| 1. |
- Al-Jubair, Tamim, et al.
(författare)
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Characterization of human aquaporin protein-protein interactions using microscale thermophoresis (MST)
- 2022
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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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).
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| 2. |
- Al-Jubair, Tamim, et al.
(författare)
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High-yield overproduction and purification of human aquaporins from Pichia pastoris
- 2022
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 3:2
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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).
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| 3. |
- 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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| 4. |
- Kitchen, Philip, et al.
(författare)
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Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells
- 2020
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Ingår i: STAR Protocols. - : Elsevier BV. - 2666-1667. ; 1:3
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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).
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| 5. |
- Kitchen, Philip, et al.
(författare)
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Targeting Aquaporin-4 Subcellular Localization to Treat Central Nervous System Edema
- 2020
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Ingår i: Cell. - : Elsevier BV. - 0092-8674. ; 181:4, s. 19-799
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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.
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| 6. |
- Kreida, Stefan, et al.
(författare)
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The role of phosphorylation in calmodulin-mediated gating of human AQP0
- 2024
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Ingår i: The Biochemical journal. - 0264-6021. ; 481:1, s. 17-32
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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.
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| 7. |
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| 8. |
- Rodríguez-Arce, Irene, et al.
(författare)
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Moonlighting of Haemophilus influenzae heme acquisition systems contributes to the host airway-pathogen interplay in a coordinated manner
- 2019
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Ingår i: Virulence. - : Informa UK Limited. - 2150-5608 .- 2150-5594. ; 10:1, s. 315-333
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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.
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| 9. |
- Singh, Birendra, et al.
(författare)
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A fine-tuned interaction between the trimeric autotransporter Haemophilus surface fibrils and vitronectin leads to serum resistance and adherence to respiratory epithelial cells.
- 2014
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Ingår i: Infection and Immunity. - 1098-5522. ; 82:6, s. 2378-2389
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Tidskriftsartikel (refereegranskat)abstract
- Haemophilus influenzae type b (Hib) escapes the host immune system by recruitment of the complement regulator vitronectin that inhibits the formation of the membrane attack complex (MAC) by inhibiting C5b-C7 complex formation and C9 polymerization. We previously reported that Hib acquires vitronectin at the surface by using Haemophilus surface fibrils (Hsf). Here we studied in detail the interaction between Hsf and vitronectin and its role in inhibition of MAC formation and invasion of lung epithelial cells. The vitronectin-binding region of Hsf was defined at the N-terminal comprising amino acids Hsf 429-652. Moreover, the Hsf recognition site on vitronectin consisted of the C-terminal amino acids 352-374. H. influenzae was killed more rapidly in vitronectin-depleted serum when compared to normal human serum (NHS), and an increased MAC deposition was observed at the surface of an Hsf-deficient H. influenzae mutant. In parallel, Hsf-expressing E. coli selectively acquired vitronectin from serum that resulted in significant inhibition of the MAC. Moreover, when vitronectin was bound to Hsf an increased bacterial adherence and internalization of epithelial cells was observed. Taken together, we have defined a fine-tuned protein-protein interaction between Hsf and vitronectin that may contribute to an increased virulence of Hib.
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| 10. |
- Singh, Birendra, et al.
(författare)
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Assays for Studying the Role of Vitronectin in Bacterial Adhesion and Serum Resistance
- 2018
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Ingår i: Journal of Visualized Experiments. - Cambride, USA : Journal of Visualized Experiments. - 1940-087X. ; :140
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Tidskriftsartikel (refereegranskat)abstract
- Bacteria utilize complement regulators as a means of evading the host immune response. Here, we describe protocols for evaluating the role vitronectin acquisition at the bacterial cell surface plays in resistance to the host immune system. Flow cytometry experiments identified human plasma vitronectin as a ligand for the bacterial receptor outer membrane protein H of Haemophilus influenzae type f. An enzyme-linked immunosorbent assay was employed to characterize the protein-protein interactions between purified recombinant protein H and vitronectin, and binding affinity was assessed using bio-layer interferometry. The biological importance of the binding of vitronectin to protein H at the bacterial cell surface in evasion of the host immune response was confirmed using a serum resistance assay with normal and vitronectin-depleted human serum. The importance of vitronectin in bacterial adherence was analyzed using glass slides with and without vitronectin coating, followed by Gram staining. Finally, bacterial adhesion to human alveolar epithelial cell monolayers was investigated. The protocols described here can be easily adapted to the study of any bacterial species of interest.
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