophorin particles remained soluble, indicating that phospholipids are not essential in maintaining the stability of the particles in aqueous solution. Complete removal of phospholipids did not affect the association of apolipophorin III with lipophorin particles. Lipophorin density increased slightly from 1.120 to 1.134 g/ml after treatment. The phospholipid-depleted particles also retained their ability to be recognized and loaded in vitro with phospholipids delivered by the fat body, thus supporting the concept of lipophorin's role as a reusable lipid shuttle for phospholipids.