| 1. |
- Frick, Anna, 1982, et al.
(författare)
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Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism
- 2013
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 454:pt 3, s. 491-499
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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.
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| 2. |
- Frick, Anna, 1982, et al.
(författare)
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X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.
- 2014
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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
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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.
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| 3. |
- Nyblom, Anna Maria, 1975, et al.
(författare)
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Structural and functional analysis of SoPIP2;1 mutants adds insight into plant aquaporin gating.
- 2009
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 387:3, s. 653-68
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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.
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| 4. |
- Frick, Anna, 1982, et al.
(författare)
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Structural basis for pH gating of plant aquaporins
- 2013
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Ingår i: Febs Letters. - : Wiley. - 0014-5793. ; 587:7, s. 989-993
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Tidskriftsartikel (refereegranskat)abstract
- Plants have evolved to cope with fluctuations in water supply by gating their water channels known
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| 5. |
- Frick, Anna, 1982
(författare)
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Structural studies of aquporins in human kidney and plant
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are key players in our biology and are links between the inside and the outside of the cell, allowing for signal transduction and transport of molecules. Aquaporins are membrane protein channels that allow water to pass in and out of the cell. Since all life depend on water, their function is vital for any type of organism. Although aquaporins are very similar, they have small but important differences in their structure and function. Understanding these subtle dissimilarities helps us understand the fundamentals of our biology and is also essential if aquaporins are to be used as drug targets. This thesis has investigated the structure and function of two aquaporins from different species; human and spinach. The spinach aquaporin SoPIP2;1 has become the structural model for gated plant aquaporins. In this thesis, structural and functional data is presented that gives further insights into the gating mechanism controlled by the physiological signals phosphorylation, pH and divalent cations. In addition, the mechanism behind the activation of SoPIP2;1 by mercury, commonly regarded as an aquaporin blocker, has been studied. Human Aquaporin 2 is crucial for the kidneys ability to concentrate primary urine, and its malfunction leads to nephrogenic diabetes insipidus. An X-ray crystallographic structure to 2.95Å is presented, which show that AQP2 is markedly different also from its most closely related homologues. These differences are mainly focused on loop D and the C-terminus and can be related to binding of Cd2+ in the structure. We present data that Cd2+ could correspond to Ca2+ in vivo, and discuss the role of the C-terminal helix as a protein interaction partner. In addition, mutations leading to nephrogenic diabetes insipidus are studied in the structural context.
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| 6. |
- Hagströmer, Carl Johan, et al.
(författare)
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Structural and functional analysis of aquaporin-2 mutants involved in nephrogenic diabetes insipidus
- 2023
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Ingår i: Scientific Reports. - 2045-2322. ; 13:1
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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.
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| 7. |
- Rebuffet, Etienne, et al.
(författare)
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Cell-free production and characterisation of human uncoupling protein 1–3
- 2017
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Ingår i: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 10, s. 276-281
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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.
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