Papers by Mats Söderström
Biochemical Journal, Aug 15, 1992
Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione tr... more Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutathione transferase subunits could be altered by continuous infusion of growth hormone to both sham-operated and hypophysectomized rats of both sexes. This treatment feminized the isoenzyme pattern in sham-operated males and a similar effect was obtained upon treating hypophysectomized rats with thyroxine, cortisone acetate and a continuous infusion of growth hormone.
Biochemical Journal, Mar 15, 1988
Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjug... more Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4: glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4. Abbreviations used: CDNB, 1-chloro-2,4-dinitrobenzene; DCNB, inhibitor that gives 50%0 inhibition.
Biochemical Journal, Feb 1, 1988
Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as w... more 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 pl 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 /aM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 /tMbromosulphophthalein 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 150 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 cooperative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.
Diabetologia, Nov 15, 2006
Aims/hypothesis The amount of visceral fat mass strongly relates to insulin resistance in humans.... more Aims/hypothesis The amount of visceral fat mass strongly relates to insulin resistance in humans. The transcription factor peroxisome proliferator activated receptor gamma (PPARG) is abundant in adipocytes and regulates genes of importance for insulin sensitivity. Our objective was to study PPARG activity in human visceral and subcutaneous adipocytes and to compare this with the most common model for human disease, the mouse. Materials and methods We transfected primary human adipocytes with a plasmid encoding firefly luciferase controlled by PPARG response element (PPRE) from the acyl-CoA-oxidase gene and measured PPRE activity by emission of light. Results We found that PPRE activity was 6.6-fold higher (median) in adipocytes from subcutaneous than from omental fat from the same subjects (n=23). The activity was also 6.2-fold higher in subcutaneous than in intraabdominal fat cells when we used a PPARG ligand-binding domain-GAL4 fusion protein as reporter, demonstrating that the difference in PPRE activity was due to different levels of activity of the PPARG receptor in the two fat depots. Stimulation with 5 μmol/l rosiglitazone did not induce a PPRE activity in visceral adipocytes that was as high as basal levels in subcutaneous adipocytes. Interestingly, in mice of two different strains the PPRE activity was similar in visceral and subcutaneous fat cells. Conclusions/interpretation We found considerably lower PPARG activity in visceral than in subcutaneous primary human adipocytes. Further studies of the molecular mechanisms behind this difference could lead to development of drugs that target the adverse effects of visceral obesity.
Blood, Apr 15, 1998
Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RAR␣ fusion p... more Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RAR␣ fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RAR␣ fusion protein. Both PML-and PLZF-RAR␣ possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RAR␣ protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RAR␣, it had a very little effect on its association with the PLZF-RAR␣ fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RAR␣ was mediated by the N-terminal PLZF moiety of the chimera. It appears that N-CoR/histone deacetylase corepressor complex interacts directly in an ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF protein and is required, at least in part, for its function as a transcriptional repressor. As the above-noted results predict, histone deacetylase inhibitors antagonize oncogenic activities of the PML-RAR␣ fusion protein and partially relieve transcriptional repression by PLZF as well as inhibitory effect of PLZF-RAR␣ on ATRA response. Taken together, our results demonstrate involvement of nuclear receptor corepressor/ histone deacetylase complex in the molecular pathogenesis of APL and provide an explanation for differential sensitivities of PML-and PLZF-RAR␣-associated leukemias to ATRA. 1998 by The American Society of Hematology. A CUTE PROMYELOCYTIC leukemia (APL) is associated with the t(15;17)(q22;q21) reciprocal chromosomal translocation that causes the fusion of the retinoic acid receptor ␣ (RAR␣) locus with a gene of unknown function called PML (for promyelocytic leukemia) and expression of PML-RAR␣ chimeric proteins in all leukemic cells. 1-4 The wild-type PML protein localizes onto nuclear bodies (NBs; also called PML oncogenic domains or PODs). 5-7 Expression of the PML-RAR␣ chimeric protein causes delocalization of PML and other components of NBs to a microspeckled nuclear structure and differentiation of APL cells with all-trans-retinoic acid (ATRA) results in restoration of their normal localization in NBs. 5-8 At present, it is not clear what relationship, if any, this phenomenon has to the pathogenesis and treatment of APL. Expression of the PML-RAR␣ protein in transgenic mice results in the development of APL, which, as the human disease,
Nature, Oct 1, 1995
Retinoic acid receptors (RARs) and retinoid-X receptors (RXRs) activate or repress transcription ... more Retinoic acid receptors (RARs) and retinoid-X receptors (RXRs) activate or repress transcription by binding as heterodimers to DNA-response elements that generally consist of two direct repeat half-sites of consensus sequence AGGTCA. On response elements consisting of direct repeats spaced by five base pairs (DR + 5 elements), RAR/RXR heterodimers activate transcription in response to RAR-specific ligands, such as all-trans-retinoic acid (RA). In contrast, on elements consisting of direct repeats spaced by one base pair (DR + 1 elements), RAR/RXR heterodimers exhibit little or no response to activating ligands and repress RXR-dependent transcription. Here we show that ligand-dependent transactivation by RAR on DR + 5 elements requires the dissociation of a new nuclear receptor co-repressor, N-CoR, and recruitment of the putative co-activators p140 and p160. Surprisingly, on DR + 1 elements, N-CoR remains associated with RAR/RXR heterodimers even in the presence of RAR ligands, resulting in constitutive repression. These observations indicate that DNA-response elements can allosterically regulate RAR-co-repressor interactions to determine positive or negative regulation of gene expression.
Biochemical and Biophysical Research Communications, Apr 1, 2009
Leukotriene C 4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5... more Leukotriene C 4 is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C 4 synthase (LTC 4 S) participate in its biosynthesis. We report evidence that LTC 4 S interacts in vitro with both FLAP and 5-LO and that these interactions involve distinct parts of LTC 4 S. FLAP bound to the N-terminal part / first hydrophobic region of LTC 4 S. This part did not bind 5-LO which bound to the second hydrophilic loop of LTC 4 S. Fluorescent FLAP-and LTC 4 S-fusion proteins co-localized at the nuclear envelope. Furthermore, GFP-FLAP and GFP-LTC 4 S colocalized with a fluorescent ER marker. In resting HEK293/T or COS-7 cells GFP-5-LO was found mainly in the nuclear matrix. Upon stimulation with calcium ionophore, GFP-5-LO
Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reac... more Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5- ...
FEBS Letters, Aug 30, 2000
We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsoma... more We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein^protein interaction may contribute to the regulation of LTC 4 production.
Journal of Endocrinology, May 1, 2003
Peroxisome proliferator-activated receptor gamma (PPAR) colocalizes with oxidized low-density lip... more Peroxisome proliferator-activated receptor gamma (PPAR) colocalizes with oxidized low-density lipoprotein (LDL) in foam cells in atherosclerotic lesions. We have explored a potential role of oxidized fatty acids in LDL as PPAR activators. LDL from patients suffering from intermittent claudication due to atherosclerosis was analyzed using HPLC and gas chromatography/mass spectrophotometry and found to contain 9-hydroxyand 13-hydroxyoctadecadienoic acid (9-and 13-HODE), as well as 5-hydroxy-, 12-hydroxy-and 15hydroxyeicosatetraenoic acid (5-, 12-and 15-HETE respectively). PPAR was potently activated by 13(S)-HODE and 15(S)-HETE, as judged by transient transfection assays in macrophages or CV-1 cells. 5(S)-and 12(S)-HETE as well as 15-deoxy-12,14-prostaglandin J 2 also activated PPAR but were less potent. Interestingly, the effect of the lipoxygenase products 13(S)-HODE and 15(S)-HETE as well as of the drug rosiglitazone were preferentially enhanced by the coactivator CREB-binding protein, whereas the effect of the cyclooxygenase product 15-deoxy-12,14-prostaglandin J 2 was preferentially enhanced by steroid receptor coactivator-1. We interpret these results, which may have relevance to the pathogenesis of atherosclerosis, to indicate that the lipoxygenase products on the one hand and the cyclooxygenase product on the other exert specific effects on the transcription of target genes through differential coactivator recruitment by PPAR /9-cis retinoic acid receptor heterodimer complexes.
Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reac... more Leukotrienes (LT) are potent pro-inflammatory mediators formed from arachidonic acid (AA) in reactions catalyzed by 5-lipoxygenase and either leukotriene A4 hydrolase or leukotriene C4 synthase. 5- ...
Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells... more Acidic preparations of lysed platelets upregulate proliferative pathways in osteoblast-like cells as demonstrated by genome-wide microarray analysis,
Biochemical Journal, 1992
Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione tr... more Immunoblot experiments and reverse-phase h.p.l.c. were used to study the levels of glutathione transferase subunits 1, 2, 3, 4, 6, 7 and 8 in the liver and adrenal of intact and hypophysectomized male and female Sprague-Dawley rats. A sexual dimorphism in the levels of several of these isoenzymes and in their responses to hypophysectomy was demonstrated. In the liver of sham-operated females and males there are differences in glutathione transferase activities and isoenzyme pattern. H.p.l.c. analysis showed higher levels of subunits 1, 3 and 4 in male rats compared with females. In contrast with the pronounced sex differences in sham-operated rats, the isoenzyme patterns of hypophysectomized males and females were very similar. In the adrenal glands, however, a sexual dimorphism became apparent only after hypophysectomy, when the level of subunit 4 was increased 14-fold in the female, whereas the corresponding increase in the male rat was only 2.7-fold. The hepatic pattern of glutat...
Molecular Endocrinology, 1997
Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of li... more Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors that stimulate the transcription of target genes in the presence of activating ligands and repress transcription in their absence. Transcriptional repression by the thyroid hormone and retinoic acid receptors has been proposed to be mediated by the nuclear receptor corepressor, N-CoR, or the related factor, SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). Recent studies have suggested that transcriptional repression by N-CoR involves a corepressor complex that also contains mSin3A/B and the histone deacetylase, RPD3. In this manuscript, we demonstrate that transcriptional repression by the retinoic acid receptor can be either positively or negatively regulated by changes in the levels of N-CoR expression, suggesting a relatively strict stoichiometric relationship between N-CoR and other components of the corepressor complex. Consistent with this interpretation, overexpression of several functionally defined domains of N-CoR also relieve repression by nuclear receptors. N-CoR is distributed throughout the nucleus in a nonuniform pattern, and a subpopulation becomes concentrated into several discrete dot structures when highly expressed. RPD3 is also widely distributed throughout the nucleus in a nonuniform pattern. Simultaneous imaging of RPD3 and N-CoR suggest that a subset of each of these proteins colocalize, consistent with the existence of coactivator complexes containing both proteins. In addition, a substantial fraction of both N-CoR and mSin3 A/B appear to be independently distributed. These observations suggest that interactions between RPD3 and Sin3/N-CoR complexes may be dynamically regulated.
Advances in …, 2002
Prostaglandin D2 (PGD2) is a major arachidonic acid metabolite in mast cells, megakaryoblastic CM... more Prostaglandin D2 (PGD2) is a major arachidonic acid metabolite in mast cells, megakaryoblastic CMK cells and antigen presenting cells, for example various types of macrophages. It induces pulmonary vasoconstriction and bronchoconstriction. Increased ...
Biochemical Journal, 1988
Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjug... more Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. Th...
The Journal of Steroid Biochemistry and Molecular Biology, 2004
Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis... more Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor  and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif. This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoR LDNLL) fused to GST interacted with retinoic acid receptor ␣ (RAR␣) and thyroid hormone receptor  (TR) in vitro. Similarly, [ 35 S-methionine]N-CoR LDNLL interacted with a RAR␣ fusion protein. N-CoR LDNLL also bound to RAR␣ in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RAR␣ in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RAR␣ and TR.
Biochemical and Biophysical Research Communications, 1996
Microsomal glutathione transferase (mGT) specifically binds leukotriene C 4 synthase in the prese... more Microsomal glutathione transferase (mGT) specifically binds leukotriene C 4 synthase in the presence of Mg 2/ ion (Söderström et al., Protein Expression and Purification (1995) 6, 352-356). To investigate if this interaction occurs in vivo we screened a human lung cDNA library with a bait vector encoding human mGT in the yeast two-hybrid system. One of the five positive clones obtained encoded leukotriene C 4 synthase. This clone was expressed in two heterologous systems. The recombinant protein cross-reacted with a guinea pig antibody raised against a Keyhole limpet hemocyanin coupled synthetic peptide corresponding to amino acids 141-150 of human leukotriene C 4 synthase.
Blood, 1998
Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARα fusion p... more Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARα fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RARα fusion protein. Both PML- and PLZF-RARα possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RARα protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RARα, it had a very little effect on its association with the PLZF-RARα fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RARα was mediated by the N-termi...
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Papers by Mats Söderström