Chemokine Receptor Binding Research Articles

Investigation of candidate long noncoding RNAs and messenger RNAs in the immediate phase of spinal cord injury based on gene expression profiles

Spinal cord injury (SCI) is a serious devastating condition and it has a high mortality rate and morbidity rate. The early pathological changes in the immediate phase of SCI may play a major part in the development of secondary injury. Alterations in the expression of many long noncoding RNAs (lncRNAs) have been shown to play fundamental roles in the diseases of the central nervous system. However, the roles of lncRNAs and messenger RNAs (mRNAs) in the immediate phase of SCI are not clear. We examined the expression of mRNAs and lncRNAs in a rat model at 2 h after SCI and identified the differentially expressed lncRNAs (DE lncRNAs) and differentially expressed mRNAs (DE mRNAs) using microarray analysis. 772 DE lncRNAs and 992 DE mRNAs were identified in spinal cord samples in the immediate phase following SCI compared with control samples. Moreover, Gene Ontology (GO) term annotation results showed that CXCR chemokine receptor binding, neutrophil apoptotic process, neutrophil migration, neutrophil extravasation, macrophage differentiation, monocyte chemotaxis and cellular response to interleukin-1 (IL-1) were the main significantly enriched GO terms. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the DEGs were enriched in toll−like receptor signaling pathway, p53 signaling pathway, MAPK signaling pathway and Jak–STAT signaling pathway. IL6, MBOAT4, FOS, TNF, JUN, STAT3, CSF2, MYC, CCL2 and FGF2 were the top 10 high-degree hub nodes and may be important targets in the immediate phase of SCI. The current study on provides novel insights into how lncRNAs and mRNAs regulate the pathogenesis of the immediate phase after SCI.

Complex interplay of kinetic factors governs the synergistic properties of HIV-1 entry inhibitors

The homotrimeric HIV-1 envelope glycoprotein (Env) undergoes receptor-triggered structural changes that mediate viral entry through membrane fusion. This process is inhibited by chemokine receptor antagonists (CoRAs) that block Env-receptor interactions and by fusion inhibitors (FIs) that disrupt Env conformational transitions. Synergy between CoRAs and FIs has been attributed to a CoRA-dependent decrease in the rate of viral membrane fusion that extends the lifetime of the intermediate state targeted by FIs. Here, we demonstrated that the magnitude of CoRA/FI synergy unexpectedly depends on FI-binding affinity and the stoichiometry of chemokine receptor binding to trimeric Env. For C-peptide FIs (clinically represented by enfuvirtide), synergy waned as binding strength decreased until inhibitor combinations behaved additively. Curiously, this affinity dependence on synergy was absent for 5-Helix-type FIs. We linked this complex behavior to the CoRA dependence of Env deactivation following FI binding. For both FI classes, reducing chemokine receptor levels on target cells or eliminating competent chemokine receptor-binding sites on Env trimers resulted in a loss of synergistic activity. These data imply that the stoichiometry required for CoRA/FI synergy exceeds that required for HIV-1 entry. Our analysis suggests two distinct roles for chemokine receptor binding, one to trigger formation of the FI-sensitive intermediate state and another to facilitate subsequent conformational transitions. Together, our results could explain the wide variety of previously reported activities for CoRA/FI combinations. These findings also have implications for the combined use of CoRAs and FIs in antiviral therapies and point to a multifaceted role for chemokine receptor binding in promoting HIV-1 entry.

Long noncoding RNA and mRNA profiling in MDA-MB-231 cells following RNAi-mediated knockdown of SIRT7.

Breast cancer is one of the most common malignant cancers among women and a major clinical obstacle. Although studies have reported the abnormal expression of SIRT7 in breast cancer, whether the function of SIRT7 regulates the expression of long noncoding RNAs (lncRNAs) in breast cancer remains unknown. We aimed to determine the differential expressions of mRNAs and lncRNAs associated with SIRT7 and understand the regulatory mechanism of SIRT7 in breast cancer. RNA sequencing was performed to explore the transcriptome in MDA-MB-231 cells after SIRT7 depletion, and a total of 50,634 different transcripts were identified. In comparison with the negative control, siSIRT7 groups showed 240 differentially expressed mRNAs and 26 differentially expressed lncRNAs. Gene ontology analysis revealed that the differentially expressed mRNAs mainly regulated DNA replication, CXCR chemokine receptor binding, and maturation of large subunit rRNA from tricistronic rRNA transcript, nucleoplasm, mitochondrion, and NAD+ ADP-ribosyltransferase activity. Kyoto Encyclopedia of Genes and Genomes analysis showed that the differentially expressed mRNAs were mainly involved in pathways associated with MAPK signaling pathway, tumor necrosis factor signaling pathway, hepatitis B, and cancer. Moreover, the target genes of the differentially expressed lncRNAs mainly regulated the carboxylic acid metabolic processes and were involved in glycolysis pathway. The mRNA-lncRNA coexpression network comprised 186 mRNAs and 23 lncRNAs. Our results provide essential data regarding differentially expressed lncRNAs and mRNAs after the depletion of SIRT7 in breast cancer cells, which may be useful to elucidate the role of SIRT7 in breast cancer development.

HIV-1 gp120 envelope glycoprotein determinants for cytokine burst in human monocytes.

The first step of HIV infection involves the interaction of the gp120 envelope glycoprotein to its receptor CD4, mainly expressed on CD4+ T cells. Besides its role on HIV-1 entry, the gp120 has been shown to be involved in the production of IL-1, IL-6, CCL20 and other innate response cytokines by bystander, uninfected CD4+ T cells and monocytes. However, the gp120 determinants involved in these functions are not completely understood. Whether signalling leading to cytokine production is due to CD4 or other receptors is still unclear. Enhanced chemokine receptor binding and subsequent clustering receptors may lead to cytokine production. By using a comprehensive panel of gp120 mutants, here we show that CD4 binding is mandatory for cytokine outburst in monocytes. Our data suggest that targeting monocytes in HIV-infected patients might decrease systemic inflammation and the potential tissue injury associated with the production of inflammatory cytokines. Understanding how gp120 mediates a cytokine burst in monocytes might help develop new approaches to improve the chronic inflammation that persists in these patients despite effective suppression of viremia by antiretroviral therapy.

Aspartate‐Based CXCR4 Chemokine Receptor Binding of Cross‐Bridged Tetraazamacrocyclic Copper(II) and Zinc(II) Complexes

The CXCR4 chemokine receptor is implicated in a number of diseases including HIV infection and cancer development and metastasis. Previous studies have demonstrated that configurationally restricted bis-tetraazamacrocyclic metal complexes are high-affinity CXCR4 antagonists. Here, we present the synthesis of Cu(2+) and Zn(2+) acetate complexes of six cross-bridged tetraazamacrocycles to mimic their coordination interaction with the aspartate side chains known to bind them to CXCR4. X-ray crystal structures for three new Cu(2+) acetate complexes and two new Zn(2+) acetate complexes demonstrate metal-ion-dependent differences in the mode of binding the acetate ligand concomitantly with the requisite cis-V-configured cross-bridged tetraazamacrocyle. Concurrent density functional theory molecular modelling studies produced an energetic rationale for the unexpected [Zn(OAc)(H2 O)](+) coordination motif present in all of the Zn(2+) cross-bridged tetraazamacrocycle crystal structures, which differs from the chelating acetate [Zn(OAc)](+) structures of known unbridged and side-bridged tetraazamacrocyclic Zn(2+) -containing CXCR4 antagonists.

Deciphering chemokine properties by a hybrid agent-based model of Aspergillus fumigatus infection in human alveoli.

The ubiquitous airborne fungal pathogen Aspergillus fumigatus is inhaled by humans every day. In the lung, it is able to quickly adapt to the humid environment and, if not removed within a time frame of 4–8 h, the pathogen may cause damage by germination and invasive growth. Applying a to-scale agent-based model of human alveoli to simulate early A. fumigatus infection under physiological conditions, we recently demonstrated that alveolar macrophages require chemotactic cues to accomplish the task of pathogen detection within the aforementioned time frame. The objective of this study is to specify our general prediction on the as yet unidentified chemokine by a quantitative analysis of its expected properties, such as the diffusion coefficient and the rates of secretion and degradation. To this end, the rule-based implementation of chemokine diffusion in the initial agent-based model is revised by numerically solving the spatio-temporal reaction-diffusion equation in the complex structure of the alveolus. In this hybrid agent-based model, alveolar macrophages are represented as migrating agents that are coupled to the interactive layer of diffusing molecule concentrations by the kinetics of chemokine receptor binding, internalization and re-expression. Performing simulations for more than a million virtual infection scenarios, we find that the ratio of secretion rate to the diffusion coefficient is the main indicator for the success of pathogen detection. Moreover, a subdivision of the parameter space into regimes of successful and unsuccessful parameter combination by this ratio is specific for values of the migration speed and the directional persistence time of alveolar macrophages, but depends only weakly on chemokine degradation rates.

Systems biology of myasthenia gravis, integration of aberrant lncRNA and mRNA expression changes.

BackgroundA novel class of transcripts, long non-coding RNAs (lncRNAs), has recently emerged as a key player in several biological processes, and important roles for these molecules have been reported in a number of complex human diseases, such as autoimmune diseases, neurological disorders, and various cancers. However, the aberrant lncRNAs implicated in myasthenia gravis (MG) remain unknown. The aim of the present study was to explore the abnormal expression of lncRNAs in peripheral blood mononuclear cells (PBMCs) and examine mRNA regulatory relationship networks among MG patients with or without thymoma.MethodsMicroarray assays were performed, and the outstanding differences between lncRNAs or mRNA expression were verified through RT-PCR. The lncRNAs functions were annotated for the target genes using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The potential regulatory relationships between the lncRNAs and target genes were analyzed using the ‘cis’ and ‘trans’ model. Outstanding lncRNAs were organized to generate a TF-lncRNA-gene network using Cytoscape software.ResultsThe lncRNA and mRNA expression profile analysis revealed subsets of differentially expressed genes in MG patients with or without thymoma. A total of 12 outstanding dysregulated expression lncRNAs, such as lncRNA oebiotech_11933, were verified through real-time PCR. Several GO terms including the cellular response to interferon-γ, platelet degranulation, chemokine receptor binding and cytokine interactions were very important in MG pathogenesis. The chromosome locations of some lncRNAs and associated co-expression genes were demonstrated using ‘cis’ analysis. The results of the ‘trans’ analysis revealed that some TFs (i.e., CTCF, TAF1and MYC) regulate lncRNA and gene expression. The outstanding lncRNAs in each group were implicated in the regulation of the TF-lncRNA-target gene network.ConclusionThe results of the present study provide a perspective on lncRNA expression in MG. We identify a subset of aberrant lncRNAs and mRNAs as potential biomarkers for the diagnosis of MG. The GO and KEGG pathway analysis provides an annotation to determine the functions of these lncRNAs. The results of the ‘cis’ and ‘trans’ analyses provide information concerning the modular regulation of lncRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0087-z) contains supplementary material, which is available to authorized users.

Expression profiling and ontology analysis of long noncoding RNAs in post-ischemic heart and their implied roles in ischemia/reperfusion injury.

Long noncoding RNAs (lncRNAs) play important regulatory roles in cellular physiology. The contributions of lncRNAs to ischemic heart disease remain largely unknown. The aim of this study was to investigate the profile of myocardial lncRNAs and their potential roles at early stage of reperfusion. lncRNAs and mRNAs were profiled by microarray and the expression of some highly-dysregulated lncRNAs was further validated using polymerase chain reaction. Our results revealed that 64 lncRNAs were up-regulated and 87 down-regulated, while 50 mRNAs were up-regulated and 60 down-regulated in infarct region at all reperfusion sampled. Gene ontology analysis indicated that dysregulated transcripts were associated with immune response, spermine catabolic process, taxis, chemotaxis, polyamine catabolic process, spermine metabolic process, chemokine activity and chemokine receptor binding. Target gene-related pathway analysis showed significant changes in cytokine-cytokine receptor interaction, the chemokine signaling pathway and nucleotide oligomerization domain (NOD)-like receptor signaling pathway which have a close relationship with myocardial ischemia/reperfusion injury (MI/RI). Besides, a gene co-expression network was constructed to identify correlated targets of 10 highly-dysregulated lncRNAs. These lncRNAs may play their roles by this network in post-ischemic heart. Such results provide a foundation for understanding the roles and mechanisms of myocardial lncRNAs at early stage of reperfusion.

Lymphatic Specific Disruption in the Fine Structure of Heparan Sulfate Inhibits Dendritic Cell Traffic and Functional T Cell Responses in the Lymph Node

Dendritic cells (DCs) are potent APCs essential for initiating adaptive immunity. Following pathogen exposure, trafficking of DCs to lymph nodes (LNs) through afferent lymphatic vessels constitutes a crucial step in the execution of their functions. The mechanisms regulating this process are poorly understood, although the involvement of certain chemokines in this process has recently been reported. In this study, we demonstrate that genetically altering the fine structure (N-sulfation) of heparan sulfate (HS) specifically in mouse lymphatic endothelium significantly reduces DC trafficking to regional LNs in vivo. Moreover, this alteration had the unique functional consequence of reducing CD8(+) T cell proliferative responses in draining LNs in an ovalbumin immunization model. Mechanistic studies suggested that lymphatic endothelial HS regulates multiple steps during DC trafficking, including optimal presentation of chemokines on the surface of DCs, thus acting as a co-receptor that may function "in trans" to mediate chemokine receptor binding. This study not only identifies novel glycan-mediated mechanisms that regulate lymphatic DC trafficking, but it also validates the fine structure of lymphatic vascular-specific HS as a novel molecular target for strategies aiming to modulate DC behavior and/or alter pathologic T cell responses in lymph nodes.

A network approach to controlling pathogenic inflammation

Aberrant inflammation appears to be a pathogenic factor in autoimmune diseases and other noxious inflammatory conditions in which the inflammatory process is misapplied, exaggerated, recurrent or chronic. The protein molecules involved in pathogenic inflammation—disease-associated proteins (DAP)—which include chemokines, cytokines, and growth factors and their receptors, appear normal; their networks of interaction are at fault. Here we demonstrate a new approach to network regulation of inflammation based on peptide sequence motifs shared by the second extra-cellular loop (ECL2) of different chemokine receptors; previously known chemokine receptor binding sites have not involved the ECL2 loop. These motifs of 9 amino acids, which we detected by sequence alignment, manifest very low E-values compared with slightly modified sequence variations, indicating that they were not likely to have evolved by chance. To test whether this shared sequence network (SSN) might serve a regulatory function, we synthesized...

Molecular Mechanisms of Gradient Sensing in Ewing's Sarcoma Cells

Tumor growth and metastasis are processes exploiting chemotaxis - directed cell movement in a chemical gradient. Currently, a lot is known about the receptors and chemokine molecules responsible for chemotaxis. One essential pair is the G-protein coupled receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1). This pair was recently shown to play an important role in Ewing's sarcoma metastasis. The pathways following chemokine-receptor binding are known as well, but there is still not enough understanding of the molecular mechanisms of gradient sensing.Here we investigate a cell line derived from Ewing's sarcoma. Using single molecule imaging and microfluidics techniques, we study the cell's response to stimulation with SDF-1. For this purpose we analyze local receptor stoichiometries, dynamics of multimerization and analyze receptor mobility. In an integral approach those molecular insights are complemented by whole cell behavior characterization, like mobility in controlled chemokine gradients. Results of this work will highlight the molecular machinery of chemokine gradient recognition and its effect on cell motility in the context of cancer spreading.

Disulfide Bond That Constrains the HIV-1 gp120 V3 Domain Is Cleaved by Thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.

Body mass index-independent inflammation in omental adipose tissue associated with insulin resistance in morbid obesity

Background Obesity is a strong risk factor for resistance to insulin-mediated glucose disposal, a precursor of type 2 diabetes and other disorders. However, not all obese individuals are insulin resistant. We sought to identify the molecular pathways that might cause obesity-associated insulin resistance in humans by studying the morbidly obese who were insulin sensitive versus insulin resistant, thereby eliminating obesity as a variable. Methods Combining gene expression profiling with computational approaches, we determined the global gene expression signatures of omental and subcutaneous adipose tissue samples obtained from similarly obese patients undergoing gastric bypass surgery. Results Gene sets related to chemokine activity and chemokine receptor binding were identified as most highly expressed in the omental tissue from insulin-resistant compared with insulin-sensitive subjects, independent of the body mass index. These upregulated genes included chemokines (C-C motif) ligand 2, 3, 4, and 18 and interleukin-8/(CC-X motif) ligand 8 and were not differentially expressed in the subcutaneous adipose tissues between the 2 groups of subjects. Insulin resistance, but not the body mass index, was associated with increased macrophage infiltration in the omental adipose tissue, as was adipocyte size, in these morbidly obese subjects. Conclusion Our findings have demonstrated that inflammation of the omental adipose tissue is strongly associated with insulin resistance in human obesity even in subjects with similar body mass index values.

Inhibition of chemokine-glycosaminoglycan interactions in donor tissue reduces mouse allograft vasculopathy and transplant rejection.

BackgroundBinding of chemokines to glycosaminoglycans (GAGs) is classically described as initiating inflammatory cell migration and creating tissue chemokine gradients that direct local leukocyte chemotaxis into damaged or transplanted tissues. While chemokine-receptor binding has been extensively studied during allograft transplantation, effects of glycosaminoglycan (GAG) interactions with chemokines on transplant longevity are less well known. Here we examine the impact of interrupting chemokine-GAG interactions and chemokine-receptor interactions, both locally and systemically, on vascular disease in allografts.Methodology/Principal FindingsAnalysis of GAG or CC chemokine receptor 2 (CCR2) deficiency were coupled with the infusion of viral chemokine modulating proteins (CMPs) in mouse aortic allograft transplants (n = 239 mice). Inflammatory cell invasion and neointimal hyperplasia were significantly reduced in N-deacetylase-N-sulfotransferase-1 (Ndst1 f/f TekCre +) heparan sulfate (GAG)-deficient (Ndst1−/−, p<0.044) and CCR2-deficient (Ccr2−/−, p<0.04) donor transplants. Donor tissue GAG or CCR2 deficiency markedly reduced inflammation and vasculopathy, whereas recipient deficiencies did not. Treatment with three CMPs was also investigated; Poxviral M-T1 blocks CC chemokine receptor binding, M-T7 blocks C, CC, and CXC GAG binding, and herpesviral M3 binds receptor and GAG binding for all classes. M-T7 reduced intimal hyperplasia in wild type (WT) (Ccr2+/+, p≤0.003 and Ccr2−/−, p≤0.027) aortic allografts, but not in Ndst1−/− aortic allografts (p = 0.933). M-T1 and M3 inhibited WT (Ccr2+/+ and Ndst1 +/+, p≤0.006) allograft vasculopathy, but did not block vasculopathy in Ccr2−/− (p = 0.61). M-T7 treatment alone, even without immunosuppressive drugs, also significantly prolonged survival of renal allograft transplants (p≤0.001).Conclusions/SignificanceInterruption of chemokine-GAG interactions, even in the absence of chemokine-receptor blockade, is a highly effective approach to reduction of allograft rejection, reducing vascular inflammation and prolonging allograft survival. Although chemokines direct both local and systemic cell migration, interruption of inherent chemokine responses in the donor tissue unexpectedly had a greater therapeutic impact on allograft vasculopathy.

A V3 Loop-Dependent gp120 Element Disrupted by CD4 Binding Stabilizes the Human Immunodeficiency Virus Envelope Glycoprotein Trimer

Human immunodeficiency virus (HIV-1) entry into cells is mediated by a trimeric complex consisting of noncovalently associated gp120 (exterior) and gp41 (transmembrane) envelope glycoproteins. The binding of gp120 to receptors on the target cell alters the gp120-gp41 relationship and activates the membrane-fusing capacity of gp41. Interaction of gp120 with the primary receptor, CD4, results in the exposure of the gp120 third variable (V3) loop, which contributes to binding the CCR5 or CXCR4 chemokine receptors. We show here that insertions in the V3 stem or polar substitutions in a conserved hydrophobic patch near the V3 tip result in decreased gp120-gp41 association (in the unliganded state) and decreased chemokine receptor binding (in the CD4-bound state). Subunit association and syncytium-forming ability of the envelope glycoproteins from primary HIV-1 isolates were disrupted more by V3 changes than those of laboratory-adapted HIV-1 envelope glycoproteins. Changes in the gp120 beta2, beta19, beta20, and beta21 strands, which evidence suggests are proximal to the V3 loop in unliganded gp120, also resulted in decreased gp120-gp41 association. Thus, a gp120 element composed of the V3 loop and adjacent beta strands contributes to quaternary interactions that stabilize the unliganded trimer. CD4 binding dismantles this element, altering the gp120-gp41 relationship and rendering the hydrophobic patch in the V3 tip available for chemokine receptor binding.

Asymmetric Deactivation of HIV-1 gp41 following Fusion Inhibitor Binding

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

Bioluminescent CXCL12 fusion protein for cellular studies of CXCR4 and CXCR7

Chemokine CXCL12 and its two known receptors, CXCR4 and CXCR7, may play a role in diseases including tumor growth and metastasis, atherosclerosis, and HIV infection. Therefore, these molecules may be promising targets for drug development. While studies of cell signaling and high-throughput screening for drug discovery increasingly are based on luminescent assays because of their high sensitivity and signal-to-background ratio, there currently is no bioluminescent assay for chemokine[#x02013]chemokine receptor binding. To develop a bioluminescent probe for chemokine binding and cellular uptake, we fused CXCL12 to Gaussia luciferase (GL), an ATP-independent enzyme that is the smallest known luciferase. Fusing CXCL12 to Gaussia luciferase (CXCL12-GL) did not alter the bioluminescence emission spectrum and only minimally affected enzyme function under varying conditions of pH, temperature, and NaCl concentration. CXCL12-GL also activated CXCR4-dependent signaling to a comparable extent as unfused CXCL12. Using multiwell plate assays, we established that CXCR7 increases cell-associated CXCL12 to a significantly greater extent than CXCR4. We also showed that CXCL12-GL can be used to quantify inhibition of chemokine receptor binding by compounds that specifically target CXCR7. These data validate CXCL12-GL as a bioluminescent probe to investigate molecular functions of CXCR4 and CXCR7 and screen for compounds that modulate ligand-receptor binding.

Sialyltransferase ST3Gal-IV controls CXCR2-mediated firm leukocyte arrest during inflammation

Recent in vitro studies have suggested a role for sialylation in chemokine receptor binding to its ligand (Bannert, N., S. Craig, M. Farzan, D. Sogah, N.V. Santo, H. Choe, and J. Sodroski. 2001. J. Exp. Med. 194:1661–1673). This prompted us to investigate chemokine-induced leukocyte adhesion in inflamed cremaster muscle venules of α2,3 sialyltransferase (ST3Gal-IV)-deficient mice. We found a marked reduction in leukocyte adhesion to inflamed microvessels upon injection of the CXCR2 ligands CXCL1 (keratinocyte-derived chemokine) or CXCL8 (interleukin 8). In addition, extravasation of ST3Gal-IV−/− neutrophils into thioglycollate-pretreated peritoneal cavities was significantly decreased. In vitro assays revealed that CXCL8 binding to isolated ST3Gal-IV−/− neutrophils was markedly impaired. Furthermore, CXCL1-mediated adhesion of ST3Gal-IV−/− leukocytes at physiological flow conditions, as well as transendothelial migration of ST3Gal-IV−/− leukocytes in response to CXCL1, was significantly reduced. In human neutrophils, enzymatic desialylation decreased binding of CXCR2 ligands to the neutrophil surface and diminished neutrophil degranulation in response to these chemokines. In addition, binding of α2,3-linked sialic acid–specific Maackia amurensis lectin II to purified CXCR2 from neuraminidase-treated CXCR2-transfected HEK293 cells was markedly impaired. Collectively, we provide substantial evidence that sialylation by ST3Gal-IV significantly contributes to CXCR2-mediated leukocyte adhesion during inflammation in vivo.

Synthesis and structure–activity relationships of heteroaryl substituted-3,4-diamino-3-cyclobut-3-ene-1,2-dione CXCR2/CXCR1 receptor antagonists

Comprehensive SAR studies were undertaken in the 3,4-diaminocyclobut-3-ene-1,2-dione class of CXCR2/CXCR1 receptor antagonists to explore the role of the heterocycle on chemokine receptor binding affinities, functional activity, as well as oral exposure in rat. The nature of the heterocycle as well as the requisite substitution pattern around the heterocycle was shown to have a dramatic effect on the overall biological profile of this class of compounds. The furyl class, particularly the 4-halo adducts, was found to possess superior binding affinities for both the CXCR2 and CXCR1 receptors, functional activity, as well as oral exposure in rat versus other heterocyclic derivatives.

Dual GPCR and GAG mimicry by the M3 chemokine decoy receptor

Viruses have evolved a myriad of evasion strategies focused on undermining chemokine-mediated immune surveillance, exemplified by the mouse γ-herpesvirus 68 M3 decoy receptor. Crystal structures of M3 in complex with C chemokine ligand 1/lymphotactin and CC chemokine ligand 2/monocyte chemoattractant protein 1 reveal that invariant chemokine features associated with G protein–coupled receptor binding are primarily recognized by the decoy C-terminal domain, whereas the N-terminal domain (NTD) reconfigures to engage divergent basic residue clusters on the surface of chemokines. Favorable electrostatic forces dramatically enhance the association kinetics of chemokine binding by M3, with a primary role ascribed to acidic NTD regions that effectively mimic glycosaminoglycan interactions. Thus, M3 employs two distinct mechanisms of chemical imitation to potently sequester chemokines, thereby inhibiting chemokine receptor binding events as well as the formation of chemotactic gradients necessary for directed leukocyte trafficking.