| 1. |
- Söndergaard Hansen, Jesper, et al.
(author)
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Perilipin 1 binds to aquaporin 7 in human adipocytes and controls its mobility via protein kinase A mediated phosphorylation
- 2016
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In: Metabolism-Clinical and Experimental. - : Elsevier BV. - 0026-0495. ; 65:12, s. 1731-1742
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Journal article (peer-reviewed)abstract
- Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.
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| 2. |
- Fischer, Gerhard, 1978, et al.
(author)
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Crystal structure of a yeast aquaporin at 1.15 angstrom reveals a novel gating mechanism.
- 2009
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In: PLoS biology. - : Public Library of Science (PLoS). - 1545-7885 .- 1544-9173. ; 7:6
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Journal article (peer-reviewed)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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| 3. |
- Geijer, Cecilia, 1980, et al.
(author)
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Yeast Aquaglyceroporins Use the Transmembrane Core to Restrict Glycerol Transport
- 2012
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 287:28, s. 23562-23570
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Journal article (peer-reviewed)abstract
- Aquaglyceroporins are transmembrane proteins belonging to the family of aquaporins, which facilitate the passage of specific uncharged solutes across membranes of cells. The yeast aquaglyceroporin Fps1 is important for osmoadaptation by regulating intracellular glycerol levels during changes in external osmolarity. Upon high osmolarity conditions, yeast accumulates glycerol by increased production of the osmolyte and by restricting glycerol efflux through Fps1. The extended cytosolic termini of Fps1 contain short domains that are important for regulating glycerol flux through the channel. Here we show that the transmembrane core of the protein plays an equally important role. The evidence is based on results from an intragenic suppressor mutation screen and domain swapping between the regulated variant of Fps1 from Saccharomyces cerevisiae and the hyperactive Fps1 ortholog from Ashbya gossypii. This suggests a novel mechanism for regulation of glycerol flux in yeast, where the termini alone are not sufficient to restrict Fps1 transport. We propose that glycerol flux through the channel is regulated by interplay between the transmembrane helices and the termini. This mechanism enables yeast cells to fine-tune intracellular glycerol levels at a wide range of extracellular osmolarities.
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| 4. |
- Palmgren, Madelene, et al.
(author)
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Quantification of the Intracellular Life Time of Water Molecules to Measure Transport Rates of Human Aquaglyceroporins
- 2017
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In: Journal of Membrane Biology. - : Springer Science and Business Media LLC. - 0022-2631 .- 1432-1424. ; 250:6, s. 629-639
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Journal article (peer-reviewed)abstract
- Orthodox aquaporins are transmembrane channel proteins that facilitate rapid diffusion of water, while aquaglyceroporins facilitate the diffusion of small uncharged molecules such as glycerol and arsenic trioxide. Aquaglyceroporins play important roles in human physiology, in particular for glycerol metabolism and arsenic detoxification. We have developed a unique system applying the strain of the yeast Pichia pastoris, where the endogenous aquaporins/aquaglyceroporins have been removed and human aquaglyceroporins AQP3, AQP7, and AQP9 are recombinantly expressed enabling comparative permeability measurements between the expressed proteins. Using a newly established Nuclear Magnetic Resonance approach based on measurement of the intracellular life time of water, we propose that human aquaglyceroporins are poor facilitators of water and that the water transport efficiency is similar to that of passive diffusion across native cell membranes. This is distinctly different from glycerol and arsenic trioxide, where high glycerol transport efficiency was recorded.
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| 5. |
- Palmgren, Madelene
(author)
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Regulation and transport mechanisms of eukaryotic aquaporins
- 2013
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Doctoral thesis (other academic/artistic)abstract
- Aquaporins are found in all kingdoms of life where they are involved in water homeostasis. They are small transmembrane water conducting channels that belong to the ancient protein family Major Intrinsic Proteins (MIP). Early on in the evolution, a gene duplication event took place that divided the aquaporin family into two subgroups; orthodox aquaporins, which are strict water facilitators, and aquaglyceroporins that except for water also transport small uncharged solutes. The main questions that I have tried to address in this thesis are which regulatory mechanisms that are involved in aquaporin gating and to investigate transport differences in solute permeation. Specifically, we have investigated yeast and human aquaporins. To find answers to our questions, we have attempted to combine structural knowledge with functional analysis. A high resolution structure of P. pastoris orthodox Aqy1 to 1.15Å generated new knowledge of regulatory mechanisms and functions of the long N-terminus that is common among fungi. We suggest that Aqy1 is gated by phosphorylation and by mechanosensation. An important functional role of Aqy1 in rapid freeze thaw cycles could be demonstrated. During this work, a single deletion strain was generated that now serves as the primary aquaporin expression platform in our laboratory. Fps1 is a regulated glycerol facilitator that is important for yeast osmo-regulation. The regulatory mechanism is still not known but here we show that a suppressor mutation within the transmembrane region restrict glycerol by its transmembrane core. Thereby, we suggest that post translational modifications in the regulatory domains of N- and C-termini fine tunes glycerol flux through Fps1. The aquaglyceroporins are classified as having a dual transport function, namely being capable of facilitating the movement of both water and glycerol over the plasma membrane. In this study, we can clearly show that there are major differences in the substrate specificity and efficiency between the different aquaglyceroporins and that small changes affect the transport efficiency and specificity of the channels.
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| 6. |
- Palmgren, Madelene, et al.
(author)
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Towards a structural and functional understanding of human aquaporin 9
- 2007
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In: Abstractbok, 5th International Conference of Aquaporin, July 13-16, Nara, Japan.
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Conference paper (other academic/artistic)abstract
- Human aquaporin 9 (hAQP9) is one of the glycerol channels that are a member of the aquaporin family. AQP9 have been shown to have the broadest specificity among the aquaporins. Except for glycerol and water it facilitates the transport of urea, arsenite, polyols and other small uncharged solutes. hAQP9 is partly expressed in hepatocytes and is thought to transport glycerol into the liver. Recently an electron microscopy projection map of AQP9 (from rat) was published to 7Å resolution indicating the typical aquaporin tetrameric structure. In our lab, we have successfully over expressed human AQP9 in Pichia pastoris. This system has showed to be a suitable host to express eukaryotic membrane proteins with high yield. The goal is to attain a high resolution structure that will reveal what lies behind the differences in specificity between the aquaporins and aquaglyceroporins. AQP9 has further been solubilised with Foscholine-12 (Fos-C12) and purified on a Ni-NTA resin followed by gelfiltration. The purified protein was detected by SDS-PAGE and estimated to be 90% pure. Transport studies by AQP9 of arsenite, glycerol and other polyols are performed in Saccharomyses cervisiae to prove functionality. We have successfully detected a clear sensitivity for arsenite of rAQP9 expressed in S.cerevisiae. Currently, the transport specificity for glycerol and other polyols are tested for both human and rat AQP9.
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| 7. |
- Palmgren, Madelene, et al.
(author)
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Towards a structural and functional understanding of human aquaporin 9
- 2008
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In: Abstractbok/Poster: 12th annual meeting of the Swedish Structural Biology Network.
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Conference paper (other academic/artistic)abstract
- Aquaporins are membrane proteins that are present in all living organisms. Today thirteen mammalian aquaporins are identified and divided into two subgroups; aquaporins, which are strict water transporters and aquaglyceroporins that apart from water also transport glycerol, urea and other small uncharged solutes. One remarkable feature with these proteins is that they exclude protons. Aquaporins are expressed in a tissue specific manner and has showed to be involved in several diseases and are therefore potential drug targets. Human aquaporin 9 is an aquaglyceroporin that has the broadest specificity among the aquporins. hAQP9 transports arsenite, glycerol, small uncharged solutes and to some extent water. It is thought to be the main rout uptake of arsenite into the liver and might play a role in hepatic arsenic toxicity. In Asia drinking water are contaminated with arsenic and millions of people are exposed to toxic levels of arsenic every day that can result in an epidemic of hepatocellular carcinoma. Today no structure with atomic resolution is available from a mammalian aquaglyceroporin. Here we report that we have successfully over expressed and purified hAQP9 in the aim to purify pure protein for further crystallisation and structure determination. We also report of point mutations that reduce arsenite transport in our initial functional studies. The aim in this project is to understand how hAQP9 can discriminate between arsenite and glycerol.
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| 8. |
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