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Query: UNIPROT:P10145 (IL-8)
 23,849 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The chemokine family forms two different types of homodimer despite members sharing nearly identical folds. To study the formation of quaternary structure in this family, rational mutagenesis was employed on a representative member of each subfamily (MIP-1beta and IL-8). The variants were studied by analytical ultracentrifugation and NMR, and it was determined that formation of a folded monomer from a natural chemokine dimer is reasonably facile, while conversion between dimer types is not. Monomeric variants of MIP-1beta and IL-8 were randomly mutated and a lambda phage-based selection system was employed in a novel way to screen for dimerization. A total of 6,000,000 random mutants were screened, but no dimers were formed, suggesting again that the chemokine fold is robust and amenable to sequence variation, while the chemokine dimer is much more difficult to attain. This work represents a biophysical analysis of an array of chemokine quaternary state variants.
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PMID:Investigation of CC and CXC chemokine quaternary state mutants. 1625 37

In this study, three novel maloyl glucans were isolated at temperatures below 15 degrees C from aloe vera gel (Aloe barbadensis Miller). These compounds were characterized using NMR spectroscopy, ESIMS, MALDITOF-MS and capillary electrophoresis. The compounds were characterized as 6-O-(1-L-maloyl)-alpha-,beta-D-Glcp (veracylglucan A), alpha-D-Glcp-(1-->4)-6-O-(1-L-maloyl)-alpha,-beta,-D-Glcp (veracylglucan B) and alpha-D-Glcp-(1-->4)-tetra-[6-O-(1-L-maloyl)-alpha-D-Glcp-(1-->4)]-6-O-(1-L-maloyl)-alpha,-beta-D-Glcp (veracylglucan C). These unusual malic acid acylated carbohydrates were then tested in vitro for effects on cell proliferation and gene expression of proinflammatory cytokines, IL-6, IL-8 and ICAM-1, using RT-PCR. Veracylglucan B demonstrated potent anti-inflammatory and anti-proliferative effects, while Veracylglucan C, on the other hand, exhibited significant cell proliferative and anti-inflammatory activities. Veracylglucan A could only be isolated in smaller quantities, and it proved to be very unstable. Thus no biological effects could be observed in this respect. The in vitro bioassays also indicated that Veracylglucan B and C are antagonistic and competitive in their effects on cell proliferation. The results of this work represent a major step forward in the research on aloe vera gel. This is the first time that two fully chemically characterized compounds are shown to be responsible for known biological activities of aloe vera gel.
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PMID:Novel bioactive maloyl glucans from aloe vera gel: isolation, structure elucidation and in vitro bioassays. 1634 66

Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.
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PMID:A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities. 1643 Nov 16

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.
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PMID:Probing receptor binding activity of interleukin-8 dimer using a disulfide trap. 1678 40

Pneumococcal lipoteichoic acid (LTA) is known to have a completely different chemical structure compared with that of Staphylococcus aureus: the polyglycerophosphate in the backbone is replaced in the pneumococcal LTA by a pentamer repeating unit consisting of one ribitol and a tetrasaccharide carrying the unusual substituents phosphocholine and N-acetyl-D-galactosamine. Neither D-alanine nor N-acetyl-D-glucosamine, which play central roles in the biological activity of the staphylococcal LTA, has been reported. The extraction using butanol is more gentle compared with the previously reported chloroform-methanol extraction and results in a higher yield of LTA. We characterized the LTA of two different strains of Streptococcus pneumoniae:R6 (serotype 2) and Fp23 (serotype 4). NMR analysis confirmed the structure of LTA from R6 but showed that its ribitol carries an N-acetyl-D-galactosamine substituent. The NMR data for the LTA from Fp23 indicate that this LTA additionally contains ribitol-bound D-alanine. Dose-response curves of the two pneumococcal LTAs in human whole blood revealed that LTA from Fp23 was significantly more potent than LTA from R6 with regard to the induction of all cytokines measured (tumor necrosis factor, interleukin-1 (IL-1), IL-8, IL-10, granulocyte colony-stimulating factor, and interferon gamma). However, other characteristics, such as lack of inhibition by endotoxin-specific LAL-F, Toll-like receptor 2 and not 4 dependence, and lack of stimulation of neutrophilic granulocytes, were shared by both LTAs. This is the first report of a difference in the structure of LTA between two pneumococcal serotypes resulting in different immunostimulatory potencies.
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PMID:Comparison of lipoteichoic acid from different serotypes of Streptococcus pneumoniae. 1694 91

We previously reported the mode of inhibition of DNA polymerase beta (pol. beta) by long chain fatty acids and a bile acid, involving binding analyses to the N-terminal 8-kDa DNA binding domain. Here we describe a site-directed mutational analysis in which the key amino acids (L11, K35, H51, K60, L77, and T79), which are direct interaction sites in the domain, were substituted with K, A, A, A, K, and A, respectively. And their pol. beta interactions with a C24-long chain fatty acid, nervonic acid (NA), and a bile acid, lithocholic acid (LCA), were investigated by gel mobility shift assay and NMR spectroscopy. In the case of K35A, there was complete loss of DNA binding activity while K60A hardly has any activity. In contrast the other mutations had no appreciable effects. Thus, K35 and K60 are key amino acid sites for binding to template DNA. The DNA binding activities of L11K, H51A, and T79A as well as the wild type were inhibited by NA to the same extent. T79A demonstrated a disturbed interaction with LCA. 1H-15N HSQC NMR analysis indicated that despite their many similarities, the wild-type and the mutant proteins displayed some significant chemical shift differences. Not only were the substituted amino acid residues three-dimensionally shifted, but some amino acids which are positioned far distant from the key amino acids showed a shift. These results suggest that the interaction surface was significantly distorted with the result that LCA could not bind to the domain. These findings confirm our previous biochemical and 3D structural proposals concerning inhibition by NA and LCA.
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PMID:Site-directed mutational analysis of structural interactions of low molecule compounds binding to the N-terminal 8 kDa domain of DNA polymerase beta. 1699 74

The three-dimensional structure of human interleukin-8 (hIL-8) was determined by the use of NMR and X-ray methodology. At high concentrations interleukin-8 and many other chemokines form a non-covalent homodimer. Several studies have been performed to investigate the relevance of the dimer on receptor activation and led to contradictory results. In order to obtain a better understanding of the dimerisation process, covalently linked homo- and heterodimers were produced by photo-induced dimerisation of hIL-8 analogues that contain the photo-activatable amino acid p-benzoyl-phenylalanine (Bpa) at different positions. Whereas the N-terminal fragment (1-54) was expressed as recombinant thioester, the C-terminal fragments (55-77) that contain Bpa either at position 65 or 74 were obtained by solid-phase peptide synthesis. The segments were combined by expressed protein ligation and led to full length IL-8 variants containing the non-proteinogenic amino acid Bpa at single positions. IP(3) activity tests showed high biological activity for the CXCR1-GFP receptor for both variants comparable to that of the native ligand. The refolded and purified ligation-products were used for dimer formation by UV-irradiation. The analysis of the reaction mixture was performed by gel-electrophoresis and mass spectrometry and showed that dimer formation of IL-8 occurred in a position dependent manner. [Bpa(74)]hIL-8 has a high tendency to form covalent dimers whereas no dimer formation was observed for the variant with Bpa at position 65. Accordingly one residue of the dimerisation interface could be identified.
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PMID:Identification of the dimerisation interface of human interleukin-8 by IL-8-variants containing the photoactivatable amino acid benzoyl-phenylalanine. 1702 63

The three-dimensional structure of IL-8/CXCL8 has been previously determined using NMR spectroscopy and X-ray crystallography, but the structure of the receptors for this chemokine has not been determined experimentally. We present here the development of a model for the structure of the IL-8/CXCL8 receptor CXCR1, using a combination of homology modeling and a molecular dynamics simulation. Based on this model, we discuss the analysis of structural, dynamic, and physicochemical properties of CXCR1. We focused on the role of pairwise ionic interactions in local structural stability of CXCR1 and the role of electrostatic potentials in recognition of CXCR1 with IL-8/CXCL8. We have performed theoretical mutations of six charged amino acids in CXCR1, which abolish binding as suggested by earlier experimental data, to shed light on the effect of charge on association ability. We propose that the observed loss of binding in the six CXCR1 mutants is owed to loss of local structural stability, rather than hindrance of the recognition process because of changes in the overall electrostatic properties of the receptor. Based on further structural analysis, we propose some mutations of charged residues involving ion pairs in different elements of transmembrane helices and extracellular loops, which are expected to alter the local structure and possibly affect binding.
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PMID:Computational studies of CXCR1, the receptor of IL-8/CXCL8, using molecular dynamics and electrostatics. 1787 99

Polypropylene glycol (PPG) is commonly added to bacterial cultures to avoid foaming. However, lipoteichoic acid (LTA) from bacteria grown with PPG lacked cytokine-inducing potency in human blood. We tested the blocking efficacy of several glycols on the cytokine response to staphylococcal LTA in human blood. PPG 1200 was the most potent inhibitor tested, shown for TNF, IL-1beta, IL-6, IL-8, IL-10 and TGF-beta induction, and displayed no cytotoxic effects. TNF induction by Staphylococcus aureus or by Toll-like receptor (TLR)2 agonists (di- and triacylated lipopeptides and LTA) was also inhibited by PPG 1200, but not that induced by Escherichia coli or TLR4 agonists. In flow cytometric studies, PPG-carrying nanobeads bound more rhodamine-labeled LTA than those with glycerol. Additionally, the methyl group peak in the (1)H-NMR of LTA shifted after incubation with increasing PPG 1200 concentrations. Sequential incubation of polystyrene plates with LTA, then PPG 1200 and then blood, with washing steps in between, showed that LTA-induced TNF release was inhibited. But when PPG 1200 was pre-incubated with blood that was washed before LTA was added, TNF induction was not repressed, demonstrating that PPG binds LTA and not cellular structures. In summary, PPG 1200 is a novel inhibitor of cytokine induction by TLR2 agonists, which interferes directly with the ligands.
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PMID:Polypropylene glycol is a selective binding inhibitor for LTA and other structurally related TLR2 agonists. 1826 69

In the chemokine family, we characterize two examples of evolutionarily conserved unfavorable sequence motifs that affect quaternary structure. In contrast to the straightforward action of favorable sequences, these unfavorable motifs produce interactions disfavoring one outcome to indirectly promote another one but should not be confused with the broad sampling produced by negative selection and/or design. To identify such motifs, we developed a statistically validated computational method combining structure and phylogeny. This approach was applied in an analysis of the alternate forms of homodimerization exhibited in the chemokine family. While the chemokine family exhibits the same tertiary fold, members of certain subfamilies, including CXCL8, form a homodimer across the beta1 strand whereas members of other subfamilies, including CCL4 and CCL2, form a homodimer on the opposite side of the chemokine fold. These alternate dimerization states suggest that CCL4 and CCL2 contain specific sequences that disfavor CXCL8 dimerization. Using our computational approach, we identified two evolutionarily conserved sequence motifs in the CC subfamilies: a drastic two-residue deletion (DeltaRV) and a simple point mutation (V27R). Cloned into the CXCL8 background, these two motifs were experimentally proven to confer a monomeric state. NMR analyses indicate that these variants are structured in solution and retain the chemokine fold. Structurally, the motifs retain a chemokine tertiary fold while introducing unfavorable quaternary interactions that inhibit CXCL8 dimerization. In demonstrating the success of our computational method, our results argue that these unfavorable motifs have been evolutionarily conserved to specifically disfavor one dimerization state and, as a result, indirectly contribute to favoring another.
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PMID:Conservation of unfavorable sequence motifs that contribute to the chemokine quaternary state. 1878 76


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