| 1. |
- Backe, Marie Balslev, et al.
(author)
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Lysine demethylase inhibition protects pancreatic β cells from apoptosis and improves β-cell function
- 2018
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In: Molecular and Cellular Endocrinology. - : Elsevier BV. - 0303-7207. ; 460, s. 47-56
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Journal article (peer-reviewed)abstract
- Transcriptional changes control β-cell survival in response to inflammatory stress. Posttranslational modifications of histone and non-histone transcriptional regulators activate or repress gene transcription, but the link to cell-fate signaling is unclear. Inhibition of lysine deacetylases (KDACs) protects β cells from cytokine-induced apoptosis and reduces type 1 diabetes incidence in animals. We hypothesized that also lysine demethylases (KDMs) regulate β-cell fate in response to inflammatory stress.Expression of the demethylase Kdm6B was upregulated by proinflammatory cytokines suggesting a possible role in inflammation-induced β-cell destruction. Inhibition of KDM6 demethylases using the selective inhibitor GSK-J4 protected insulin-producing cells and human and mouse islets from cytokine-induced apoptosis by blunting nuclear factor (NF)-κB signaling and endoplasmic reticulum (ER) stress response gene expression. GSK-J4 furthermore increased expression of insulin gene and glucose-stimulated insulin secretion. Expression of genes regulating purinergic and cytokine ligand-receptor interactions was downregulated following GSK-J4 exposure, while expression of genes involved in cell maintenance and survival was upregulated. These data suggest that KDMs are important regulators of inflammation-induced β-cell dysfunction and death.
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| 2. |
- Dupont, Daniel M, et al.
(author)
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Protein-binding RNA aptamers affect molecular interactions distantly from their binding sites.
- 2015
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In: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10:3
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Journal article (peer-reviewed)abstract
- Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site.
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| 3. |
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| 4. |
- Kjaergaard, Magnus, et al.
(author)
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Solution structure of recombinant somatomedin B domain from vitronectin produced in Pichia pastoris.
- 2007
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In: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 16:9
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Journal article (peer-reviewed)abstract
- The cysteine-rich somatomedin B domain (SMB) of the matrix protein vitronectin is involved in several important biological processes. First, it stabilizes the active conformation of the plasminogen activator inhibitor (PAI-1); second, it provides the recognition motif for cell adhesion via the cognate integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)); and third, it binds the complex between urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR). Previous structural studies on SMB have used recombinant protein expressed in Escherichia coli or SMB released from plasma-derived vitronectin by CNBr cleavage. However, different disulfide patterns and three-dimensional structures for SMB were reported. In the present study, we have expressed recombinant human SMB by two different eukaryotic expression systems, Pichia pastoris and Drosophila melanogaster S2-cells, both yielding structurally and functionally homogeneous protein preparations. Importantly, the entire population of our purified, recombinant SMB has a solvent exposure, both as a free domain and in complex with PAI-1, which is indistinguishable from that of plasma-derived SMB as assessed by amide hydrogen ((1)H/(2)H) exchange. This solvent exposure was only reproduced by one of three synthetic SMB products with predefined disulfide connectivities corresponding to those published previously. Furthermore, this connectivity was also the only one to yield a folded and functional domain. The NMR structure was determined for free SMB produced by Pichia and is largely consistent with that solved by X-ray crystallography for SMB in complex with PAI-1.
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| 5. |
- Kløveager Nielsen, Trine, et al.
(author)
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Forskningskortlægning og forskervurdering af skandinavisk forskning i året 2012 i institutioner for de 0-6-årige
- 2014
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Reports (other academic/artistic)abstract
- Sammenfatning Hvad ønsker vi at få at vide? Arten og kvaliteten af skandinavisk forskning i året 2012 i dagtilbud for de 0-6-årige gen-nem en forskningskortlægning og forskervurdering. Hvem ønsker at vide det og hvorfor? Projektet er udført af Dansk Clearinghouse for Uddannelsesforskning som et samarbejds-projekt med Danmarks Evalueringsinstitut. Projektet støttes af Utdanningsdirektoratet i Norge og Skolverket i Sverige. EVA anvender resultatet af den gennemførte forskningskortlægning og -vurdering til ud-arbejdelse af årsrapporten Bakspejlet og som baggrundsdata i Nordic Base of Early Childhood Education and Care (NB-ECEC). Hvad fandt vi frem til? Først og fremmest blev det konstateret, at den øgede opmærksomhed på dagtilbud, som har kunnet iagttages gennem de senere år i de skandinaviske lande, nu er slået igennem også i forskningen i form af en kraftig forøgelse af forskningen regnet i antallet af pro-jekter. Forøgelsen i antallet af projekter er hovedsageligt forbundet med en øgning i antallet af undersøgelser fra Norge. Der blev i året Der blev i året 2012 publiceret 83 undersøgelser om dagtilbud for de 0-6-årige. Heraf havde 16 data fra Danmark, 47 fra Norge og 25 fra Sverige, mens fem udover skandinavi-ske data også havde data fra ikke-skandinaviske lande. 67 af undersøgelserne har god videnskabelig kvalitet. Det pædagogiske personale er undersøgelsesgenstand i 70 % af årets studier. I 48 % ind-går børnene. Forældre indgår i 9 % af undersøgelserne. Flertallet (84 %) af årets undersøgelser inddrager et bredere samfundsmæssigt perspek-tiv i undersøgelsens analyser. Et sådant perspektiv findes ikke i de resterende 16 % af undersøgelserne. 40 % af undersøgelserne anvender et etnografisk design, efterfulgt af tværsnitsstudier (25 %) og holdningsundersøgelser (17 %). I årets forskning er der 31 % af studierne, som anvender kvantitativ analyse. Feltets typiske forskning har et kvalitativt design. De 67 undersøgelser om institutioner for 0-6-årige i Skandinavien, udgivet i 2012 med tilfredsstillende forskningskvalitet, er inddelt efter fem prædefinerede temaer, som der er opmærksomhed på i praksis og policy på feltet. De valgte temaer berøres i 44 af de 67 undersøgelser. Temaerne er: De yngste børn • Sprogudvikling • Dagtilbuddets strukturelle og fysiske rammer som læringsmiljø • Udvikling af dagtilbud • Dagtilbuddets betydning
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| 6. |
- Kovrov, Oleg, et al.
(author)
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On the mechanism of angiopoietin-like protein 8 for control of lipoprotein lipase activity
- 2019
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In: Journal of Lipid Research. - : American Society for Biochemistry and Molecular Biology. - 0022-2275 .- 1539-7262. ; 60:4, s. 783-793
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Journal article (peer-reviewed)abstract
- Angiopoietin-like (ANGPTL) 8 is a secreted inhibitor of LPL, a key enzyme in plasma triglyceride metabolism. It was previously reported that ANGPTL8 requires another member of the ANGPTL family, ANGPTL3, to act on LPL. ANGPTL3, much like ANGPTL4, is a physiologically relevant regulator of LPL activity, which causes irreversible inactivation of the enzyme. Here, we show that ANGPTL8 can form complexes with either ANGPTL3 or ANGPTL4 when the proteins are refolded together from their denatured states. In contrast to the augmented inhibitory effect of the ANGPTL3/ANGPTL8 complex on LPL activity, the ANGPTL4/ANGPTL8 complex is less active compared with ANGPTL4 alone. In our experiments, all three members of the ANGPTL family use the same mechanism to inactivate LPL, which involves dissociation of active dimeric LPL to monomers. This inactivation can be counteracted by the presence of glycosylphosphatidylinositol-anchored HDL binding protein 1, the endothelial LPL transport protein previously known to protect LPL from spontaneous and ANGPTL4-catalyzed inactivation. Our data demonstrate that ANGPTL8 may function as an important metabolic switch, by forming complexes with ANGPTL3, or with ANGPTL4, in order to direct the flow of energy from triglycerides in blood according to the needs of the body.
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| 7. |
- Kristensen, Kristian K., et al.
(author)
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A disordered acidic domain in GPIHBP1 harboring a sulfated tyrosine regulates lipoprotein lipase
- 2018
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 115:26, s. E6020-E6029
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Journal article (peer-reviewed)abstract
- The intravascular processing of triglyceride-rich lipoproteins depends on lipoprotein lipase (LPL) and GPIHBP1, a membrane protein of endothelial cells that binds LPL within the subendothelial spaces and shuttles it to the capillary lumen. In the absence of GPIHBP1, LPL remains mislocalized within the subendothelial spaces, causing severe hypertriglyceridemia (chylomicronemia). The N-terminal domain of GPIHBP1, an intrinsically disordered region (IDR) rich in acidic residues, is important for stabilizing LPL's catalytic domain against spontaneous and ANGPTL4-catalyzed unfolding. Here, we define several important properties of GPIHBP1's IDR. First, a conserved tyrosine in the middle of the IDR is posttranslationally modified by O-sulfation; this modification increases both the affinity of GPIHBP1-LPL interactions and the ability of GPIHBP1 to protect LPL against. ANGPTL4-catalyzed unfolding. Second, the acidic IDR of GPIHBP1 increases the probability of a GPIHBP1-LPL encounter via electrostatic steering, increasing the association rate constant (k(on)) for LPL binding by >250-fold. Third, we show that LPL accumulates near capillary endothelial cells even in the absence of GPIHBP1. In wild-type mice, we expect that the accumulation of LPL in close proximity to capillaries would increase interactions with GPIHBP1. Fourth, we found that GPIHBP1's IDR is not a key factor in the pathogenicity of chylomicronemia in patients with the GPIHBP1 autoimmune syndrome. Finally, based on biophysical studies, we propose that the negatively charged IDR of GPIHBP1 traverses a vast space, facilitating capture of LPL by capillary endothelial cells and simultaneously contributing to GPIHBP1's ability to preserve LPL structure and activity.
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| 8. |
- Kristensen, Kristian K., et al.
(author)
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Unfolding of monomeric lipoprotein lipase by ANGPTL4 : Insight into the regulation of plasma triglyceride metabolism
- 2020
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In: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 117:8, s. 4337-4346
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Journal article (peer-reviewed)abstract
- The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.
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| 9. |
- Luchini, Alessandra, et al.
(author)
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Peptide Disc Mediated Control of Membrane Protein Orientation in Supported Lipid Bilayers for Surface-Sensitive Investigations
- 2020
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:1, s. 1081-1088
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Journal article (peer-reviewed)abstract
- In vitro characterization of membrane proteins requires experimental approaches providing mimics of the microenvironment that proteins encounter in native membranes. In this context, supported lipid bilayers provide a suitable platform to investigate membrane proteins by a broad range of surface-sensitive techniques such as neutron reflectometry (NR), quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), and fluorescence microscopy. Nevertheless, the successful incorporation of membrane proteins in lipid bilayers with sufficiently high concentration and controlled orientation relative to the bilayer remains challenging. We propose the unconventional use of peptide discs made by phospholipids and amphipathic 18A peptides to mediate the formation of supported phospholipid bilayers with two different types of membrane proteins, CorA and tissue factor (TF). The membrane proteins are reconstituted in peptide discs, deposited on a solid surface, and the peptide molecules are then removed with extensive buffer washes. This leaves a lipid bilayer with a relatively high density of membrane proteins on the support surface. As a very important feature, the strategy allows membrane proteins with one large extramembrane domain to be oriented in the bilayer, thus mimicking the in vivo situation. The method is highly versatile, and we show its general applicability by characterizing with the above-mentioned surface-sensitive techniques two different membrane proteins, which were efficiently loaded in the supported bilayers with similar to 0.6% mol/mol (protein/lipid) concentration corresponding to 35% v/v for CorA and 8% v/v for TF. Altogether, the peptide disc mediated formation of supported lipid bilayers with membrane proteins represents an attractive strategy for producing samples for structural and functional investigations of membrane proteins and for preparation of suitable platforms for drug testing or biosensor development.
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| 10. |
- Mysling, Simon, et al.
(author)
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The angiopoietin-like protein ANGPTL4 catalyzes unfolding of the hydrolase domain in lipoprotein lipase and the endothelial membrane protein GPIHBP1 counteracts this unfolding
- 2016
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In: eLIFE. - 2050-084X. ; 5
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Journal article (peer-reviewed)abstract
- Lipoprotein lipase (LPL) undergoes spontaneous inactivation via global unfolding and this unfolding is prevented by GPIHBP1 (Mysling et al., 2016). We now show: (1) that ANGPTL4 inactivates LPL by catalyzing the unfolding of its hydrolase domain; (2) that binding to GPIHBP1 renders LPL largely refractory to this inhibition; and (3) that both the LU domain and the intrinsically disordered acidic domain of GPIHBP1 are required for this protective effect. Genetic studies have found that a common polymorphic variant in ANGPTL4 results in lower plasma triglyceride levels. We now report: (1) that this ANGPTL4 variant is less efficient in catalyzing the unfolding of LPL; and (2) that its Glu-to-Lys substitution destabilizes its N-terminal alpha-helix. Our work elucidates the molecular basis for regulation of LPL activity by ANGPTL4, highlights the physiological relevance of the inherent instability of LPL, and sheds light on the molecular defects in a clinically relevant variant of ANGPTL4.
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