| 1. |
- Chivasso, Clara, et al.
(author)
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Unraveling human aqp5-pip molecular interaction and effect on aqp5 salivary glands localization in ss patients
- 2021
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In: Cells. - : MDPI AG. - 2073-4409. ; 10:8
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Journal article (peer-reviewed)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.
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| 2. |
- Frick, Anna, 1982, et al.
(author)
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Mercury increases water permeability of a plant aquaporin through a non-cysteine-related mechanism
- 2013
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In: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 454:pt 3, s. 491-499
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Journal article (peer-reviewed)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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| 3. |
- Frick, Anna, 1982, et al.
(author)
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Structural basis for pH gating of plant aquaporins
- 2013
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In: Febs Letters. - : Wiley. - 0014-5793. ; 587:7, s. 989-993
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Journal article (peer-reviewed)abstract
- Plants have evolved to cope with fluctuations in water supply by gating their water channels known
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| 4. |
- Järvå, Michael A., et al.
(author)
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Polymer Adhesin Domains in Gram-Positive Cell Surface Proteins
- 2020
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In: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 11
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Research review (peer-reviewed)abstract
- Surface proteins in Gram-positive bacteria are often involved in biofilm formation, host-cell interactions, and surface attachment. Here we review a protein module found in surface proteins that are often encoded on various mobile genetic elements like conjugative plasmids. This module binds to different types of polymers like DNA, lipoteichoic acid and glucans, and is here termed polymer adhesin domain. We analyze all proteins that contain a polymer adhesin domain and classify the proteins into distinct classes based on phylogenetic and protein domain analysis. Protein function and ligand binding show class specificity, information that will be useful in determining the function of the large number of so far uncharacterized proteins containing a polymer adhesin domain.
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| 5. |
- Järvå, Michael
(author)
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Plant aquaporin regulation: Structural and functional studies using diffraction and scattering techniques
- 2015
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Doctoral thesis (other academic/artistic)abstract
- Water is the basis for life as we know it. It is only logical then that all organisms have evolved specialized proteins, aquaporins, which regulate water flow across their membranes. Plants, which are immobile, depend more on their environment and also use water flows to move, to breathe, and to grow. This is reflected by the much more diverse set of aquaporins plants facilitate. These work in cohort to tightly control the water flow throughout the plant. The aim of this thesis has been to deepen the understanding of a spinach leaf aquaporin, SoPIP2;1 and to develop new tools for structural studies of membrane proteins. We have studied how the SoPIP2;1 function is modulated by pH, calcium and mercury using X-ray crystallography and water transport assays in proteoliposomes. We elucidated the pH gating mechanism, discovered an additional binding site for calcium, found an unusual activating effect of mercury and hypothesized a novel mechanism by which this occurs. We have also used X-ray scattering techniques for structural studies of SoPIP2;1 in solution, thereby circumventing the need for crystallization. Using WAXS we studied the calcium-induced structural changes of SoPIP2;1 in detergent micelles. However, solvation in detergent micelles is a problem in many ways, both for the protein and for many research tools. To deal with this we explored the nanodisc system, which is a soluble discoidal bilayer in which membrane proteins can be reconstituted – thus creating a homogenous population of soluble membrane proteins without the need for detergent. We then used this tool to extract useful structural data from SoPIP2;1 using SAXS/SANS.
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| 6. |
- Lind, Ulrika, et al.
(author)
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Analysis of aquaporins from the euryhaline barnacle Balanus improvisus reveals differential expression in response to changes in salinity
- 2017
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 12:7
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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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| 7. |
- Rebuffet, Etienne, et al.
(author)
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Cell-free production and characterisation of human uncoupling protein 1–3
- 2017
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In: Biochemistry and Biophysics Reports. - : Elsevier BV. - 2405-5808. ; 10, s. 276-281
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Journal article (peer-reviewed)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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| 8. |
- Sainas, Stefano, et al.
(author)
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Targeting myeloid differentiation using potent 2-hydroxypyrazolo[1,5-a]pyridine scaffold-based human dihydroorotate dehydrogenase (hDHODH) inhibitors.
- 2018
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In: Journal of medicinal chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 61:14, s. 6034-6055
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Journal article (peer-reviewed)abstract
- Human dihydroorotate dehydrogenase (hDHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis, the conversion of dihydroorotate to orotate. hDHODH has recently been found to be associated with acute myelogenous leukemia, a disease for which the standard of intensive care has not changed over decades. This work presents a novel class of hDHODH inhibitors, which are based on an unusual carboxylic group bioisostere 2-hydroxypyrazolo[1,5-a]pyridine, that has been designed starting from brequinar, one of the most potent hDHODH inhibitors. A combination of structure-based and ligand-based strategies produced compound 4, which shows brequinar-like hDHODH potency in vitro and is superior in terms of cytotoxicity and immunosuppression. Compound 4 also restores myeloid differentiation in leukemia cell lines at concentrations that are one log digit lower than those achieved in experiments with brequinar. This paper reports the design, synthesis, SAR, X-ray crystallography, biological assays and physicochemical characterization of the new class of hDHODH inhibitors.
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| 9. |
- Schmitt, Andreas, et al.
(author)
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Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation
- 2020
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In: Journal of Molecular Biology. - : Elsevier. - 0022-2836 .- 1089-8638. ; 432:20, s. 5681-5695
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Journal article (peer-reviewed)abstract
- Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.
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| 10. |
- Sharma, Amit, et al.
(author)
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A simple adaptation to a protein crystallography station to facilitate difference X-ray scattering studies
- 2019
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In: Journal of Applied Crystallography. - : International Union of Crystallography (IUCr). - 0021-8898 .- 1600-5767. ; 52, s. 378-386
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Journal article (peer-reviewed)abstract
- The X-ray crystallography station I911-2 at MAXLab II (Lund, Sweden) has been adapted to enable difference small- and wide-angle X-ray scattering (SAXS/WAXS) data to be recorded. Modifications to the beamline included a customized flow cell, a motorized flow cell holder, a helium cone, a beam stop, a sample stage and a sample delivery system. This setup incorporated external devices such as infrared lasers, LEDs and reaction mixers to induce conformational changes in macromolecules. This platform was evaluated through proof-of-principle experiments capturing light-induced conformational changes in phytochromes. A difference WAXS signature of conformational changes in a plant aquaporin was also demonstrated using caged calcium.
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