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1.
  • Backmark, Anna, 1979, et al. (author)
  • Affinity tags can reduce merohedral twinning of membrane protein crystals
  • 2008
  • In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
  • Journal article (peer-reviewed)abstract
    • This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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2.
  • Fischer, Gerhard, 1978, et al. (author)
  • Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.
  • 2009
  • In: PLoS biology. - : Public Library of Science (PLoS). - 1545-7885 .- 1544-9173. ; 7:6
  • Journal article (peer-reviewed)abstract
    • Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
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3.
  • Frick, Anna, 1982, et al. (author)
  • X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.
  • 2014
  • In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:17, s. 6305-10
  • Journal article (peer-reviewed)abstract
    • Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.
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4.
  • Jansson, Anna, 1985, et al. (author)
  • Monitoring the osmotic response of single yeast cells through force measurement in the environmental scanning electron microscope
  • 2014
  • In: Measurement science and technology. - : IOP Publishing. - 0957-0233 .- 1361-6501. ; 25:2
  • Journal article (peer-reviewed)abstract
    • We present a measurement system that combines an environmental scanning electron microscope (ESEM) and an atomic force microscope (AFM). This combination enables studies of static and dynamic mechanical properties of hydrated specimens, such as individual living cells. The integrated AFM sensor provides direct and continuous force measurement based on piezoresistive force transduction, allowing the recording of events in the millisecond range. The in situ ESEM-AFM setup was used to study Pichia pastoris wild-type yeast cells. For the first time, a quantified measure of the osmotic response of an individual yeast cell inside an ESEM is presented. With this technique, cell size changes due to humidity variations can be monitored with nanometre accuracy. In addition, mechanical properties were extracted from load-displacement curves. A Young's modulus of 13-15 MPa was obtained for the P. pastoris yeast cells. The developed method is highly interesting as a complementary tool for the screening of drugs directed towards cellular water transport activity and provides new possibilities of studying mechanosensitive regulation of aquaporins.
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5.
  • Lind, Ulrika, et al. (author)
  • Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity
  • 2017
  • In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:7
  • Journal article (peer-reviewed)abstract
    • Barnacles are sessile macro-invertebrates, found along rocky shores in coastal areas worldwide. The euryhaline bay barnacle Balanus improvisus (Darwin, 1854) (= Amphibalanus improvisus) can tolerate a wide range of salinities, but the molecular mechanisms underlying the osmoregulatory capacity of this truly brackish species are not well understood. Aquaporins are pore-forming integral membrane proteins that facilitate transport of water, small solutes and ions through cellular membranes, and that have been shown to be important for osmoregulation in many organisms. The knowledge of the function of aquaporins in crustaceans is, however, limited and nothing is known about them in barnacles. We here present the repertoire of aquaporins from a thecostracan crustacean, the barnacle B. improvisus, based on genome and transcriptome sequencing. Our analyses reveal that B. improvisus contains eight genes for aquaporins. Phylogenetic analysis showed that they represented members of the classical water aquaporins (Aqp1, Aqp2), the aquaglyceroporins (Glp1, Glp2), the unorthodox aquaporin (Aqp12) and the arthropod-specific big brain aquaporin (Bib). Interestingly, we also found two big brain-like proteins (BibL1 and BibL2) constituting a new group of aquaporins not yet described in arthropods. In addition, we found that the two water-specific aquaporins were expressed as C-terminal splice variants. Heterologous expression of some of the aquaporins followed by functional characterization showed that Aqp1 transported water and Glp2 water and glycerol, agreeing with the predictions of substrate specificity based on 3D modeling and phylogeny. To investigate a possible role for the B. improvisus aquaporins in osmoregulation, mRNA expression changes in adult barnacles were analysed after long-term acclimation to different salinities. The most pronounced expression difference was seen for AQP1 with a substantial (>100-fold) decrease in the mantle tissue in low salinity (3 PSU) compared to high salinity (33 PSU). Our study provides a base for future mechanistic studies on the role of aquaporins in osmoregulation. © 2017 Lind et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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6.
  • Nyblom, Anna Maria, 1975, et al. (author)
  • Exceptional overproduction of a functional human membrane protein
  • 2007
  • In: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 56:1, s. 110-120
  • Journal article (peer-reviewed)abstract
    • Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQPI in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. (c) 2007 Elsevier Inc. All rights reserved.
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7.
  • Nyblom, Anna Maria, 1975, et al. (author)
  • Structural and functional analysis of SoPIP2;1 mutants adds insight into plant aquaporin gating.
  • 2009
  • In: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:3, s. 653-68
  • Journal article (peer-reviewed)abstract
    • Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca(2+) can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C(alpha) atom of Glu31 10 A away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.
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8.
  • Schmitz, Florian, et al. (author)
  • A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions
  • 2021
  • In: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
  • Journal article (peer-reviewed)abstract
    • Interactions between membrane proteins within a cellular environment are crucial for all living cells. Robust methods to screen and analyse membrane protein complexes are essential to shed light on the molecular mechanism of membrane protein interactions. Most methods for detecting protein:protein interactions (PPIs) have been developed to target the interactions of soluble proteins. Bimolecular fluorescence complementation (BiFC) assays allow the formation of complexes involving PPI partners to be visualized in vivo, irrespective of whether or not these interactions are between soluble or membrane proteins. In this study, we report the development of a screening approach which utilizes BiFC and applies flow cytometry to characterize membrane protein interaction partners in the host Saccharomyces cerevisiae. These data allow constructive complexes to be discriminated with statistical confidence from random interactions and potentially allows an efficient screen for PPIs in vivo within a high-throughput setup.
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9.
  • Sjöhamn, Jennie, 1986, et al. (author)
  • Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes.
  • 2016
  • In: Protein science : a publication of the Protein Society. - : Wiley. - 1469-896X. ; 25:12, s. 2196-2208
  • Journal article (peer-reviewed)abstract
    • Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high-resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0-CaM complex on the AQP0 C-terminus is also demonstrated since the C-terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X-ray crystallography or single particle electron microscopy.
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10.
  • Tornroth-Horsefield, S, et al. (author)
  • Structural mechanism of plant aquaporin gating
  • 2006
  • In: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 439:7077, s. 688-694
  • Journal article (peer-reviewed)abstract
    • 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 angstrom resolution and in its open conformation at 3.9 angstrom 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 angstrom 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.
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11.
  • Törnroth-Horsefield, Susanna, 1973, et al. (author)
  • Aquaporin gating
  • 2007
  • In: Journal of Biomolecular Structure & Dynamics. ; 24:6, s. 719-721
  • Journal article (peer-reviewed)
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12.
  • Törnroth-Horsefield, Susanna, 1973, et al. (author)
  • Structural insights into eukaryotic aquaporin regulation.
  • 2010
  • In: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:12, s. 2580-8
  • Journal article (peer-reviewed)abstract
    • Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions.
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14.
  • Zeng, Jiao, et al. (author)
  • High-resolution structure of a fish aquaporin reveals a novel extracellular fold
  • 2022
  • In: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
  • Journal article (peer-reviewed)abstract
    • Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (Anabas testudineus), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.
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15.
  • Öberg, Fredrik, 1982, et al. (author)
  • Glycosylation increases the thermostability of human aquaporin 10 protein.
  • 2011
  • In: The Journal of biological chemistry. - 1083-351X. ; 286:36, s. 31915-23
  • Journal article (peer-reviewed)abstract
    • Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.
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16.
  • Öberg, Fredrik, 1982, et al. (author)
  • Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris.
  • 2009
  • In: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 26:4, s. 215-27
  • Journal article (peer-reviewed)abstract
    • Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.
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17.
  • Aricescu, A R, et al. (author)
  • Eukaryotic expression: developments for structural proteomics.
  • 2006
  • In: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 62, s. 1114-1124
  • Journal article (peer-reviewed)abstract
    • The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
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18.
  • Bill, Roslyn M., et al. (author)
  • Analysis of the pore of the unusual major intrinsic protein channel, yeast Fps1p.
  • 2001
  • In: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 276:39, s. 36543-9
  • Journal article (peer-reviewed)abstract
    • Fps1p is a glycerol efflux channel from Saccharomyces cerevisiae. In this atypical major intrinsic protein neither of the signature NPA motifs of the family, which are part of the pore, is preserved. To understand the functional consequences of this feature, we analyzed the pseudo-NPA motifs of Fps1p by site-directed mutagenesis and assayed the resultant mutant proteins in vivo. In addition, we took advantage of the fact that the closest bacterial homolog of Fps1p, Escherichia coli GlpF, can be functionally expressed in yeast, thus enabling the analysis in yeast cells of mutations that make this typical major intrinsic protein more similar to Fps1p. We observed that mutations made in Fps1p to "restore" the signature NPA motifs did not substantially affect channel function. In contrast, when GlpF was mutated to resemble Fps1p, all mutants had reduced activity compared with wild type. We rationalized these data by constructing models of one GlpF mutant and of the transmembrane core of Fps1p. Our model predicts that the pore of Fps1p is more flexible than that of GlpF. We discuss the fact that this may accommodate the divergent NPA motifs of Fps1p and that the different pore structures of Fps1p and GlpF may reflect the physiological roles of the two glycerol facilitators.
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19.
  • Bill, R. M., et al. (author)
  • Aquaporins - Expression, purification and characterization
  • 2021
  • In: Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier BV. - 0005-2736. ; 1863:9
  • Journal article (peer-reviewed)abstract
    • Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.
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21.
  • Caddeo, Andrea, et al. (author)
  • LPIAT1/MBOAT7 contains a catalytic dyad transferring polyunsaturated fatty acids to lysophosphatidylinositol.
  • 2021
  • In: Biochimica et biophysica acta. Molecular and cell biology of lipids. - : Elsevier BV. - 1879-2618 .- 1388-1981. ; 1866:5
  • Journal article (peer-reviewed)abstract
    • Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished. Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A+H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors. MBOAT7 preferentially transferred 20:4 and 20:5 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A+H356A) result in a loss of O-acyltransferase activity. Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased understanding of the enzymatic activity of MBOAT7 give insights into the understanding on the basis of FLD.
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22.
  • Carlesso, Antonio, 1990, et al. (author)
  • Yeast as a tool for membrane protein production and structure determination
  • 2022
  • In: Fems Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
  • Research review (peer-reviewed)abstract
    • Although the majority of eukaryotic MEMBRANE PROTEIN structures are DERIVED FROM PROTEINS produced in HEK293 and insect cells, the authors show here the importance of yeast as a production host and its role as an essential player in the production of eukaryotic membrane proteins for structural and functional analysis. Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of alpha-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of alpha-helical membrane proteins.
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23.
  • Fantoni, A, et al. (author)
  • Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris
  • 2007
  • In: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 52:1, s. 31-39
  • Journal article (peer-reviewed)abstract
    • We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1.
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24.
  • Ferndahl, Cecilia, 1975, et al. (author)
  • Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory
  • 2010
  • In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 9
  • Journal article (peer-reviewed)abstract
    • Background: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. Results: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. Conclusions: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.
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25.
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26.
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27.
  • Hedfalk, Kristina, 1969, et al. (author)
  • A Regulatory Domain in the C-terminal Extension of the Yeast Glycerol Channel Fps1p
  • 2004
  • In: Journal of biological chemistry. ; 279:15, s. 14954-14960
  • Journal article (peer-reviewed)abstract
    • The Saccharomyces cerevisiae gene FPS1 encodes an aquaglyceroporin of the major intrinsic protein (MIP) family. The main function of Fps1p seems to be the efflux of glycerol in the adaptation of the yeast cell to lower external osmolarity. Fps1p is an atypical member of the family, because the protein is much larger (669 amino acids) than most MIPs due to long hydrophilic extensions in both termini. We have shown previously that a short domain in the N-terminal extension of the protein is required for restricting glycerol transport through the channel (Tamás, M. J., Karlgren, S., Bill, R. M., Hedfalk, K., Allegri, L., Ferreira, M., Thevelein, J. M., Rydström, J., Mullins, J. G. L., and Hohmann, S. (2003) J. Biol. Chem. 278, 63376345). Deletion of the N-terminal domain results in an unregulated channel, loss of glycerol, and osmosensitivity. In this work we have investigated the role of the Fps1p C terminus (139 amino acids). A set of eight truncations has been constructed and tested in vivo in a yeast fps1 strain. We have performed growth tests, membrane localization following cell fractionation, and glycerol accumulation measurements as well as an investigation of the osmotic stress response. Our results show that the C-terminal extension is also involved in restricting transport through Fps1p. We have identified a sequence of 12 amino acids, residues 535546, close to the sixth transmembrane domain. This element seems to be important for controlling Fps1p function. Similar to the N-terminal domain, the C-terminal domain is amphiphilic and has a potential to dip into the membrane
  •  
28.
  • Hedfalk, Kristina, et al. (author)
  • Aquaporin gating
  • 2006
  • In: Current Opinion in Structural Biology. - : Elsevier BV. - 1879-033X .- 0959-440X. ; 16, s. 447-456
  • Research review (peer-reviewed)abstract
    • An acceleration in the rate at which new aquaporin structures are determined means that structural models are now available for mammalian AQP0, AQP1, AQP2 and AQP4, bacterial GlpF, AqpM and AQPZ, and the plant SoPIP2;1. With an apparent consensus emerging concerning the mechanism of selective water transport and proton extrusion, emphasis has shifted towards the issues of substrate selectivity and the mechanisms of aquaporin regulation. In particular, recently determined structures of plant SoPIP2;1, sheep and bovine AQP0, and Escherichia coli AQPZ provide new insights into the underlying structural mechanisms by which water transport rates are regulated in diverse organisms. From these results, two distinct pictures of 'capping' and 'pinching' have emerged to describe aquaporin gating.
  •  
29.
  • Hedfalk, Kristina, 1969 (author)
  • Codon optimisation for heterologous gene expression in yeast.
  • 2012
  • In: Recombinant Protein Production in Yeast / ed. by Roslyn M. Bill. - London : Springer. - 9781617797699 ; , s. 47-55
  • Book chapter (peer-reviewed)abstract
    • Heterologous protein production is used to amplify the yield of a desired protein target. To date, however, this is not a streamlined process: the factors defining an optimal protein production experiment are still poorly understood. This empirical exercise is particularly challenging for proteins of eukaryotic origin as well as those located in cellular membranes. The strong interest in structural and functional characterisation of eukaryotic membrane proteins—of which many are targets for different drugs—means that large amounts of pure protein, and hence high production levels in a suitable host, are required. On the genetic level, there are mainly two ways to positively influence the final yield of a desired protein target. First, the sequence surrounding the starting ATG can be altered and second the genetic code itself can be optimised to suit the selected host for production. The practical aspects of these two strategies will be discussed and exemplified in further detail in this chapter together with some hints and troubleshooting around different stages of the procedure.
  •  
30.
  • Hedfalk, Kristina, 1969 (author)
  • Further advances in the production of membrane proteins in Pichia pastoris.
  • 2013
  • In: Bioengineered. - : Informa UK Limited. - 2165-5987 .- 2165-5979. ; 4:6, s. 363-367
  • Journal article (peer-reviewed)abstract
    • Membrane proteins have essential cellular functions and are therefore of high interest in both academia and industry. Many efforts have been made on producing those targets in yields allowing crystallization experiments aiming for high resolution structures and mechanistic understanding. The first step of production provides a crucial barrier to overcome, but what we now see, is great progress in membrane protein structural determination in a relatively short time. Achievements on recombinant protein production have been essential for this development and the yeast Pichia pastoris is the most commonly used host for eukaryotic membrane proteins. High-resolution structures nicely illustrate the successes in protein production, and this is the measure used by Ramón and Marin in their review "Advances in the production of membrane proteins in Pichia pastoris" from 2011. Here, additional advances on production and crystallization of eukaryotic membrane proteins are described and reflected on.
  •  
31.
  • Hedfalk, Kristina, 1969, et al. (author)
  • Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
  • 2008
  • In: Protein expression and purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 59:1, s. 69-78
  • Journal article (peer-reviewed)abstract
    • 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.
  •  
32.
  • Hedfalk, Kristina, 1969, et al. (author)
  • Recombinant production of the human aquaporins in the yeast Pichia pastoris (Invited Review).
  • 2013
  • In: Molecular membrane biology. - : Informa UK Limited. - 0968-7688 .- 1464-5203. ; 30:1, s. 15-31
  • Journal article (peer-reviewed)abstract
    • Abstract Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism - including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris.
  •  
33.
  • Hedfalk, Kristina, 1969 (author)
  • Structure, Function and Regulation of Fps1p. A Eukaryotic Solute Efflux Channel
  • 2002
  • Doctoral thesis (other academic/artistic)abstract
    • Integral membrane proteins belonging to the Major Intrinsic Protein (MIP) family can be found in all kingdoms of life. MIPs are transporters of water, small neutral solutes and possibly ions, and hence are important for osmoregulation at different levels. The aim of this thesis has been to study the structure-function relationship of an atypical member of this family, Fps1p, which is a glycerol facilitator from the yeast Saccharomyces cerevisiae. Fps1p is located in the plasma membrane and mediates efflux of the compatible solute glycerol in the cell's adaptation to lower external osmolarity. Fps1p does not contain the two highly conserved NPA motifs in its channel forming loops, which might have consequences for the transport function and/or the specificity of this MIP. Furthermore, Fps1p is exceptionally large compared to other MIPs due to long hydrophilic extensions in both termini, and these domains have been shown to be crucial for the regulation of glycerol transport. Fps1p can be characterised in vivo in whole yeast cells, since its function is reflected in cell growth under different osmotic conditions. A short domain, twelve amino acids from the transmembrane domain in each terminus, has been found to be important for proper regulation. Moreover, the N-terminus seems to act as a restriction domain, since higher activity is observed when this domain is deleted. The mechanism is not clear, but Fps1p is probably gated by conformational changes upon shifts in external osmolarity. The Fps1p membrane core has been modelled based on the known structure of the Escherichia coli glycerol facilitator, GlpF, which is its close homologue. Much effort has been invested in production of pure Fps1p for functional and structural studies. Fps1p has been overexpressed in several systems using the hosts S. cerevisiae, Pichia pastoris and E. coli. In this thesis Fps1p has been compared with MIP proteins to be able to understand more about the structure, transport and the specificity as well as with other solute efflux channels in order to understand more about its regulation.
  •  
34.
  • Henricsson, Cecilia, 1975, et al. (author)
  • Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations
  • 2005
  • In: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 71:10, s. 6185-6192
  • Journal article (peer-reviewed)abstract
    • The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*, encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7 strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol- producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7 strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain.
  •  
35.
  • Isaksson, Simon, 1988, et al. (author)
  • Protein-Containing Lipid Bilayers Intercalated with Size-Matched Mesoporous Silica Thin Films
  • 2017
  • In: Nano Letters. - : American Chemical Society (ACS). - 1530-6992 .- 1530-6984. ; 17:1, s. 476-485
  • Journal article (peer-reviewed)abstract
    • Proteins are key components in a multitude of biological processes, of which the functions carried out by transmembrane (membrane-spanning) proteins are especially demanding for investigations. This is because this class of protein needs to be incorporated into a lipid bilayer representing its native environment, and in addition, many experimental conditions also require a solid support for stabilization and analytical purposes. The solid support substrate may, however, limit the protein functionality due to protein material interactions and a lack of physical space. We have in this work tailored the pore size and pore ordering of a mesoporous silica thin film to match the native cell-membrane arrangement of the transmembrane protein human aquaporin 4 (hAQP4). Using neutron reflectivity (NR), we provide evidence of how substrate pores host the bulky water-soluble domain of hAQP4, which is shown to extend 7.2 nm into the pores of the substrate. Complementary surface analytical tools, including quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence microscopy, revealed successful protein-containing supported lipid bilayer (pSLB) formation on mesoporous silica substrates, whereas pSLB formation was hampered on nonporous silica. Additionally, electron microscopy (TEM and SEM), light scattering (DLS and stopped-flow), and small-angle X-ray scattering (SAXS) were employed to provide a comprehensive characterization of this novel hybrid organic-inorganic interface, the tailoring of which is likely to be generally applicable to improve the function and stability of a broad range of membrane proteins containing water-soluble domains.
  •  
36.
  • Itel, F., et al. (author)
  • CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels
  • 2012
  • In: Faseb Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:12, s. 5182-5191
  • Journal article (peer-reviewed)abstract
    • Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO2, using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO2 permeability (PCO2) of similar to 0.01 cm/s, which is 2 orders of magnitude lower than the PCO2 of pure planar phospholipid bilayers (similar to 1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit PCO2 approximate to 0.01 cm/s, identifying cholesterol as the major determinant of membrane PCO2. This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in PCO2, respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in PCO2, indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.-Itel, F., Al-Samir, S., Oberg, F., Chami, M., Kumar, M., Supuran, C. T., Deen, P. M. T., Meier, W., Hedfalk, K., Gros, G., Endeward, V. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels. FASEB J. 26, 5182-5191 (2012). www.fasebj.org
  •  
37.
  • Jungersted, Jakob Mutanu, et al. (author)
  • In vivo studies of aquaporins 3 and 10 in human stratum corneum.
  • 2013
  • In: Archives of dermatological research. - : Springer Science and Business Media LLC. - 1432-069X .- 0340-3696. ; 305:8, s. 699-704
  • Journal article (peer-reviewed)abstract
    • Aquaporins (AQPs) constitute one family of transmembrane proteins facilitating transport of water across cell membranes. Due to their specificity, AQPs have a broad spectrum of physiological functions, and for keratinocytes there are indications that these channel proteins are involved in cell migration and proliferation with consequences for the antimicrobial defense of the skin. AQP3 and AQP10 are aqua-glyceroporins, known to transport glycerol as well as water. AQP3 is the predominant AQP in human skin and has previously been demonstrated in the basal layer of epidermis in normal human skin, but not in stratum corneum (SC). AQP10 has not previously been identified in human skin. Previous studies have demonstrated the presence of AQP3 and AQP10 mRNA in keratinocytes. In this study, our aim was to investigate if these aquaporin proteins were actually present in human SC cells. This can be seen as a first step toward elucidating the possible functional role of AQP3 and AQP10 in SC hydration. Specifically we investigate the presence of AQP3 and AQP10 in vivo in human SC using "minimal-invasive" technique for obtaining SC samples. SC samples were obtained from six healthy volunteers. Western blotting and immunohistochemistry were used to demonstrate the presence of AQP3 as well as AQP10. The presence of AQP3 and AQP10 was verified by Western blotting, allowing for detection of proteins by specific antibodies. Applying immunohistochemistry, cell-like structures in the shape of corneocytes were identified in all samples by AQP3 and AQP10 antibodies. In conclusion, identification of AQP3 and AQP10 protein in SC in an in vivo model is new. Together with the new "minimal-invasive" method for SC collection presented, this opens for new possibilities to study the role of AQPs in relation to function of the skin barrier.
  •  
38.
  • Karlsson, Maria, 1985, et al. (author)
  • Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris.
  • 2003
  • In: FEBS Letters. - 1873-3468 .- 0014-5793. ; 537:1-3, s. 68-72
  • Journal article (peer-reviewed)abstract
    • The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.
  •  
39.
  • Kitchen, Philip, et al. (author)
  • Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.
  • 2015
  • In: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 10:11
  • Journal article (peer-reviewed)abstract
    • Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.
  •  
40.
  • Li, J., et al. (author)
  • Molecular Level Characterization of Circulating Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder
  • 2021
  • In: Neurology(R) neuroimmunology & neuroinflammation. - 2332-7812. ; 8:5
  • Journal article (peer-reviewed)abstract
    • OBJECTIVE: To determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD. METHODS: We combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro. RESULTS: Multiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01). CONCLUSIONS: The composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
  •  
41.
  • Pingitore, Piero, 1986, et al. (author)
  • Large scale production of the active human ASCT2 (SLC1A5) transporter in Pichia pastoris - functional and kinetic asymmetry revealed in proteoliposomes
  • 2013
  • In: Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier BV. - 0005-2736. ; 1828:9, s. 2238-2246
  • Journal article (peer-reviewed)abstract
    • The human glutamine/neutral amino acid transporter ASCT2 (hASCT2) was over-expressed in Pichia pastoris and purified by Ni2+-chelating and gel filtration chromatography. The purified protein was reconstituted in liposomes by detergent removal with a batch-wise procedure. Time dependent [H-3]glutamine/glutamine antiport was measured in proteoliposomes which was active only in the presence of external Na+. Internal Na+ slightly stimulated the antiport. Optimal activity was found at pH 7.0. A substantial inhibition of the transport was observed by Cys, Thr, Ser, Ala, Asn and Met (>= 70%) and by mercurials and methanethiosulfonates (>= 80%). Heterologous antiport of [H-3]glutamine with other neutral amino acids was also studied. The transporter showed asymmetric specificity for amino acids: Ala, Cys, Val, Met were only inwardly transported, while Gln, Ser, Asn, and Thr were transported bi-directionally. From kinetic analysis of [H-3]glutamine/glutamine antiport Km values of 0.097 and 1.8 mM were measured on the external and internal sides of proteoliposomes, respectively. The Km for Na+ on the external side was 32 mM. The homology structural model of the hASCT2 protein was built using the GltPh of Pyrococcus horikoshii as template. Cys395 was the only Cys residue externally exposed, thus being the potential target of SH reagents inhibition and, hence, potentially involved in the transport mechanism. (C) 2013 The Authors. Published by Elsevier B.V. All rights reserved.
  •  
42.
  • Pingitore, Piero, 1986, et al. (author)
  • Recombinant PNPLA3 protein shows triglyceride hydrolase activity and its I148M mutation results in loss of function.
  • 2014
  • In: Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids. - : Elsevier BV. - 1388-1981. ; 1841:4, s. 574-580
  • Journal article (peer-reviewed)abstract
    • The patatin-like phospholipase domain containing 3 (PNPLA3, also called adiponutrin, ADPN) is a membrane-bound protein highly expressed in the liver. The genetic variant I148M (rs738409) was found to be associated with progression of chronic liver disease. We aimed to establish a protein purification protocol in a yeast system (Pichia pastoris) and to examine the human PNPLA3 enzymatic activity, substrate specificity and the I148M mutation effect. hPNPLA3 148I wild type and 148M mutant cDNA were cloned into P. pastoris expression vectors. Yeast cells were grown in 3L fermentors. PNPLA3 protein was purified from membrane fractions by Ni-affinity chromatography. Enzymatic activity was assessed using radiolabeled substrates. Both 148I wild type and 148M mutant proteins are localized to the membrane. The wild type protein shows a predominant lipase activity with mild lysophosphatidic acid acyl transferase activity (LPAAT) and the I148M mutation results in a loss of function of both these activities. Our data show that PNPLA3 has a predominant lipase activity and I148M mutation results in a loss of function.
  •  
43.
  • Pirazzi, Carlo, et al. (author)
  • PNPLA3 has retinyl-palmitate lipase activity in human hepatic stellate cells
  • 2014
  • In: Human Molecular Genetics. - : Oxford University Press (OUP). - 0964-6906 .- 1460-2083. ; 23:15, s. 4077-4085
  • Journal article (peer-reviewed)abstract
    • Retinoids are micronutrients that are stored as retinyl esters in the retina and hepatic stellate cells (HSCs). HSCs are key players in fibrogenesis in chronic liver diseases. The enzyme responsible for hydrolysis and release of retinyl esters from HSCs is unknown and the relationship between retinoid metabolism and liver disease remains unclear. We hypothesize that the patatin-like phospholipase domain-containing 3 (PNPLA3) protein is involved in retinol metabolism in HSCs. We tested our hypothesis both in primary human HSCs and in a human cohort of subjects with non-alcoholic fatty liver disease (N = 146). Here we show that PNPLA3 is highly expressed in human HSCs. Its expression is regulated by retinol availability and insulin, and increased PNPLA3 expression results in reduced lipid droplet content. PNPLA3 promotes extracellular release of retinol from HSCs in response to insulin. We also show that purified wild-type PNPLA3 hydrolyzes retinyl palmitate into retinol and palmitic acid. Conversely, this enzymatic activity is markedly reduced with purified PNPLA3 148M, a common mutation robustly associated with liver fibrosis and hepatocellular carcinoma development. We also find the PNPLA3 I148M genotype to be an independent (P = 0.009 in a multivariate analysis) determinant of circulating retinol-binding protein 4, a reliable proxy for retinol levels in humans. This study identifies PNPLA3 as a lipase responsible for retinyl-palmitate hydrolysis in HSCs in humans. Importantly, this indicates a potential novel link between HSCs, retinoid metabolism and PNPLA3 in determining the susceptibility to chronic liver disease.
  •  
44.
  • Scalise, M., et al. (author)
  • Cys Site-Directed Mutagenesis of the Human SLC1A5 (ASCT2) Transporter: Structure/Function Relationships and Crucial Role of Cys467 for Redox Sensing and Glutamine Transport
  • 2018
  • In: International Journal of Molecular Sciences. - : MDPI AG. - 1422-0067. ; 19:3
  • Journal article (peer-reviewed)abstract
    • The human plasma membrane transporter ASCT2 is responsible for mediating Na-dependent antiport of neutral amino acids. New insights into structure/function relationships were unveiled by a combined approach of recombinant over-expression, site-directed mutagenesis, transport assays in proteoliposomes and bioinformatics. WT and Cys mutants of hASCT2 were produced in P. pastoris and purified for functional assay. The reactivity towards SH reducing and oxidizing agents of WT protein was investigated and opposite effects were revealed; transport activity increased upon treatment with the Cys reducing agent DTE, i.e., when Cys residues were in thiol (reduced) state. Methyl-Hg, which binds to SH groups, was able to inhibit WT and seven out of eight Cys to Ala mutants. On the contrary, C467A loses the sensitivity to both DTE activation and Methyl-Hg inhibition. The C467A mutant showed a Km for Gln one order of magnitude higher than that of WT. Moreover, the C467 residue is localized in the substrate binding region of the protein, as suggested by bioinformatics on the basis of the EAAT1 structure comparison. Taken together, the experimental data allowed identifying C467 residue as crucial for substrate binding and for transport activity modulation of hASCT2.
  •  
45.
  • Scalise, Mariafrancesca, et al. (author)
  • Cysteine is not a substrate but a specific modulator of human ASCT2 (SLC1A5) transporter.
  • 2015
  • In: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 589:23, s. 3617-23
  • Journal article (peer-reviewed)abstract
    • The Alanine Serine Cysteine Transporter 2 (ASCT2) is involved in balancing the intracellular amino acid pool. This function is allowed by the antiport mechanism and the asymmetric specificity towards different neutral amino acids, distinctive of this transporter. In the present work, the interaction of the putative substrate Cys with the human ASCT2 has been studied using the recombinant hASCT2 over-produced in Pichia pastoris and the native ASCT2 extracted from HeLa in both proteoliposomes and intact cells. It was found that Cys is a potent competitive inhibitor of hASCT2 but is not a substrate. Moreover, Cys binding to a second site, different from that of substrate, triggers a protein-mediated unidirectional Gln efflux.
  •  
46.
  • Scalise, Mariafrancesca, et al. (author)
  • Transport mechanism and regulatory properties of the human amino acid transporter ASCT2 (SLC1A5).
  • 2014
  • In: Amino acids. - : Springer Science and Business Media LLC. - 1438-2199 .- 0939-4451. ; 46:11, s. 2463-75
  • Journal article (peer-reviewed)abstract
    • The kinetic mechanism of the transport catalyzed by the human glutamine/neutral amino acid transporter hASCT2 over-expressed in P. pastoris was determined in proteoliposomes by pseudo-bi-substrate kinetic analysis of the Na(+)-glutamineex/glutaminein transport reaction. A random simultaneous mechanism resulted from the experimental analysis. Purified functional hASCT2 was chemically cross-linked to a stable dimeric form. The oligomeric structure correlated well with the kinetic mechanism of transport. Half-saturation constants (Km) of the transporter for the other substrates Ala, Ser, Asn and Thr were measured both on the external and internal side. External Km were much lower than the internal ones confirming the asymmetry of the transporter. The electric nature of the transport reaction was determined imposing a negative inside membrane potential generated by K(+) gradients in the presence of valinomycin. The transport reaction resulted to be electrogenic and the electrogenicity originated from external Na(+). Internal Na(+) exerted a stimulatory effect on the transport activity which could be explained by a regulatory, not a counter-transport, effect. Native and deglycosylated hASCT2 extracted from HeLa showed the same transport features demonstrating that the glycosyl moiety has no role in transport function. Both in vitro and in vivo interactions of hASCT2 with the scaffold protein PDZK1 were revealed.
  •  
47.
  • Sjöhamn, Jennie, 1986, et al. (author)
  • Unraveling aquaporin interaction partners.
  • 2014
  • In: Biochimica et Biophysica Acta (BBA) General Subjects. - : Elsevier BV. - 0304-4165. ; 1840:5, s. 1614-1623
  • Journal article (peer-reviewed)abstract
    • Insight into protein-protein interactions (PPIs) is highly desirable in order to understand the physiology of cellular events. This understanding is one of the challenges in biochemistry and molecular biology today, especially for eukaryotic membrane proteins where hurdles of production, purification and structural determination must be passed.
  •  
48.
  • Tamás, Markus J., 1970, et al. (author)
  • A Short Regulatory Domain Restricts Glycerol Transport through Yeast Fps1p
  • 2003
  • In: J. Biol. Chem. ; 278, s. 6337-6345
  • Journal article (peer-reviewed)abstract
    • The controlled export of solutes is crucial for cellular adaptation to hypotonic conditions. In the yeast Saccharomyces cerevisiae glycerol export is mediated by Fps1p, a member of the major intrinsic protein (MIP) family of channel proteins. Here we describe a short regulatory domain that restricts glycerol transport through Fps1p. This domain is required for retention of cellular glycerol under hypertonic stress and hence acquisition of osmotolerance. It is located in the N-terminal cytoplasmic extension close to the first transmembrane domain. Several residues within that domain and its precise position are critical for channel control while the proximal residues 13-215 of the N-terminal extension are not required. The sequence of the regulatory domain and its position are perfectly conserved in orthologs from other yeast species. The regulatory domain has an amphiphilic character, and structural predictions indicate that it could fold back into the membrane bilayer. Remarkably, this domain has structural similarity to the channel forming loops B and E of Fps1p and other glycerol facilitators. Intragenic second-site suppressor mutations of the sensitivity to high osmolarity conferred by truncation of the regulatory domain caused diminished glycerol transport, confirming that elevated channel activity is the cause of the osmosensitive phenotype.
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49.
  • Tsiavaliaris, G., et al. (author)
  • Low CO2 permeability of cholesterol-containing liposomes detected by stopped-flow fluorescence spectroscopy
  • 2015
  • In: Faseb Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 29:5, s. 1780-1793
  • Journal article (peer-reviewed)abstract
    • Here we ask the following: 1) what is the CO2 permeability (Pco(2)) of unilamellar liposomes composed of l--phosphatidylcholine (PC)/l--phosphatidylserine (PS) = 4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco(2)? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric O-18-exchange measurements (Chol reduces Pco(2) 100-fold, AQP1 increases Pco(2) 10-fold) vs. from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO(2)) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a Pco(2) = 0.01cm/s, and Chol-free vesicles exhibit approximate to 1 cm/s, and 2) the Pco(2) of 0.01 cm/s is increased 10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco(2) that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1.Tsiavaliaris, G., Itel, F., Hedfalk, K., Al-Samir, S., Meier, W., Gros, G., Endeward, V. Low CO2 permeability of cholesterol-containing liposomes detected by stopped-flow fluorescence spectroscopy.
  •  
50.
  • Wang, Hao, et al. (author)
  • Characterization of aquaporin-driven hydrogen peroxide transport.
  • 2020
  • In: Biochimica et biophysica acta. Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1862:2
  • Journal article (peer-reviewed)abstract
    • Aquaporins are membrane-intrinsic proteins initially defined as water (H2O) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (H2O2). H2O2 is a signaling molecule that partakes in immune responses where its transport is mediated by aquaporins. To shed further light on the molecular basis of the aquaporin function in H2O2 transport, we have characterized an Arabidopsis thaliana aquaporin, AtPIP2;4, recombinantly produced to high yields in Pichia pastoris. Here, we present a newly established assay that allows detection of H2O2 transport by purified aquaporins reconstituted into liposomes, enabling us to compare aquaporin homologues with respect to substrate specificity. To get additional insight into the structural determinants for aquaporin-mediated H2O2 transport, we solved the 3D-structure of AtPIP2;4 to 3.7Å resolution and found structural identity to the water channel from spinach (SoPIP2;1), with the difference that Cd2+ cation is not required to retain the closed conformation. The transport specificities of the two plant aquaporins were compared to a human homologue, AQP1. Overall, we conclude that AtPIP2;4, SoPIP2;1 and hAQP1 are all transporters of both H2O and H2O2, but have different efficiencies for various specificities. Notably, all three homologues expedite H2O transport equally well while the plant aquaporins are more permeable to H2O2 than hAQP1. Comparison of the structures indicates that the observed variations in H2O and H2O2 transport cannot be explained by differences in the monomeric pore. Possibly, the determinants for transport specificities reside in the flexible domains outside the membrane core of these channels.
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