| 1. |
- Frick, Anna, 1982, et al.
(författare)
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X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking.
- 2014
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 1091-6490 .- 0027-8424. ; 111:17, s. 6305-10
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin 2 (AQP2) is a water channel found in the kidney collecting duct, where it plays a key role in concentrating urine. Water reabsorption is regulated by AQP2 trafficking between intracellular storage vesicles and the apical membrane. This process is tightly controlled by the pituitary hormone arginine vasopressin and defective trafficking results in nephrogenic diabetes insipidus (NDI). Here we present the X-ray structure of human AQP2 at 2.75 Å resolution. The C terminus of AQP2 displays multiple conformations with the C-terminal α-helix of one protomer interacting with the cytoplasmic surface of a symmetry-related AQP2 molecule, suggesting potential protein-protein interactions involved in cellular sorting of AQP2. Two Cd(2+)-ion binding sites are observed within the AQP2 tetramer, inducing a rearrangement of loop D, which facilitates this interaction. The locations of several NDI-causing mutations can be observed in the AQP2 structure, primarily situated within transmembrane domains and the majority of which cause misfolding and ER retention. These observations provide a framework for understanding why mutations in AQP2 cause NDI as well as structural insights into AQP2 interactions that may govern its trafficking.
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| 2. |
- Ericsson, Per, et al.
(författare)
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Toward 17µm pitch heterogeneously integrated Si/SiGe quantum well bolometer focal plane arrays
- 2011
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Ingår i: Infrared Technology and Applications XXXVII. - : SPIE - International Society for Optical Engineering. ; , s. 801216-1-801216-9
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Konferensbidrag (refereegranskat)abstract
- Most of today's commercial solutions for un-cooled IR imaging sensors are based on resistive bolometers using either Vanadium oxide (VOx) or amorphous Silicon (a-Si) as the thermistor material. Despite the long history for both concepts, market penetration outside high-end applications is still limited. By allowing actors in adjacent fields, such as those from the MEMS industry, to enter the market, this situation could change. This requires, however, that technologies fitting their tools and processes are developed. Heterogeneous integration of Si/SiGe quantum well bolometers on standard CMOS read out circuits is one approach that could easily be adopted by the MEMS industry. Due to its mono crystalline nature, the Si/SiGe thermistor material has excellent noise properties that result in a state-ofthe- art signal-to-noise ratio. The material is also stable at temperatures well above 450°C which offers great flexibility for both sensor integration and novel vacuum packaging concepts. We have previously reported on heterogeneous integration of Si/SiGe quantum well bolometers with pitches of 40μm x 40μm and 25μm x 25μm. The technology scales well to smaller pixel pitches and in this paper, we will report on our work on developing heterogeneous integration for Si/SiGe QW bolometers with a pixel pitch of 17μm x 17μm.
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| 3. |
- Hedfalk, Kristina, 1969, et al.
(författare)
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Recombinant production of the human aquaporins in the yeast Pichia pastoris (Invited Review).
- 2013
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 0968-7688 .- 1464-5203. ; 30:1, s. 15-31
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Tidskriftsartikel (refereegranskat)abstract
- Abstract Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism - including humans. These proteins are vital molecules and their malfunction can lead to several severe disorders and diseases. Hence, an increased understanding of their structure, function and regulation is of the utmost importance for developing current and future drugs. Heading towards this goal, the first problem to overcome is to acquire the proteins in sufficient amounts to enable functional and structural characterization. Using a suitable host organism, large amounts of target molecules can possibly be produced, but for membrane proteins limitations are frequently encountered. In the work described here, we have produced the 13 human aquaporins (hAQPs) in one of the most successful hosts for recombinant overproduction of eukaryotic proteins; the yeast Pichia pastoris, in order to explore the underlying bottleneck to a successful membrane protein production experiment. Here we present exceptional yield of hAQP1, whereas some other hAQPs were below the threshold needed for scaled up production. In the overproduction process, we have established methods for efficient production screening as well as for accurate determination of the initial production yield. Furthermore, we have optimized the yield of low producing targets, enabling studies of proteins previously out of reach, exemplified with hAQP4 as well as the homologue PfAQP. Taken together, our results. present insight into factors directing high production of eukaryotic membrane proteins together with suggestions on ways to optimize the recombinant production in the yeast P. pastoris.
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| 4. |
- Itel, F., et al.
(författare)
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CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels
- 2012
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Ingår i: Faseb Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 26:12, s. 5182-5191
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Tidskriftsartikel (refereegranskat)abstract
- Recent observations that some membrane proteins act as gas channels seem surprising in view of the classical concept that membranes generally are highly permeable to gases. Here, we study the gas permeability of membranes for the case of CO2, using a previously established mass spectrometric technique. We first show that biological membranes lacking protein gas channels but containing normal amounts of cholesterol (30-50 mol% of total lipid), e.g., MDCK and tsA201 cells, in fact possess an unexpectedly low CO2 permeability (PCO2) of similar to 0.01 cm/s, which is 2 orders of magnitude lower than the PCO2 of pure planar phospholipid bilayers (similar to 1 cm/s). Phospholipid vesicles enriched with similar amounts of cholesterol also exhibit PCO2 approximate to 0.01 cm/s, identifying cholesterol as the major determinant of membrane PCO2. This is confirmed by the demonstration that MDCK cells depleted of or enriched with membrane cholesterol show dramatic increases or decreases in PCO2, respectively. We demonstrate, furthermore, that reconstitution of human AQP-1 into cholesterol-containing vesicles, as well as expression of human AQP-1 in MDCK cells, leads to drastic increases in PCO2, indicating that gas channels are of high functional significance for gas transfer across membranes of low intrinsic gas permeability.-Itel, F., Al-Samir, S., Oberg, F., Chami, M., Kumar, M., Supuran, C. T., Deen, P. M. T., Meier, W., Hedfalk, K., Gros, G., Endeward, V. CO2 permeability of cell membranes is regulated by membrane cholesterol and protein gas channels. FASEB J. 26, 5182-5191 (2012). www.fasebj.org
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| 5. |
- Kitchen, Philip, et al.
(författare)
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Plasma Membrane Abundance of Human Aquaporin 5 Is Dynamically Regulated by Multiple Pathways.
- 2015
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 10:11
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.
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| 6. |
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| 7. |
- Nyblom, Anna Maria, 1975, et al.
(författare)
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Exceptional overproduction of a functional human membrane protein
- 2007
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928 .- 1096-0279. ; 56:1, s. 110-120
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryotic-especially human-membrane protein overproduction remains a major challenge in biochemistry. Heterologously overproduced and purified proteins provide a starting point for further biochemical, biophysical and structural studies, and the lack of sufficient quantities of functional membrane proteins is frequently a bottleneck hindering this. Here, we report exceptionally high production levels of a correctly folded and crystallisable recombinant human integral membrane protein in its active form; human aquaporin 1 (hAQP1) has been heterologously produced in the membranes of the methylotrophic yeast Pichia pastoris. After solubilisation and a two step purification procedure, at least 90 mg hAQP1 per liter of culture is obtained. Water channel activity of this purified hAQP was verified by reconstitution into proteoliposomes and performing stopped-flow vesicle shrinkage measurements. Mass spectrometry confirmed the identity of hAQPI in crude membrane preparations, and also from purified protein reconstituted into proteoliposomes. Furthermore, crystallisation screens yielded diffraction quality crystals of untagged recombinant hAQP1. This study illustrates the power of the yeast P. pastoris as a host to produce exceptionally high yields of a functionally active, human integral membrane protein for subsequent functional and structural characterization. (c) 2007 Elsevier Inc. All rights reserved.
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| 8. |
- Wöhri, Annemarie, 1976, et al.
(författare)
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A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization
- 2008
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 16:7, s. 1003-1009
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Tidskriftsartikel (refereegranskat)abstract
- A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.
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| 9. |
- Öberg, Fredrik, 1982
(författare)
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AQUAPORINS: Production Optimization and Characterization
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Aquaporins are water facilitating proteins embedded in the cellular membranes. Such channels have been identified in almost every living organism – including humans. They are vital molecules and their malfunction can lead to several severe disorders. An increased understanding of their structure, function and regulation is of utmost importance for developing current and future drugs. The first problem to overcome is to acquire the proteins in sufficient amounts to enable characterization. To achieve this, proteins are often produced in a host organism. One of the most successful hosts for recombinant overproduction is the yeast Pichia pastoris. Using this yeast we could obtain exceptional yield of aquaporin 1, whereas some others were below the threshold needed for successful subsequent characterization. In this process, we have established methods allowing fast and accurate determination of the initial production yield. Furthermore, we optimized the yield for low producing targets, enabling studies of proteins previously out of reach, exemplified with human aquaporin 4. Characterization has been performed on aquaporins obtained in sufficient quantities, and the functionality of aquaporin 1, 5 and 10 has been assessed. Furthermore, a glycosylation was found to stabilize the aquaporin 10 tetramer although only a minority of the monomers where modified. Moreover, we used protein crystallography to determine the three dimensional structure of a hAQP5 mutant, providing insight into regulation of the protein by trafficking. Taken together, these results provide insight into factors directing high production of eukaryotic membrane proteins. The subsequent characterization, including functional and structural determination, reveals new knowledge about aquaporin activity and regulation.
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| 10. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Glycosylation increases the thermostability of human aquaporin 10 protein.
- 2011
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 286:36, s. 31915-23
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Tidskriftsartikel (refereegranskat)abstract
- Human aquaporin10 (hAQP10) is a transmembrane facilitator of both water and glycerol transport in the small intestine. This aquaglyceroporin is located in the apical membrane of enterocytes and is believed to contribute to the passage of water and glycerol through these intestinal absorptive cells. Here we overproduced hAQP10 in the yeast Pichia pastoris and observed that the protein is glycosylated at Asn-133 in the extracellular loop C. This finding confirms one of three predicted glycosylation sites for hAQP10, and its glycosylation is unique for the human aquaporins overproduced in this host. Nonglycosylated protein was isolated using both glycan affinity chromatography and through mutating asparagine 133 to a glutamine. All three forms of hAQP10 where found to facilitate the transport of water, glycerol, erythritol, and xylitol, and glycosylation had little effect on functionality. In contrast, glycosylated hAQP10 showed increased thermostability of 3-6 °C compared with the nonglycosylated protein, suggesting a stabilizing effect of the N-linked glycan. Because only one third of hAQP10 was glycosylated yet the thermostability titration was mono-modal, we suggest that the presence of at least one glycosylated protein within each tetramer is sufficient to convey an enhanced structural stability to the remaining hAQP10 protomers of the tetramer.
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| 11. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Improving recombinant eukaryotic membrane protein yields in Pichia pastoris: the importance of codon optimization and clone selection.
- 2011
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 28:6, s. 398-411
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Tidskriftsartikel (refereegranskat)abstract
- In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.
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| 12. |
- Öberg, Fredrik, 1982, et al.
(författare)
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Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris.
- 2009
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Ingår i: Molecular membrane biology. - : Informa UK Limited. - 1464-5203 .- 0968-7688. ; 26:4, s. 215-27
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.
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