| 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. |
- Nesverova, Veronika
(author)
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Structural Details of Human Aquaporin Regulation : Three stories of three aquaporins
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Every cell is surrounded by a thin plasma membrane, protecting it from its surrounding. Specialized protein channels and transporters are embedded in this membrane, ensuring selective transport of molecules in and out of the cells. To maintain optimal hydration, water channels called aquaporins (AQPs) are present in every cell of our body. Thirteen isoforms of aquaporins exist in humans of which three, AQP2, AQP5 and AQP0, were studied in this thesis. The water passage through aquaporins needs to be very tightly regulated which happens by two distinct post-translational mechanisms. Either, the protein can be trafficked to the plasma membrane from storage vesicles when needed, like the case of AQP2 and AQP5. Alternatively, the channel might open and close based on the needs of the cell, which is a preferred way of regulation for AQP0. The need for regulation is signaled by a trigger – for example by changes in osmolarity and pH or by a hormone binding to its receptor. This signal is then carried out by binding to other regulatory proteins and/or by post- translational modifications, specifically phosphorylation.In this thesis we studied the structural details of three protein-protein interactions involved in regulation of human AQP2, AQP5 and AQP0 by trafficking or gating. The binding of AQP2 to lysosomal trafficking regulator- interacting protein 5 (LIP5) targets AQP2 to multivesicular bodies of the endosomal sorting pathway. We quantified the effect of phosphorylation on this interaction using microscale thermophoresis (MST) and we studied the binding interface using molecular docking, mutational studies and fluorescence quenching. Our results reveal that LIP5 binds AQP2 in a same way as it binds the ESCRT-III complex of the endosomal sorting machinery. We have identified residues important for the interaction and showed that AQP2 phosphorylation at specific sites located outside the LIP5 binding site impairs the binding to LIP5. Moreover, we have obtained an 8Å cryo-EM model of the complex in a phospholipid nanodisc, which confirms our previous results and shows that two molecules of LIP5 bind each AQP2 tetramer. This structure and the method used to obtain it serve as a stepping stone towards structure determination of other aquaporin complexes.For AQP5, the interaction with prolactin-inducible protein (PIP) is suggested to be important for its trafficking to the plasma membrane. We have characterized the interaction between the full-length proteins using MST and showed that it is mediated by the distal C-terminus and that one PIP molecule binds the AQP5 tetramer. In case of AQP0, the binding of calmodulin (CaM) upon increased intracellular calcium concentrations closes the channel. Using MST and a liposome-based water permeability assay, we showed that binding of CaM is inhibited by AQP0 phosphorylation at two different sites but that phosphorylation of a third site allows CaM to bind in a manner that keeps the channel open.Our studies give new insights into the role of protein-protein interactions and phosphorylation in two distinct AQP regulatory mechanisms, thereby significantly increasing our understanding of how cellular water permeability can be controlled in a tissue-dependent manner.
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| 3. |
- Roche, Jennifer Virginia, et al.
(author)
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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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In: Journal of Biological Chemistry. - 0021-9258. ; 292:35, s. 14636-14648
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Journal article (peer-reviewed)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, et al.
(author)
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Structural insights into AQP2 targeting to multivesicular bodies
- 2019
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 20:21
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Journal article (peer-reviewed)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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