Gene annotation of nuclear receptor superfamily genes in the kissing bug Rhodnius prolixus and the effects of 20-hydroxyecdysone on lipid metabolism.

The hormone 20-hydroxyecdysone is fundamental for regulating moulting and metamorphosis in immature insects, and it plays a role in physiological regulation in adult insects. This hormone acts by binding and activating a receptor, the ecdysone receptor, which is part of the nuclear receptor gene superfamily. Here, we analyse the genome of the kissing bug Rhodnius prolixus to annotate the nuclear receptor superfamily genes. The R. prolixus genome displays a possible duplication of the HNF4 gene. All the analysed insect organs express most nuclear receptor genes as shown by RT-PCR. The quantitative PCR analysis showed that the RpEcR and RpUSP genes are highly expressed in the testis, while the RpHNF4-1 and RpHNF4-2 genes are more active in the fat body and ovaries and in the anterior midgut, respectively. Feeding does not induce detectable changes in the expression of these genes in the fat body. However, the expression of the RpHNF4-2 gene is always higher than that of RpHNF4-1. Treating adult females with 20-hydroxyecdysone increased the amount of triacylglycerol stored in the fat bodies by increasing their lipogenic capacity. These results indicate that 20-hydroxyecdysone acts on the lipid metabolism of adult insects, although the underlying mechanism is not clear.

Urea increases tolerance of yeast inorganic pyrophosphatase activity to ethanol: the other side of urea interaction with proteins.

Ethanol is the major product of yeast sugar fermentation and yet, at certain concentrations, it is very toxic to yeast cells. The major targets for ethanol's toxicity are the plasma membrane and the cytosolic enzymes: ethanol alters membrane organization and permeability and inactivates and unfolds globular cytosolic enzymes. The effects of ethanol on the plasma membrane are attenuated by the presence of trehalose, a disaccharide of glucose that is accumulated simultaneously with urea. The data presented in this paper show that trehalose is not effective at protecting yeast cytosolic inorganic pyrophosphatase against the inactivation of its catalytic activity promoted by alcohols. In contrast, 1 M trehalose increased the toxicity of alcohols against pyrophosphatase by at least 34%. On the other hand, 1.5 M urea attenuated the inactivation of pyrophosphatase promoted by alcohols by approximately 50%. Here we propose that, in the presence of alcohols, urea functions as a molecular filter, enriching the vicinity of the protein with water and excluding alcohol molecules. Conversely, trehalose tends to increase the interaction of alcohols with protein molecules, by withdrawing water, leading to a stronger inactivation promoted for a given concentration of alcohol in the bulk solution on pyrophosphatase activity.

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+).

The plasma membrane (Ca(2+) + Mg(2+))ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca(2+) ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca(2+) strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca(2+)-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca(2+) is related to opposite long-term hydration effects on the substrate binding domain and the Ca(2+) binding domain.

Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment.

Yeast cells have had to develop mechanisms in order to protect themselves from chemical and physical agents of the environment to which they are exposed. One of these physical agents is thermal variation. Some yeast cells are known to accumulate high concentrations of trehalose when submitted to heat shock. In this work, we have studied the effect of trehalose on the protection against thermal inactivation of purified plasma membrane H+-ATPase from Schizosaccharomyces pombe, in the solubilized and in the reconstituted state. We observed that after 1 min of incubation at 51 degrees C in the presence of 1 M trehalose, about 50% of soluble enzyme remains active. In the same conditions, but in the absence of trehalose, the activity was completely abolished. The t0.5 for the enzyme inactivation increased from 10 to 50 s after reconstitution into asolectin liposomes. Curiously, in the presence of 1 M trehalose, the t0.5 for inactivation of the reconstituted enzyme was further increased to higher than 300 s, regardless of whether trehalose was added inside or outside the liposome. Additionally, the concentration that confers 50% for the protection by trehalose (K0.5) decreased from 0.5 M, in the solubilized state, to 0.04 M in the reconstituted state, suggesting a synergetic effect between sugar and lipids. Gel electrophoresis revealed that the pattern of H+-ATPase cleavage by trypsin changed when 1 M trehalose was present in the buffer. It is suggested that both in a soluble and in a phospholipid environment, accumulation of trehalose leads to a more heat-stable conformation of the enzyme, probably an E2-like form.

Effects of trehalose and ethanol on yeast cytosolic pyrophosphatase.

Trehalose has been described to protect several enzymes against destabilizing conditions. This sugar is naturally accumulated by yeast as a stress protectant. A common stress condition that yeast is normally submitted is the presence of ethanol, the by-product of fermentation process of several yeast. In this paper we show the effects of trehalose and ethanol, alone or together, on yeast pyrophosphatase, and the effects of these compounds on inhibition and unfolding of pyrophosphatase promoted by urea. We show that both trehalose and ethanol inhibit pyrophosphatase in a dose-dependent manner, and that the presence of ethanol does not modify the inhibition promoted by trehalose as well as the presence of trehalose does not modify the inhibition promoted by ethanol. The effects of trehalose on pyrophosphatase are completely reversible, but the inhibition caused by ethanol was only partially reversible. Incubation of pyrophosphatase with 10% (v/v) ethanol promoted an inhibition of 15%, and the control activity was completely recovered after removal of ethanol. On the other hand, when pyrophosphatase was incubated with 20% (v/v) ethanol an inhibition of 40% of the control activity was observed which persisted after removal of ethanol. Ethanol also potentiates the inhibition of pyrophosphatase promoted by urea, and contributes for an irreversible inactivation and unfolding of pyrophosphatase in the presence of urea. Trehalose, that protects this enzyme against the inhibition and unfolding promoted by the chaotropic compound urea, was inefficient to protect against the effects of ethanol. Trehalose was also efficient to prevent an irreversible inactivation induced by urea.

Stabilization against thermal inactivation promoted by sugars on enzyme structure and function: why is trehalose more effective than other sugars?

Trehalose has been described to act as the best stabilizer of structure and function of several macromolecules. Although other sugars also stabilize macromolecules, none of them are as effective as trehalose. The extraordinary effect of trehalose has been attributed to several of its properties such as making hydrogen bonds with membranes or the ability to modify the solvation layer of proteins. However, the explanations always result in a question: Why is trehalose more effective than other sugars? Here, we show that trehalose has a larger hydrated volume than other related sugars. According to our results, trehalose occupies at least 2.5 times larger volume than sucrose, maltose, glucose, and fructose. We correlate this property with the ability to protect the structure and function of enzymes against thermal inactivation. When the concentrations of all sugars were corrected by the percentage of the occupied volume, they presented the same effectiveness. Our results suggest that because of this larger hydrated volume, trehalose can substitute more water molecules in the solution, and this property is very close to its effectiveness. Finally, these data drive us to conclude that the higher size exclusion effect is responsible for the difference in efficiency of protection against thermal inactivation of enzymes.

Biosynthesis of O-N-acetylglucosamine-linked glycans in Trypanosoma cruzi. Characterization of the novel uridine diphospho-N-acetylglucosamine:polypeptide N-acetylglucosaminyltransferase-catalyzing formation of N-acetylglucosamine alpha1-->O-threonine.

In this study, we have characterized the activity of a uridine diphospho-N-acetylglucosamine:polypeptide-alpha-N-acetylglucosaminylt ransferase (O-alpha-GlcNAc-transferase) from Trypanosoma cruzi. The activity is present in microsomal membranes and is responsible for the addition of O-linked alpha-N-acetylglucosamine to cell surface proteins. This preparation adds N-acetylglucosamine to a synthetic peptide KPPTTTTTTTTKPP containing the consensus threonine-rich dodecapeptide encoded by T. cruzi MUC gene (Di Noia, J. M., Sánchez D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146-24149). Incorporation of N-[3H]acetylglucosamine is linearly dependent on incubation time and concentration of enzyme and substrate. The transferase activity has an optimal pH of 7.5- 8.5, requires Mn2+, is unaffected by tunicamycin or amphomycin, and is strongly inhibited by UDP. The optimized synthetic peptide acceptor for the cytosolic O-GlcNAc-transferase (YSDSPSTST) (Haltiwanger, R. S., Holt, G. D., and Hart, G. W. (1990) J. Biol. Chem. 265, 2563-2568) is not a substrate for this enzyme. The glycosylated KPPTTTTTTTTKPP product is susceptible to base-catalyzed beta-elimination, and the presence of N-acetylglucosamine alpha-linked to threonine is supported by enzymatic digestion and nuclear magnetic resonance data. These results describe a unique biosynthetic pathway for T. cruzi surface mucin-like molecules, with potential chemotherapeutic implications.

Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme.

Baker's yeast cells accumulate osmolytes as a response to several stress conditions such as high-temperature and low-temperature shifts, dehydration, or osmotic stress. One of the major osmolytes that accumulates is trehalose, which plays an essential role affecting the survival of yeast at the time of stress. In this report, we show that trehalose efficiently protects the function and the structure of two yeast cytosolic enzymes against chemical denaturation by guanidinium chloride. Other sugars tested also protected yeast pyrophosphatase and glucose-6-phosphate dehydrogenase structure against guanidinium chloride effects, but were not as efficient at protecting enzyme activity. The thermostable pyrophosphatase from Bacillus stearothermophilus was also protected by several sugars against the chaotropic properties of guanidinium chloride, but was only protected by trehalose against functional inactivation. The function of the membrane-embedded H+-ATPase from yeast could not be protected by any of the tested sugars, although all of the sugars protected its structure from guanidinium-chloride-induced unfolding. The results presented in this study suggest that several sugars are able to prevent protein unfolding induced by a chaotropic compound. However, prevention of functional inactivation depends on the nature of the sugar. Trehalose was the most efficient, being able to protect many cytosolic enzymes against guanidinium chloride. The efficiency of protection also depended on the nature of the protein tested. This might explain why trehalose is one of the osmolytes accumulated in yeast and also why it is not the only osmolyte to accumulate.

Trehalose protects yeast pyrophosphatase against structural and functional damage induced by guanidinium chloride.

Trehalose is accumulated at very high concentrations in yeasts when this organism is submitted to a stress condition. This report approaches the question on the protective effect of trehalose and its degradation product, glucose, against structural and functional damage promoted by guanidinium on yeast cytosolic pyrophosphatase. Here it is shown that both, 1 M trehalose or 2 M glucose, are able to attenuate at almost the same extent the conformational changes promoted by guanidinium chloride on the pyrophosphatase structure. On the other hand while 1 M trehalose increases 3.8 times the Ki (from 0.15 to 0.57 M) for guanidinium chloride inhibition of pyrophosphatase activity, 2 M glucose did not even duplicate this parameter (from 0.15 to 0.25 M). These data support evidences for a functional reason for the accumulation by yeasts of trehalose, and not other compound, during stress conditions.

Glycerol inhibits or uncouples the plasma membrane (Ca(2+)+Mg2+)ATPase of kidney proximal tubules depending on the Ca2+ concentration.

In this report it is shown that glycerol (0.5-10% v/v) stimulate the C12-E9-solubilized renal (Ca(2+)+Mg2+)ATPase in the presence of low concentrations of free Ca2+ (< 10(-6) M). At 4% (v/v), the polyol decreases the K0.5 for Ca2+ from 1.15 to 0.22 microM at the high-affinity site, and a very-high-affinity Ca2+ component appears. This component has a K0.5 < or = 10(-9) M and its maximal velocity is about one-third that of the fully activated enzyme (at 10-20 microM Ca2+), which is not affected by glycerol (21.1 and 20.2 nmollmg-1.min-1 in the absence and presence of the polyol, respectively). The low-affinity, inhibitory component of the Ca2+ curve (50-1000 microM) is also unaffected by glycerol. With 0.07 microM free Ca2+ and soluble enzyme, the stimulatory effect of glycerol saturates at approximately 10% (v/v). In contrast, with 17 microM free Ca2+, glycerol has little effect up to 10% (v/v), and then progressively inhibits ATPase activity. These data indicate that the effect of the polyol is modulated by the occupancy of the high-affinity Ca2+ sites. In native vesicles, the stimulation promoted by low concentrations of glycerol at low concentrations of Ca2+ is accompanied by inhibition of active Ca2+ transport, indicating that, in these conditions, the polyol uncouples ATPase activity and ATP-driven Ca2+ influx.

Polyols that accumulate in renal tissue uncouple the plasma membrane calcium pump and counteract the inhibition by urea and guanidine hydrochloride.

Sorbitol and mannitol, two stereoisomeric osmolytes, inhibit the ATP-dependent Ca2+ transport in inside-out vesicles derived from basolateral membranes from kidney proximal tubules. This inhibition (I0.5 = 400 and 390 mM respectively) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by these osmolytes. In the presence of 1 M sorbitol or mannitol, Ca2+ uptake is inhibited by 70 and 75%, respectively. Since the Ca(2+)-stimulated ATPase activity is unaffected, sorbitol and mannitol uncouple the Ca2+ transport from the ATPase activity. The inhibition of Ca2+ transport by these osmolytes is reversible, since the inhibition disappears when the vesicles are preincubated with 1 M sorbitol or mannitol and then diluted 25-fold in reaction medium to measure Ca2+ accumulation. On the other hand, these osmolytes protect the (Ca2+ + Mg2+) ATPase from the inhibition of Ca2+ transport and ATPase activity by urea and guanidinium. These data suggest that the high concentrations of polyols that renal cells accumulate during antidiuresis, may regulate Ca2+ transport across the plasma membrane. In addition, polyols may protect the (Ca2+ + Mg2+) ATPase from the deleterious structural effects of urea, a compound that also accumulates during antidiuresis.

Protective role of trehalose in thermal denaturation of yeast pyrophosphatase.

Trehalose, a disaccharide of glucose, is accumulated in yeast cytosol when this organism is submitted to a stress condition. Recently it was shown that the level of trehalose increase up to 15 times when yeast cells are submitted to heat shock (De Virgilio et al., 1991). In this report we give evidence how trehalose may play an important role on the stress-survival of yeasts when submitted to a heat shock. We show that 1.5 M trehalose increases 13-fold the half-time for thermal inactivation (t0.5) of yeast cytosolic pyrophosphatase at 50 degrees C. This thermal protection conferred by trehalose is dose-dependent, after 10 min at 50 degrees C, a condition which inactivated pyrophosphatase, the presence of 2 M trehalose preserves 95% of total activity. Other carbohydrates were tested but were not so effective as trehalose. The presence of trehalose at high concentrations in the reaction medium at 35 degrees C inhibits pyrophosphatase activity. This inhibition is less effective at 50 degrees C suggesting that under this condition the enzyme is temperature-protected and active.

Uncoupling by trehalose of Ca2+ transport and ATP hydrolysis by the plasma membrane (Ca2+ + Mg2+) ATPase of kidney tubules.

Trehalose, the disaccharide of glucose, inhibits both initial rate and maximal capacity of ATP-dependent Ca2+ transport in inside-out vesicles of basolateral membrane from kidney proximal tubules. This inhibition (I0.5 = 60 mM) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by trehalose. In the presence of 600 mM trehalose, Ca2+ uptake was almost completely inhibited, but the Ca(2+)-stimulated ATPase activity was unaffected; thus trehalose uncouples the Ca2+ transport from the ATPase activity. The Ca2+ transport inhibition by trehalose is reversible, since the inhibition disappeared when the vesicles were pre-incubated with 600 mM trehalose and then diluted in reaction medium to measure Ca2+ accumulation. Other mono- and disaccharides such as glucose, fructose, galactose, sucrose, maltose and lactose were tested but were not so effective as trehalose. The uncoupling of Ca2+ transport from hydrolysis can be explained by an interaction of trehalose with the phospholipid environment of the enzyme that induces conformational changes in specific domains of the enzyme so as to impair the coupling process.

Monosaccharides and disaccharides decrease the Km for phosphorylation of a membrane-bound enzyme ATPase.

The disaccharides trehalose and sucrose, and to a lesser extent the monosaccharides glucose and fructose, decrease the apparent Km of the Ca2+, Mg(2+)-ATPase of sarcoplasmic reticulum for Pi. This effect is more pronounced at pH 7.4 than at pH 6.2. The enzyme is not phosphorylated by Pi when the temperature of the medium is decreased to 0 degree C, but when 1.5 M trehalose or sucrose is present phosphoenzyme formation increases to 0.5 mumol E-P/g protein.