Analysis of the calcium-induced arachidonic acid (AA) binding to S100A8/A9 revealed that maximal AA binding was achieved at molar ratios of 1 mol S100A8 and 1 mol S100A9 and for values greater than 3 ...calciums per EF-hand. The AA binding capacity was not induced by the binding of other bivalent cations, such as Zn
2+, Cu
2+, and Mg
2+, to the protein complex. In contrast, the binding of AA was prevented by the addition of either Zn
2+ or Cu
2+ in the presence of calcium, whereas Mg
2+ failed to abrogate the AA binding capacity. The inhibitory effect was not due to blocking the formation of S100A8/A9 as demonstrated by a protein-protein interaction assay. Fluorescence measurements gave evidence that both Zn
2+ and Cu
2+ induce different conformational changes thereby affecting the calcium-induced formation of the AA binding pocket within the protein complex. Due to the fact that the inhibitory effect of Zn
2+ was present at physiological serum concentrations, it is assumed that released S100A8/A9 may carry AA at inflammatory lesions, but not within the blood compartment.
Peroxisome proliferator-activated receptors (PPARs) play a role in inflammation and, in particular, PPARγ is involved in monocyte/macrophage differentiation. Members of the fatty acid-binding protein ...(FABP) family have been reported to function as transactivators for PPARs. Therefore, the expression of PPARs and FABPs in the myeloid lineage was investigated by real-time PCR and immunofluorescence analysis. We found adipocyte-, epidermal-, and heart-type FABP to be ubiquitously expressed within the myeloid lineage. In contrast, liver-type FABP was exclusively detected in murine alveolar macrophages (AM), confirmed on protein level by double fluorescence analysis. The PPAR subtypes also showed a temporally and spatially regulated expression pattern in myeloid cells: the β-subtype was expressed in bone marrow, peritoneal, and alveolar macrophages, whereas it was not detected in dendritic cells (DCs). The γ
1-isoform was present in all cells, however, at different levels, whereas the γ
2-isoform was expressed in alveolar macrophages and dendritic cells. A low level PPARα mRNA could be detected in peritoneal macrophages and immature dendritic cells but not in mature dendritic cells and bone marrow macrophages. Interestingly, PPARα mRNA was also absent in the alveolar macrophages although liver-type FABP was expressed, indicating that gene expression of liver-type FABP was independent of PPARα. Since liver-type FABP is known as transactivator of PPARγ the simultaneous expression of both proteins may have general implications for the activation of PPARγ in alveolar macrophages.
S100A9, also referred to as MRP14, is a calcium-binding protein whose expression is tightly regulated during differentiation
of myeloid cells. The present study was performed to study the cell type- ...and differentiation-specific transcriptional regulation
of the S100A9 gene. Analysis of the S100A9 promoter in MonoMac-6 cells revealed evidence for a novel regulatory region from
position â400 to â374 bp, termed myeloid-related protein regulatory element (MRE). MRE deletion resulted in a 5.2-fold reduction
of promoter activity. By electrophoretic mobility shift analysis two nuclear complexes binding to this region were identified
and referred to as MRE-binding complex A (MbcA) and MRE-binding complex B (MbcB). By mutagenesis the MRE-binding motif could
be narrowed to a 12-bp region. The relevance of MRE is deduced from the observations that the formation of either MRE-binding
complex A or MRE-binding complex B strongly correlated with S100A9 gene expression in a cell type-specific, activation- and
differentiation-dependent manner. Moreover, DNA affinity chromatography and Western blot studies indicate that a Kruppel-related
zinc finger protein and the transcriptional intermediary factor 1β (TIF1β) are involved in an MRE-binding complex, thereby
regulating the S100A9 gene expression.
The interaction of the Ca2+-binding protein S100A12 with RAGE (receptor of advanced glycation endproducts) has been considered as a novel proinflammatory axis, since blockage of RAGE/S100A12 ligation ...suppresses chronic cellular activation and tissue injury in mouse models. However, the existence of a murine S100A12 ortholog is unknown. Because experimental approaches failed to identify it, we started an analysis of gene locus evolution. Human S100A12 is localized in the S100 gene cluster between S100A8 and S100A9, which are neighbors in both mouse and human. Confirming identical gene order, we found a DNA region between the murine S100A8 and S100A9 genes that is 60.9% identical to a region of the human S100A12 gene, including the first exon. Instead of the second and third exon, we found homology to a region close to the human S100A9 locus. To exclude a murine S100A12 ortholog elsewhere in the genome, we used human S100A12 as query for TBlastN homology searches. The matches were either too short, or identity was too low, or they could clearly be identified as distinct S100 genes. Obviously, an S100A12 ortholog is neither present in mouse nor rat, indicating that S100A12 has been lost during rodent evolution, probably due to a deletion.
The two calcium- and zinc-binding proteins, S100A9 and S100 A8, abundant in myeloid cells are considered to play important roles in both calcium signalling and zinc homeostasis. Polymorphonuclear ...neutrophils from S100A9 ko mice are also devoid of S100A8. Therefore, S100A9-deficient neutrophils were used as a model to study the role of the two S100 proteins in the neutrophils's calcium and zinc metabolism. Analysis of the intracellular zinc level upon pyrithione and (±)-(E)-methyl-2-(E)-hydroxyimino-5-nitro-6-methoxy-3-hexeneamide (NOR-1) treatment revealed no differences between S100A9-deficient and wildtype neutrophils. Similar, the calcium signals were not distinguishable from S100A9-deficient and wildtype neutrophils upon stimulation with platelet activating factor (PAF), thapsigargin or macrophage inflammatory protein 1 alpha (MIP-1 alpha), indicating despite their massive expression S100A8/A9 do neither serve as calcium nor as zinc buffering proteins in granulocytes. In contrast, stimulation with adenosine-5′-triphosphate (ATP) induces a significant stronger increase of the intracellular free calcium level in S100A9-deficient cells compared to wildtype cells. Moreover, the ATP-induced calcium signal was still different when the cells were incubated in calcium free buffer suggesting that pirinergic receptors of the P
2Y class could be involved in this signalling pathway.
The hallmark of developing inflammatory lesions is the excess migration of recruited phagocytes together with the enhanced cell surface expression of adhesion molecules. Recent investigations give ...evidence that the two myeloid-related proteins MRP8 (S100A8) and MRP14 (S100A9), which are abundant in activated or recruited phagocytes, may have a modulatory role in inflammatory responses. S100A9 displays a regulatory role in the transendothelial migration of human monocytes, and the secreted S100A8/A9 complex may serve as a transport protein to move arachidonic acid to its target cells.
In order to evaluate the age dependency of enzymes involved in the energy-generating system, skeletal muscle specimens from rats of different ages were investigated for several mitochondrial enzymes. ...1-14Cpyruvate (+/- ADP) oxidation rates and pyruvate dehydrogenase complex (PDHC) activity increased significantly from low early values during the neonatal period to nearly adult values at the end of the suckling period. Other enzymes of the pyruvate oxidation route such as citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC detected a clear increase in the intensity of the bands of the alpha subunits of E1 (pyruvate dehydrogenase) and E2 (dihydrolipoyl transacetylase) within the first 3 weeks of life. The ratio between the individual PDHC proteins indicated that E1 alpha, the regulatory subunit of the multienzyme complex, is the most rapidly increasing protein with age.
Due to the low degree of sequence similarity it has been speculated that murine and human S100A9 (MRP14), an inflammatory marker protein belonging to the S100 protein family, may have different ...cellular functions in mouse and man. The present study was undertaken to investigate the murine S100A9 protein (mS100A9) biochemically. We demonstrate that in murine peripheral CD11b+ cells up to 20% of the protein of the cytosolic fraction consists of mS100A9 and that several minor mS100A9 isoforms are present. Cell fractionation experiments with CD11b+ murine leukocytes showed that mS100A9 is found in the cytosol as well as in the insoluble fraction. Transient expression of a green fluorescence protein–mS100A9 fusion in mammalian cells revealed that mS100A9 is localized in neither the nucleus nor the vesicles. Recombinantly expressed murine S100A9 interacts in vitro with murine and human S100A8 in an in vitro glutathione S‐transferase pull‐down assay. Homodimerization was not observed. For further biochemical analysis the myeloid 32D cell line is presented as a suitable model, to study murine myeloid expressed S100 proteins. Both murine S100A9 and its dimerization partner mS100A8 are expressed at the onset of granulocyte‐colony stimulating factor induced myeloid differentiation. Substantial amounts of this complex are constitutively secreted by granulocytic 32D cells into the medium. In summary, these data suggest, that the human and murine S100A9 may share a higher degree of functional homology than of sequence similarity.
The present investigation was undertaken to gain insights into the nature of both substrate binding sites of acyl-CoA:lysophospholipid acyltransferase (LAT) which could be potentially useful for the ...identification and purification of this specific acyltransferase. Therefore, we have investigated the specificity of LAT from crude membranes of pig spleen toward various 1-palmitoyl-glycerophospholipids and 1-acyl-glycerophosphocholines (1-acyl-GPC). The enzyme showed the highest specificity toward 1-acyl-GPC and was able to distinguish between the acyl-chain length of the 1-acyl group within the 1-acyl-GPC molecule. We found preferential reactivity in the order C10:0 < C12:0 << C14:0, C18:0, C16:0 < C18:1 of 1-acyl-GPC. Lysophosphatidic acid or 1-O-alkyl-GPC were only poor substrates for the enzyme. In competition studies we could show that palmitic acid, oleic acid, arachidonic acid, and palmitoyl-CoA competitively inhibited LAT activity, whereas the coenzyme A failed to inhibit LAT enzyme activity in a concentration-dependent manner. We concluded that the ligand acyl-CoA is bound via its acyl chain. The finding that palmitoyl-CoA was a poor substrate as well as an inhibitor was the basis for protein purification. When palmitoyl-CoA–agarose was used as matrix for affinity chromatography, LAT enzyme activity was bound and eluted by high salt concentrations yielding an estimated 10-fold purification of the solubilized LAT enzyme.
One of the earliest changes observed in activated lymphocytes is the enhanced incorporation of unsaturated fatty acids into membrane phospholipids catalyzed by phospholipases and acyltransferases. ...This early membrane phospholipid remodeling has been shown to be independent from protein synthesis. We have investigated the oleic acid incorporation into phospholipids of activated T-lymphocytes within hours and present data that the sustained membrane phospholipid remodeling in activated T-lymphocytes was largely decreased by cycloheximide and actinomycin D treatment while neither protein synthesis inhibitor had an effect on the fatty acid incorporation into phospholipids in resting T-lymphocytes. Lisofylline, an inhibitor of lysophosphatidic acid:acyl-CoA acyltransferase, had no inhibitory activity, indicating that the membrane lipid remodeling was not due to fatty acid incorporation into de novo-synthesized phospholipids. The membrane phospholipid alteration induced by mitogens was also diminished by hydrocortisone (HC) in a concentration-dependent manner. The steroid hormone antagonist RU486 failed to reverse but potentiated this inhibitory activity of HC. HC did not affect the fatty acid uptake, and the decrease of fatty acid incorporation into phospholipids induced by HC was accompanied by an increase of fatty acid incorporation into triglycerides, indicating that the inhibitory activity of HC was specific for fatty acid incorporation into phospholipids catalyzed by lysophospholipid:acyl-CoA acyltransferase (LAT). HC did not directly inhibit the LAT enzyme activity. From these data we conclude that LAT gene transcription is induced as an early event following T-cell activation. The inhibitory action of hydrocortisone may give new insights into the regulatory mechanisms involved in LAT expression.
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