| 1. |
- Schmitz, Florian, et al.
(författare)
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A bimolecular fluorescence complementation flow cytometry screen for membrane protein interactions
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- Interactions between membrane proteins within a cellular environment are crucial for all living cells. Robust methods to screen and analyse membrane protein complexes are essential to shed light on the molecular mechanism of membrane protein interactions. Most methods for detecting protein:protein interactions (PPIs) have been developed to target the interactions of soluble proteins. Bimolecular fluorescence complementation (BiFC) assays allow the formation of complexes involving PPI partners to be visualized in vivo, irrespective of whether or not these interactions are between soluble or membrane proteins. In this study, we report the development of a screening approach which utilizes BiFC and applies flow cytometry to characterize membrane protein interaction partners in the host Saccharomyces cerevisiae. These data allow constructive complexes to be discriminated with statistical confidence from random interactions and potentially allows an efficient screen for PPIs in vivo within a high-throughput setup.
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| 2. |
- Zeng, Jiao, et al.
(författare)
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High-resolution structure of a fish aquaporin reveals a novel extracellular fold
- 2022
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Ingår i: Life Science Alliance. - : Life Science Alliance, LLC. - 2575-1077. ; 5:12
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are protein channels embedded in the lipid bilayer in cells from all organisms on earth that are crucial for water homeostasis. In fish, aquaporins are believed to be important for osmoregulation; however, the molecular mechanism behind this is poorly understood. Here, we present the first structural and functional characterization of a fish aquaporin; cpAQP1aa from the fresh water fish climbing perch (Anabas testudineus), a species that is of high osmoregulatory interest because of its ability to spend time in seawater and on land. These studies show that cpAQP1aa is a water-specific aquaporin with a unique fold on the extracellular side that results in a constriction region. Functional analysis combined with molecular dynamic simulations suggests that phosphorylation at two sites causes structural perturbations in this region that may have implications for channel gating from the extracellular side.
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| 3. |
- Bill, R. M., et al.
(författare)
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Aquaporins - Expression, purification and characterization
- 2021
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Ingår i: Biochimica Et Biophysica Acta-Biomembranes. - : Elsevier BV. - 0005-2736. ; 1863:9
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin water channels facilitate the bi-directional flow of water and small, neutral solutes down an osmotic gradient in all kingdoms of life. Over the last two decades, the availability of high-quality protein has underpinned progress in the structural and functional characterization of these water channels. In particular, recombinant protein technology has guaranteed the supply of aquaporin samples that were of sufficient quality and quantity for further study. Here we review the features of successful expression, purification and characterization strategies that have underpinned these successes and that will drive further breakthroughs in the field. Overall, Escherichia coli is a suitable host for prokaryotic isoforms, while Pichia pastoris is the most commonly-used recombinant host for eukaryotic variants. Generally, a two-step purification procedure is suitable after solubilization in glucopyranosides and most structures are determined by X-ray following crystallization.
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| 4. |
- Caddeo, Andrea, et al.
(författare)
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LPIAT1/MBOAT7 contains a catalytic dyad transferring polyunsaturated fatty acids to lysophosphatidylinositol.
- 2021
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Ingår i: Biochimica et biophysica acta. Molecular and cell biology of lipids. - : Elsevier BV. - 1879-2618 .- 1388-1981. ; 1866:5
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Tidskriftsartikel (refereegranskat)abstract
- Human membrane bound O-acyltransferase domain-containing 7 (MBOAT7), also known as lysophosphatidylinositol acyltransferase 1 (LPIAT1), is an enzyme involved in the acyl-chain remodeling of phospholipids via the Lands' cycle. The MBOAT7 rs641738 variant has been associated with the entire spectrum of fatty liver disease (FLD) and neurodevelopmental disorders, but the exact enzymatic activity and the catalytic site of the protein are still unestablished. Human wild type MBOAT7 and three MBOAT7 mutants missing in the putative catalytic residues (N321A, H356A, N321A+H356A) were produced into Pichia pastoris, and purified using Ni-affinity chromatography. The enzymatic activity of MBOAT7 wild type and mutants was assessed measuring the incorporation of radiolabeled fatty acids into lipid acceptors. MBOAT7 preferentially transferred 20:4 and 20:5 polyunsaturated fatty acids (PUFAs) to lysophosphatidylinositol (LPI). On the contrary, MBOAT7 showed weak enzymatic activity for transferring saturated and unsaturated fatty acids, regardless the lipid substrate. Missense mutations in the putative catalytic residues (N321A, H356A, N321A+H356A) result in a loss of O-acyltransferase activity. Thus, MBOAT7 catalyzes the transfer of PUFAs to lipid acceptors. MBOAT7 shows the highest affinity for LPI, and missense mutations at the MBOAT7 putative catalytic dyad inhibit the O-acyltransferase activity of the protein. Our findings support the hypothesis that the association between the MBOAT7 rs641738 variant and the increased risk of NAFLD is mediated by changes in the hepatic phosphatidylinositol acyl-chain remodeling. Taken together, the increased understanding of the enzymatic activity of MBOAT7 give insights into the understanding on the basis of FLD.
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| 5. |
- Carlesso, Antonio, 1990, et al.
(författare)
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Yeast as a tool for membrane protein production and structure determination
- 2022
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Ingår i: Fems Yeast Research. - : Oxford University Press (OUP). - 1567-1356 .- 1567-1364. ; 22:1
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Forskningsöversikt (refereegranskat)abstract
- Although the majority of eukaryotic MEMBRANE PROTEIN structures are DERIVED FROM PROTEINS produced in HEK293 and insect cells, the authors show here the importance of yeast as a production host and its role as an essential player in the production of eukaryotic membrane proteins for structural and functional analysis. Membrane proteins are challenging targets to functionally and structurally characterize. An enduring bottleneck in their study is the reliable production of sufficient yields of stable protein. Here, we evaluate all eukaryotic membrane protein production experiments that have supported the deposition of a high-resolution structure. We focused on the most common yeast host systems, Saccharomyces cerevisiae and Pichia pastoris. The first high-resolution structure of a membrane protein produced in yeast was described in 1999 and today there are 186 structures of alpha-helical membrane proteins, representing 101 unique proteins from 37 families. Homologous and heterologous production are equally common in S. cerevisiae, while heterologous production dominates in P. pastoris, especially of human proteins, which represent about one-third of the total. Investigating protein engineering approaches (78 proteins from seven families) demonstrated that the majority contained a polyhistidine tag for purification, typically at the C-terminus of the protein. Codon optimization and truncation of hydrophilic extensions were also common approaches to improve yields. We conclude that yeast remains a useful production host for the study of alpha-helical membrane proteins.
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| 6. |
- Li, J., et al.
(författare)
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Molecular Level Characterization of Circulating Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder
- 2021
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Ingår i: Neurology(R) neuroimmunology & neuroinflammation. - 2332-7812. ; 8:5
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: To determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD. METHODS: We combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro. RESULTS: Multiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01). CONCLUSIONS: The composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones. Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
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| 7. |
- Wang, Hao, et al.
(författare)
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Characterization of aquaporin-driven hydrogen peroxide transport.
- 2020
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Ingår i: Biochimica et biophysica acta. Biomembranes. - : Elsevier BV. - 1879-2642 .- 0005-2736. ; 1862:2
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are membrane-intrinsic proteins initially defined as water (H2O) channels in all organisms and subsequently found to have multiple substrate specificities, such as hydrogen peroxide (H2O2). H2O2 is a signaling molecule that partakes in immune responses where its transport is mediated by aquaporins. To shed further light on the molecular basis of the aquaporin function in H2O2 transport, we have characterized an Arabidopsis thaliana aquaporin, AtPIP2;4, recombinantly produced to high yields in Pichia pastoris. Here, we present a newly established assay that allows detection of H2O2 transport by purified aquaporins reconstituted into liposomes, enabling us to compare aquaporin homologues with respect to substrate specificity. To get additional insight into the structural determinants for aquaporin-mediated H2O2 transport, we solved the 3D-structure of AtPIP2;4 to 3.7Å resolution and found structural identity to the water channel from spinach (SoPIP2;1), with the difference that Cd2+ cation is not required to retain the closed conformation. The transport specificities of the two plant aquaporins were compared to a human homologue, AQP1. Overall, we conclude that AtPIP2;4, SoPIP2;1 and hAQP1 are all transporters of both H2O and H2O2, but have different efficiencies for various specificities. Notably, all three homologues expedite H2O transport equally well while the plant aquaporins are more permeable to H2O2 than hAQP1. Comparison of the structures indicates that the observed variations in H2O and H2O2 transport cannot be explained by differences in the monomeric pore. Possibly, the determinants for transport specificities reside in the flexible domains outside the membrane core of these channels.
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| 8. |
- Wang, Hao, et al.
(författare)
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Quantitative analysis of H2O2 transport through purified membrane proteins
- 2020
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Ingår i: MethodsX. - : Elsevier BV. - 2215-0161. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Hydrogen peroxide (H2O2) is an important signal molecule produced in animal and plant cells. The balance of H2O2 between the intra- and extracellular space is regulated by integral membrane proteins, which thereby modulate signaling. Several methods have been established to analyze aquaporin mediated transport of H2O2 in whole cells with the intrinsic limitation that the amount of protein responsible for a certain activity cannot be standardized. As a consequence, the quantification of the transport and specific activity is difficult to extract making it problematic to compare isoforms and mutated variants of one specific target. Moreover, in cell-based assays, the expression of the target protein may alter the physiological processes of the host cell providing a complication and the risk of misleading results. To improve the measurements of protein based H2O2 transport, we have developed an assay allowing quantitative measurements. • Using purified aquaporin reconstituted in proteoliposomes, transport of H2O2 can be accurately measured. • Inside the liposomes, H2O2 catalyzes the reaction between Amplex Red and horseradish peroxidase (HRP) giving rise to the fluorescent product resorufin. • Analysing pure protein provides direct biochemical evidence of a specific transport excluding putative cellular background. © 2020 The Author(s)
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