| 1. |
- Agemark, Maria, et al.
(author)
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Reconstitution of water channel function and 2D-crystallization of human aquaporin 8.
- 2012
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In: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 0005-2736. ; 1818:3, s. 839-850
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Journal article (peer-reviewed)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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| 2. |
- Alexandersson, Erik, et al.
(author)
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Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress.
- 2010
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In: Plant Journal. - 1365-313X. ; 61, s. 650-660
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Journal article (peer-reviewed)abstract
- Summary Aquaporins facilitate water transport over cellular membranes and are therefore believed to play an important role in water homeostasis. In higher plants aquaporin-like proteins, also called major intrinsic proteins (MIPs), are divided into 5 subfamilies. We have previously shown that MIP transcription in Arabidopsis thaliana generally is down-regulated in leaves upon drought stress, apart from two members of the Plasma membrane Intrinsic Protein (PIP) subfamily, AtPIP1;4 and AtPIP2;5, which are up-regulated. In order to assess if this regulation is general or accession-specific we monitored gene expression of all PIPs in five Arabidopsis accessions. Overall drought regulation of PIPs was well conserved for all five accessions tested suggesting a general and fundamental physiological role of this drought response. In addition, significant differences among accessions were identified for transcripts of three PIP genes. Principal component analysis showed that most of the PIP transcriptional variation during drought stress could be explained by one variable linked to leaf water content. Promoter-GUS constructs of AtPIP1;4, AtPIP2;5 and also AtPIP2;6, which is unresponsive to drought stress, had distinct expression patterns concentrated to the base of the leaf petioles and parts of the flowers. The presence of drought stress response elements within the 1.6 kb promoter regions of AtPIP1;4 and AtPIP2;5, was demonstrated by comparing transcription of the promoter reporter construct and the endogenous gene upon drought stress. Analysis by ATTED-II and other web-based bioinformatical tools showed that several of the MIPs down-regulated upon drought are strongly co-expressed, whereas AtPIP1;4, AtPIP2;5 and AtPIP2;6 are not co-expressed.
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| 3. |
- Alexandersson, Erik, et al.
(author)
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Whole gene family expression and drought stress regulation of aquaporins
- 2005
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In: Plant Molecular Biology. - : Springer Science and Business Media LLC. - 1573-5028 .- 0167-4412. ; 59:3, s. 469-484
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Journal article (peer-reviewed)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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| 4. |
- Ampah-Korsah, Henry, et al.
(author)
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Single amino acid substitutions in the selectivity filter render NbXIP1;1α aquaporin water permeable
- 2017
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In: BMC Plant Biology. - : Springer Science and Business Media LLC. - 1471-2229. ; 17:1, s. 61-61
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Journal article (peer-reviewed)abstract
- BACKGROUND: Aquaporins (AQPs) are integral membrane proteins that facilitate transport of water and/or other small neutral solutes across membranes in all forms of life. The X Intrinsic Proteins (XIPs) are the most recently recognized and the least characterized aquaporin subfamily in higher plants. XIP1s have been shown to be impermeable to water but permeable to boric acid, glycerol, hydrogen peroxide and urea. However, uncertainty regarding the determinants for selectivity and lack of an activity that is easy to quantify have hindered functional investigations. In an effort to resolve these issues, we set out to introduce water permeability in Nicotiana benthamiana XIP1;1α (NbXIP1;1α), by exchanging amino acid residues of predicted alternative aromatic/arginine (ar/R) selectivity filters of NbXIP1;1α for residues constituting the water permeable ar/R selectivity filter of AtTIP2;1.RESULTS: Here, we present functional results regarding the amino acid substitutions in the putative filters as well as deletions in loops C and D of NbXIP1;1α. In addition, homology models were created based on the high resolution X-ray structure of AtTIP2;1 to rationalize the functional properties of wild-type and mutant NbXIP1;1α. Our results favour Thr 246 rather than Val 242 as the residue at the helix 5 position in the ar/R filter of NbXIP1;1α and indicate that the pore is not occluded by the loops when heterologously expressed in Pichia pastoris. Moreover, our results show that a single amino acid substitution in helix 1 (L79G) or in helix 2 (I102H) is sufficient to render NbXIP1;1α water permeable. Most of the functional results can be rationalized from the models based on a combination of aperture and hydrophobicity of the ar/R filter.CONCLUSION: The water permeable NbXIP1;1α mutants imply that the heterologously expressed proteins are correctly folded and offer means to explore the structural and functional properties of NbXIP1;1α. Our results support that Thr 246 is part of the ar/R filter. Furthermore, we suggest that a salt bridge to an acidic residue in helix 1, conserved among the XIPs in clade B, directs the orientation of the arginine in the ar/R selectivity filter and provides a novel approach to tune the selectivity of AQPs.
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| 5. |
- Ampah-Korsah, Henry, et al.
(author)
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The aquaporin splice variant NbXIP1;1α is permeable to boric acid and is Phosphorylated in the N-terminal domain
- 2016
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In: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 7:JUNE2016
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Journal article (peer-reviewed)abstract
- Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.
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| 6. |
- Anderberg, Hanna, et al.
(author)
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Algal MIPs, high diversity and conserved motifs
- 2011
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In: BMC Evolutionary Biology. - : Springer Science and Business Media LLC. - 1471-2148. ; 11
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Journal article (peer-reviewed)abstract
- Background: Major intrinsic proteins (MIPs) also named aquaporins form channels facilitating the passive transport of water and other small polar molecules across membranes. MIPs are particularly abundant and diverse in terrestrial plants but little is known about their evolutionary history. In an attempt to investigate the origin of the plant MIP subfamilies, genomes of chlorophyte algae, the sister group of charophyte algae and land plants, were searched for MIP encoding genes. Results: A total of 22 MIPs were identified in the nine analysed genomes and phylogenetic analyses classified them into seven subfamilies. Two of these, Plasma membrane Intrinsic Proteins (PIPs) and GlpF-like Intrinsic Proteins (GIPs), are also present in land plants and divergence dating support a common origin of these algal and land plant MIPs, predating the evolution of terrestrial plants. The subfamilies unique to algae were named MIPA to MIPE to facilitate the use of a common nomenclature for plant MIPs reflecting phylogenetically stable groups. All of the investigated genomes contained at least one MIP gene but only a few species encoded MIPs belonging to more than one subfamily. Conclusions: Our results suggest that at least two of the seven subfamilies found in land plants were present already in an algal ancestor. The total variation of MIPs and the number of different subfamilies in chlorophyte algae is likely to be even higher than that found in land plants. Our analyses indicate that genetic exchanges between several of the algal subfamilies have occurred. The PIP1 and PIP2 groups and the Ca2+ gating appear to be specific to land plants whereas the pH gating is a more ancient characteristic shared by all PIPs. Further studies are needed to discern the function of the algal specific subfamilies MIPA-E and to fully understand the evolutionary relationship of algal and terrestrial plant MIPs.
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| 7. |
- Anderberg, Hanna, et al.
(author)
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Annotation of Selaginella moellendorffii Major Intrinsic Proteins and the Evolution of the Protein Family in Terrestrial Plants.
- 2012
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In: Frontiers in Plant Science. - : Frontiers Media SA. - 1664-462X. ; 3
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Journal article (peer-reviewed)abstract
- Major intrinsic proteins (MIPs) also called aquaporins form pores in membranes to facilitate the permeation of water and certain small polar solutes across membranes. MIPs are present in virtually every organism but are uniquely abundant in land plants. To elucidate the evolution and function of MIPs in terrestrial plants, the MIPs encoded in the genome of the spikemoss Selaginella moellendorffii were identified and analyzed. In total 19 MIPs were found in S. moellendorffii belonging to 6 of the 7 MIP subfamilies previously identified in the moss Physcomitrella patens. Only three of the MIPs were classified as members of the conserved water specific plasma membrane intrinsic protein (PIP) subfamily whereas almost half were found to belong to the diverse NOD26-like intrinsic protein (NIP) subfamily permeating various solutes. The small number of PIPs in S. moellendorffii is striking compared to all other land plants and no other species has more NIPs than PIPs. Similar to moss, S. moellendorffii only has one type of tonoplast intrinsic protein (TIP). Based on ESTs from non-angiosperms we conclude that the specialized groups of TIPs present in higher plants are not found in primitive vascular plants but evolved later in a common ancestor of seed plants. We also note that the silicic acid permeable NIP2 group that has been reported from angiosperms appears at the same time. We suggest that the expansion of the number MIP isoforms in higher plants is primarily associated with an increase in the different types of specialized tissues rather than the emergence of vascular tissue per se and that the loss of subfamilies has been possible due to a functional overlap between some subfamilies.
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| 8. |
- Backmark, Anna, 1979, et al.
(author)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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In: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Journal article (peer-reviewed)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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| 9. |
- Barfod, Anders, et al.
(author)
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In Vitro Selection of RNA Aptamers Directed Against Protein E: A Haemophilus influenzae Adhesin. : a Haemophilus influenzae adhesin
- 2014
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In: Molecular Biotechnology. - : Springer Science and Business Media LLC. - 1559-0305 .- 1073-6085. ; 56:8, s. 714-725
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Journal article (peer-reviewed)abstract
- Protein E (PE) of Haemophilus influenzae is a highly conserved ubiquitous surface protein involved in adhesion to and activation of epithelial cells. The host proteins-vitronectin, laminin, and plasminogen are major targets for PE-dependent interactions with the host. To identify novel inhibitory molecules of PE, we used an in vitro selection method based on systematic evolution of ligands by exponential enrichment known as SELEX in order to select 2'F-modified RNA aptamers that specifically bind to PE. Fourteen selection cycles were performed with decreasing concentrations of PE. Sequencing of clones from the 14th selection round revealed the presence of semiconserved sequence motifs in loop regions of the RNA aptamers. Among these, three aptamers showed the highest affinity to PE in electrophoretic mobility shift assays and in dot blots. These three aptamers also inhibited the interaction of PE with vitronectin as revealed by ELISA. Moreover, pre-treatment of H. influenzae with the aptamers significantly inhibited binding of vitronectin to the bacterial surface. Biacore experiments indicated that one of the aptamers had a higher binding affinity for PE as compared to the other aptamers. Our results show that it is possible to select RNA inhibitors against bacterial adhesins using SELEX in order to inhibit interactions with target proteins.
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| 10. |
- Carlsbecker, A, et al.
(author)
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The DAL10 gene from Norway spruce (Picea abies) belongs to a potentially gymnosperm-specific subclass of MADS-box genes and is specifically active in seed cones and pollen cones.
- 2003
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In: Evolution & Development. - 1525-142X. ; 5:6, s. 551-561
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Journal article (peer-reviewed)abstract
- Transcription factors encoded by different members of the MADS-box gene family have evolved central roles in the regulation of reproductive organ development in the flowering plants, the angiosperms. Development of the stamens and carpels, the pollen- and seed-bearing organs, involves the B- and C-organ-identity MADS-box genes. B- and C-type gene orthologs with activities specifically in developing pollen- and seed-bearing organs are also present in the distantly related gymnosperms: the conifers and the gneto-phytes. We now report on the characterization of DAL10, a novel MADS-box gene from the conifer Norway spruce, which unlike the B- and C-type conifer genes shows no distinct orthology relationship to any angiosperm gene or clade in phylogenetic analyses. Like the B- and C-type genes, it is active specifically in developing pollen cones and seed cones. In situ RNA localization experiments show DAL10 to be expressed in the cone axis, which carry the microsporophylls of the young pollen cone. In contrast, in the seed cone it is expressed both in the cone axis and in the bracts, which subtend the ovuliferous scales. Expression data and the phenotype of transgenic Arabidopsis plants expressing DAL10 suggest that the gene may act upstream to or in concert with the B- and C-type genes in the establishment of reproductive identity of developing cones.
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