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1.
The aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 μM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance. 相似文献
2.
Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore. 相似文献
3.
We describe a single aquaporin gene in Toxoplasma gondii which, surprisingly, has only 28% sequence similarity to the aquaglyceroporin of another apicomplexan parasite, Plasmodium falciparum. Sequence comparisons showed 47% similarity to water-specific plant aquaporins and the conservation of typical pore-forming residues. We established that the Toxoplasma aquaporin protein is a bifunctional membrane pore with intermediate water and high glycerol permeability. Furthermore, we identified hydroxyurea, an antineoplastic agent with inhibitory effects on parasite proliferation, as a permeant of this channel. 相似文献
4.
Martin Hansen Jürgen F J Kun Joachim E Schultz Eric Beitz 《The Journal of biological chemistry》2002,277(7):4874-4882
The malaria parasite Plasmodium falciparum faces drastic osmotic changes during kidney passages and is engaged in the massive biosynthesis of glycerolipids during its development in the blood-stage. We identified a single aquaglyceroporin (PfAQP) in the nearly finished genome of P. falciparum with highest similarity to the Escherichia coli glycerol facilitator (50.4%), but both canonical Asn-Pro-Ala (NPA) motifs in the pore region are changed to Asn-Leu-Ala (NLA) and Asn-Pro-Ser (NPS), respectively. Expression in Xenopus oocytes renders them highly permeable for both water and glycerol. Sugar alcohols up to five carbons and urea pass the pore. Mutation analyses of the NLA/NPS motifs showed their structural importance, but the symmetrical pore properties were maintained. PfAQP is expressed in blood-stage parasites throughout the development from rings via trophozoites to schizonts and is localized to the parasite but not to the erythrocyte cytoplasm or membrane. Its unique bi-functionality indicates functions in the protection from osmotic stress and efficiently provides access to the serum glycerol pool for the use in ATP generation and primarily in the phospholipid synthesis. 相似文献
5.
Pavlovic-Djuranovic S Kun JF Schultz JE Beitz E 《Biochimica et biophysica acta》2006,1758(8):1012-1017
The aquaglyceroporin of Plasmodium falciparum (PfAQP) is a bi-functional channel with permeability for water and solutes. Its functions supposedly are in osmotic protection of parasites and in facilitation of glycerol permeation for glycerolipid biosynthesis. Here, we show PfAQP permeability for the glycolysis-related metabolites methylglyoxal, a cytotoxic byproduct, and dihydroxyacetone, a ketotriose. AQP3, the red cell aquaglyceroporin, also passed dihydroxacetone but excluded methylglyoxal. Proliferation of malaria parasites was inhibited by methylglyoxal with an IC50 around 200 microM. Surprisingly, also dihydroxyacetone, which is an energy source in human cells, was antiproliferative in chloroquine-sensitive and resistant strains with an IC50 around 3 mM. We expressed P. falciparum glyceraldehyde 3-phosphate dehydrogenase (PfGAPDH) to examine whether it is inhibited by either carbonyl compound. Methylglyoxal did not affect PfGAPDH on incubation with 2.5 mM for 20 h. Treatment with 2.5 mM dihydroxyacetone, however, abolished PfGAPDH activity within 6 h. Aquaglyceroporin permeability for glycolytic metabolites may thus be of physiological significance. 相似文献
6.
Hedfalk K Pettersson N Oberg F Hohmann S Gordon E 《Protein expression and purification》2008,59(1):69-78
The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms. 相似文献
7.
1,3‐propanediol binds deep inside the channel to inhibit water permeation through aquaporins
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Lili Yu Roberto A. Rodriguez L. Laurie Chen Liao Y. Chen George Perry Stanton F. McHardy Chih‐Ko Yeh 《Protein science : a publication of the Protein Society》2016,25(2):433-441
Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all‐atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3‐propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro‐homeostasis indirectly by altering the osmotic gradients. 相似文献
8.
The glycerol uptake facilitator, GlpF, a major intrinsic protein found in Escherichia coli, selectively conducts water and glycerol across the inner membrane. The free energy landscape characterizing the assisted transport of glycerol by this homotetrameric aquaglyceroporin has been explored by means of equilibrium molecular dynamics over a timescale spanning 0.12 μs. To overcome the free energy barriers of the conduction pathway, an adaptive biasing force is applied to the glycerol molecule confined in each of the four channels. The results illuminate the critical role played by intramolecular relaxation on the diffusion properties of the permeant. These free energy calculations reveal that glycerol tumbles and isomerizes on a timescale comparable to that spanned by its adaptive-biasing-force-assisted conduction in GlpF. As a result, reorientation and conformational equilibrium of glycerol in GlpF constitute a bottleneck in the molecular simulations of the permeation event. A profile characterizing the position-dependent diffusion of the permeant has been determined, allowing reaction rate theory to be applied for investigating conduction kinetics based on the measured free energy landscape. 相似文献
9.
The aquaglyceroporins of Escherichia coli, EcGlpF, and of Plasmodium falciparum, PfAQP, are probably the best characterized members of the solute-conducting aquaporin (AQP) subfamily. Their crystal structures have been elucidated and numerous experimental and theoretical analyses have been conducted. However, opposing reports on their rates of water permeability require clarification. Hence, we expressed EcGlpF and PfAQP in yeast, prepared protoplasts, and compared water and glycerol permeability of both aquaglyceroporins in the presence of different osmolytes, i.e. sucrose, sorbitol, PEG300, and glycerol. We found that water permeability of PfAQP strongly depends on the external osmolyte, with full inhibition by sorbitol, and increasing water permeability when glycerol, PEG300, and sucrose were used. EcGlpF expression did not enhance water permeability over that of non-expressing control protoplasts regardless of the osmolyte. Glycerol permeability of PfAQP was also inhibited by sorbitol, but to a smaller extent, whereas EcGlpF conducted glycerol independently of the osmolyte. Mixtures of glycerol and urea passed PfAQP equally well under isosmotic conditions, whereas under hypertonic conditions in a countercurrent with water, glycerol was clearly preferred over urea. We conclude that PfAQP has high and EcGlpF low water permeability, and explain the inhibiting effect of sorbitol on PfAQP by its binding to the extracellular vestibule. The preference for glycerol under hypertonic conditions implies that in a physiological setting, PfAQP mainly acts as a water/glycerol channel rather than a urea facilitator. 相似文献
10.
Aquaporins are protein channels located across the cell membrane with the role of conducting water or other small sugar alcohol molecules (aquaglyceroporins). The high-resolution X-ray structure of the human aquaporin 5 (HsAQP5) shows that HsAQP5, as all the other known aquaporins, exhibits tetrameric structure. By means of molecular dynamics simulations we analyzed the role of spontaneous fluctuations on the structural behavior of the human AQP5. We found that different conformations within the tetramer lead to a distribution of monomeric channel structures, which can be characterized as open or closed. The switch between the two states of a channel is a tap-like mechanism at the cytoplasmic end which regulates the water passage through the pore. The channel is closed by a translation of the His67 residue inside the pore. Moreover, water permeation rate calculations revealed that the selectivity filter, located at the other end of the channel, regulates the flow rate of water molecules when the channel is open, by locally modifying the orientation of His173. Furthermore, the calculated permeation rates of a fully open channel are in good agreement with the reported experimental value. 相似文献
11.
Hubert JF Duchesne L Delamarche C Vaysse A Gueuné H Raguénès-Nicol C 《Biology of the cell / under the auspices of the European Cell Biology Organization》2005,97(9):675-686
Background information. Transport of water and small neutral solutes across plasma membranes is facilitated by AQP (aquaporin) and aquaglyceroporin channels, which belong to the MIP (major intrinsic protein) family. So far, more than 800 MIP proteins have been identified on the basis of sequence homology, but only less than 10% of them have been functionally characterized. In most studies, the channel properties of MIP proteins have been determined by using Xenopus oocyte swelling assays or stopped-flow spectrophotometry on proteoliposomes. As both methods sometimes present disadvantages, we developed an alternative method for analysing MIP function.Results. The kinetics of plasmolysis or deplasmolysis of Escherichia coli cells in suspension, in response to osmotic challenges, was analysed by stopped-flow spectrophotometry. Cytoplasmic volume variations were monitored either by GFP (green fluorescent protein) fluorescence quenching or by 90 degrees scattered light. The single exponential response to up-shocks in the impermeant solute mannitol was strongly accelerated when the cells expressed the native E. coli AQP AqpZ (rate constant 37.24 versus 3.05 s(-1) for control cells). The responses to hyperosmotic shocks realized with glycerol were biphasic. First, a light-scattering increase corresponded to cell plasmolysis. Secondly, deplasmolysis occurred when glycerol entered into the cell. Both phases were accelerated when the aquaglyceroporin GlpF was present in cell membranes. We concluded that the behaviour of MIP-expressing bacteria in the stopped-flow system was qualitatively identical with that reported for MIP-expressing oocytes or MIP-containing proteoliposomes. We then used this system to analyse the effects of mutations in the pore constriction of Gla(Llac), the aquaglyceroporin from Lactococcus lactis. In the present study, we show that Gla(Llac) loses its ability to transport glycerol but retains its ability to transport water when Val(223) was replaced by a histidine, the residue at the equivalent position in strict AQPs.Conclusions. These results show that stopped-flow spectrophotometry performed on E. coli cell suspensions is a useful experimental system to analyse the selectivity of wild-type or mutant MIP proteins and that a bifunctional aquaglyceroporin switches to an AQP by a single amino acid mutation in the pore constriction. 相似文献
12.
Three aspects have to be taken into consideration when discussing cellular water and solute permeability of fungal cells: cell wall properties, membrane permeability, and transport through proteinaceous pores (the main focus of this review). Yet, characterized major intrinsic proteins (MIPs) can be grouped into three functional categories: (mainly) water transporting aquaporins, aquaglyceroporins that confer preferentially solute permeability (e.g., glycerol and ammonia), and bifunctional aquaglyceroporins that can facilitate efficient water and solute transfer. Two ancestor proteins, a water (orthodox aquaporin) and a solute facilitator (aquaglyceroporin), are supposed to give rise to today’s MIPs. Based on primary sequences of fungal MIPs, orthodox aquaporins/X-intrinsic proteins (XIPs) and FPS1-like/Yfl054-like/other aquaglyceroporins are supposed to be respective sister groups. However, at least within the fungal kingdom, no easy functional conclusion can be drawn from the phylogenetic position of a given protein within the MIP pedigree. In consequence, ecophysiological prediction of MIP relevance is not feasible without detailed functional analysis of the respective protein and expression studies. To illuminate the diverse MIP implications in fungal lifestyle, our current knowledge about protein function in two organisms, baker’s yeast and the Basidiomycotic Laccaria bicolor, an ectomycorrhizal model fungus, was exemplarily summarized in this review. MIP function has been investigated in such a depth in Saccharomyces cerevisiae that a system-wide view is possible. Yeast lifestyle, however, is special in many circumstances. Therefore, L. bicolor as filamentous Basidiomycete was added and allows insight into a very different way of life. Special emphasis was laid in this review onto ecophysiological interpretation of MIP function. 相似文献
13.
《生物化学与生物物理学报:生物膜》2022,1864(1):183795
14.
Michael Lever 《Biochimica et Biophysica Acta (BBA)/General Subjects》1998,1425(1):61-73
With water as the elution solvent, zwitterionic solutes and polyols were retained on HPLC columns, more than was water, by totally hydrophobic packing materials. Relative retentions were systematically affected by oxygen functional groups in the packing material, explicable as specific retention of water. Reproducible elution sequences of 20 solutes at a variety of hydrophobic surfaces (aromatic and both long- and short-alkyl aliphatic surfaces) showed there is a general process, consistent with interactions with hydration water at the surface having solvent properties distinct from bulk water. Early eluting solutes included glycine, sarcosine and taurine. Glycine betaine followed both these and N,N-dimethylglycine. The natural betaines propionobetaine and dimethylsulfoniopropionate also preceded glycine betaine. Dimethylsulfoxide was strongly retained, as (to a lesser extent) was proline betaine. Polyols eluted in the sequence sorbitol, trehalose, glycerol. Changes in the chemical nature of the surface or base material affected relative retentions of water and solutes. The presence of hydrogen-bonding functions increased retention of polyols, as well as water, relative to zwitterionic solutes. Specific effects with some solutes may be related to inconsistencies seen in biological systems. Pressures up to 8 MPa did not affect relative retention, constraining models based on the formation of low-density water. 相似文献
15.
Background
Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.Methods
The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.Results
Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. 14C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.Conclusions
P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.General significance
These studies provide new insight into glycerolipid metabolism in P. falciparum. 相似文献16.
W. Cheng C. X. Wang W. Z. Chen Ying Wu Xu Yun Yu Shi 《European biophysics journal : EBJ》1998,27(2):105-112
In this paper, the finite difference Poisson-Boltzmann (FDPB) method with four dielectric constants is developed to study
the effect of dielectric saturation on the electrostatic barriers of the permeation ion. In this method, the inner shape of
the channel pore is explicitly represented, and the fact that the dielectric constant inside the channel pore is different
from that of bulk water is taken into account. A model channel system which is a right-handed twist bundle with four α-helical segments is provided for this study. From the FDPB calculations, it is found that the difference of the ionic electrostatic
solvation energy for wider domains depends strongly on the pore radius in the vicinity of the ion when the pore dielectric
constant is changed from 78 to 5. However, the electrostatic solvation energy of the permeation ion can not be significantly
affected by the dielectric constant in regions with small pore radii. Our results indicate that the local electrostatic interactions
inside the ion channel are of major importance for ion electrostatic solvation energies, and the effect of dielectric saturation
on the electrostatic barriers is coupled to the interior channel dimensions.
Received: 28 January 1997 / Accepted: 24 September 1997 相似文献
17.
John G. Baust 《Cryobiology》1983,20(3):357-364
The exogenous cues to overwintering adaptations vary not just between components of hardening but between species. One species, P. brevicornis, initiates glycerol synthesis in response to 0 °C exposures while a second species, E. solidaginis, increases glycerol levels not in response to temperature but in apparent association with changes in total body mass. This species maintains a constant annual percentage of water while occupying a hibernaculum that dries considerably. During overwintering, E. solidaginis losses approximately 50% of its total body mass. In addition to the changes described, this species (northern populations) increases the amount of water bound to both protein and low-molecular-weight compounds during hardening. The increase in binding exceeds threefold between 25 and ?30 °C (0.193 to 0.633 g/g dry wt) (29). These data do not unequivocally demonstrate the existence of a hydration trigger to glycerol synthesis but are adequate to put forth such a hypothesis. A decrease in total bulk water levels due to both wet weight loss and increases in bound water may provide conditions of functionally reduced intracellular metabolic water. Since polyol production necessitates the disruption of carbon flow between glucose-6-phosphate and pyruvate, one or more enzymes may be sensitive to water reductions. Pyruvate kinase is sensitive to available water levels. Inhibition of this enzyme would likely cause a shunting of carbon metabolism to glycerol production. This hypothesis becomes attractive in light of the observation that in a variety of species, glycerol accumulations have been correlated with dehydration and hyperosmotic conditions. A common adaptative mechanism may exist in response to apparently different environmental perturbations. 相似文献
18.
19.
Nestor L. Uzcategui Yao Zhou Katherine Figarella Jun Ye Rita Mukhopadhyay Hiranmoy Bhattacharjee 《Molecular microbiology》2008,70(6):1477-1486
The Leishmania major aquaglyceroporin, LmAQP1, is responsible for the transport of antimonite [Sb(III)], an activated form of Pentostam or Glucantime. Downregulation of LmAQP1 provides resistance to trivalent antimony compounds and increased expression of LmAQP1 in drug‐resistant parasites can reverse the resistance. Besides metalloid transport, LmAQP1 is also permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. LmAQP1 also plays a physiological role in volume regulation and osmotaxis. In this study, we examined the role of extracellular C‐loop glutamates (Glu143, Glu145 and Glu152) in LmAQP1 activity. Alteration of both Glu143 and Glu145 to alanines did not affect either the biochemical or physiological properties of the protein, suggesting that neither residue is critical for LmAQP1 activity. Alteration of Glu152 to alanine, aspartate and glutamine affected metalloid transport in the order, wild‐type > E152Q > E152D > E152A. In fact, axenic amastigotes expressing E152A LmAQP1 accumulated negligible levels of either arsenite [As(III)] or Sb(III). Alteration of Glu152 significantly affected volume regulation and osmotaxis, suggesting that Glu152 is critical for the physiological activity of the parasite. More importantly, alteration of Glu152 to alanine did not affect glycerol permeability. Although the metalloids, As(III) and Sb(III), are believed to be transported through aquaglyceroporin channels as they behave as inorganic molecular mimic of glycerol, this is the first report where metalloid and glycerol transport can be dissected by a single mutation at the extracellular pore entry of LmAQP1 channel. 相似文献
20.
This study is an investigation of the ability of the bacterial channel alpha-hemolysin to facilitate water permeation across biological membranes. alpha-Hemolysin channels were incorporated into rabbit erythrocyte ghosts at varying concentrations, and water permeation was induced by mixing the ghosts with hypertonic sucrose solutions. The resulting volume decrease of the ghosts was followed by time-resolved optical absorption at pH 5, 6, and 7. The average single-channel permeability coefficient of alpha-hemolysin for water ranged between 1.3x10-12 cm/s and 1.5x10-12 cm/s, depending on pH. The slightly increased single-channel permeability coefficient at lower pH-values was attributed to an increase in the effective pore size. The activation energy of water transport through the channel was low (Ea=5.4 kcal/mol), suggesting that the properties of water inside the alpha-hemolysin channel resemble those of bulk water. This conclusion was supported by calculations based on macroscopic hydrodynamic laws of laminar water flow. Using the known three-dimensional structure of the channel, the calculations accurately predicted the rate of water flow through the channel. The latter finding also indicated that water permeation data can provide a good estimate of the pore size for large channels. 相似文献