| 1. |
- Brändén, Magnus, 1971, et al.
(författare)
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Refractive-Index-Based Screening of Membrane-Protein-Mediated Transfer across Biological Membranes
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 0006-3495 .- 1542-0086. ; 99:1, s. 124-133
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Tidskriftsartikel (refereegranskat)abstract
- Numerous membrane-transport proteins are major drug targets, and therefore a key ingredient in pharmaceutical development is the availability of reliable, efficient tools for membrane transport characterization and inhibition. Here, we present the use of evanescent-wave sensing for screening of membrane-protein-mediated transport across lipid bilayer membranes. This method is based on a direct recording of the temporal variations in the refractive index that occur upon a transfer-dependent change in the solute concentration inside liposomes associated to a surface plasmon resonance (SPR) active sensor surface. The applicability of the method is demonstrated by a functional study of the aquaglyceroporin PfAQP from the malaria parasite Plasmodium falciparum. Assays of the temperature dependence of facilitated diffusion of sugar alcohols on a single set of PfAQP-reconstituted liposomes reveal that the activation energies for facilitated diffusion of xylitol and sorbitol are the same as that previously measured for glycerol transport in the aquaglyceroporin of Escherichia coli (5 kcal/mole). These findings indicate that the aquaglyceroporin selectivity filter does not discriminate sugar alcohols based on their length, and that the extra energy cost of dehydration of larger sugar alcohols, upon entering the pore, is compensated for by additional hydrogen-bond interactions within the aquaglyceroporin pore.
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| 2. |
- Aponte-Santamaria, C., et al.
(författare)
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Temperature dependence of protein-water interactions in a gated yeast aquaporin
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Regulation of aquaporins is a key process of living organisms to counteract sudden osmotic changes. Aqy1, which is a water transporting aquaporin of the yeast Pichia pastoris, is suggested to be gated by chemo-mechanical stimuli as a protective regulatory-response against rapid freezing. Here, we tested the influence of temperature by determining the X-ray structure of Aqy1 at room temperature (RT) at 1.3 angstrom resolution, and by exploring the structural dynamics of Aqy1 during freezing through molecular dynamics simulations. At ambient temperature and in a lipid bilayer, Aqy1 adopts a closed conformation that is globally better described by the RT than by the low-temperature (LT) crystal structure. Locally, for the blocking-residue Tyr31 and the water molecules inside the pore, both LT and RT data sets are consistent with the positions observed in the simulations at room-temperature. Moreover, as the temperature was lowered, Tyr31 adopted a conformation that more effectively blocked the channel, and its motion was accompanied by a temperature-driven rearrangement of the water molecules inside the channel. We therefore speculate that temperature drives Aqy1 from a loosely-to a tightly-blocked state. This analysis provides high-resolution structural evidence of the influence of temperature on membrane-transport channels.
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| 3. |
- Fischer, Gerhard, 1978, et al.
(författare)
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Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.
- 2009
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Ingår i: PLoS biology. - : Public Library of Science (PLoS). - 1545-7885 .- 1544-9173. ; 7:6
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
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| 4. |
- Fischer, Gerhard, 1978
(författare)
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Structure and Function of Aqua(glycero)porins - A Path to Drug Design
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are a major class of the drug targets. Rational design of drugs requires knowledge about the structure and function of these proteins. Working with membrane proteins, however, is experimentally challenging as the required technology is still emerging. Water being the medium of life, Nature has evolved specialized water channels called aquaporins. These transporters are found in all kingdoms of life – from simple microorganisms to humans – and function to maintain water homeostasis. These water channels are surprisingly similar in structure and are divided into two major groups: While orthodox aquaporins transport water only, the closely related aquaglyceroporins also transport small solutes like glycerol or other sugar alcohols. Aquaporins have been suggested to be involved in a wide variety of human disorders, but also as a means to combat malicious parasites. During the course of this work, we discovered the only aquaporin from the yeast Pichia pastoris. We determined its atomic structure using X-ray crystallography to 0.90 Å resolution, the highest resolution for any membrane protein to date. Surprisingly, this structure showed the water pore being closed by its elongated N-terminus and provided in detail information on the water transport mechanism through this pore. Further functional characterization by a combination of size-based water conduction assays and temperature-dependent crystallography revealed that the channel can be opened. We suggested a gating mechanism via phosphorylation and mechanosensing, which was found to be beneficial to the survival of the organism. Medically, protein structures like these can also be used for rational drug design. For aqua(glycero)porins, however, the discovery of inhibitors is hampered by the lack of a medium- to high-throughput assay to test potential channel blockers. Using novel technology based on surface plasmon resonance (Biacore™), we established a fully automated drug screening assay with the capability to test up to 50 compounds/day. Application of this method in combination with virtual screening yielded a drug lead with an IC50-value of 1-10 μM against the aquaglyceroporin from the malaria parasite Plasmodium falciparum.
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| 5. |
- Kosinska-Eriksson, Urszula, 1976, et al.
(författare)
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Subangstrom resolution X-ray structure details aquaporin-water interactions.
- 2013
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Ingår i: Science (New York, N.Y.). - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 340:6138, s. 1346-9
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporins are membrane channels that facilitate the flow of water across biological membranes. Two conserved regions are central for selective function: the dual asparagine-proline-alanine (NPA) aquaporin signature motif and the aromatic and arginine selectivity filter (SF). Here, we present the crystal structure of a yeast aquaporin at 0.88 angstrom resolution. We visualize the H-bond donor interactions of the NPA motif's asparagine residues to passing water molecules; observe a polarized water-water H-bond configuration within the channel; assign the tautomeric states of the SF histidine and arginine residues; and observe four SF water positions too closely spaced to be simultaneously occupied. Strongly correlated movements break the connectivity of SF waters to other water molecules within the channel and prevent proton transport via a Grotthuss mechanism.
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| 6. |
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| 7. |
- Rödström, Karin, 1986, et al.
(författare)
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Structural studies of staphylococcal enterotoxin H in complex with T cell receptor and major histocompatibility complex class II
- 2010
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Ingår i: FEBS JOURNAL. - 1742-464X. ; 2010, 277, s. 53-54
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Superantigens (SAgs) are bacterial toxins capable of cross-linking the immune receptors of the host, the T cell receptor (TCR) and major histocompatibility complex (MHC) class II, and thereby trigger a massive release of cytokines. This could lead to toxic shock syndrome, which can have a fatal outcome. Here, we present the crystal structure of the ternary complex between the superantigen, staphylococcal enterotoxin H (SEH), TCR and MHC, as well as the dimer complex, including only TCR and SEH. It is evident that SEH interacts with the variable α domain (Vα) of TCR, in sharp contrast to previously studied SAgs that interact with the Vβ domain. Due to the high structural conservation of amino acids in SEH that are crucial for the interaction, we propose that in addition to Vβ activation of T cells, there are SAgs, in addition to SEH, which are able to activate T cells through Vα as well. In addition to providing crucial information regarding the nature of TCR-mediated recognition of superantigens, the finding have central implications for future strategies aimed at preventing or modulating the often pathogenic response to superantigens.
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| 8. |
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| 9. |
- Saline, Maria, et al.
(författare)
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The structure of superantigen complexed with TCR and MHC reveals novel insights into superantigenic T cell activation.
- 2010
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Ingår i: Nature communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 1:8
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Tidskriftsartikel (refereegranskat)abstract
- Superantigens (SAgs) are bacterial toxins that interact with immunoreceptors, T cell receptor (TCR) and major histocompatibility complex (MHC) class II, conventionally through the variable β-domain of TCR (TCRVβ). They induce a massive release of cytokines, which can lead to diseases such as food poisoning and toxic shock syndrome. In this study, we report the X-ray structure of the ternary complex between staphylococcal enterotoxin H (SEH) and its human receptors, MHC class II and TCR. The structure demonstrates that SEH predominantly interacts with the variable α-domain of TCR (TCRVα), which is supported by nuclear magnetic resonance (NMR) analyses. Furthermore, there is no contact between MHC and TCR upon complex formation. Structural analyses suggest that the major contact points to TCRVα are conserved among other bacterial SAgs. Consequently, a new dimension of SAg biology emerges, suggesting that in addition to the conventional interactions with the TCRVβ domain, SAgs can also activate T cells through the TCRVα domain.
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| 10. |
- Schmit, Stephanie L, et al.
(författare)
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Novel Common Genetic Susceptibility Loci for Colorectal Cancer.
- 2019
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Ingår i: Journal of the National Cancer Institute. - : Oxford University Press (OUP). - 0027-8874 .- 1460-2105. ; 111:2, s. 146-157
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Tidskriftsartikel (refereegranskat)abstract
- Background: Previous genome-wide association studies (GWAS) have identified 42 loci (P < 5 × 10-8) associated with risk of colorectal cancer (CRC). Expanded consortium efforts facilitating the discovery of additional susceptibility loci may capture unexplained familial risk.Methods: We conducted a GWAS in European descent CRC cases and control subjects using a discovery-replication design, followed by examination of novel findings in a multiethnic sample (cumulative n = 163 315). In the discovery stage (36 948 case subjects/30 864 control subjects), we identified genetic variants with a minor allele frequency of 1% or greater associated with risk of CRC using logistic regression followed by a fixed-effects inverse variance weighted meta-analysis. All novel independent variants reaching genome-wide statistical significance (two-sided P < 5 × 10-8) were tested for replication in separate European ancestry samples (12 952 case subjects/48 383 control subjects). Next, we examined the generalizability of discovered variants in East Asians, African Americans, and Hispanics (12 085 case subjects/22 083 control subjects). Finally, we examined the contributions of novel risk variants to familial relative risk and examined the prediction capabilities of a polygenic risk score. All statistical tests were two-sided.Results: The discovery GWAS identified 11 variants associated with CRC at P < 5 × 10-8, of which nine (at 4q22.2/5p15.33/5p13.1/6p21.31/6p12.1/10q11.23/12q24.21/16q24.1/20q13.13) independently replicated at a P value of less than .05. Multiethnic follow-up supported the generalizability of discovery findings. These results demonstrated a 14.7% increase in familial relative risk explained by common risk alleles from 10.3% (95% confidence interval [CI] = 7.9% to 13.7%; known variants) to 11.9% (95% CI = 9.2% to 15.5%; known and novel variants). A polygenic risk score identified 4.3% of the population at an odds ratio for developing CRC of at least 2.0.Conclusions: This study provides insight into the architecture of common genetic variation contributing to CRC etiology and improves risk prediction for individualized screening.
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| 11. |
- Törnroth-Horsefield, Susanna, 1973, et al.
(författare)
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Structural insights into eukaryotic aquaporin regulation.
- 2010
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Ingår i: FEBS letters. - : Wiley. - 1873-3468 .- 0014-5793. ; 584:12, s. 2580-8
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin-mediated water transport across cellular membranes is an ancient, ubiquitous mechanism within cell biology. This family of integral membrane proteins includes both water selective pores (aquaporins) and transport facilitators of other small molecules such as glycerol and urea (aquaglyceroporins). Eukaryotic aquaporins are frequently regulated post-translationally by gating, whereby the rate of flux through the channel is controlled, or by trafficking, whereby aquaporins are shuttled from intracellular storage sites to the plasma membrane. A number of high-resolution X-ray structures of eukaryotic aquaporins have recently been reported and the new structural insights into gating and trafficking that emerged from these studies are described. Basic structural themes reoccur, illustrating how the problem of regulation in diverse biological contexts builds upon a limited set of possible solutions.
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| 12. |
- 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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| 13. |
- 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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