| 1. |
- Alexandersson, Erik, et al.
(författare)
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Whole gene family expression and drought stress regulation of aquaporins
- 2005
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Ingår i: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 1573-5028 .- 0167-4412. ; 59:3, s. 469-484
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Tidskriftsartikel (refereegranskat)abstract
- Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level.
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| 2. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 3. |
- Horsefield, Rob, 1977, et al.
(författare)
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High-resolution x-ray structure of human aquaporin 5
- 2008
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Ingår i: Proceedings of the National Academy of Sciences. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 105:36, s. 13327-13332
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 5 (HsAQP5)facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-angstrom resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.
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| 4. |
- Agemark, Maria
(författare)
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Expression and Function of Human and Plant Aquaporins
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aquaporins (AQPs) belong to a family of water permeable membrane channels found in virtually all living organisms. Thirteen isoforms of mammalian AQPs are found, whereas in Arabidopsis thaliana 35 genes encoding AQPs are found. The AQPs are distributed in different organs, cell types and in different subcellular membranes. The aquaporins form homotetramers where each monomer functions as an independent pore. In addition to water, some aquaporins are permeable to other small solutes, such as glycerol, urea, H2O2 and NH3. The objective of the work described in this thesis is to increase the knowledge of expression and function of human and plant aquaporins. Human AQP8 and AQP5 were heterologously expressed in Pichia pastoris. The proteins were purified and reconstituted into proteoliposomes to verify the functionality and were subsequently crystallized. The 2-D crystals of human AQP8 were analyzed by electron diffraction and diffracted to 3 Å, whereas the 3-D crystals of human AQP5 were analyzed by X-ray crystallography resulting in a high-resolution structure of 2.0 Å. A large amount of pure protein is needed in order to be able to crystallize a protein. Therefore, a method was developed to screen for multi-copy P. pastoris clones expressing selected aquaporins at a high level. The number of gene copies was verified by quantitative PCR and for each isoform it was correlated to the aquaporin protein expression level, although there is a large variation in expression between isoforms with the same copy number. Many aquaporins are known to be regulated at a post-translational level. Based on the high-resolution structure of the plant SoPIP2;1, we wanted to explore amino acid residues involved in stabilizing the open or closed conformation of the protein. The mutations we made were located in the D-loop, known to be involved in the gating mechanism of SoPIP2;1, or immediately after the D-loop. The mutant proteins were expressed in Xenopus laevis oocytes to determine their water transport rate. We identified several amino acid residues involved in stabilizing an open conformation or a closed conformation. In order for maintaining water homeostasis in chloroplasts and in order for the light-dependent photosynthetic reaction to occur, water needs to be transported into the chloroplast and into the thylakoid lumen. Arabidopsis chloroplast membranes were analyzed by immunoblots and mass spectrometric analysis to elucidate the aquaporin isoforms present in the chloroplast membranes. We identified the PIP2;1 isoform, one or several PIP1 isoforms and the TIP2;1 isoform in the thylakoid membranes, and the PIP2;1 isoform and the TIP2;1 isoform in the inner envelope membranes.
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| 5. |
- Agemark, Maria, et al.
(författare)
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Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
- 2012
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2736. ; 1818:3, s. 839-850
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Tidskriftsartikel (refereegranskat)abstract
- Among the thirteen human aquaporins (AQP0-12), the primary structure of AQP8 is unique. By sequence alignment it is evident that mammalian AQP8s form a separate subfamily distinct from the other mammalian aquaporins. The constriction region of the pore determining the solute specificity deviates in AQP8 making it permeable to both ammonia and H(2)O(2) in addition to water. To better understand the selectivity and gating mechanism of aquaporins, high-resolution structures are necessary. So far, the structure of one human aquaporin (HsAQP5) has been solved at atomic resolution. For mammalian aquaporins in general, high-resolution structures are only available for those belonging to the water-specific subfamily (including HsAQP5). Thus, it is of interest to solve structures of other aquaporin subfamily members with different solute specificities. To achieve this the aquaporins need to be overexpressed heterologously and purified. Here we use the methylotrophic yeast Pichia pastoris as a host for the overexpression. A wide screen of different detergents and detergent-lipid combinations resulted in the solubilization of functional human AQP8 protein and in well-ordered 2D crystals. It also became evident that removal of amino acids constituting affinity tags was crucial to achieve highly ordered 2D crystals diffracting to 3Å.
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| 6. |
- Badn, Wiaam, et al.
(författare)
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Inhibition of Inducible Nitric Oxide Synthase Enhances Anti-tumour Immune Responses in Rats Immunized with IFN-gamma-Secreting Glioma Cells.
- 2007
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Ingår i: Scandinavian Journal of Immunology. - : Wiley. - 1365-3083 .- 0300-9475. ; 65:3, s. 289-297
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Tidskriftsartikel (refereegranskat)abstract
- Interferon gamma (IFN-gamma) has successfully been used in immunotherapy of different experimental tumours. Mechanistically, IFN-gamma has extensive effects on the immune system including release of nitric oxide (NO) by upregulation of the inducible nitric oxide synthase (iNOS). NO has putative immunosuppressive effects but could also play a role in killing of tumour cells. Therefore, the aim of the present study was to clarify whether inhibition of iNOS in rats immunized with glioma cells (N32) producing IFN-gamma (N32-IFN-gamma), could enhance the anti-tumour immune response. Initially, both a selective iNOS, L-N-6-(I-Iminoethyl)-L-lysine (L-NIL), and non-selective, N-nitro-L-arginine methyl ester (L-NAME), inhibitor of NOS were tested in vitro. After polyclonal stimulation with LPS and SEA, both L-NIL and L-NAME enhanced proliferation and production of IFN-gamma from activated rat splenocytes and this effect was inversely correlated to the production of NO. However, L-NIL had a broader window of efficacy and a lower minimal effective dose. When rats were immunized with N32-IFN-gamma), and administered NOS inhibitors by intraperitoneal (i.p.) mini-osmotic pumps, only splenocytes of rats treated with L-NIL, but not L-NAME, displayed an enhanced proliferation and production of IFN-gamma when re-stimulated with N32 tumour cells. Based on these findings, L-NIL was administered concurrently with N32-IFN-gamma cells to rats with intracerebral (i.c.) tumours resulting in a prolonged survival. These results show that inhibition of iNOS can enhance an IFN-gamma-based immunotherapy of experimental i.c. tumours implying that NO released after immunization has mainly immunosuppressive net effects.
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| 7. |
- Johansson, Henrik, et al.
(författare)
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Evaluation of the GARD assay in a blind cosmetics Europe study
- 2017
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Ingår i: ALTEX Alternatives To Animal Experimentation. - : ALTEX Edition. - 1868-596X. ; 34:4, s. 515-523
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Tidskriftsartikel (refereegranskat)abstract
- Chemical hypersensitivity is an immunological response towards foreign substances, commonly referred to as sensitizers, which gives rise primarily to the clinical symptoms known as allergic contact dermatitis. For the purpose of mitigating risks associated with consumer products, chemicals are screened for sensitizing effects. Historically, such predictive screenings have been performed using animal models. However, due to industrial and regulatory demand, animal models for the purpose of sensitization assessment are being replaced by non-animal testing methods, a global trend that is spreading across industries and market segments. To meet this demand, the Genomic Allergen Rapid Detection (GARD) assay was developed. GARD is a novel, cell-based assay that utilizes the innate recognition of xenobiotic substances by dendritic cells, as measured by a multivariate readout of genomic biomarkers. Following cellular stimulation, chemicals are classified as sensitizers or non-sensitizers based on induced transcriptional profiles. Recently, a number of non-animal methods were comparatively evaluated by Cosmetics Europe, using a coherent and blinded test panel of reference chemicals with human and local lymph node assay data, comprising a wide range of sensitizers and non-sensitizers. The outcome of the GARD assay is presented in this paper. It was demonstrated that GARD is a highly functional assay with a predictive performance of 83% in this Cosmetics Europe dataset. The average accumulated predictive accuracy of GARD across independent datasets was 86% for skin sensitization hazard.
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| 8. |
- Nordén, Kristina, et al.
(författare)
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Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris
- 2011
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Ingår i: BMC Biotechnology. - : Springer Science and Business Media LLC. - 1472-6750. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: When performing functional and structural studies, large quantities of pure protein are desired. Most membrane proteins are however not abundantly expressed in their native tissues, which in general rules out purification from natural sources. Heterologous expression, especially of eukaryotic membrane proteins, has also proven to be challenging. The development of expression systems in insect cells and yeasts has resulted in an increase in successful overexpression of eukaryotic proteins. High yields of membrane protein from such hosts are however not guaranteed and several, to a large extent unexplored, factors may influence recombinant expression levels. In this report we have used four isoforms of aquaporins to systematically investigate parameters that may affect protein yield when overexpressing membrane proteins in the yeast Pichia pastoris. Results: By comparing clones carrying a single gene copy, we show a remarkable variation in recombinant protein expression between isoforms and that the poor expression observed for one of the isoforms could only in part be explained by reduced transcript levels. Furthermore, we show that heterologous expression levels of all four aquaporin isoforms strongly respond to an increase in recombinant gene dosage, independent of the amount of protein expressed from a single gene copy. We also demonstrate that the increased expression does not appear to compromise the protein folding and the membrane localisation. Conclusions: We report a convenient and robust method based on qPCR to determine recombinant gene dosage. The method is generic for all constructs based on the pPICZ vectors and offers an inexpensive, quick and reliable means of characterising recombinant P. pastoris clones. By using this method we show that: (1) heterologous expression of all aquaporins investigated respond strongly to an increase in recombinant gene dosage (2) expression from a single recombinant gene copy varies in an isoform dependent manner (3) the poor expression observed for AtSIP1;1 is mainly caused by posttranscriptional limitations. The protein folding and membrane localisation seems to be unaffected by increased expression levels. Thus a screen for elevated gene dosage can routinely be performed for identification of P. pastoris clones with high expression levels of aquaporins and other classes of membrane proteins.
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