Arachidonate 15-lipoxygenase (arachidonate:oxygen 15-oxidoreductase, EC 1.13.11.33) is a lipidperoxidating enzyme that is implicated in oxidizing low density lipoprotein to its atherogenic form. Monocyte/macrophage 15-lipoxygenase is present in human atherosclerotic lesions. To pursue a basis for induction of the enzyme, which is not present in blood monocytes, the ability of relevant cytokines to regulate its expression was investigated. Interleukin 4 (IL4), among 16 factors tested, specifically induced 15-lipoxygenase mRNA and protein in cultured human monocytes. Interferon y and hydrocortisone inhibited this induction. High-performance liquid chromatography analysis of lipid extracts from IL-4-treated monocytes detected 15-lipoxygenase products esterified to the cellular membrane lipids, indicating enzymatic action on endogenous substrates. Stimulation of IL-4-treated monocytes with calcium ionophore or opsonized zymosan A enhanced the formation of 15-lipoxygenase products. These data identify 1L14 and interferon y as physiological regulators of lipoxygenase expression and suggest an important link between 15-lipoxygenase function and the immune/inflammatory response in atherosclerosis as well as other diseases.
To address the role of dimerization in the function of the monocyte chemoattractant protein-1, MCP-1, we mutated residues that comprise the core of the dimerization interface and characterized the ability of these mutants to dimerize and to bind and activate the MCP-1 receptor, CCR2b. One mutant, P8A*, does not dimerize. However, it has wild type binding affinity, stimulates chemotaxis, inhibits adenylate cyclase, and stimulates calcium influx with wild type potency and efficacy. These data suggest that MCP-1 binds and activates its receptor as a monomer. In contrast, Y13A*, another monomeric mutant, has a 100-fold weaker binding affinity, is a much less potent inhibitor of adenylate cyclase and stimulator of calcium influx, and is unable to stimulate chemotaxis. Thus Tyr 13 may make important contacts with the receptor that are required for high affinity binding and signal transduction. We also explored whether a mutant, Chemokines are small secreted proteins that function as intercellular messengers to control migration and activation of specific subsets of leukocytes (1-3). This process is mediated by the interaction of chemokines with seven transmembrane Gprotein-coupled receptors on the surface of target cells. Interest in these proteins was first stimulated by the observation of elevated levels in a number of inflammatory diseases (4, 5) including rheumatoid arthritis (6, 7), arteriosclerosis (8, 9), and asthma (10). Although it is not clear whether excessive production is the cause or consequence of these diseases, the demonstration that neutralizing antibodies (11-13) reduced symptoms in a number of animal models generated optimism that receptor antagonists may have therapeutic benefit (14). Recently, it has been shown that certain chemokine receptors also serve as obligate coreceptors for entry of the human immunodeficiency virus into CD4ϩ cells (15)(16)(17) and that viral replication can be inhibited by the ligands of the coreceptors (18 -21). Thus a wide range of clinically important diseases are associated with chemokines and their receptors, motivating many studies to understand the molecular details of chemokine function.Chemokines have been classified into two major families based on their pattern of cysteine residues, their chromosomal location, and their cell specificities (22). ␣-Chemokines such as IL-8 1 have a conserved CXC cysteine motif and act predominantly on neutrophils, whereas -chemokines have a CC signature and attract monocytes and T-cells. The recently discovered chemokines lymphotactin (23) and fractalkine/neurotactin (24,25) are characterized by C and CX 3 C motifs, respectively, and chemoattract T-cells and NK cells. Mutagenesis studies, particularly of ␣-and -chemokines, have provided some insight into the structural determinants of receptor binding and the specificity of these proteins, but many details have yet to emerge.Considerable effort has been devoted to characterizing the stochiometry of chemokine-receptor complexes because most chemokines oligomerize to an extent tha...
Monocyte chemoattracant-1 (MCP-1) stimulates leukocyte chemotaxis to inflammatory sites, such as rheumatoid arthritis, atherosclerosis, and asthma, by use of the MCP-1 receptor, CCR2, a member of the G-proteincoupled seven-transmembrane receptor superfamily. These studies identified a family of antagonists, spiropiperidines. One of the more potent compounds blocks MCP-1 binding to CCR2 with a K d of 60 nM, but it is unable to block binding to CXCR1, CCR1, or CCR3. These compounds were effective inhibitors of chemotaxis toward MCP-1 but were very poor inhibitors of CCR1-mediated chemotaxis. The compounds are effective blockers of MCP-1-driven inhibition of adenylate cyclase and MCP-1-and MCP-3-driven cytosolic calcium influx; the compounds are not agonists for these pathways. We showed that glutamate 291 (Glu 291 ) of CCR2 is a critical residue for high affinity binding and that this residue contributes little to MCP-1 binding to CCR2. The basic nitrogen present in the spiropiperidine compounds may be the interaction partner for Glu 291 , because the basicity of this nitrogen was essential for affinity; furthermore, a different class of antagonists, a class that does not have a basic nitrogen (2-carboxypyrroles), were not affected by mutations of Glu 291 . In addition to the CCR2 receptor, spiropiperidine compounds have affinity for several biogenic amine receptors. Receptor models indicate that the acidic residue, Glu 291, from transmembrane-7 of CCR2 is in a position similar to the acidic residue contributed from transmembrane-3 of biogenic amine receptors, which may account for the shared affinity of spiropiperidines for these two receptor classes. The models suggest that the acid-base pair, Glu 291 to piperidine nitrogen, anchors the spiropiperidine compound within the transmembrane ovoid bundle. This binding site may overlap with the space required by MCP-1 during binding and signaling; thus the small molecule ligands act as antagonists. An acidic residue in transmembrane region 7 is found in most chemokine receptors and is rare in other serpentine receptors. The model of the binding site may suggest ways to make new small molecule chemokine receptor antagonists, and it may rationalize the design of more potent and selective antagonists.Chemokines are a large family of small proteins that mediate attraction of leukocytes to inflammatory sites (1-3). The chemokine family shares a common pattern of disulfide bonds and a common overall tertiary structure as shown in solution or crystallographically determined structures (4 -6). The chemokine family is divided into four subfamilies based on the number of residues between the first and second cysteine. Among the chemokines, the CC chemokine monocyte chemoattracant-1 (MCP-1) 1 has received a great deal of attention because of its involvement in diseases. MCP-1 expression is elevated in the inflamed synovium of rheumatoid arthritis, and its expression is reduced by anti-arthritic drugs (7,8). Other work has shown that MCP-1 is elevated in asthmatic patients; the am...
The CC chemokine, MCP-1, has been identified as a major chemoattractant for T cells and monocytes, and plays a significant role in the pathology of inflammatory diseases. To identify the regions of MCP-1 that contact its receptor, CCR2, we substituted all surface-exposed residues with alanine. Some residues were also mutated to other amino acids to identify the importance of charge, hydrophobicity, or aromaticity at specific positions. The binding affinity of each mutant for CCR2 was assayed with THP-1 and CCR2-transfected CHL cells. The majority of point mutations had no effect. Residues at the N-terminus of the protein, known to be crucial for signaling, contribute less than a factor of 10 to the binding affinity. However, two clusters of primarily basic residues (R24, K35, K38, K49, and Y13), separated by a 35 A hydrophobic groove, reduced the level of binding by 15-100-fold. A peptide fragment encompassing residues 13-35 recapitulated some of the mutational data derived from the intact protein. It exhibited modest binding as a linear peptide and dramatically improved affinity when the region which adopts a single turn of a 3(10)-helix in the protein, which includes R24, was constrained by a disulfide bond. Additional constraints at the ends of the peptide, corresponding to the disulfide between the first and third cysteines in MCP-1, yielded further improvements in affinity. Together, these data suggest a model in which a large surface area of MCP-1 contacts the receptor, and the accumulation of a number of weak interactions results in the 35 pM affinity observed for the wild-type (WT) protein. The receptor binding site of MCP-1 also is significantly different from the binding sites of RANTES and IL-8, providing insight into the issue of receptor specificity. It was previously shown that the N-terminus of CCR2 is critical for binding MCP-1 [Monteclaro, F. S., and Charo, I. F. (1996) J. Biol. Chem. 271, 19084-92; Monteclaro, F. S., and Charo, I. F. (1997) J. Biol. Chem. 272, 23186-90]. Point mutations of six acidic residues in this region of the receptor were made to test their role in ligand binding. This identified D25 and D27 of the DYDY motif as being important. On the basis of our data, we propose a model in which the receptor N-terminus lies along the hydrophobic groove in an extended fashion, placing the DYDY motif near the basic cluster involving R24 and K49 of MCP-1. This in turn orients the signaling residues (Y13 and the N-terminus) for productive interaction with the receptor.
Abstract-To study the possible role of the human lipid-oxidizing enzyme 15-lipoxygenase (15-LO) in atherosclerosis, we overexpressed it specifically in the vascular wall of C57B6/SJL mice by using the murine preproendothelin-1 promoter. The mice overexpressing 15-LO were crossbred with low density lipoprotein (LDL) receptor-deficient mice to investigate atherogenesis. High levels of 15-LO were expressed in the atherosclerotic lesion in the double-transgenic mice as assessed by immunohistochemistry. The double-transgenic, 15-LO-overexpressing, LDL receptor-deficient mice (LDLR Ϫ/Ϫ /15LO) developed significantly larger atherosclerotic lesions at the aortic sinus compared with lesions in the LDL receptor-deficient (LDLR Ϫ/Ϫ ) mice after 3 and 6 weeks (107 000 versus 28 000 m 2 [PϽ0.001] and 121 000 versus 87 000 m 2 [PϽ0.05], respectively) of an atherogenic diet. LDL from the LDLR Ϫ/Ϫ /15LO mice was more susceptible to oxidation than was the LDL from the control LDLR Ϫ/Ϫ mice, as shown by a shorter lag period for copper-induced conjugated diene formation. On the other hand, no differences were found in the levels of serum anti-oxidized LDL antibodies between the study groups. There were also no differences with respect to the density of macrophages and T lymphocytes infiltrating the lesions in both experimental groups. Taken together, these results support the hypothesis that 15-LO overexpression in the vessel wall is associated with enhanced atherogenesis.
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