| 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. |
- Anisimova, Tatiana, et al.
(författare)
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Ego-depletion is in the way : the challenges of controlled communication and the role of the regulatory focus theory in sustainable goals pursuit
- 2024
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Ingår i: Journal of Communication Management. - : Emerald Group Publishing Limited. - 1363-254X .- 1478-0852. ; 28:1, s. 134-146
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Tidskriftsartikel (refereegranskat)abstract
- Purpose - Via the lens of the ego-depletion phenomenon (Baumeister et al., 1998) and Higgins' (1998) regulatory focus theory, this paper seeks to explain why current controlled communications are failing to achieve coherence between people's free will and their actions pursuing sustainable goals in a society. This paper explains how ego-depletion triggered by controlled communications can lead to confusion and decision fatigue in a society, thus potentially sabotaging people's participation in sustainable behaviour. Design/methodology/approach - The authors applied Jaakkola's (2020) theory synthesis approach to integrate concepts from previously unconnected disciplines in order to generate novel insights in the area of controlled communication management. Findings- The authors develop a theoretical framework and present research propositions that can help advance research and the discourse at the intersection of controlled communication and self-regulation theories. Research limitations/implications - This paper possesses the limitations associated with conceptual papers, e.g. the lack of empirical support of the study's conceptual arguments. Practical implications - This paper generates novel insights to assist communication practitioners and policymakers to improve vehicles and mechanisms of controlled communication with the public regarding sustainable goals pursuit. Originality/value - To the best of authors' knowledge, this is one of the first papers that has merged the domains of self-regulation, ego-depletion, and controlled communication in an integrative framework in order to explain the mechanisms of how to enhance the effectiveness of controlled communication associated with sustainable goals pursuit.
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| 4. |
- Brynildsrud, Ola B., et al.
(författare)
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Global expansion of Mycobacterium tuberculosis lineage 4 shaped by colonial migration and local adaptation
- 2018
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Ingår i: Science Advances. - : AMER ASSOC ADVANCEMENT SCIENCE. - 2375-2548. ; 4:10
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Tidskriftsartikel (refereegranskat)abstract
- On the basis of population genomic and phylogeographic analyses of 1669 Mycobacterium tuberculosis lineage 4 (L4) genomes, we find that dispersal of L4 has been completely dominated by historical migrations out of Europe. We demonstrate an intimate temporal relationship between European colonial expansion into Africa and the Americas and the spread of L4 tuberculosis (TB). Markedly, in the age of antibiotics, mutations conferring antimicrobial resistance overwhelmingly emerged locally (at the level of nations), with minimal cross-border transmission of resistance. The latter finding was found to reflect the relatively recent emergence of these mutations, as a similar degree of local restriction was observed for susceptible variants emerging on comparable time scales. The restricted international transmission of drug-resistant TB suggests that containment efforts at the level of individual countries could be successful.
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| 5. |
- 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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| 6. |
- Kitchen, Philip, et al.
(författare)
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Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.
- 2015
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.
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| 7. |
- 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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| 8. |
- Kitchen, Philip, et al.
(författare)
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Water channel pore size determines exclusion properties but not solute selectivity
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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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.
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| 9. |
- Markou, Andrea, et al.
(författare)
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Molecular mechanisms governing aquaporin relocalisation
- 2022
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736. ; 1864:4
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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.
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| 10. |
- Salman, Mootaz M., et al.
(författare)
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Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis
- 2022
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Ingår i: Brain. - : Oxford University Press (OUP). - 0006-8950 .- 1460-2156. ; 145:1, s. 64-75
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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.
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