RESUMO
BACKGROUND: The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. RESULTS: The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. CONCLUSIONS: The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.
Assuntos
Saccharomycetales , Gorgulhos , Animais , Proteínas de Fluorescência Verde/genética , Citometria de FluxoRESUMO
The Ezrin/Radixin/Moesin (ERM) family of proteins act as cross-linkers between the plasma membrane and the actin cytoskeleton. This mechanism plays an essential role in processes related to membrane remodeling and organization, such as cell polarization, morphogenesis and adhesion, as well as in membrane protein trafficking and signaling pathways. For several human aquaporin (AQP) isoforms, an interaction between the ezrin band Four-point-one, Ezrin, Radixin, Moesin (FERM)-domain and the AQP C-terminus has been demonstrated, and this is believed to be important for AQP localization in the plasma membrane. Here, we investigate the structural basis for the interaction between ezrin and two human AQPs: AQP2 and AQP5. Using microscale thermophoresis, we show that full-length AQP2 and AQP5 as well as peptides corresponding to their C-termini interact with the ezrin FERM-domain with affinities in the low micromolar range. Modelling of the AQP2 and AQP5 FERM complexes using ColabFold reveals a common mode of binding in which the proximal and distal parts of the AQP C-termini bind simultaneously to distinct binding sites of FERM. While the interaction at each site closely resembles other FERM-complexes, the concurrent interaction with both sites has only been observed in the complex between moesin and its C-terminus which causes auto-inhibition. The proposed interaction between AQP2/AQP5 and FERM thus represents a novel binding mode for extrinsic ERM-interacting partners.
Assuntos
Aquaporina 2 , Aquaporina 5 , Proteínas do Citoesqueleto , Ligação Proteica , Humanos , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/química , Aquaporina 5/metabolismo , Aquaporina 5/química , Aquaporina 2/metabolismo , Aquaporina 2/química , Sítios de Ligação , Aquaporinas/metabolismo , Aquaporinas/química , Domínios Proteicos , Modelos Moleculares , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/químicaRESUMO
Aquaporins (AQPs), membrane proteins responsible for facilitating water transport, found in plant membrane vesicles (MV), have been related to the functionality and stability of MV. We focused on AQPs obtained from broccoli, as they show potential for biotechnological applications. To gain further insight into the role of AQPs in MV, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in their purification with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2). We reconstituted AQPs in liposomes to study their functionality, and the size of proteoliposomes did not change concerning liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4 °C and at room temperature but not at 37 °C. BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract, resulting in increased entrapment efficiency (EE) compared to conventional liposomes. Molecular docking was utilized to identify binding sites in PIP2s for resveratrol, highlighting the role of aquaporins in the improved EE. Moreover, interactions between plant AQP and human integrin were shown, which may increase internalization by the human target cells. Our results suggest AQP-based alternative encapsulation systems can be used in specifically targeted biotechnological applications.
Assuntos
Aquaporinas , Brassica , Proteolipídeos , Humanos , Lipossomos/metabolismo , Resveratrol/metabolismo , Simulação de Acoplamento Molecular , Aquaporinas/metabolismo , Brassica/genética , Brassica/metabolismo , Água/químicaRESUMO
BACKGROUND: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. RESULTS: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. CONCLUSIONS: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations.
Assuntos
Proteínas de Insetos/química , Receptores Odorantes/química , Gorgulhos/química , Animais , Sítios de Ligação , Feminino , Proteínas de Insetos/genética , Ligantes , Masculino , Receptores Odorantes/genética , Gorgulhos/genéticaRESUMO
The aquaglyceroporins are a subfamily of aquaporins that conduct both water and glycerol. Aquaporin-3 (AQP3) has an important physiological function in renal water reabsorption, and AQP3-mediated hydrogen peroxide (H2O2) permeability can enhance cytokine signaling in several cell types. The related aquaglyceroporin AQP7 is required for dendritic cell chemokine responses and antigen uptake. Selective small-molecule inhibitors are desirable tools for investigating the biological and pathological roles of these and other AQP isoforms. Here, using a calcein fluorescence quenching assay, we screened a library of 7360 drug-like small molecules for inhibition of mouse AQP3 water permeability. Hit confirmation and expansion with commercially available substances identified the ortho-chloride-containing compound DFP00173, which inhibited mouse and human AQP3 with an IC50 of â¼0.1-0.4 µm but had low efficacy toward mouse AQP7 and AQP9. Surprisingly, inhibitor specificity testing revealed that the methylurea-linked compound Z433927330, a partial AQP3 inhibitor (IC50, â¼0.7-0.9 µm), is a potent and efficacious inhibitor of mouse AQP7 water permeability (IC50, â¼0.2 µm). Stopped-flow light scattering measurements confirmed that DFP00173 and Z433927330 inhibit AQP3 glycerol permeability in human erythrocytes. Moreover, DFP00173, Z433927330, and the previously identified AQP9 inhibitor RF03176 blocked aquaglyceroporin H2O2 permeability. Molecular docking to AQP3, AQP7, and AQP9 homology models suggested interactions between these inhibitors and aquaglyceroporins at similar binding sites. DFP00173 and Z433927330 constitute selective and potent AQP3 and AQP7 inhibitors, respectively, and contribute to a set of isoform-specific aquaglyceroporin inhibitors that will facilitate the evaluation of these AQP isoforms as drug targets.
Assuntos
Aquaporina 3/antagonistas & inibidores , Aquaporinas/antagonistas & inibidores , Tiofenos/farmacologia , Animais , Células CHO , Permeabilidade da Membrana Celular , Cricetulus , Eritrócitos/metabolismo , Glicerol/metabolismo , Humanos , Camundongos , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Tiofenos/química , Água/metabolismoRESUMO
The human Transient Receptor Potential A1 (hTRPA1) ion channel, also known as the wasabi receptor, acts as a biosensor of various potentially harmful stimuli. It is activated by a wide range of chemicals, including the electrophilic compound N-methylmaleimide (NMM), but the mechanism of activation is not fully understood. Here, we used mass spectrometry to map and quantify the covalent labeling in hTRPA1 at three different concentrations of NMM. A functional truncated version of hTRPA1 (Δ1-688 hTRPA1), lacking the large N-terminal ankyrin repeat domain (ARD), was also assessed in the same way. In the full length hTRPA1, the labeling of different cysteines ranged from nil up to 95% already at the lowest concentration of NMM, suggesting large differences in reactivity of the thiols. Most important, the labeling of some cysteine residues increased while others decreased with the concentration of NMM, both in the full length and the truncated protein. These findings indicate a conformational switch of the proteins, possibly associated with activation or desensitization of the ion channel. In addition, several lysines in the transmembrane domain and the proximal N-terminal region were labeled by NMM, raising the possibility that lysines are also key targets for electrophilic activation of hTRPA1.
Assuntos
Canal de Cátion TRPA1/metabolismo , Repetição de Anquirina/fisiologia , Cisteína/metabolismo , Humanos , Ativação do Canal Iônico/fisiologia , Lisina/metabolismo , Espectrometria de Massas/métodos , Domínios Proteicos/fisiologia , Compostos de Sulfidrila/metabolismoRESUMO
Aquaporins of the TIP subfamily (Tonoplast Intrinsic Proteins) have been suggested to facilitate permeation of water and ammonia across the vacuolar membrane of plants, allowing the vacuole to efficiently sequester ammonium ions and counteract cytosolic fluctuations of ammonia. Here, we report the structure determined at 1.18 Å resolution from twinned crystals of Arabidopsis thaliana aquaporin AtTIP2;1 and confirm water and ammonia permeability of the purified protein reconstituted in proteoliposomes as further substantiated by molecular dynamics simulations. The structure of AtTIP2;1 reveals an extended selectivity filter with the conserved arginine of the filter adopting a unique unpredicted position. The relatively wide pore and the polar nature of the selectivity filter clarify the ammonia permeability. By mutational studies, we show that the identified determinants in the extended selectivity filter region are sufficient to convert a strictly water-specific human aquaporin into an AtTIP2;1-like ammonia channel. A flexible histidine and a novel water-filled side pore are speculated to deprotonate ammonium ions, thereby possibly increasing permeation of ammonia. The molecular understanding of how aquaporins facilitate ammonia flux across membranes could potentially be used to modulate ammonia losses over the plasma membrane to the atmosphere, e.g., during photorespiration, and thereby to modify the nitrogen use efficiency of plants.
Assuntos
Amônia/metabolismo , Aquaporinas/química , Proteínas de Arabidopsis/química , Aquaporinas/metabolismo , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Cristalização , Estrutura MolecularRESUMO
BACKGROUND: Aquaporins (AQPs) facilitate the passage of small neutral polar molecules across membranes of the cell. In animals there are four distinct AQP subfamilies, whereof AQP8 homologues constitute one of the smallest subfamilies with just one member in man. AQP8 conducts water, ammonia, urea, glycerol and H2O2 through various membranes of animal cells. This passive channel has been connected to a number of phenomena, such as volume change of mitochondria, ammonia neurotoxicity, and mitochondrial dysfunction related to oxidative stress. Currently, there is no experimentally determined structure of an AQP8, hence the structural understanding of this subfamily is limited. The recently solved structure of the plant AQP, AtTIP2;1, which has structural and functional features in common with AQP8s, has opened up for construction of homology models that are likely to be more accurate than previous models. RESULTS: Here we present homology models of seven vertebrate AQP8s. Modeling based on the AtTIP2;1 structure alone resulted in reasonable models except for the pore being blocked by a phenylalanine that is not present in AtTIP2;1. To achieve an open pore, these models were supplemented with models based on the bacterial water specific AQP, EcAqpZ, creating a chimeric monomeric model for each AQP8 isoform. The selectivity filter (also named the aromatic/arginine region), which defines the permeant substrate profile, comprises five amino acid residues in AtTIP2;1, including a histidine coming from loop C. Compared to AtTIP2;1, the selectivity filters of modelled AQP8s only deviates in that they are slightly more narrow and more hydrophobic due to a phenylalanine replacing the histidine from loop C. Interestingly, the models do not exclude the existence of a side pore beneath loop C similar to that described in the structure of AtTIP2;1. CONCLUSIONS: Our models concur that AQP8s are likely to have an AtTIP2;1-like selectivity filter. The detailed description of the expected configuration of residues in the selectivity filters of AQP8s provides an excellent starting point for planning of as well as rationalizing the outcome of mutational studies. Our strategy to compile hybrid models based on several templates may prove useful also for other AQPs for which structural information is limited.
Assuntos
Aquaporinas/química , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Vertebrados/metabolismo , Animais , Arabidopsis/química , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Isoformas de Proteínas/química , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli.
Assuntos
Repetição de Anquirina , Culicidae/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potencial de Receptor Transitório/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Temperatura Baixa , Cristalografia por Raios X , Temperatura Alta , Humanos , Ligação Proteica , Conformação Proteica , Homologia de Sequência de Aminoácidos , Canais de Potencial de Receptor Transitório/isolamento & purificaçãoRESUMO
BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1. RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter. CONCLUSION: The water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.
Assuntos
Aquaporinas/metabolismo , Nicotiana/química , Proteínas de Plantas/metabolismo , Substituição de Aminoácidos , Aquaporinas/química , Aquaporinas/genética , Proteínas de Arabidopsis/química , Modelos Moleculares , Mutação , Pichia/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformação Proteica , Deleção de Sequência , Homologia Estrutural de Proteína , Água/química , Água/metabolismoRESUMO
We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).
Assuntos
Repetição de Anquirina , Canais de Cálcio/fisiologia , Temperatura Baixa , Proteínas do Tecido Nervoso/fisiologia , Canais de Potencial de Receptor Transitório/fisiologia , Canais de Cálcio/química , Humanos , Proteínas do Tecido Nervoso/química , Técnicas de Patch-Clamp , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/químicaRESUMO
As ectotherms, insects need heat-sensitive receptors to monitor environmental temperatures and facilitate thermoregulation. We show that TRPA5, a class of ankyrin transient receptor potential (TRP) channels absent in dipteran genomes, may function as insect heat receptors. In the triatomine bug Rhodnius prolixus (order: Hemiptera), a vector of Chagas disease, the channel RpTRPA5B displays a uniquely high thermosensitivity, with biophysical determinants including a large channel activation enthalpy change (72 kcal/mol), a high temperature coefficient (Q10 = 25), and in vitro temperature-induced currents from 53°C to 68°C (T0.5 = 58.6°C), similar to noxious TRPV receptors in mammals. Monomeric and tetrameric ion channel structure predictions show reliable parallels with fruit fly dTRPA1, with structural uniqueness in ankyrin repeat domains, the channel selectivity filter, and potential TRP functional modulator regions. Overall, the finding of a member of TRPA5 as a temperature-activated receptor illustrates the diversity of insect molecular heat detectors.
RESUMO
Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of three human aquaporins (HsAQP1, HsAQP4, and HsAQP5) have been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP1, HsAQP4 and HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.
Assuntos
Aquaporinas/química , Aquaporinas/biossíntese , Aquaporinas/genética , Aquaporinas/isolamento & purificação , Cristalografia por Raios X , Detergentes/química , Expressão Gênica , Humanos , Lipídeos/química , Pichia/genética , Pichia/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Relação Estrutura-AtividadeRESUMO
It has been hypothesized that aquaporin-9 (AQP9) is part of the unknown route of hepatocyte glycerol uptake. In a previous study, leptin receptor-deficient wild-type mice became diabetic and suffered from fasting hyperglycemia whereas isogenic AQP9(-/-) knock-out mice remained normoglycemic. The reason for this improvement in AQP9(-/-) mice was not established before. Here, we show increased glucose output (by 123% ± 36% S.E.) in primary hepatocyte culture when 0.5 mM extracellular glycerol was added. This increase depended on AQP9 because it was absent in AQP9(-/-) cells. Likewise, the increase was abolished by 25 µM HTS13286 (IC(50) ~ 2 µM), a novel AQP9 inhibitor, which we identified in a small molecule library screen. Similarly, AQP9 deletion or chemical inhibition eliminated glycerol-enhanced glucose output in perfused liver preparations. The following control experiments suggested inhibitor specificity to AQP9: (i) HTS13286 affected solute permeability in cell lines expressing AQP9, but not in cell lines expressing AQPs 3, 7, or 8. (ii) HTS13286 did not influence lactate- and pyruvate-dependent hepatocyte glucose output. (iii) HTS13286 did not affect glycerol kinase activity. Our experiments establish AQP9 as the primary route of hepatocyte glycerol uptake for gluconeogenesis and thereby explain the previously observed, alleviated diabetes in leptin receptor-deficient AQP9(-/-) mice.
Assuntos
Aquaporinas/metabolismo , Gluconeogênese/fisiologia , Glucose/metabolismo , Glicerol/metabolismo , Hepatócitos/metabolismo , Animais , Aquaporinas/genética , Células CHO , Cricetinae , Cricetulus , Crioprotetores/metabolismo , Crioprotetores/farmacologia , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Gluconeogênese/efeitos dos fármacos , Glucose/genética , Glicerol/farmacologia , Hepatócitos/citologia , Ácido Láctico/metabolismo , Camundongos , Camundongos Knockout , Ácido Pirúvico/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismoRESUMO
Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 A resolution and in its open conformation at 3.9 A resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16 A and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.
Assuntos
Aquaporinas/química , Aquaporinas/metabolismo , Ativação do Canal Iônico , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Spinacia oleracea/química , Simulação por Computador , Modelos Moleculares , Fosforilação , Fosfosserina/metabolismo , Conformação Proteica , Spinacia oleracea/metabolismo , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Aquaporin-9 (AQP9) is a facilitator of glycerol and other small neutral solute transmembrane diffusion. Identification of specific inhibitors for aquaporin family proteins has been difficult, due to high sequence similarity between the 13 human isoforms, and due to the limited channel surface areas that permit inhibitor binding. The few AQP9 inhibitor molecules described to date were not suitable for in vivo experiments. We now describe the characterization of a new small molecule AQP9 inhibitor, RG100204 in cell-based calcein-quenching assays, and by stopped-flow light-scattering recordings of AQP9 permeability in proteoliposomes. Moreover, we investigated the effects of RG100204 on glycerol metabolism in mice. In cell-based assays, RG100204 blocked AQP9 water permeability and glycerol permeability with similar, high potency (~5 × 10-8 M). AQP9 channel blocking by RG100204 was confirmed in proteoliposomes. After oral gavage of db/db mice with RG100204, a dose-dependent elevation of plasma glycerol was observed. A blood glucose-lowering effect was not statistically significant. These experiments establish RG100204 as a direct blocker of the AQP9 channel, and suggest its use as an experimental tool for in vivo experiments on AQP9 function.
Assuntos
Aquaporinas , Glicerol , Animais , Humanos , Camundongos , Aquaporinas/metabolismo , Glicemia/metabolismo , Glicerol/metabolismo , Glicerol/farmacologia , Fígado/metabolismo , Camundongos Endogâmicos , Água/metabolismoRESUMO
Aquaporins facilitate water transport over cellular membranes, and are therefore believed to play an important role in water homeostasis. In higher plants aquaporin-like proteins, also called major intrinsic proteins (MIPs), are divided into five subfamilies. We have previously shown that MIP transcription in Arabidopsis thaliana is generally downregulated in leaves upon drought stress, apart from two members of the plasma membrane intrinsic protein (PIP) subfamily, AtPIP1;4 and AtPIP2;5, which are upregulated. In order to assess whether this regulation is general or accession-specific we monitored the gene expression of all PIPs in five Arabidopsis accessions. The overall drought regulation of PIPs was well conserved for all five accessions tested, suggesting a general and fundamental physiological role of this drought response. In addition, significant differences among accessions were identified for transcripts of three PIP genes. Principal component analysis showed that most of the PIP transcriptional variation during drought stress could be explained by one variable linked to leaf water content. Promoter-GUS constructs of AtPIP1;4, AtPIP2;5 and also AtPIP2;6, which is unresponsive to drought stress, had distinct expression patterns concentrated in the base of the leaf petioles and parts of the flowers. The presence of drought stress response elements within the 1.6-kb promoter regions of AtPIP1;4 and AtPIP2;5 was demonstrated by comparing transcription of the promoter reporter construct and the endogenous gene upon drought stress. Analysis by ATTED-II and other web-based bioinformatical tools showed that several of the MIPs downregulated upon drought are strongly co-expressed, whereas AtPIP1;4, AtPIP2;5 and AtPIP2;6 are not co-expressed.
Assuntos
Aquaporinas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Secas , Regulação da Expressão Gênica de Plantas , Aquaporinas/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Regulação para Baixo , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Componente Principal , Regiões Promotoras Genéticas , RNA de Plantas/genética , Estresse Fisiológico , Transformação GenéticaRESUMO
BACKGROUND: Major intrinsic proteins (MIPs) also named aquaporins form channels facilitating the passive transport of water and other small polar molecules across membranes. MIPs are particularly abundant and diverse in terrestrial plants but little is known about their evolutionary history. In an attempt to investigate the origin of the plant MIP subfamilies, genomes of chlorophyte algae, the sister group of charophyte algae and land plants, were searched for MIP encoding genes. RESULTS: A total of 22 MIPs were identified in the nine analysed genomes and phylogenetic analyses classified them into seven subfamilies. Two of these, Plasma membrane Intrinsic Proteins (PIPs) and GlpF-like Intrinsic Proteins (GIPs), are also present in land plants and divergence dating support a common origin of these algal and land plant MIPs, predating the evolution of terrestrial plants. The subfamilies unique to algae were named MIPA to MIPE to facilitate the use of a common nomenclature for plant MIPs reflecting phylogenetically stable groups. All of the investigated genomes contained at least one MIP gene but only a few species encoded MIPs belonging to more than one subfamily. CONCLUSIONS: Our results suggest that at least two of the seven subfamilies found in land plants were present already in an algal ancestor. The total variation of MIPs and the number of different subfamilies in chlorophyte algae is likely to be even higher than that found in land plants. Our analyses indicate that genetic exchanges between several of the algal subfamilies have occurred. The PIP1 and PIP2 groups and the Ca2+ gating appear to be specific to land plants whereas the pH gating is a more ancient characteristic shared by all PIPs. Further studies are needed to discern the function of the algal specific subfamilies MIPA-E and to fully understand the evolutionary relationship of algal and terrestrial plant MIPs.
Assuntos
Aquaporinas/genética , Clorófitas/genética , Proteínas de Plantas/genética , Plantas/genética , Sequência de Aminoácidos , Sequência Conservada , Genoma de Planta , Dados de Sequência Molecular , Filogenia , Alinhamento de SequênciaRESUMO
BACKGROUND: When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris. RESULTS: By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation. CONCLUSIONS: We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.
Assuntos
Aquaporinas/metabolismo , Dosagem de Genes , Pichia/metabolismo , Proteínas Recombinantes/metabolismo , Aquaporinas/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Clonagem Molecular , Regulação Fúngica da Expressão Gênica , Variação Genética , Humanos , Pichia/genética , Reação em Cadeia da Polimerase , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-A resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.