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1.	Törnroth-Horsefield, Susanna, 1973, et al. (författare) Aquaporin gating 2007 Ingår i: Journal of Biomolecular Structure & Dynamics. ; 24:6, s. 719-721 Tidskriftsartikel (refereegranskat)
2.	Törnroth-Horsefield, Susanna, 1973, et al. (författare) Crystal Structure of AcrB in Complex with a Single Transmembrane Subunit Reveals Another Twist. 2007 Ingår i: Structure (London, England : 1993). - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 15:12, s. 1663-73 Tidskriftsartikel (refereegranskat)abstract Bacterial drug resistance is a serious concern for human health. Multidrug efflux pumps export a broad variety of substrates out of the cell and thereby convey resistance to the host. In Escherichia coli, the AcrB:AcrA:TolC efflux complex forms a principal transporter for which structures of the individual component proteins have been determined in isolation. Here, we present the X-ray structure of AcrB in complex with a single transmembrane protein, assigned by mass spectrometry as YajC. A specific rotation of the periplasmic porter domain of AcrB is also revealed, consistent with the hypothesized "twist-to-open" mechanism for TolC activation. Growth experiments with yajc-deleted E. coli reveal a modest increase in the organism's susceptibility to beta-lactam antibiotics, but this effect could not conclusively be attributed to the loss of interactions between YajC and AcrB.
3.	Adawy, Alaa, et al. (författare) High Resolution Protein Crystals Using an Efficient Convection-Free Geometry 2013 Ingår i: Crystal Growth & Design. - : American Chemical Society (ACS). - 1528-7483 .- 1528-7505. ; 13:2, s. 775-781 Tidskriftsartikel (refereegranskat)abstract Macromolecular crystallography is the most direct and accurate approach to determine the three-dimensional structure of biological macromolecules. The growth of high quality single crystals, yielding diffraction to the highest X-ray resolution, remains a bottleneck in this methodology. Here we show that through a modification of the batch crystallization method, an entirely convection-free crystallization environment is achieved, which enhances the purity and crystallinity of protein crystals. This is accomplished by using an upside-down geometry, where crystals grow at the “ceiling” of a growth-cell completely filled with the crystallization solution. The “ceiling crystals” experience the same diffusion-limited conditions as in space microgravity experiments. The new method was tested on bovine insulin and two hen egg-white lysozyme polymorphs. In all cases, ceiling crystals diffracted X-rays to resolution limits beyond that for other methods using similar crystallization conditions without further optimization. In addition, we demonstrate that the ceiling crystallization method leads to crystals with much lower impurity incorporation.
4.	Al-Jubair, Tamim, et al. (författare) Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST) 2022 Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2 Tidskriftsartikel (refereegranskat)abstract Aquaporin water channels (AQPs) are membrane proteins that maintain cellular water homeostasis. The interactions between human AQPs and other proteins play crucial roles in AQP regulation by both gating and trafficking. Here, we describe a protocol for characterizing the interaction between a human AQP and a soluble interaction partner using microscale thermophoresis (MST). MST has the advantage of low sample consumption and high detergent compatibility enabling AQP protein-protein interaction investigation with a high level of control of components and environment. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Roche et al. (2017).
5.	Al-Jubair, Tamim, et al. (författare) High-yield overproduction and purification of human aquaporins from Pichia pastoris 2022 Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2 Tidskriftsartikel (refereegranskat)abstract Aquaporins (AQPs) are membrane-bound water channels that play crucial roles in maintaining the water homeostasis of the human body. Here, we present a protocol for high-yield recombinant expression of human AQPs in the methylotropic yeast Pichia pastoris and subsequent AQP purification. The protocol typically yields 1–5 mg AQP per g of yeast cell at >95% purity and is compatible with any membrane protein cloned into Pichia pastoris, although expression levels may vary. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Frick et al. (2014).
6.	Chivasso, Clara, et al. (författare) Ezrin is a novel protein partner of aquaporin-5 in human salivary glands and shows altered expression and cellular localization in sjögren’s syndrome 2021 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 22:17 Tidskriftsartikel (refereegranskat)abstract Sjögren’s syndrome (SS) is an exocrinopathy characterized by the hypofunction of salivary glands (SGs). Aquaporin-5 (AQP5); a water channel involved in saliva formation; is aberrantly dis-tributed in SS SG acini and contributes to glandular dysfunction. We aimed to investigate the role of ezrin in AQP5 mislocalization in SS SGs. The AQP5–ezrin interaction was assessed by immuno-precipitation and proteome analysis and by proximity ligation assay in immortalized human SG cells. We demonstrated, for the first time, an interaction between ezrin and AQP5. A model of the complex was derived by computer modeling and in silico docking; suggesting that AQP5 interacts with the ezrin FERM-domain via its C-terminus. The interaction was also investigated in human minor salivary gland (hMSG) acini from SS patients (SICCA-SS); showing that AQP5–ezrin complexes were absent or mislocalized to the basolateral side of SG acini rather than the apical region compared to controls (SICCA-NS). Furthermore, in SICCA-SS hMSG acinar cells, ezrin immunore-activity was decreased at the acinar apical region and higher at basal or lateral regions, accounting for altered AQP5–ezrin co-localization. Our data reveal that AQP5–ezrin interactions in human SGs could be involved in the regulation of AQP5 trafficking and may contribute to AQP5-altered localization in SS patients.
7.	Chivasso, Clara, et al. (författare) Unraveling human aqp5-pip molecular interaction and effect on aqp5 salivary glands localization in ss patients 2021 Ingår i: Cells. - : MDPI AG. - 2073-4409. ; 10:8 Tidskriftsartikel (refereegranskat)abstract Saliva secretion requires effective translocation of aquaporin 5 (AQP5) water channel to the salivary glands (SGs) acinar apical membrane. Patients with Sjögren’s syndrome (SS) display abnormal AQP5 localization within acinar cells from SGs that correlate with sicca manifestation and glands hypofunction. Several proteins such as Prolactin-inducible protein (PIP) may regulate AQP5 trafficking as observed in lacrimal glands from mice. However, the role of the AQP5-PIP complex remains poorly understood. In the present study, we show that PIP interacts with AQP5 in vitro and in mice as well as in human SGs and that PIP misexpression correlates with an altered AQP5 distribution at the acinar apical membrane in PIP knockout mice and SS hMSG. Furthermore, our data show that the protein-protein interaction involves the AQP5 C-terminus and the N-terminal of PIP (one molecule of PIP per AQP5 tetramer). In conclusion, our findings highlight for the first time the role of PIP as a protein controlling AQP5 localization in human salivary glands but extend beyond due to the PIP-AQP5 interaction described in lung and breast cancers.
8.	Frick, Anna, 1982, et al. (författare) Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism 2013 Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 454:pt 3, s. 491-499 Tidskriftsartikel (refereegranskat)abstract Water transport across cellular membranes is mediated by a family of membrane proteins known as AQPs (aquaporins). AQPs were first discovered on the basis of their ability to be inhibited by mercurial compounds, an experiment which has followed the AQP field ever since. Although mercury inhibition is most common, many AQPs are mercury insensitive. In plants, regulation of AQPs is important in order to cope with environmental changes. Plant plasma membrane AQPs are known to be gated by phosphorylation, pH and Ca2+. We have previously solved the structure of the spinach AQP SoPIP2;1 (Spinacia oleracea plasma membrane intrinsic protein 2; 1) in closed and open conformations and proposed a mechanism for how this gating can be achieved. To study the effect of mercury on SoPIP2; 1 we solved the structure of the SoPIP2;1-mercury complex and characterized the water transport ability using proteoliposomes. The structure revealed mercury binding to three out of four cysteine residues. In contrast to what is normally seen for AQPs, mercury increased the water transport rate of SoPIP2; 1, an effect which could not be attributed to any of the cysteine residues. This indicates that other factors might influence the effect of mercury on SoPIP2; 1, one of which could be the properties of the lipid bilayer.
9.	Frick, Anna, 1982, et al. (författare) Structural basis for pH gating of plant aquaporins 2013 Ingår i: Febs Letters. - : Wiley. - 0014-5793. ; 587:7, s. 989-993 Tidskriftsartikel (refereegranskat)abstract Plants have evolved to cope with fluctuations in water supply by gating their water channels known
10.	Frick, Anna, 1982, et al. (författare) X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking. 2014 Ingår i: 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 Tidskriftsartikel (refereegranskat)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.
11.	Hagströmer, Carl Johan, et al. (författare) Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus 2023 Ingår i: Scientific Reports. - 2045-2322. ; 13:1 Tidskriftsartikel (refereegranskat)abstract Aquaporins are water channels found in the cell membrane, where they allow the passage of water molecules in and out of the cells. In the kidney collecting duct, arginine vasopressin-dependent trafficking of aquaporin-2 (AQP2) fine-tunes reabsorption of water from pre-urine, allowing precise regulation of the final urine volume. Point mutations in the gene for AQP2 may disturb this process and lead to nephrogenic diabetes insipidus (NDI), whereby patients void large volumes of highly hypo-osmotic urine. In recessive NDI, mutants of AQP2 are retained in the endoplasmic reticulum due to misfolding. Here we describe the structural and functional characterization of three AQP2 mutations associated with recessive NDI: T125M and T126M, situated close to a glycosylation site and A147T in the transmembrane region. Using a proteoliposome assay, we show that all three mutants permit the transport of water. The crystal structures of T125M and T126M together with biophysical characterization of all three mutants support that they retain the native structure, but that there is a significant destabilization of A147T. Our work provides unique molecular insights into the mechanisms behind recessive NDI as well as deepens our understanding of how misfolded proteins are recognized by the ER quality control system.
12.	Johansson, Tomas, et al. (författare) X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli. 2005 Ingår i: Journal of molecular biology. - : Elsevier BV. - 0022-2836 .- 1089-8638. ; 352:2, s. 299-312 Tidskriftsartikel (refereegranskat)abstract The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II.
13.	Kitchen, Philip, et al. (författare) Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells 2020 Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:3 Tidskriftsartikel (refereegranskat)abstract Aquaporins (AQPs) are membrane channel proteins that facilitate the movement of water down osmotic gradients across biological membranes. This protocol allows measurements of AQP-mediated water transport across the plasma membrane of live mammalian cells. Calcein is a fluorescent dye that is quenched in a concentration-dependent manner. Therefore, on short timescales, its concentration-dependent fluorescence can be used as a probe of cell volume, and therefore a probe of water transport into or out of cells. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020) and Kitchen and Conner (2015). For the underlying methodology development, please refer to Fenton et al. (2010) and Solenov et al. (2004).
14.	Kitchen, Philip, et al. (författare) Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema 2020 Ingår i: Cell. - : Elsevier BV. - 0092-8674. ; 181:4, s. 19-799 Tidskriftsartikel (refereegranskat)abstract Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.
15.	Kitchen, Philip, et al. (författare) Water channel pore size determines exclusion properties but not solute selectivity 2019 Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1 Tidskriftsartikel (refereegranskat)abstract Aquaporins (AQPs) are a ubiquitous family of transmembrane water channel proteins. A subgroup of AQP water channels also facilitates transmembrane diffusion of small, polar solutes. A constriction within the pore, the aromatic/arginine (ar/R) selectivity filter, is thought to control solute permeability: previous studies on single representative water channel proteins suggest narrow channels conduct water, whilst wider channels permit passage of solutes. To assess this model of selectivity, we used mutagenesis, permeability measurements and in silico comparisons of water-specific as well as glycerol-permeable human AQPs. Our studies show that single amino acid substitutions in the selectivity filters of AQP1, AQP4 and AQP3 differentially affect glycerol and urea permeability in an AQP-specific manner. Comparison between in silico-calculated channel cross-sectional areas and in vitro permeability measurements suggests that selectivity filter cross-sectional area predicts urea but not glycerol permeability. Our data show that substrate discrimination in water channels depends on a complex interplay between the solute, pore size, and polarity, and that using single water channel proteins as representative models has led to an underestimation of this complexity.
16.	Kreida, Stefan, et al. (författare) The role of phosphorylation in calmodulin-mediated gating of human AQP0 2024 Ingår i: The Biochemical journal. - 0264-6021. ; 481:1, s. 17-32 Tidskriftsartikel (refereegranskat)abstract Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.
17.	Krmar, Rafael T., et al. (författare) Effect of controlled hypotensive hemorrhage on plasma sodium levels in anesthetized pigs : An exploratory study 2023 Ingår i: Physiological Reports. - : John Wiley & Sons. - 2051-817X. ; 11:22 Tidskriftsartikel (refereegranskat)abstract Perioperative hyponatremia, due to non-osmotic release of the antidiuretic hormone arginine vasopressin, is a serious electrolyte disorder observed in connection with many types of surgery. Since blood loss during surgery contributes to the pathogenesis of hyponatremia, we explored the effect of bleeding on plasma sodium using a controlled hypotensive hemorrhage pig model. After 30-min baseline period, hemorrhage was induced by aspiration of blood during 30 min at mean arterial pressure <50 mmHg. Thereafter, the animals were resuscitated with retransfused blood and a near-isotonic balanced crystalloid solution and monitored for 180 min. Electrolyte and water balances, cardiovascular response, renal hemodynamics, and markers of volume regulation and osmoregulation were investigated. All pigs (n = 10) developed hyponatremia. All animals retained hypotonic fluid, and none could excrete net-free water. Urinary excretion of aquaporin 2, a surrogate marker of collecting duct responsiveness to antidiuretic hormone, was significantly reduced at the end of the study, whereas lysine vasopressin, i.e., the pig antidiuretic hormone remained high. In this animal model, hyponatremia developed due to net positive fluid balance and generation of electrolyte-free water by the kidneys. A decreased urinary aquaporin 2 excretion may indicate an escape from antidiuresis.
18.	Lieske, Julia, et al. (författare) On-chip crystallization for serial crystallography experiments and on-chip ligand-binding studies 2019 Ingår i: IUCrJ. - 2052-2525. ; 6:4, s. 714-728 Tidskriftsartikel (refereegranskat)abstract Efficient and reliable sample delivery has remained one of the bottlenecks for serial crystallography experiments. Compared with other methods, fixed-target sample delivery offers the advantage of significantly reduced sample consumption and shorter data collection times owing to higher hit rates. Here, a new method of on-chip crystallization is reported which allows the efficient and reproducible growth of large numbers of protein crystals directly on micro-patterned silicon chips for in-situ serial crystallography experiments. Crystals are grown by sitting-drop vapor diffusion and previously established crystallization conditions can be directly applied. By reducing the number of crystal-handling steps, the method is particularly well suited for sensitive crystal systems. Excessive mother liquor can be efficiently removed from the crystals by blotting, and no sealing of the fixed-target sample holders is required to prevent the crystals from dehydrating. As a consequence, 'naked' crystals are obtained on the chip, resulting in very low background scattering levels and making the crystals highly accessible for external manipulation such as the application of ligand solutions. Serial diffraction experiments carried out at cryogenic temperatures at a synchrotron and at room temperature at an X-ray free-electron laser yielded high-quality X-ray structures of the human membrane protein aquaporin 2 and two new ligand-bound structures of thermolysin and the human kinase DRAK2. The results highlight the applicability of the method for future high-throughput on-chip screening of pharmaceutical compounds.
19.	Markou, Andrea, et al. (författare) Molecular mechanisms governing aquaporin relocalisation 2022 Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1864:4 Tidskriftsartikel (refereegranskat)abstract The aquaporins (AQPs) form a family of integral membrane proteins that facilitate the movement of water across biological membrane by osmosis, as well as facilitating the diffusion of small polar solutes. AQPs have been recognised as drug targets for a variety of disorders associated with disrupted water or solute transport, including brain oedema following stroke or trauma, epilepsy, cancer cell migration and tumour angiogenesis, metabolic disorders, and inflammation. Despite this, drug discovery for AQPs has made little progress due to a lack of reproducible high-throughput assays and difficulties with the druggability of AQP proteins. However, recent studies have suggested that targetting the trafficking of AQP proteins to the plasma membrane is a viable alternative drug target to direct inhibition of the water-conducting pore. Here we review the literature on the trafficking of mammalian AQPs with a view to highlighting potential new drug targets for a variety of conditions associated with disrupted water and solute homeostasis.
20.	Nilsson, Daniel (författare) Insane in the membrane: : Insertion of marginally hyrdophobic transmembrane helices and global analysis of membrane protein topology 2014 Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract Proteins are responsible for carrying out most of the tasks in a living cell; transcription, translation, replication, movement, catalysis and communication to mention a few. A subset of proteins is the integral membrane proteins, IMPs, which play an important role in governing communication across membranes, whether it is the plasma membrane or an organelle membrane. As IMPs are often responsible for uptake of substances such as hormones and pharmaceuticals they are also very often prime targets in medical research. In order for an IMP to function correctly it must fold and insert into the membrane properly as well as display the correct motifs to its surroundings, on either side of the membrane. IMPs cannot spontaneously insert into the membrane, the insertion is assisted by the Sec-translocon machinery. The Sec-translocon creates a pore in the membrane enabling nascent polypeptides to translocate across the membrane. This thesis will cover both the insertion of IMP segments as well as an evaluation of different approaches on how to investigate the topology of integral membrane proteins.The first question to be addressed is whether there are any specific sequence motifs within the sequence context that can improve the co-translational insertion of a marginally hydrophobic transmembrane helix, mTMH, into the membrane. A mTMH is a protein segment that would not insert by itself into the membrane. It has however been shown that these mTMHs can insert effectively into the membrane using their neighboring helices and loops, referred to as its sequence context, to compensate for the unfavorable insertion of only the mTMH. We show for a number of mTMHs that disrupting the sequence context motifs, usually lowering the ΔG-values for insertion by introducing more hydrophobic residues through substitutions in the sequence context, does not by itself improve the insertion of a given mTMH. It can however be concluded that the positive inside rule is of great importance to improve recognition and co-translational insertion of these mTMHs as it provides an oriental preference of the subsequent helix. This oriental preference will enable the mTMH to insert. This means that the positive inside rule it stronger when followed by a transmembrane helix, at least for the insertion of mTMHs.The second question addressed is that of how to design a method to analyze the topology of membrane proteins in a high-throughput proteomic fashion. In order to extract information on membrane protein topology a protease can be used to degrade the exposed parts of the integral membrane protein, known as shaving. These peptides can then subsequently be degraded and analyzed using MS and bioinformatics. To compare different proteases, we first apply our shaving experiment on two over-expressed proteins and analyze the detected peptides using MS. Secondly; we run the same experiment on non-over-expressed Escherichia coli membrane proteins with known structure. Finally, the results from the above experiments were used to test the accuracy of a number of topology predictors. We can conclude that the use of the protease Thermolysin does show promising results when compared to for instance trypsin. Even though the two proteases show somewhat similar output on the proteins used in this study, Thermolysin does produce fewer peptides originating from the transmembrane region. This is most likely due to the milder, more native like reaction conditions combined with the shorter incubation time used for Thermolysin as compared to trypsin. These properties are believed to greatly improve the output and accuracy when applied on large scale global analysis.
21.	Nyblom, Anna Maria, 1975, et al. (författare) Structural and functional analysis of SoPIP2;1 mutants adds insight into plant aquaporin gating. 2009 Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:3, s. 653-68 Tidskriftsartikel (refereegranskat)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.
22.	Nyblom, Maria, et al. (författare) Regulation of eukaryotic aquaporins 2016 Ingår i: Aquaporins in Health and Disease : New Molecular Targets for Drug Discovery - New Molecular Targets for Drug Discovery. - 9781498707831 - 9781498707848 ; , s. 53-76 Bokkapitel (refereegranskat)abstract Membrane-bound water channels known as aquaporins (AQPs) facilitate water transport across biological membranes along osmotic gradients. Since all living cells depend on their ability to maintain water homeostasis, this must be tightly regulated. In eukaryotes, this is achieved by gating, which involves a conformational change of the protein, thereby physically blocking water transport, or by trafficking in which AQPs are shuttled between intracellular storage sites and the plasma membrane. Gating is common amongst plant AQPs in response to environmental stress and has been shown to be triggered by phosphorylation, pH and binding of divalent cations. Gating has been demonstrated for yeast AQPs for which it is believed to confer protection against osmotic shock and rapid freezing. In mammals, AQP regulation is mainly achieved through trafficking. Thirteen AQPs have been identified in humans, the majority of which are regulated by trafficking in response to a wide range of stimuli. The far best characterized trafficking mechanism is that of AQP2 in the kidney collecting duct where it plays a key role in urine concentration. AQP2 trafficking is controlled by the pituitary hormone vasopressin that stimulates phosphorylation of the AQP2 C-terminus, triggering translocation of AQP2 from intracellular storage vesicles to the apical membrane. Defective trafficking of human AQPs can lead to several disease states, for example nephrogenic diabetes insipidus (AQP2) and Sjögren's syndrome (AQP5). In this chapter, we give an overview of what is known about the regulation of eukaryotic AQPs, focusing particularly on structure-function relationships. We discuss the physiological role of AQP regulation, specific regulatory mechanisms and reoccurring themes in both gating and trafficking.
23.	Oswald, Christine, et al. (författare) Crystallization and preliminary crystallographic analysis of the NAD(H)-binding domain of Escherichia coli transhydrogenase 2004 Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 60:4, s. 743-745 Tidskriftsartikel (refereegranskat)abstract Transhydrogenase is a proton-pumping membrane protein that is required for the cellular regeneration of NADPH. The NAD(H)-binding domain (domain I) of transhydrogenase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique at room temperature. The crystals, which were grown from PEG 4000 and ammonium acetate in citrate buffer, belong to the triclinic space group P1, with unit-cell parameters a = 38.8, b = 66.8, c = 76.4 Å, α = 67.5, β = 80.8, γ = 81.5°. X-ray diffraction data were collected to 1.9 Å resolution using synchrotron radiation. The crystals contain one dimer of transhydrogenase domain I per asymmetric unit. © 2004 International Union of Crystallography. Printed in Denmark - all rights reserved.
24.	Rebuffet, Etienne, et al. (författare) Cell-free production and characterisation of human uncoupling protein 1–3 2017 Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 10, s. 276-281 Tidskriftsartikel (refereegranskat)abstract The uncoupling proteins (UCPs) leak protons across the inner mitochondrial membrane, thus uncoupling the proton gradient from ATP synthesis. The main known physiological role for this is heat generation by UCP1 in brown adipose tissue. However, UCPs are also believed to be important for protection against reactive oxygen species, fine-tuning of metabolism and have been suggested to be involved in disease states such as obesity, diabetes and cancer. Structural studies of UCPs have long been hampered by difficulties in sample preparation with neither expression in yeast nor refolding from inclusion bodies in E. coli yielding sufficient amounts of pure and stable protein. In this study, we have developed a protocol for cell-free expression of human UCP1, 2 and 3, resulting in 1 mg pure protein per 20 mL of expression media. Lauric acid, a natural UCP ligand, significantly improved protein thermal stability and was therefore added during purification. Secondary structure characterisation using circular dichroism spectroscopy revealed the proteins to consist of mostly α-helices, as expected. All three UCPs were able to bind GDP, a well-known physiological inhibitor, as shown by the Fluorescence Resonance Energy Transfer (FRET) technique, suggesting that the proteins are in a natively folded state.
25.	Roche, Jennifer Virginia, et al. (författare) Aquaporin protein-protein interactions 2017 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 18:11 Forskningsöversikt (refereegranskat)abstract Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein-protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein-protein interactions; dividing the interactions into three types: (1) interactions between aquaporin tetramers; (2) interactions between aquaporin monomers within a tetramer (hetero-tetramerization); and (3) transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissueand trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.
26.	Roche, Jennifer Virginia, et al. (författare) Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5 2017 Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 292:35, s. 14636-14648 Tidskriftsartikel (refereegranskat)abstract The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser256, Ser261, Ser264, and Thr269), of which Ser256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications.
27.	Roche, Jennifer Virginia, et al. (författare) Structural insights into AQP2 targeting to multivesicular bodies 2019 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:21 Tidskriftsartikel (refereegranskat)abstract Vasopressin-dependent trafficking of AQP2 in the renal collecting duct is crucial for the regulation of water homeostasis. This process involves the targeting of AQP2 to the apical membrane during dehydration as well as its removal when hydration levels have been restored. The latter involves AQP2 endocytosis and sorting into multivesicular bodies (MVB), from where it may be recycled, degraded in lysosomes, or released into urine via exosomes. The lysosomal trafficking regulator-interacting protein 5 (LIP5) plays a crucial role in this by coordinating the actions of the endosomal sorting complex required for transport III (ESCRT-III) and vacuolar protein sorting 4 (Vps4) ATPase, resulting in the insertion of AQP2 into MVB inner vesicles. While the interaction between LIP5 and the ESCRT-III complex and Vps4 is well characterized, very little is known about how LIP5 interacts with AQP2 or any other membrane protein cargo. Here, we use a combination of fluorescence spectroscopy and computer modeling to provide a structural model of how LIP5 interacts with human AQP2. We demonstrate that, the AQP2 tetramer binds up to two LIP5 molecules and that the interaction is similar to that seen in the complex between LIP5 and the ESCRT-III component, charged multivesicular body protein 1B (CHMP1B). These studies give the very first structural insights into how LIP5 enables membrane protein insertion into MVB inner vesicles and significantly increase our understanding of the AQP2 trafficking mechanism.
28.	Rodríguez-Arce, Irene, et al. (författare) Moonlighting of Haemophilus influenzae heme acquisition systems contributes to the host airway-pathogen interplay in a coordinated manner 2019 Ingår i: Virulence. - : Informa UK Limited. - 2150-5608 .- 2150-5594. ; 10:1, s. 315-333 Tidskriftsartikel (refereegranskat)abstract Nutrient iron sequestration is the most significant form of nutritional immunity and causes bacterial pathogens to evolve strategies of host iron scavenging. Cigarette smoking contains iron particulates altering lung and systemic iron homeostasis, which may enhance colonization in the lungs of patients suffering chronic obstructive pulmonary disease (COPD) by opportunistic pathogens such as nontypeable. NTHi is a heme auxotroph, and the NTHi genome contains multiple heme acquisition systems whose role in pulmonary infection requires a global understanding. In this study, we determined the relative contribution to NTHi airway infection of the four heme-acquisition systems HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF that are located at the bacterial outer membrane or the periplasm. Our computational studies provided plausible 3D models for HbpA, SapA, PE, and HxuA interactions with heme. Generation and characterization of single mutants in the hxuCBA, hpe, sapA, and hbpA genes provided evidence for participation in heme binding-storage and inter-bacterial donation. The hxuA, sapA, hbpA, and hpe genes showed differential expression and responded to heme. Moreover, HxuCBA, PE, SapABCDFZ, and HbpA-DppBCDF presented moonlighting properties related to resistance to antimicrobial peptides or glutathione import, together likely contributing to the NTHi-host airway interplay, as observed upon cultured airway epithelia and in vivo lung infection. The observed multi-functionality was shown to be system-specific, thus limiting redundancy. Together, we provide evidence for heme uptake systems as bacterial factors that act in a coordinated and multi-functional manner to subvert nutritional- and other sources of host innate immunity during NTHi airway infection.
29.	Salman, Mootaz M., et al. (författare) Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis 2022 Ingår i: Brain. - : Oxford University Press (OUP). - 0006-8950 .- 1460-2156. ; 145:1, s. 64-75 Tidskriftsartikel (refereegranskat)abstract Aquaporin channels facilitate bidirectional water flow in all cells and tissues. AQP4 is highly expressed in astrocytes. In the CNS, it is enriched in astrocyte endfeet, at synapses, and at the glia limitans, where it mediates water exchange across the blood-spinal cord and blood-brain barriers (BSCB/BBB), and controls cell volume, extracellular space volume, and astrocyte migration. Perivascular enrichment of AQP4 at the BSCB/BBB suggests a role in glymphatic function. Recently, we have demonstrated that AQP4 localization is also dynamically regulated at the subcellular level, affecting membrane water permeability. Ageing, cerebrovascular disease, traumatic CNS injury, and sleep disruption are established and emerging risk factors in developing neurodegeneration, and in animal models of each, impairment of glymphatic function is associated with changes in perivascular AQP4 localization. CNS oedema is caused by passive water influx through AQP4 in response to osmotic imbalances. We have demonstrated that reducing dynamic relocalization of AQP4 to the BSCB/BBB reduces CNS oedema and accelerates functional recovery in rodent models. Given the difficulties in developing pore-blocking AQP4 inhibitors, targeting AQP4 subcellular localization opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental questions about water homeostasis in health and disease.
30.	Steffen, Jonas Hyld, et al. (författare) Assessing water permeability of aquaporins in a proteoliposome-based stopped-flow setup 2022 Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2 Tidskriftsartikel (refereegranskat)abstract Aquaporins (AQPs) are water channels embedded in the cell membrane that are critical in maintaining water homeostasis. We describe a protocol for determining the water permeation capacity of AQPs reconstituted into proteoliposomes. Using a stopped-flow setup, AQP embedded in proteoliposomes are exposed to an osmogenic gradient that triggers water flux. The consequent effects on proteoliposome size can be tracked using the fluorescence of an internalized fluorophore. This enables controlled characterization of water flux by AQPs. For complete details on the use and execution of this protocol, please refer to Kitchen et al. (2020).
31.	Tamim, Al-Jubair, et al. (författare) Haemophilus influenzae Type f Hijacks Vitronectin Using Protein H To Resist Host Innate Immunity and Adhere to Pulmonary Epithelial Cells. 2015 Ingår i: Journal of Immunology. - : The American Association of Immunologists. - 1550-6606 .- 0022-1767. ; 195:12, s. 5688-5695 Tidskriftsartikel (refereegranskat)abstract The incidence of invasive Haemophilus influenzae type b (Hib) disease has significantly decreased since the introduction of an efficient vaccine against Hib. However, in contrast to Hib, infections caused by H. influenzae serotype f (Hif) are emerging. We recently did a whole genome sequencing of an invasive Hif isolate, and reported that Hif interacts with factor H by expressing protein H (PH). In this study, upon screening with various human complement regulators, we revealed that PH is also a receptor for vitronectin (Vn), an abundant plasma protein that regulates the terminal pathway of the human complement system in addition to being a component of the extracellular matrix. Bacterial Vn binding was significantly reduced when the lph gene encoding PH was deleted in an invasive Hif isolate. The dissociation constant (KD) of the interaction between recombinant PH and Vn was 2.2 μM, as revealed by Biolayer interferometry. We found that PH has different regions for simultaneous interaction with both Vn and factor H, and that it recognized the C-terminal part of Vn (aa 352-362). Importantly, PH-dependent Vn binding resulted in better survival of the wild-type Hif or PH-expressing Escherichia coli when exposed to human serum. Finally, we observed that PH mediated an increased bacterial adherence to alveolar epithelial cells in the presence of Vn. In conclusion, our study reveals that PH most likely plays an important role in Hif pathogenesis by increasing serum resistance and adhesion to the airways.
32.	Törnroth-Horsefield, Susanna, et al. (författare) Insight into the Mammalian Aquaporin Interactome 2022 Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 23:17 Forskningsöversikt (refereegranskat)abstract Aquaporins (AQPs) are a family of transmembrane water channels expressed in all living organisms. AQPs facilitate osmotically driven water flux across biological membranes and, in some cases, the movement of small molecules (such as glycerol, urea, CO2, NH3, H2O2). Protein–protein interactions play essential roles in protein regulation and function. This review provides a comprehensive overview of the current knowledge of the AQP interactomes and addresses the molecular basis and functional significance of these protein–protein interactions in health and diseases. Targeting AQP interactomes may offer new therapeutic avenues as targeting individual AQPs remains challenging despite intense efforts.
33.	Törnroth-Horsefield, Susanna, 1973, et al. (författare) Opening and closing the metabolite gate. 2008 Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490. ; 105:50, s. 19565-6 Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
34.	Törnroth-Horsefield, Susanna, et al. (författare) Phosphorylation of human AQP2 and its role in trafficking 2020 Ingår i: Vitamins and Hormones. - : Elsevier. - 0083-6729. - 9780128208090 ; 112, s. 95-117 Bokkapitel (refereegranskat)abstract Human Aquaporin 2 (AQP2) is a membrane-bound water channel found in the kidney collecting duct whose regulation by trafficking plays a key role in regulating urine volume. AQP2 trafficking is tightly controlled by the pituitary hormone arginine vasopressin (AVP), which stimulates translocation of AQP2 residing in storage vesicles to the apical membrane. The AVP-dependent translocation of AQP2 to and from the apical membrane is controlled by multiple phosphorylation sites in the AQP2 C-terminus, the phosphorylation of which alters its affinity to proteins within the cellular membrane protein trafficking machinery. The aim of this chapter is to provide a summary of what is currently known about AVP-mediated AQP2 trafficking, dissecting the roles of individual phosphorylation sites, kinases and phosphatases and interacting proteins. From this, the picture of an immensely complex process emerges, of which many structural and molecular details remains to be elucidated.
35.	Törnroth-Horsefield, Susanna, 1973, et al. (författare) Structural insights into eukaryotic aquaporin regulation. 2010 Ingår i: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:12, s. 2580-8 Tidskriftsartikel (refereegranskat)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.
36.	Zeng, Jiao, et al. (författare) High-resolution structure of a fish aquaporin reveals a novel extracellular fold 2022 Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12 Tidskriftsartikel (refereegranskat)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.
37.	Zhang, Yuewen, et al. (författare) A microfluidic strategy for the detection of membrane protein interactions 2020 Ingår i: Lab on a Chip. - : Royal Society of Chemistry (RSC). - 1473-0189 .- 1473-0197. ; 20:17, s. 3230-3238 Tidskriftsartikel (refereegranskat)abstract Membrane proteins perform a vast range of vital biological functions and are the gatekeepers for exchange of information and matter between the intracellular and extracellular environment. However, membrane protein interactions can be challenging to characterise in a quantitative manner due to the low solubility and large size of the membrane protein complex with associated lipid or detergent molecules. Here, we show that measurements of the changes in charge and diffusivity on the micron scale allow for non-disruptive studies of membrane protein interactions in solution. The approach presented here uses measurements of key physical properties of membrane proteins and their ligands to characterise the binding equilibrium parameters. We demonstrate this approach for human aquaporins (AQPs), key membrane proteins in the regulation of water homeostasis in cells. We perform quantitative measurements to characterise the interactions between two full-length AQP isoforms and the regulatory protein, calmodulin (CaM), and show that CaM selectively binds AQP0. Through direct measurements of the diffusivity and mobility in an external electric field, the diffusion coefficients and electrophoretic mobilities are determined for the individual components and the resulting AQP0-CaM complex. Furthermore, we obtain directly the binding equilibrium parameters and effective charge of each component. These results open up a route towards the use of microfluidics as a general platform in protein science and open up new possibilities for the characterisation of membrane protein interactions in solution.

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