| 51. |
- Reyhani, Vahid, et al.
(författare)
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Fibrin binds to collagen and provides a bridge for alpha V beta 3 integrin-dependent contraction of collagen gels
- 2014
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462, s. 113-123
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Tidskriftsartikel (refereegranskat)abstract
- The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through alpha V beta 3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via alpha V beta 3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects alpha V beta 3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.
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| 52. |
- Samyn, Dieter R., et al.
(författare)
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Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways
- 2012
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 445, s. 413-422
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure–function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg168 and Lys492, and Asp178, Asp358 and Glu473 as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp358 (helix 7) and Lys492 (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp358 are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.
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| 53. |
- Schneider, Hannah, et al.
(författare)
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The human transmembrane mucin MUC17 responds to TNF alpha by increased presentation at the plasma membrane
- 2019
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 476, s. 2281-2295
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Tidskriftsartikel (refereegranskat)abstract
- Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNF alpha resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.
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| 54. |
- Schönherr, Christina, et al.
(författare)
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Activating ALK mutations found in neuroblastoma are inhibited by Crizotinib and NVP-TAE684
- 2011
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 440, s. 405-413
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the kinase domain of ALK (anaplastic lymphoma kinase) have recently been shown to be important for the progression of the childhood tumour neuroblastoma. In the present study we investigate six of the putative reported constitutively active ALK mutations, in positions G1128A, I1171N, F1174L, R1192P, F1245C and R1275Q. Our analyses were performed in cell-culture-based systems with both mouse and human ALK mutant variants and subsequently in a Drosophila melanogaster model system. Our investigation addressed the transforming potential of the putative gain-of-function ALK mutations as well as their signalling potential and the ability of two ATP-competitive inhibitors, Crizotinib (PF-02341066) and NVP-TAE684, to abrogate the activity of ALK. The results of the present study indicate that all mutations tested are of an activating nature and thus are implicated in tumour initiation or progression of neuroblastoma. Importantly for neuroblastoma patients, all ALK mutations used in the present study can be blocked by the inhibitors, although some mutants exhibited higher levels of drug sensitivity than others.
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| 55. |
- Selles, Benjamin, et al.
(författare)
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Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5 : kinetics, catalytic mechanism and oxidative inactivation.
- 2012
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 442, s. 369-380
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Tidskriftsartikel (refereegranskat)abstract
- Glutathione peroxidases constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms ((SeCys- or Cys-Gpxs) which are regenerated by glutathione or thioredoxins, (Trxs) respectively. We present here new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. This study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1 s-1. In PtGpx5, Glu79, which replaces the Gln usually found in Gpx catalytic tetrad, is likely involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide between the Trx catalytic cysteine and the Gpx5 resolving cysteine. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Besides, mass spectrometry analysis of in vitro oxidized PtGpx5 demonstrated that the peroxidatic cysteine can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.
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| 56. |
- Shaikhali, Jehad, et al.
(författare)
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Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein
- 2015
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 468:3, s. 385-400
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Tidskriftsartikel (refereegranskat)abstract
- The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.
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| 57. |
- Silkstone, Gary G A, et al.
(författare)
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Engineering tyrosine electron transfer pathways decreases oxidative toxicity in hemoglobin : Implications for blood substitute design
- 2016
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Ingår i: Biochemical Journal. - 0264-6021. ; 473:19, s. 3371-3383
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Tidskriftsartikel (refereegranskat)abstract
- Hemoglobin (Hb)-based oxygen carriers (HBOC) have been engineered to replace or augment the oxygen-carrying capacity of erythrocytes. However, clinical results have generally been disappointing due to adverse side effects linked to intrinsic hememediated oxidative toxicity and nitric oxide (NO) scavenging. Redox-Active tyrosine residues can facilitate electron transfer between endogenous antioxidants and oxidative ferryl heme species. A suitable residue is present in the α-subunit (Y42) of Hb, but absent from the homologous position in the β-subunit (F41). We therefore replaced this residue with a tyrosine (βF41Y, Hb Mequon). The βF41Y mutation had no effect on the intrinsic rate of lipid peroxidation as measured by conjugated diene and singlet oxygen formation following the addition of ferric(met) Hb to liposomes. However, βF41Y significantly decreased these rates in the presence of physiological levels of ascorbate. Additionally, heme damage in the β-subunit following the addition of the lipid peroxide hydroperoxyoctadecadieoic acid was five-fold slower in βF41Y. NO bioavailability was enhanced in βF41Y by a combination of a 20% decrease in NO dioxygenase activity and a doubling of the rate of nitrite reductase activity. The intrinsic rate of heme loss from methemoglobin was doubled in the β-subunit, but unchanged in the α-subunit. We conclude that the addition of a redox-Active tyrosine mutation in Hb able to transfer electrons from plasma antioxidants decreases heme-mediated oxidative reactivity and enhances NO bioavailability. This class of mutations has the potential to decrease adverse side effects as one component of a HBOC product.
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| 58. |
- Sjögren, Jonathan, et al.
(författare)
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EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein
- 2013
-
Ingår i: Biochemical Journal. - 0264-6021. ; 455:1, s. 107-118
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Tidskriftsartikel (refereegranskat)abstract
- Many bacteria have evolved ways to interact with glycosylation functions of the immune system of their hosts. Streptococcus pyogenes [GAS (group A Streptococcus)] secretes the enzyme EndoS that cleaves glycans on human IgG and impairs the effector functions of the antibody. The ndoS gene, encoding EndoS, has, until now, been thought to be conserved throughout the serotypes. However, in the present study, we identify EndoS2, an endoglycosidase in serotype M49 GAS strains. We characterized EndoS2 and the corresponding ndoS2 gene using sequencing, bioinformatics, phylogenetic analysis, recombinant expression and LC–MS analysis of glycosidic activity. This revealed that EndoS2 is present exclusively, and highly conserved, in serotype M49 of GAS and is only 37% identical with EndoS. EndoS2 showed endo-β-N-acetylglucosaminidase activity on all N-linked glycans of IgG and on biantennary and sialylated glycans of AGP (α1-acid glycoprotein). The enzyme was found to act only on native IgG and AGP and to be specific for free biantennary glycans with or without terminal sialylation. GAS M49 expression of EndoS2 was monitored in relation to carbohydrates present in the culture medium and was linked to the presence of sucrose. We conclude that EndoS2 is a unique endoglycosidase in serotype M49 and differs from EndoS of other GAS strains by targeting both IgG and AGP. EndoS2 expands the repertoire of GAS effectors that modify key glycosylated molecules of host defence.
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| 59. |
- Spégel, Peter, et al.
(författare)
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Metabolomic analyses reveal profound differences in glycolytic and tricarboxylic acid cycle metabolism in glucose-responsive and -unresponsive clonal beta-cell lines
- 2011
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Ingår i: Biochemical Journal. - 0264-6021. ; 435, s. 277-284
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Tidskriftsartikel (refereegranskat)abstract
- Insulin secretion from pancreatic beta-cells is controlled by complex metabolic and energetic changes provoked by exposure to metabolic fuels. Perturbations in these processes lead to impaired insulin secretion, the ultimate cause of T2D (Type 2 diabetes). To increase our understanding of stimulus secretion coupling and metabolic processes potentially involved in the pathogenesis of T2D, a comprehensive investigation of the metabolic response in the glucose-responsive INS-1 832/13 and glucose-unresponsive INS-1 832/2 beta-cell lines was performed. For this metabolomics analysis, we used GC/MS (gas chromatography/mass spectrometry) combined with multivariate statistics. We found that perturbed secretion in the 832/2 line was characterized by disturbed coupling of glycolytic and TCA (tricarboxylic acid)-cycle metabolism. The importance of this metabolic coupling was reinforced by our observation that insulin secretion partially could be reinstated by stimulation of the cells with mitochondrial fuels which bypass glycolytic metabolism. Furthermore, metabolic and functional profiling of additional beta-cell lines (INS-1, INS-1 832/1) confirmed the important role of coupled glycolytic and TCA-cycle metabolism in stimulus-secretion coupling. Dependence of the unresponsive clones on glycolytic metabolism was paralleled by increased stabilization of HIF-1 alpha (hypoxia-inducible factor 1 alpha). The relevance of a similar perturbation for human T2D was suggested by increased expression of HIF-1 alpha target genes in islets from T2D patients.
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| 60. |
- Spégel, Peter, et al.
(författare)
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Time-resolved metabolomics analysis of beta-cells implicates the pentose phosphate pathway in the control of insulin release
- 2013
-
Ingår i: Biochemical Journal. - 0264-6021. ; 450, s. 595-605
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Tidskriftsartikel (refereegranskat)abstract
- Insulin secretion is coupled with changes in beta-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP(+), NADPH and insulin secretion were measured within 15 mm of stimulation of clonal INS-1 832/13 beta-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the beta-cell metabolic response. This was fulfilled by the NADPH/NADP(+) ratio, which was elevated (8-fold; P < 0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P < 0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 beta-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in beta-cell stimulus-secretion coupling.
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