Molecular-size reduction of a potent CXCR4-chemokine antagonist using orthogonal combination of conformation- and sequence-based libraries.
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Amino Acid Sequence↗
Biomedical subjects
Stephen C Peiper
Publications and source records attributed to Stephen C Peiper.
Constitutive activation of CCR5 and CCR2 induced by conformational changes in the conserved TXP motif in transmembrane helix 2.
CCR5 is a G protein-coupled receptor for RANTES, MIP-1alpha, MIP-1beta, and MCP-2 that functions as the front line coreceptor for human immunodeficiency virus type 1 infection. To elucidate the mechanism for CCR5 activation, this coreceptor was expressed in yeast coupled to the pheromone response pathway and a constitutively active mutant (CAM) was derived by random mutagenesis. Conversion of Thr-82 in the highly conserved TXP motif in transmembrane helix 2 to Pro, His, Tyr, Arg, or Lys conferred autonomous signaling activity in yeast and mammalian cells. This substitution also imparted constitutive signaling to CCR2 in yeast and mammalian cells, but not CCR1, CCR3, CCR4, CXCR2, or CXCR4. The CCR5-CAM, but not the CCR2-CAM had a reduction in ligand binding affinity. Whereas the amplitude of calcium mobilization induced by RANTES stimulation was lower in the CCR5-CAM than the wild-type (WT) receptor, MCP-1 induced a higher signal in the CCR2-CAM than in CCR2-WT. The chemotactic response of CCR5-CAM(T82P) to RANTES was similar to that of CCR5-WT, but CCR5-CAM(T82K) was dramatically decreased. The chemotactic response of CCR2-WT and CCR2-CAM(T94K) were similar. These findings extend insight into the role of the TXP motif in the mechanism for CCR5 signaling. CCR2, the receptor most closely genetically related to CCR5, shared a similar signaling mechanism, but other receptors containing the TXP motif did not. The expression of CCR5 and CCR2 in yeast and the availability of variants with autonomous signaling represent critical tools for characterizing receptor antagonists and developing approaches to block their role in human diseases.
Amino Acid Motifs↗
Duffy antigen facilitates movement of chemokine across the endothelium in vitro and promotes neutrophil transmigration in vitro and in vivo.
The Duffy Ag expressed on RBCs, capillaries, and postcapillary venular endothelial cells binds selective CXC and CC chemokines with high affinity. Cells transfected with the Duffy Ag internalize but do not degrade chemokine ligand. It has been proposed that Duffy Ag transports chemokines across the endothelium. We hypothesized that Duffy Ag participates in the movement of chemokines across the endothelium and, by doing so, modifies neutrophil transmigration. We found that the Duffy Ag transfected into human endothelial cells facilitates movement of the radiolabeled CXC chemokine, growth related oncogene-alpha/CXC chemokine ligand 1 (GRO-alpha/CXCL1), across an endothelial monolayer. In addition, neutrophil migration toward GRO-alpha/CXCL1 and IL-8 (IL-8/CXCL8) was enhanced across an endothelial monolayer expressing the Duffy Ag. Furthermore, GRO-alpha/CXCL1 stimulation of endothelial cells expressing the Duffy Ag did not affect gene expression by oligonucleotide microarray analysis. These in vitro observations are supported by the finding that IL-8/CXCL8-driven neutrophil recruitment into the lungs was markedly attenuated in transgenic mice lacking the Duffy Ag. We conclude that Duffy Ag has a role in enhancing leukocyte recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium.
Amino Acid Sequence↗
Encephalitogenic activity of truncated myelin oligodendrocyte glycoprotein (MOG) peptides and their recognition by CD8+ MOG-specific T cells on oligomeric MHC class I molecules.
We have previously demonstrated that the 21-residue peptide pMOG(35-55) from myelin oligodendrocyte glycoprotein (MOG) contains an antigenic epitope that activates CD8(+) encephalitogenic T cells in C57BL/6 (B6) mice. To identify the core encephalitogenic epitope of CD8(+) MOG-specific T cells, we have prepared a panel of highly purified peptides of varying lengths, which span the entire length of pMOG(35-55), and tested their binding to recombinant H-2D(b) dimers and their ability to induce EAE. Two of the truncated peptides, pMOG(40-54) and pMOG(44-54), strongly bound recombinant H-2D(b) protein and this complex bound MOG-specific CD8(+) T cells. Interestingly, pMOG(40-54) retained the full capability of inducing paralytic disease, whereas only a part of the B6 mice immunized with pMOG(44-54) developed clinical paralysis and central nervous system (CNS) inflammation. Further deletion of 1 amino acid from either the N- or C-terminus of the peptide pMOG(44-54) dramatically reduced binding to recombinant H-2D(b), and abolished the induction of paralysis and CNS inflammation. Our results demonstrate that the ability of truncated pMOG(35-55) peptides to bind recombinant H-2D(b) protein does not always correlate with their ability of inducing encephalomyelitis. This approach enables the further identification of the core pathogenic epitope within the pMOG(35-55) that activates MOG-specific encephalitogenic CD8(+) T cells.
Animals↗
Fiber optic immunosensors for cardiovascular disease diagnosis: quantification of Protein C, Factor V Leiden, and cardiac Troponin T in plasma.
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Biomarkers↗
CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion.
We hypothesized that the CXC chemokine receptor-4 (CXCR4)-stromal-derived factor-1 (SDF-1) axis may be involved in metastasis of CXCR4(+) tumor cells into the bone marrow and lymph nodes, which secrete the alpha-chemokine SDF-1. To explore this hypothesis, we phenotyped by fluorescence-activated cell sorter analysis various human tumor cell lines for expression of CXCR4 and found that it was highly expressed on several rhabdomyosarcoma (RMS) cell lines. We also observed that cell lines derived from alveolar RMS, which is characterized by recurrent PAX3- and PAX7-FKHR gene fusions and is associated with a poor prognosis, expressed higher levels of CXCR4 than lines derived from embryonal RMS. Furthermore, transfer of a PAX3-FKHR gene into embryonal RMS cell activates CXCR4 expression. Because alveolar RMS frequently metastasizes to the bone marrow and lymph nodes, it seems that the CXCR4-SDF-1 axis could play an important role in this process. These findings prompted us to determine whether SDF-1 regulates the metastatic behavior of RMS cells. Accordingly, we found that, although SDF-1 did not affect proliferation or survival of these cell lines, it induced in several of them (1) phosphorylation of mitogen-activated protein kinase p42/44; (2) locomotion; (3) directional chemotaxis across membranes covered by laminin, fibronectin, or Matrigel; (4) adhesion to laminin, fibronectin, and endothelial cells; and (5) increased MMP-2 and diminished tissue inhibitors of metalloproteinases secretion. The small-molecule CXCR4-specific inhibitor, T140, effectively blocked the in vitro responses of RMS cells to SDF-1. On the basis of these observations we suggest that the CXCR4-SDF-1 axis may play an important role in tumor spread and metastasis of RMS cells to bone marrow and that molecular strategies aimed at inhibiting this axis could thus prove to be useful therapeutic measures.
Apoptosis↗
Plasmodium yoelii uses the murine Duffy antigen receptor for chemokines as a receptor for normocyte invasion and an alternative receptor for reticulocyte invasion.
Erythrocyte invasion by malaria parasites is a complex multistep process involving parasite and erythrocyte receptors. It is a critical stage in the parasite life cycle and, therefore, a logical step in which to intervene to prevent or ameliorate disease. Rodent models of malaria, commonly Plasmodium yoelii, are frequently used for studies of malaria pathogenesis. Little is known, however, about the invasion machinery of rodent malaria parasites. We have found previously that mice congenic for a region of chromosome 1, containing the Duffy antigen/receptor for chemokines (DARC), have different susceptibility to P yoelii infection. Because P vivax, a human parasite, and P knowlesi, a simian parasite, use DARC to enter human erythrocytes, we sought to identify the role of the murine DARC in P yoelii invasion. Using a novel in vivo invasion assay and DARC knock-out mice, we found that DARC knock-out normocytes (mature erythrocytes) had negligible levels of P yoelii invasion compared with wild-type normocytes, demonstrating that DARC is a receptor for invasion of murine erythrocytes. In contrast, DARC knock-out reticulocytes were invaded at a rate similar to that for wild-type reticulocytes. We conclude that there is a DARC- independent pathway for reticulocyte invasion. These findings represent the first identification of a murine malaria receptor on erythrocytes and the first determination that different pathways of invasion exist on normocytes and reticulocytes. Because we show conservation of host-receptor interactions between rodent and human malaria, we can now use this model to identify how immunity can interfere with the invasion process.
Animals↗
Role of CD4 hinge region in GP120 utilization by immunoglobulin domain 1.
Immunoglobulin-like domain 1 of CD4 (D1-CD4) promotes HIV infection by binding the envelope glycoprotein (ENV) and exposing its coreceptor-binding site. To study CD4-ENV-coreceptor interactions, we characterized hybrid receptors having domains 1 and 2 of CD4 (D1D2-CD4) joined to the N-terminus of chemokine receptors CCR5, CXCR4, CXCR2, and DARC. Hybrid receptors showed conserved ENV-coreceptor specificity in cell-cell fusion assays. Although D1D2-CD4-CCR5 was sufficient to permit ENV-mediated fusion, D1-CD4-CCR5 and human D1/mouse D2-CD4-CCR5 lacked CD4 function and binding to a neutralizing antibody mapped to D1-CD4. Chimeric D1D2-CD4 joined to CCR5 revealed that the C-terminal 20 residues of human D2-CD4 are required for efficient ENV-mediated fusion. Mutagenesis of hybrid receptors showed the importance of residues forming D1-D2 CD4 interdomain contacts and hinge region proximal residues. Mutagenesis of WT human CD4 confirmed that residues forming D1-D2 interdomain contacts and hinge-region proximal residues contribute positively to CD4 activity in the full-length receptor.
Amino Acid Sequence↗
A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists.
CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.
Amino Acid Substitution↗
Changes in gene expression of ARPE-19 cells in response to vitreous treatment.
PURPOSES: The purpose of this study is to determine the changes in gene expression by a human retinal pigment epithelium (RPE) cell line (ARPE-19) in response to vitreous treatment, which induces RPE proliferation and phenotypic changes in vitro that mimic the repair response observed in vivo during proliferative vitreoretinopathy. METHODS: ARPE-19 cells were grown for more than 4 weeks and their gene expression studied in: (1) subconfluent cultures treated with 50% human vitreous for 72 h; (2) subconfluent cultures without vitreous treatment, and (3) a confluent, nondividing monolayer. Total RNA was extracted from RPE cells and differential gene expression between each condition was determined using gene arrays (Clontech, Palo Alto, Calif., USA). Semiquantitative RT-PCR was used to confirm the upregulation of 4 genes related to vitreous treatment. In addition, the secretion of 1 of these upregulated gene products, monocyte chemotactic protein 1 (MCP-1), was confirmed by ELISA. RESULTS: A greater than threefold increase in the expression of mRNA for cell cycle regulators, intracellular transducers, cell adhesion proteins, growth factors and chemokines was observed following vitreous treatment of the RPE cell line and a corresponding decrease in the expression of genes related to apoptosis. RT-PCR confirmed the increased gene expression of MCP-1, intercellular adhesion molecule-1, vascular cell adhesion protein 1 precursor and vascular endothelial growth factor in vitreous-treated cells. Immunoassays further showed an increased MCP-1 secretion by vitreous-treated RPE. CONCLUSIONS: These findings suggest that in this in vitro vitreous treatment model, ARPE-19 cells participate in a mock repair response by upregulating genes encoding for proteins associated with inflammation and wound healing.
Cell Line↗