| 1. |
- Kågedal, Katarina, 1970-, et al.
(författare)
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Sphingosine-induced apoptosis is dependent on lysosomal proteases
- 2001
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 359:2, s. 335-343
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Tidskriftsartikel (refereegranskat)abstract
- We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes.
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| 2. |
- Andersson, C, et al.
(författare)
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Activation and inhibition of microsomal glutathione transferase from mouse liver.
- 1988
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 249:3, s. 819-23
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Tidskriftsartikel (refereegranskat)abstract
- Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.
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| 3. |
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| 4. |
- Baird, Sarah K, et al.
(författare)
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Metallothionein protects against oxidative stress-induced lysosomal destabilization
- 2006
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 394:1, s. 275-283
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Tidskriftsartikel (refereegranskat)abstract
- The introduction of apo-ferritin or the iron chelator DFO (desferrioxamine) conjugated to starch into the lysosomal compartment protects cells against oxidative stress, lysosomal rupture and ensuing apoptosis/necrosis by binding intralysosomal redox-active iron, thus preventing Fenton-type reactions and ensuing peroxidation of lysosomal membranes. Because up-regulation of MTs (metallothioneins) also generates enhanced cellular resistance to oxidative stress, including X-irradiation, and MTs were found to be capable of iron binding in an acidic and reducing lysosomal-like environment, we propose that these proteins might similarly stabilize lysosomes following autophagocytotic delivery to the lysosomal compartment. Here, we report that Zn-mediated MT up-regulation, assayed by Western blotting and immunocytochemistry, results in lysosomal stabilization and decreased apoptosis following oxidative stress, similar to the protection afforded by fluid-phase endocytosis of apo-ferritin or DFO. In contrast, the endocytotic uptake of an iron phosphate complex destabilized lysosomes against oxidative stress, but this was suppressed in cells with up-regulated MT. It is suggested that the resistance against oxidative stress, known to occur in MT-rich cells, may be a consequence of autophagic turnover of MT, resulting in reduced iron-catalysed intralysosomal peroxidative reactions. © 2006 Biochemical Society.
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| 5. |
- Berndt, Carsten, et al.
(författare)
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Chelation of lysosomal iron protects against ionizing radiation.
- 2010
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 432:2, s. 295-301
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Tidskriftsartikel (refereegranskat)abstract
- Ionizing radiation causes DNA damage and consequent apoptosis, mainly due to the production of hydroxyl radicals (HO•) that follows radiolytic splitting of water. However, superoxide (O2•-) and H2O2 also form and induce oxidative stress with resulting LMP (lysosomal membrane permeabilization) arising from iron-catalysed oxidative events. The latter will contribute significantly to radiation-induced cell death and its degree largely depends on the quantities of lysosomal redox-active iron present as a consequence of autophagy and endocytosis of iron-rich compounds. Therefore radiation sensitivity might be depressed by lysosome-targeted iron chelators. In the present study, we have shown that cells in culture are significantly protected from ionizing radiation damage if initially exposed to the lipophilic iron chelator SIH (salicylaldehyde isonicotinoyl hydrazone), and that this effect is based on SIH-dependent lysosomal stabilization against oxidative stress. According to its dose-response-modifying effect, SIH is a most powerful radioprotector and a promising candidate for clinical application, mainly to reduce the radiation sensitivity of normal tissue. We propose, as an example, that inhalation of SIH before each irradiation session by patients undergoing treatment for lung malignancies would protect normally aerated lung tissue against life-threatening pulmonary fibrosis, whereas the sensitivity of malignant lung tumours, which usually are non-aerated, will not be affected by inhaled SIH.
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| 6. |
- Dunlop, Rachael A, et al.
(författare)
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Proteins containing oxidized amino acids induce apoptosis in human monocytes.
- 2011
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 435:1, s. 207-216
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Tidskriftsartikel (refereegranskat)abstract
- Cellular deposits of oxidized and aggregated proteins are hallmarks of a variety of age-related disorders, but whether such proteins contribute to pathology is not well understood. We previously reported that oxidized proteins form lipofuscin/ceroid-like bodies with a lysosomal-type distribution and up-regulate the transcription and translation of proteolytic lysosomal enzymes in cultured J774 mouse macrophages. Given the recently identified role of lysosomes in the induction of apoptosis, we have extended our studies to explore a role for oxidized proteins in apoptosis. Oxidized proteins were biosynthetically generated in situ by substituting oxidized analogues for parent amino acids. Apoptosis was measured with Annexin-V/PI (propidium iodide), TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling), MMP (mitochondrial membrane permeabilization), caspase activation and cytochrome c release, and related to lysosomal membrane permeabilization. Synthesized proteins containing the tyrosine oxidation product L-DOPA (L-3,4-dihydroxyphenylalanine) were more potent inducers of apoptosis than proteins containing the phenylalanine oxidation product o-tyrosine. Apoptosis was dependent upon incorporation of oxidized residues, as indicated by complete abrogation in cultures incubated with the non-incorporation control D-DOPA (D-3,4-dihydroxyphenylalanine) or when incorporation was competed out by parent amino acids. The findings of the present study suggest that certain oxidized proteins could play an active role in the progression of age-related disorders by contributing to LMP (lysosomal membrane permeabilization)-initiated apoptosis and may have important implications for the long-term use of L-DOPA as a therapeutic agent in Parkinson's disease.
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| 7. |
- Favre, Cécile J., et al.
(författare)
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Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
- 1996
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 316:1, s. 137-142
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Tidskriftsartikel (refereegranskat)abstract
- In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.
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| 8. |
- Fälker, Knut, 1971-, et al.
(författare)
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Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets
- 2011
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Ingår i: Biochemical Journal. - : Portland Press -- London. - 0264-6021 .- 1470-8728. ; 436:2, s. 469-480
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Tidskriftsartikel (refereegranskat)abstract
- PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both G(α12/13) and G(αq) signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca²⁺ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y₁₂ receptor-induced G(αi) signalling accounted for the loss of the aggregation response, as mimicking G(αi/z) signalling with 2-MeS-ADP (2-methylthioadenosine-5'-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.
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| 9. |
- Jufvas, Åsa, et al.
(författare)
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Scaffolding protein IQGAP1: an insulin-dependent link between caveolae and the cytoskeleton in primary human adipocytes?
- 2016
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Ingår i: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 473:19, s. 3177-3188
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitously expressed IQ motif-containing GTPase activating protein-1 (IQGAP1) is a scaffolding protein implicated in an array of cellular functions, in particular by binding to cytoskeletal elements and signaling proteins. A role of IQGAP1 in adipocytes has not been reported. We therefore investigated the cellular IQGAP1 interactome in primary human adipocytes. Immunoprecipitation and quantitative mass spectrometry identified caveolae and caveolae-associated proteins as the major IQGAP1 interactors alongside cytoskeletal proteins. We confirmed co-localization of IQGAP1 with the defining caveolar marker protein caveolin-1 by confocal microscopy and proximity ligation assay. Most interestingly, insulin enhanced the number of IQGAP1 interactions with caveolin-1 by five-fold. Moreover, we found a significantly reduced abundance of IQGAP1 in adipocytes from patients with type 2 diabetes compared with cells from nondiabetic control subjects. Both the abundance of IQGAP1 protein and mRNA were reduced, indicating a transcriptional defect in diabetes. Our findings suggest a novel role of IQGAP1 in insulin-regulated interaction between caveolae and cytoskeletal elements of the adipocyte, and that this is quelled in the diabetic state.
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| 10. |
- Jönsson, Cecilia, et al.
(författare)
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Insulin and beta-adrenergic receptors mediate lipolytic and anti-lipolytic signalling that is not altered by type 2 diabetes in human adipocytes
- 2019
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Ingår i: Biochemical Journal. - : PORTLAND PRESS LTD. - 0264-6021 .- 1470-8728. ; 476, s. 2883-2908
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Tidskriftsartikel (refereegranskat)abstract
- Control of fatty acid storage and release in adipose tissue is fundamental in energy homeostasis and the development of obesity and type 2 diabetes. We here take the whole signalling network into account to identify how insulin and beta-adrenergic stimulation in concert controls lipolysis in mature subcutaneous adipocytes obtained from non-diabetic and, in parallel, type 2 diabetic women. We report that, and show how, the anti-lipolytic effect of insulin can be fully explained by protein kinase B (PKB/Akt)-dependent activation of the phosphodiesterase PDE3B. Through the same PKB-dependent pathway beta-adrenergic receptor signalling, via cAMP and PI3K alpha, is anti-lipolytic and inhibits its own stimulation of lipolysis by 50%. Through this pathway both insulin and beta-adrenergic signalling control phosphorylation of FOXO1. The dose-response of lipolysis is bell-shaped, such that insulin is anti-lipolytic at low concentrations, but at higher concentrations of insulin lipolysis was increasingly restored due to inhibition of PDE3B. The control of lipolysis was not altered in adipocytes from diabetic individuals. However, the release of fatty acids was increased by 50% in diabetes due to reduced reesterification of lipolytically liberated fatty acids. In conclusion, our results reveal mechanisms of control by insulin and beta-adrenergic stimulation - in human adipocytes - that define a network of checks and balances ensuring robust control to secure uninterrupted supply of fatty acids without reaching concentrations that put cellular integrity at risk. Moreover, our results define how selective insulin resistance leave lipolytic control by insulin unaltered in diabetes, while the fatty acid release is substantially increased.
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