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Rita Mukhopadhyay - One of the best experts on this subject based on the ideXlab platform.
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an Aquaglyceroporin as a new drug target in leishmania
2014Co-Authors: Rita Mukhopadhyay, Herbert WertheimAbstract:Aquaporins belong to MIPs (Major Intrinsic Protein), a large super-family of integral membrane proteins. They are present throughout the five kingdoms, including bacteria, archaea, protista, fungi, plantae, animalia and even viruses [1]. The ubiquitous nature of this channel underlines its importance in life processes. However, most of these aquaporins are redundant and null mutants for any one of them from bacteria to mammals do not cause lethality [2,3] with one exception from the parasite protozoan Leishmania (Mukhopadhyay and Ouellette, unpublished observation). The aquaporin family can be functionally divided into two sub-groups, the orthodox aquaporins, which are water-specific channels, and Aquaglyceroporins, which allow the transport of water, glycerol, metalloids and other small, uncharged solutes. These channels are all tetrameric complexes with each monomer functioning independently; unlike ion channels the center of the quaternary structure is impermeable. Each monomer contains six transmembrane helices and two short half-helices which meet in the middle of the membrane to form a pseudo-transmembrane span. The comparison between the crystal structures of E. coli Aquaglyceroporin GlpF and human aquaporin AQP1 showed a larger pore for Aquaglyceroporins accommodating the larger glycerol molecule [4]. The constriction at the pore mouth is termed as aromatic arginine (ar/R) constriction. An additional constriction in the middle of the pore is the Asn-Ala-Pro (NPA) region. These two NPA motifs form the central of the pore and act as a proton filter acting as the capping amino acids at the positive ends of helices B and E and act as hydrogen donors to the oxygen atoms of passing permeants [5]. The Aquaglyceroporins facilitate the transport of wide variety of uncharged solutes in addition to water. These include glycerol, urea, dihydroxyacetone (DHA), methylglyoxal (MG), polyols and metalloids such as trivalent arsenic (AsIII) and antimony (SbIII) [6]. In mammals, thirteen AQPs (0-12) have been identified so far. Among them four are classical Aquaglyceroporins AQP3, 7, 9 and 10 [7]. A number of aquaporins have been identified in parasitic protozoa from a single Aquaglyceroporin in Plasmodium, to three in Trypanosoma brucei, five in Leishmania, two in Toxoplasma and one in Cryptosporifium [8-11]. A common feature of the parasite AQPs is that they are generally better water transporters when compared to E. coli (GlpF) and mammalian ones. It appears that protozoal Aquaglyceroporins are bifunctional and conduct both water and glycerol at reasonable rates [12]. Among five members of the AQP family in Leishmania, we identified and characterized the first Aquaglyceroporin AQP1. AQP1 is involved in the accumulation of metalloids in Leishmania promastigotes and amastigotes along with water, glycerol, methylglyoxal, glyceraldehyde to name a few [9]. The other four AQPs are not involved in metalloid transport (Mukhopadhyay, unpublished data) and are closer to classical aquaporins. Channels such as AQPs are at the interphase of host parasite interactions and could be attractive drug targets and/or mediator of specific drugs such as arsenic and antimony. Protozoan parasites cause deadliest of diseases throughout the world. The situation is worse in developing countries because of poor hygiene and infrastructure.
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Aquaglyceroporins: Generalized metalloid channels
Biochimica et Biophysica Acta, 2013Co-Authors: Rita Mukhopadhyay, Hiranmoy Bhattacharjee, Barry P. RosenAbstract:Abstract Background Aquaporins (AQPs), members of a superfamily of transmembrane channel proteins, are ubiquitous in all domains of life. They fall into a number of branches that can be functionally categorized into two major sub-groups: i) orthodox aquaporins, which are water-specific channels, and ii) Aquaglyceroporins, which allow the transport of water, non-polar solutes, such as urea or glycerol, the reactive oxygen species hydrogen peroxide, and gases such as ammonia, carbon dioxide and nitric oxide and, as described in this review, metalloids. Scope of review This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells. Major conclusions As(OH)3 and Sb(OH)3 behave as inorganic molecular mimics of glycerol, a property that allows their passage through AQP channels. Plant AQPs also allow the passage of boron and silicon as their hydroxyacids, boric acid (B(OH)3) and orthosilicic acid (Si(OH)4), respectively. Genetic analysis suggests that germanic acid (GeO2) is also a substrate. While As(III), Sb(III) and Ge(IV) are toxic metalloids, borate (B(III)) and silicate (Si(IV)) are essential elements in higher plants. General significance The uptake of environmental metalloids by aquaporins provides an understanding of (i) how toxic elements such as arsenic enter the food chain; (ii) the delivery of arsenic and antimony containing drugs in the treatment of certain forms of leukemia and chemotherapy of diseases caused by pathogenic protozoa; and (iii) the possibility that food plants such as rice could be made safer by genetically modifying them to exclude arsenic while still accumulating boron and silicon. This article is part of a Special Issue entitled Aquaporins.
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Trypanosoma brucei Aquaglyceroporins facilitate the uptake of arsenite and antimonite in a pH dependent way.
Cellular Physiology and Biochemistry, 2013Co-Authors: Néstor L. Uzcátegui, Bjoern Bassarak, Nestor W. Meza, Katherine Figarella, José Luis Ramírez, Rita Mukhopadhyay, Michael DuszenkoAbstract:Background: Trypanosoma brucei is a primitive parasitic protozoan that thrives in diverse environments such as the midgut of the tsetse fly and the blood of a mammalian host. For an adequate adaptation to these environments, the parasites Aquaglyceroporins play an important role. Methods and Results: In order to test their ability to transport trivalent arsenic and antimony, we expressed the three known Trypanosoma brucei Aquaglyceroporins (TbAQPs) in the heterologous systems of yeast null aquaporin mutant and Xenopus laevis oocytes. For both expression systems, we found a pH dependent intracellular accumulation of As(III) or Sb(III) mediated by all of the three TbAQPs, with the exception of TbAQP1-As(III) uptake. Additionally, we observed that Trypanosoma brucei Aquaglyceroporins allow the passage of As(III) in both directions. Conclusion: Taken together, these results demonstrated that T. brucei Aquaglyceroporins can serve as entry routes for As(III) and Sb(III) into the parasitic cell, and that this uptake is pH sensitive. Therefore, aquaporins of protozoan parasites may be considered useful as a vehicle for drug delivery.
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the role of alanine 163 in solute permeability of leishmania major Aquaglyceroporin lmaqp1
Molecular and Biochemical Parasitology, 2011Co-Authors: Rita Mukhopadhyay, Néstor L. Uzcátegui, Katherine Figarella, Eric Beitz, Goutam Mandal, Venkata Subba Rao Atluri, Yao Zhou, Abdul A Ajees, Hiranmoy BhattacharjeeAbstract:Leishmania major Aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an Aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum Aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg(2+). This inhibition could be reversed upon addition of β-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance.
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alteration in glycerol and metalloid permeability by a single mutation in the extracellular c loop of leishmania major Aquaglyceroporin lmaqp1
Molecular Microbiology, 2008Co-Authors: Néstor L. Uzcátegui, Katherine Figarella, Rita Mukhopadhyay, Yao Zhou, Hiranmoy BhattacharjeeAbstract: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.
Eric Beitz - One of the best experts on this subject based on the ideXlab platform.
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Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2
PLoS pathogens, 2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific Aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of Aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.
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Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.
2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:(A) Shown are the crystal structure of the prototypical Aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.
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structural determinants of the hydrogen peroxide permeability of aquaporins
FEBS Journal, 2014Co-Authors: Abdulnasser Almasalmeh, Dawid Krenc, Eric BeitzAbstract:Aquaporins (AQP) conduct small, uncharged molecules, such as water (orthodox AQPs), ammonia (aquaammoniaporins) or glycerol (Aquaglyceroporins). The physiological functions of AQPs are involved in osmotic volume regulation or the transport of biochemical precursors and metabolic waste products. The recent identification of hydrogen peroxide (H₂O₂) as a permeant of certain AQPs suggests additional roles in mitigating oxidative stress or enabling paracrine H₂O₂ signalling. Yet, an analysis of the structural requirements of the H₂O₂ permeability of AQPs is missing. We subjected a representative set of wild-type and mutant AQPs to a newly established quantitative phenotypic assay. We confirmed high H₂O₂ permeability of the human aquaammoniaporin AQP8 and found intermediate H₂O₂ permeability of the prototypical orthodox water channel AQP1 from the rat. Differences from an earlier report showing an absence of H₂O₂ permeability of human AQP1 can be explained by expression levels. By generating point mutations in the selectivity filter of rat orthodox aquaporin AQP1, we established a correlation of H₂O₂ permeability primarily with water permeability and secondarily with the pore diameter. Even the narrowest pore of the test set (i.e. rat orthodox aquaporin AQP1 H180F with a pore diameter smaller than that of natural orthodox AQPs) conducted water and H₂O₂. We further found that H₂O₂ permeability of the Aquaglyceroporin from the malaria parasite Plasmodium falciparum was lower despite its wider pore diameter. The data suggest that all water-permeable AQPs are H₂O₂ channels, yet H₂O₂ permeability varies with the isoform. Thus, generally, AQPs must be considered as putative players in situations of oxidative stress (e.g. in Plasmodium-infected red blood cells, immune cells, the cardiovascular system or cells expressing AQP8 in their mitochondria).
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preparative scale production and functional reconstitution of a human Aquaglyceroporin aqp3 using a cell free expression system
New Biotechnology, 2013Co-Authors: Annika Mullerlucks, Eric Beitz, Patrizia Gena, Daniele Frascaria, Nicola Altamura, Maria Svelto, Giuseppe CalamitaAbstract:Understanding the selectivity of aquaporin (AQP) membrane channels and exploiting their biotechnological potential will require structural and functional studies of wild type and modified proteins; however, expression systems have not previously yielded AQPs in the necessary milligrams quantities. Cell free (CF) systems have emerged in recent years as fast, efficient and versatile technologies for the production of high quality membrane proteins. Here, we establish a convenient method to synthesize large amounts of functional human Aquaglyceroporin 3 protein (AQP3), an AQP of physiological relevance conducting glycerol and some small neutral solutes besides water. Milligram amounts of AQP3 were produced as a histidine-tagged protein (hAQP3-6His) in an Escherichia coli extract-based CF system in the presence of the non-ionic detergent Brij-98. The recombinant AQP3 was purified by affinity chromatography, incorporated into liposomes and evaluated functionally by stopped-flow light scattering. Correct protein folding was indicated by the high glycerol and water permeability exhibited by the hAQP3-6His proteoliposomes as compared to empty control liposomes. Functionality of hAQP3-6His was further confirmed by the strong inhibition of the glycerol and water permeability by phloretin and HgCl2, respectively, two blockers of AQP3. Fast and convenient CF production of functional AQP3 may serve as basis for further structural/functional assessment of Aquaglyceroporins and help boosting the AQP-based biomimetic technologies.
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molar concentrations of sorbitol and polyethylene glycol inhibit the plasmodium Aquaglyceroporin but not that of e coli involvement of the channel vestibules
Biochimica et Biophysica Acta, 2012Co-Authors: Jie Song, Abdulnasser Almasalmeh, Dawid Krenc, Eric BeitzAbstract: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.
Hiranmoy Bhattacharjee - One of the best experts on this subject based on the ideXlab platform.
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Aquaglyceroporins: Generalized metalloid channels
Biochimica et Biophysica Acta, 2013Co-Authors: Rita Mukhopadhyay, Hiranmoy Bhattacharjee, Barry P. RosenAbstract:Abstract Background Aquaporins (AQPs), members of a superfamily of transmembrane channel proteins, are ubiquitous in all domains of life. They fall into a number of branches that can be functionally categorized into two major sub-groups: i) orthodox aquaporins, which are water-specific channels, and ii) Aquaglyceroporins, which allow the transport of water, non-polar solutes, such as urea or glycerol, the reactive oxygen species hydrogen peroxide, and gases such as ammonia, carbon dioxide and nitric oxide and, as described in this review, metalloids. Scope of review This review summarizes the key findings that AQP channels conduct bidirectional movement of metalloids into and out of cells. Major conclusions As(OH)3 and Sb(OH)3 behave as inorganic molecular mimics of glycerol, a property that allows their passage through AQP channels. Plant AQPs also allow the passage of boron and silicon as their hydroxyacids, boric acid (B(OH)3) and orthosilicic acid (Si(OH)4), respectively. Genetic analysis suggests that germanic acid (GeO2) is also a substrate. While As(III), Sb(III) and Ge(IV) are toxic metalloids, borate (B(III)) and silicate (Si(IV)) are essential elements in higher plants. General significance The uptake of environmental metalloids by aquaporins provides an understanding of (i) how toxic elements such as arsenic enter the food chain; (ii) the delivery of arsenic and antimony containing drugs in the treatment of certain forms of leukemia and chemotherapy of diseases caused by pathogenic protozoa; and (iii) the possibility that food plants such as rice could be made safer by genetically modifying them to exclude arsenic while still accumulating boron and silicon. This article is part of a Special Issue entitled Aquaporins.
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the role of alanine 163 in solute permeability of leishmania major Aquaglyceroporin lmaqp1
Molecular and Biochemical Parasitology, 2011Co-Authors: Rita Mukhopadhyay, Néstor L. Uzcátegui, Katherine Figarella, Eric Beitz, Goutam Mandal, Venkata Subba Rao Atluri, Yao Zhou, Abdul A Ajees, Hiranmoy BhattacharjeeAbstract:Leishmania major Aquaglyceroporin LmAQP1 allows adventitious passage of antimonite, an activated form of the drug Pentostam, which is used as the first line treatment for leishmaniasis. The extracellular C-loop of an Aquaglyceroporin confers substrate specificity. Alteration of Glu125 to serine in the Plasmodium falciparum Aquaglyceroporin PfAQP has been shown to selectively affect water but not glycerol permeability. The C-loop of LmAQP1 is twelve residues longer than PfAQP, and Ala163 is at an equivalent position as Glu125 of PfAQP. The role of Ala163 in LmAQP1 solute permeability was investigated. Alteration of Ala163 to serine or threonine did not significantly affect conduction of solutes. However, alteration to aspartate, glutamate, and glutamine blocked passage of water, glycerol, and other organic solutes. While LmAQP1 is a mercurial insensitive water channel, mutation of the adjacent threonine (Thr164) to cysteine led to inhibition of water passage by Hg(2+). This inhibition could be reversed upon addition of β-mercaptoethanol. These data suggest that, unlike Glu125 (PfAQP), Ala163 is not involved in stabilization of the C-loop and selective solute permeability. Ala163 is located near the pore mouth of the channel, and replacement of Ala163 by bulkier residue sterically hinders the passage of solutes. Alteration of Ala163 to serine or threonine affected metalloid uptake in the order, wild-type>A163S>A163T. Metalloid conduction was near completely blocked when Ala163 was mutagenized to aspartate, glutamate, or glutamine. Mutations such as A163S and A163T that reduced the permeability to antimonite, without a significant loss in water or solute conductivity raises the possibility that, subtle changes in the side chain of the amino acid residue in position 163 of LmAQP1 may play a role in drug resistance.
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alteration in glycerol and metalloid permeability by a single mutation in the extracellular c loop of leishmania major Aquaglyceroporin lmaqp1
Molecular Microbiology, 2008Co-Authors: Néstor L. Uzcátegui, Katherine Figarella, Rita Mukhopadhyay, Yao Zhou, Hiranmoy BhattacharjeeAbstract: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.
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biochemical characterization of leishmania major Aquaglyceroporin lmaqp1 possible role in volume regulation and osmotaxis
Molecular Microbiology, 2007Co-Authors: Katherine Figarella, Néstor L. Uzcátegui, Hiranmoy Bhattacharjee, Marc Ouellette, Yao Zhou, Ann Lefurgey, Rita MukhopadhyayAbstract:Summary The Leishmania major Aquaglyceroporin, LmAQP1, is responsible for the transport of trivalent metalloids, arsenite and antimonite. We have earlier shown that downregulation of LmAQP1 provides resistance to trivalent antimony compounds whereas increased expression of LmAQP1 in drug-resistant parasites can reverse the resistance. In this paper we describe the biochemical characterization of LmAQP1. Expression of LmAQP1 in Xenopus oocytes rendered them permeable to water, glycerol, methylglyoxal, dihydroxyacetone and sugar alcohols. The transport property of LmAQP1 was severely affected when a critical Arg230, located inside the pore of the channel, was altered to either alanine or lysine. Immunofluorescence and immuno-electron microscopy revealed LmAQP1 to be localized to the flagellum of Leishmania promastigotes and in the flagellar pocket membrane and contractile vacuole/spongiome complex of amastigotes. This is the first report of an Aquaglyceroporin being localized to the flagellum of any microbe. Leishmania promastigotes and amastigotes expressing LmAQP1 could regulate their volume in response to hypoosmotic stress. Additionally, Leishmania promastigotes overexpressing LmAQP1 were found to migrate faster towards an osmotic gradient. These results taken together suggest that Leishmania LmAQP1 has multiple physiological roles, being involved in solute transport, volume regulation and osmotaxis.
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novel pathway for arsenic detoxification in the legume symbiont sinorhizobium meliloti
Journal of Bacteriology, 2005Co-Authors: Hungchi Yang, Barry P. Rosen, Jiujun Cheng, Turlough M Finan, Hiranmoy BhattacharjeeAbstract:We report a novel pathway for arsenic detoxification in the legume symbiont Sinorhizobium meliloti. Although a majority of ars operons consist of three genes, arsR (transcriptional regulator), arsB [As(OH)3/H+ antiporter], and arsC (arsenate reductase), the S. meliloti ars operon includes an Aquaglyceroporin (aqpS) in place of arsB. The presence of AqpS in an arsenic resistance operon is interesting, since Aquaglyceroporin channels have previously been shown to adventitiously facilitate uptake of arsenite into cells, rendering them sensitive to arsenite. To understand the role of aqpS in arsenic resistance, S. meliloti aqpS and arsC were disrupted individually. Disruption of aqpS resulted in increased tolerance to arsenite but not arsenate, while cells with an arsC disruption showed selective sensitivity to arsenate. The results of transport experiments in intact cells suggest that AqpS is the only protein of the S. meliloti ars operon that facilitates transport of arsenite. Coexpression of S. meliloti aqpS and arsC in a strain of E. coli lacking the ars operon complemented arsenate but not arsenite sensitivity. These results imply that, when S. meliloti is exposed to environmental arsenate, arsenate enters the cell through phosphate transport systems and is reduced to arsenite by ArsC. Internally generated arsenite flows out of the cell by downhill movement through AqpS. Thus, AqpS confers arsenate resistance together with ArsC-catalyzed reduction. This is the first report of an Aquaglyceroporin with a physiological function in arsenic resistance.
Nicola Baker - One of the best experts on this subject based on the ideXlab platform.
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pentamidine and
2016Co-Authors: Jane C. Munday, Nicola Baker, Lucy Glover, Anthonius A. Eze, Caroline Clucas, David Aguinaga Andrés, Manal J. Natto, Ibrahim A. Teka, Jennifer Mcdonald, Rebecca S. LeeAbstract:Trypanosoma brucei Aquaglyceroporin 2 is a high-affinity transporter fo
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Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2
PLoS pathogens, 2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific Aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of Aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.
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Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.
2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:(A) Shown are the crystal structure of the prototypical Aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.
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Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates
International journal for parasitology. Drugs and drug resistance, 2015Co-Authors: Fabrice E. Graf, Jane C. Munday, Nicola Baker, David Horn, Harry P. De Koning, Pascal MäserAbstract:Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.
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Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Nicola Baker, Michael P. Barrett, Jane C. Munday, Harry P. De Koning, Lucy Glover, David Aguinaga Andrés, David HornAbstract:African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related Aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional “selectivity filter.” AQP2-specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3. AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle–stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional Aquaglyceroporin, AQP2, renders cells sensitive to both melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.
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Pentamidine Is Not a Permeant but a Nanomolar Inhibitor of the Trypanosoma brucei Aquaglyceroporin-2
PLoS pathogens, 2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:The chemotherapeutic arsenal against human African trypanosomiasis, sleeping sickness, is limited and can cause severe, often fatal, side effects. One of the classic and most widely used drugs is pentamidine, an aromatic diamidine compound introduced in the 1940s. Recently, a genome-wide loss-of-function screen and a subsequently generated trypanosome knockout strain revealed a specific Aquaglyceroporin, TbAQP2, to be required for high-affinity uptake of pentamidine. Yet, the underlying mechanism remained unclear. Here, we show that TbAQP2 is not a direct transporter for the di-basic, positively charged pentamidine. Even though one of the two common cation filters of Aquaglyceroporins, i.e. the aromatic/arginine selectivity filter, is unconventional in TbAQP2, positively charged compounds are still excluded from passing the channel. We found, instead, that the unique selectivity filter layout renders pentamidine a nanomolar inhibitor of TbAQP2 glycerol permeability. Full, non-covalent inhibition of an aqua(glycero)porin in the nanomolar range has not been achieved before. The remarkable affinity derives from an electrostatic interaction with Asp265 and shielding from water as shown by structure-function evaluation and point mutation of Asp265. Exchange of the preceding Leu264 to arginine abolished pentamidine-binding and parasites expressing this mutant were pentamidine-resistant. Our results indicate that TbAQP2 is a high-affinity receptor for pentamidine. Taken together with localization of TbAQP2 in the flagellar pocket of bloodstream trypanosomes, we propose that pentamidine uptake is by endocytosis.
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Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.
2016Co-Authors: Jie Song, Nicola Baker, David Horn, Monja Rothert, Björn Henke, Laura Jeacock, Eric BeitzAbstract:(A) Shown are the crystal structure of the prototypical Aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.
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Chimerization at the AQP2-AQP3 locus is the genetic basis of melarsoprol-pentamidine cross-resistance in clinical Trypanosoma brucei gambiense isolates
International journal for parasitology. Drugs and drug resistance, 2015Co-Authors: Fabrice E. Graf, Jane C. Munday, Nicola Baker, David Horn, Harry P. De Koning, Pascal MäserAbstract:Aquaglyceroporin-2 is a known determinant of melarsoprol–pentamidine cross-resistance in Trypanosoma brucei brucei laboratory strains. Recently, chimerization at the AQP2–AQP3 tandem locus was described from melarsoprol–pentamidine cross-resistant Trypanosoma brucei gambiense isolates from sleeping sickness patients in the Democratic Republic of the Congo. Here, we demonstrate that reintroduction of wild-type AQP2 into one of these isolates fully restores drug susceptibility while expression of the chimeric AQP2/3 gene in aqp2–aqp3 null T. b. brucei does not. This proves that AQP2–AQP3 chimerization is the cause of melarsoprol–pentamidine cross-resistance in the T. b. gambiense isolates.
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Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes.
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Nicola Baker, Michael P. Barrett, Jane C. Munday, Harry P. De Koning, Lucy Glover, David Aguinaga Andrés, David HornAbstract:African trypanosomes cause sleeping sickness in humans, a disease that is typically fatal without chemotherapy. Unfortunately, drug resistance is common and melarsoprol-resistant trypanosomes often display cross-resistance to pentamidine. Although melarsoprol/pentamidine cross-resistance (MPXR) has been an area of intense interest for several decades, our understanding of the underlying mechanisms remains incomplete. Recently, a locus encoding two closely related Aquaglyceroporins, AQP2 and AQP3, was linked to MPXR in a high-throughput loss-of-function screen. Here, we show that AQP2 has an unconventional “selectivity filter.” AQP2-specific gene knockout generated MPXR trypanosomes but did not affect resistance to a lipophilic arsenical, whereas recombinant AQP2 reversed MPXR in cells lacking native AQP2 and AQP3. AQP2 was also shown to be disrupted in a laboratory-selected MPXR strain. Both AQP2 and AQP3 gained access to the surface plasma membrane in insect life-cycle–stage trypanosomes but, remarkably, AQP2 was specifically restricted to the flagellar pocket in the bloodstream stage. We conclude that the unconventional Aquaglyceroporin, AQP2, renders cells sensitive to both melarsoprol and pentamidine and that loss of AQP2 function could explain cases of innate and acquired MPXR.