| 1. |
- 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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| 2. |
- Roche, Jennifer Virginia, et al.
(författare)
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Aquaporin protein-protein interactions
- 2017
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 18:11
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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.
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| 3. |
- Roche, Jennifer Virginia, et al.
(författare)
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Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5
- 2017
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 292:35, s. 14636-14648
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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.
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| 4. |
- Roche, Jennifer Virginia
(författare)
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Protein-Protein Interactions in Human Aquaporin Regulation
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Water is an essential compoment of every living orgamism and forms a major part of the human body. Regulated water transport is crucial for proper cell functioning and body homeostasis. Cell, the smallest structural and functional unit of life, uses specialized water conducting pores made up of proteins to transport water across the plasma membrane by facilitated diffusion.The discovery of one of these proteins in red blood cells was the first evidence of cellular water conducting channels and the protein was later named aquaporin 1 (AQP1). Since then, thirteen human aquaporin isoforms have been identified and classified as orthodox AQPs, those that transport water only, and aquaglyceroporins, those that also transport glycerol, ammonia, urea or other small solutes.In this thesis, we focus on human AQP0 and AQP2, with an aim to deepen our understanding of how they are regulated by protein-protein interactions and the effect of phosphorylation, a post translational modification. Human AQP2 is found in the kidney where it plays an important role in regulating urine volume and is regulated by trafficking. The phosphorylation of the C-terminal residues in AQP2 and its interaction with lysosomal trafficking regulator interacting protein 5 (LIP5) plays a major role in AQP2 trafficking and targeting for lysosomal degradation. AQP0 has dual role in the eye lens, functioning as a water transporter as well as an adhesion protein in cell junctions in lens fibre cells. Previous studies have demonstrated that the binding of calmodulin (CaM) to the C-terminal of AQP0 in the presence of calcium inhibits water transport in a calcium dependent manner.In our studies, the interaction between interacting proteins (LIP5 and CaM) and full length (FL) AQPs and AQP peptides was measured using Micro Scale Thermophoresis (MST) and fluorescence anisotropy. While we were able to show the interaction and determine the binding affinities for both FL AQPs and peptides, we also could demonstrate that using FL proteins was advantageous compared to peptides. Interaction studies between FL AQP2 and LIP5 showed that AQP2 binds the LIP5 N-terminal domain (ND-LIP5) and that one ND-LIP5 molecule bound per AQP2 tetramer. We also showed that phosphorylation of the distal C-terminus of AQP2 allosterically regulates its interaction with LIP5. The stability of AQP2 and AQP2 phoshpho mimics as well as soluble proteins in the presence of detergents was demonstrated by Circular Dichroism (CD). Small angle X-ray scattering (SAXS) measurements provided information on AQP2 in nanodiscs. However, more experimental data would be required to characterize the structural aspects of AQP2:LIP5 interaction. Our data shows that recombinantly expressed AQP0 functions as a water channel when reconstituted into proteoliposomes. We demonstrated for the first time that direct binding of CaM reduces its water permeability in vitro and that phosphorylation of S229 and S235 in AQP0 abolishes the interaction with CaM while S231 shared similar binding affinities as FL AQP0. We further showed that CaM binds AQP0 in a cooperative manner.
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| 5. |
- Roche, Jennifer Virginia, et al.
(författare)
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Structural insights into AQP2 targeting to multivesicular bodies
- 2019
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Ingår i: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:21
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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.
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