Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes.

Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

Zn(2+)-dependent surface behavior of diacylglycerol pyrophosphate and its mixtures with phosphatidic acid at different pHs.

Diacylglycerol pyrophosphate (DGPP) is a minor lipid that attenuates the phosphatidic acid (PA) signal, and also DGPP itself would be a signaling lipid. Diacylglycerol pyrophosphate is an anionic phospholipid with a pyrophosphate group attached to diacylglycerol that was shown to respond to changes of pH, thus affecting the surface organization of DGPP and their interaction with PA. In this work, we have investigated how the presence of Zn(2+) modulates the surface organization of DGPP and its interaction with PA at acidic and basic pHs. Both lipids formed expanded monolayers at pHs 5 and 8. At pH 5, monolayers formed by DGPP became stiffer when Zn(2+)was added to the subphase, while the surface potential decreased. At this pH, Zn(2+) induced a phase transition from an expanded to a condensed-phase state in monolayers formed by PA. Conversely, at pH 8 the effects induced by the presence of Zn(2+) on the surface behaviors of the pure lipids were smaller. Thus, the interaction of the bivalent cation with both lipids was modulated by pH and by the ionization state of the polar head groups. Mixed monolayers of PA and DGPP showed a non-ideal behavior and were not affected by the presence of Zn(2+) at pH 8. This could be explained considering that when mixed, the lipids formed a closely packed monolayer that could not be further modified by the cation. Our results indicate that DGPP and PA exhibit expanded- and condensed-phase states depending on pH, on the proportion of each lipid in the film and on the presence of Zn(2+). This may have implications for a possible role of DGPP as a signaling lipid molecule.

Differences in phosphatidic acid signalling and metabolism between ABA and GA treatments of barley aleurone cells.

Phosphatidic acid (PA) is the common lipid product in abscisic acid (ABA) and gibberellic acid (GA) response. In this work we investigated the lipid metabolism in response to both hormones. We could detect an in vivo phospholipase D activity (PLD, EC 3.1.4.4). This PLD produced [(32)P]PA (phosphatidic acid) rapidly (minutes) in the presence of ABA, confirming PA involvement in signal transduction, and transiently, indicating rapid PA removal after generation. The presence of PA removal by phosphatidate phosphatase 1 and 2 isoforms (E.C. 3.1.3.4) was verified in isolated aleurone membranes in vitro, the former but not the latter being specifically responsive to the presence of GA or ABA. The in vitro DGPP phosphatase activity was not modified by short time incubation with GA or ABA while the in vitro PA kinase - that allows the production of 18:2-DGPP from 18:2-PA - is stimulated by ABA. The long term effects (24 h) of ABA or GA on lipid and fatty acid composition of aleurone layer cells were then investigated. An increase in PC and, to a lesser extent, in PE levels is the consequence of both hormone treatments. ABA, in aleurone layer cells, specifically activates a PLD whose product, PA, could be the substrate of PAP1 and/or PAK activities. Neither PLD nor PAK activation can be monitored by GA treatment. The increase in PAP1 activity monitored after ABA or GA treatment might participate in the increase in PC level observed after 24 h hormone incubation.

Stress response to high osmolarity in Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi undergoes differentiation in the rectum of triatomine, where increased osmolarity is caused mainly by elevated content of NaCl from urine. Early biochemical events in response to high osmolarity in this parasite have not been totally elucidated. In order to clarify the relationship between these events and developmental stages of T. cruzi, epimastigotes were subjected to hyperosmotic stress, which caused activation of Na(+)/H(+) exchanger from acidic vacuoles and accumulation of inositol trisphosphate (InsP(3)). Suppression of InsP(3) levels was observed in presence of intracellular Ca(2+) chelator or pre-treatment with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), which also inhibited the alkalinization of acidic vacuoles via a Na(+)/H(+) exchanger and the consequent increase in cytosolic calcium. These effects were activated and inhibited by PMA and Chelerythrine respectively, suggesting regulation by protein kinase C. The T. cruzi Na(+)/H(+) exchanger, TcNHE1, has 11 transmembrane domains and is localized in acidic vacuoles of epimastigotes. The analyzed biochemical changes were correlated with morphological changes, including an increase in the size of acidocalcisomes and subsequent differentiation to an intermediate form. Both processes were delayed when TcNHE1 was inhibited by EIPA, suggesting that these early biochemical events allow the parasite to adapt to conditions faced in the rectum of the insect vector.

Comparative phytohormone profiles, lipid kinase and lipid phosphatase activities in barley aleurone, coleoptile, and root tissues.

We analyzed lipid kinase and lipid phosphatase activities and determined endogenous phytohormone levels by liquid chromatography-tandem mass spectrometry in root and coleoptile tissues following germination of barley (Hordeum vulgare) seeds. The enzymes showing highest activity in aleurone cells were diacylglycerol kinase (DAG-k, EC 2.7.1.107) and phosphatidate kinase (PA-k). The ratio of gibberellins (GAs) to abscisic acid (ABA) was 2-fold higher in aleurone than in coleoptile or root tissues. In coleoptiles, phosphatidylinositol 4-kinase (PI4-k, EC 2.7.1.67) showed the highest enzyme activity, and jasmonic acid (JA) level was higher than in aleurone. In roots, activities of PI4-k, DAG-k, and PA-k were similar, and salicylic acid (SA) showed the highest concentration. In the assays to evaluate the hydrolysis of DGPP (diacylglycerol pyrophosphate) and PA (phosphatidic acid) we observed that PA hydrolysis by LPPs (lipid phosphate phosphatases) was not modified; however, the diacylglycerol pyrophosphate phosphatase (DGPPase) was strikingly higher in coleoptile and root tissues than to aleurone. Relevance of these findings in terms of signaling responses and seedling growth is discussed.

Regulation of phosphatidic acid levels in Trypanosoma cruzi.

Lipid kinases and phosphatases play essential roles in signal transduction processes involved in cytoskeletal rearrangement, membrane trafficking, and cellular differentiation. Phosphatidic acid (PtdOH) is an important mediator lipid in eukaryotic cells, but little is known regarding its regulation in the parasite Trypanosoma cruzi, an agent of Chagas disease. In order to clarify the relationship between PtdOH metabolism and developmental stages of T. cruzi, epimastigotes in culture were subjected to hyperosmotic stress (~1,000 mOsm/L), mimicking the environment in the rectum of vector triatomine bugs. These experimental conditions resulted in differentiation to an intermediate form between epimastigotes and trypomastigotes. Morphological changes of epimastigotes were correlated with an increase in PtdOH mass accomplished by increased enzyme activity of diacylglycerol kinase (DAGK, E.C. 2.7.1.107) and concomitant decreased activity of phosphatidate phosphatases type 1 and type 2 (PAP1, PAP2, E.C. 3.1.3.4). Our results indicate progressive increases of PtdOH levels during the differentiation process, and suggest that the regulation of PtdOH metabolism is an important mechanism in the transition from T. cruzi epimastigote to intermediate form.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves.

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

The surface organization of diacylglycerol pyrophosphate and its interaction with phosphatidic acid at the air-water interface.

Diacylglycerol pyrophosphate (DGPP), a phosphorylated form of phosphatidic acid (PA), gained attention recently due to its role as signaling lipid. However, little is known about its surface organization and potential impact on membrane-mediated function. In this work we investigated the interfacial behavior of Langmuir monolayers formed with pure DGPP and of its mixtures with PA. We found that changes of the subphase pH affect the surface behavior of DGPP. At pH 8, DGPP forms liquid expanded monolayers with a compressibility modulus of about 60mNm⁻¹ at collapse. On acidic solutions, the compressibility modulus increases to 90mNm⁻¹ and the average molecular area is smaller. At pH 8, DGPP and its precursor PA form thermodynamically favored topographically homogeneous non-ideal mixtures. The interaction among these lipids leads to a non-ideal diminution of the mean molecular area and consequently, to an increase of the compressibility modulus, with variations of the surface electrostatics. The favorable interaction of PA and DGPP, leading to changes of the film packing suggest that DGPP may act as a structural signal transducer in membrane-mediated cellular processes.

Fetal bovine serum concentration affects delta9 desaturase activity of Trypanosoma cruzi.

Fetal bovine serum (FBS) is an important factor in the culture of Trypanosoma cruzi, since this parasite obtains and metabolizes fatty acids (FAs) from the culture medium, and changes in FBS concentration reduce the degree of unsaturation of FAs in phosphoinositides. When T. cruzi epimastigotes were cultured with 5% instead of 10% FBS, and stearic acid was used as the substrate, (9) desaturase activity decreased by 50%. Apparent K (m) and V (m) values for stearic acid, determined from Lineaweaver-Burk plots, were 2 microM and 219 pmol/min/mg of protein, respectively. In studies of the requirement for reduced pyridine nucleotide, (9) desaturase activity reached a maximum with 8 microM NADH and then remained constant; the apparent K (m) and V (m) were 4.3 microM and 46.8 pmol/min/mg of protein, respectively. The effect of FBS was observed only for (9) desaturase activity; (12) desaturase activity was not affected. The results suggest that decreased FBS in culture medium is a signal that modulates (9) desaturase activity in T. cruzi epimastigotes.

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels.

BACKGROUND AND AIMS: Experimental evidence in the literature suggests that O(2)(*-) produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O(2)(*-). METHODS: Stress treatments were imposed using 150 mm NaCl or 300 mM sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O(2)(*-) was determined using nitro blue tetrazolium, and H(2)O(2) was determined using 2', 7'-dichlorofluorescin. KEY RESULTS: In non-stressed plants, the distribution of accelerating growth and highest O(2)(*-) levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O(2)(*-) levels increased in sorbitol-treated roots and decreased in NaCl-treated roots. CONCLUSIONS: The lack of association between apoplastic O(2)(*-) levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O(2)(*-) may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.

Diacylglycerol pyrophosphate inhibits the alpha-amylase secretion stimulated by gibberellic acid in barley aleurone.

ABA plays an important regulatory role in seed germination because it inhibits the response to GA in aleurone, a secretory tissue surrounding the endosperm. Phosphatidic acid (PA) is a well-known intermediary in ABA signaling, but the role of diacylglycerol pyrophosphate (DGPP) in germination processes is not clearly established. In this study, we show that PA produced by phospholipase D (E.C. 3.1.4.4) during the antagonist effect of ABA in GA signaling is rapidly phosphorylated by phosphatidate kinase (PAK) to DGPP. This is a crucial fact for aleurone function because exogenously added dioleoyl-DGPP inhibits secretion of alpha-amylase (E.C. 3.2.1.1). Aleurone treatment with ABA and 1-butanol results in normal secretory activity, and this effect is reversed by addition of dioleoyl-DGPP. We also found that ABA decreased the activity of an Mg2+-independent, N-ethylmaleimide-insensitive form of phosphatidate phosphohydrolase (PAP2) (E.C. 3.1.3.4), leading to reduction of PA dephosphorylation and increased PAK activity. Sequence analysis using Arabidopsis thaliana lipid phosphate phosphatase (LPP) sequences as queries identified two putative molecular homologues, termed HvLPP1 and HvLPP2, encoding putative Lpps with the presence of well-conserved structural Lpp domains. Our results are consistent with a role of DGPP as a regulator of ABA antagonist effect in GA signaling and provide evidence about regulation of PA level by a PAP2 during ABA response in aleurone.

Phosphatidylinositol kinases as regulators of GA-stimulated alpha-amylase secretion in barley (Hordeum vulgare).

Phosphorylated derivatives of phosphatidylinositol, in association with phosphatidylinositol 3-kinase (PI3 kinase, EC 2.7.1.137) and phosphatidylinositol 4-kinase (PI4 kinase, EC 2.7.1.67), play a key role in regulation of fundamental cell processes. We present evidence for a relationship between alpha-amylase (EC 3.2.1.1) secretion regulated by GA and levels of phosphatidylinositol 3-phosphate and phosphatidylinositol 4-phosphate (PtdIns(4)P) in barley (Hordeum vulgare). Microsomal membranes were incubated in the presence of [gamma-(32)P]ATP, and radiolabeled membrane lipids were extracted and separated by TLC using a boric acid system. Treatment of aleurone layers with GA for short or long periods of time increased PI4 kinase activity. To evaluate the effect of PtdIns(4)P levels on GA signaling, we used phenylarsine oxide (PAO), an inhibitor of PI4 kinase activity. PAO reversibly reduced the alpha-amylase secretion and protoplast cell vacuolation in a dose-dependent manner. Wortmannin showed a similar inhibitory effect on alpha-amylase secretion and PI4 kinase activity. GA evoked only a long-term increase in PI3 kinase activity, which was also affected by PAO. The effect of PAO was suppressed by the reducing agent 2,3-dimercapto-1-propanol (BAL), leading to restoration of secretion, vacuolation and PI4 kinase activity. In contrast, the effect of PAO on PI3 kinase activity was not abolished by BAL, suggesting that PI3 kinase is not involved in the secretion process. Likewise, the compound LY294002 inhibited PI3 kinase but had no effect on the secretion process. These findings indicate that PI4 kinase acts as a positive regulator of early GA signaling in aleurone.

Involvement of Na+/H+ exchanger in the calcium signaling in epimastigotes of Trypanosoma cruzi.

We studied the effect of Na(+) extracellular on Ca(2+) mobilization from intracellular store evoked by carbachol in Trypanosoma cruzi. We report that slow component of Ca(2+) signaling evoked by agonist is dependent on extracellular Na(+) but not on InsP(3) increase. Moreover, this Ca(2+) signaling progressively increased when pH of the medium changed from 7.0 to 7.8. In addition, we found that it was regulated by PKC. The agonist was also able to induce the alkalinization of the acidic compartment, and both Ca(2+) signaling and alkalinization were inhibited by the EIPA-inhibitor of the Na(+)/H(+) exchanger. These results demonstrated the alkalinization of acidic vacuoles and PKC are involved in the triggering of the epimastigote Ca(2+) signaling.