12 research outputs found
Differences in Sulfotyrosine Binding amongst CXCR1 and CXCR2 Chemokine Ligands
Tyrosine sulfation, a post-translational modification found on many chemokine receptors, typically increases receptor affinity for the chemokine ligand. A previous bioinformatics analysis suggested that a sulfotyrosine (sY)-binding site on the surface of the chemokine CXCL12 may be conserved throughout the chemokine family. However, the extent to which receptor tyrosine sulfation contributes to chemokine binding has been examined in only a few instances. Computational solvent mapping correctly identified the conserved sulfotyrosine-binding sites on CXCL12 and CCL21 detected by nuclear magnetic resonance (NMR) spectroscopy, demonstrating its utility for hot spot analysis in the chemokine family. In this study, we analyzed five chemokines that bind to CXCR2, a subset of which also bind to CXCR1, to identify hot spots that could participate in receptor binding. A cleft containing the predicted sulfotyrosine-binding pocket was identified as a principal hot spot for ligand binding on the structures of CXCL1, CXCL2, CXCL7, and CXCL8, but not CXCL5. Sulfotyrosine titrations monitored via NMR spectroscopy showed specific binding to CXCL8, but not to CXCL5, which is consistent with the predictions from the computational solvent mapping. The lack of CXCL5âsulfotyrosine interaction and the presence of CXCL8âsulfotyrosine binding suggests a role for receptor post-translational modifications regulating ligand selectivity
New paradigms in chemokine receptor signal transduction: Moving beyond the two-site model.
Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions
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Investigations on T cell transmigration in a human skin-on-chip (SoC) model.
A microfluidics-based three-dimensional skin-on-chip (SoC) model is developed in this study to enable quantitative studies of transendothelial and transepithelial migration of human T lymphocytes in mimicked skin inflammatory microenvironments and to test new drug candidates. The keys results include 1) CCL20-dependent T cell transmigration is significantly inhibited by an engineered CCL20 locked dimer (CCL20LD), supporting the potential immunotherapeutic use of CCL20LD for treating skin diseases such as psoriasis; 2) transepithelial migration of T cells in response to a CXCL12 gradient mimicking T cell egress from the skin is significantly reduced by a sphingosine-1-phosphate (S1P) background, suggesting the role of S1P for T cell retention in inflamed skin tissues; and 3) T cell transmigration is induced by inflammatory cytokine stimulated epithelial cells in the SoC model. Collectively, the developed SoC model recreates a dynamic multi-cellular micro-environment that enables quantitative studies of T cell transmigration at a single cell level in response to physiological cutaneous inflammatory mediators and potential drugs
Structural Analysis of a Novel Small Molecule Ligand Bound to the CXCL12 Chemokine
CXCL12
binds to CXCR4, promoting both chemotaxis of lymphocytes
and metastasis of cancer cells. We previously identified small molecule
ligands that bind CXCL12 and block CXCR4-mediated chemotaxis. We now
report a 1.9 Ă
resolution X-ray structure of CXCL12 bound by
such a molecule at a site normally bound by sY21 of CXCR4. The complex
structure reveals binding hot spots for future inhibitor design and
suggests a new approach to targeting CXCL12âCXCR4 signaling
in drug discovery
CCR7 Sulfotyrosine Enhances CCL21 Binding
Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the ι-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and β3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect
Sulfopeptide probes of the CXCR4/CXCL12 interface reveal oligomer-specific contacts and chemokine allostery
Tyrosine sulfation is a post-translational modification that enhances proteinâprotein interactions and may identify druggable sites in the extracellular space. The G protein-coupled receptor CXCR4 is a prototypical example with three potential sulfation sites at positions 7, 12, and 21. Each receptor sulfotyrosine participates in specific contacts with its chemokine ligand in the structure of a soluble, dimeric CXCL12:CXCR4(1â38) complex, but their relative importance for CXCR4 binding and activation by the monomeric chemokine remains undefined. NMR titrations with short sulfopeptides showed that the tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12 binding affinity and site specificity. Whereas the Tyr21 sulfopeptide bound the same site as in previously solved structures, the Tyr7 and Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12 monomer that is inaccessible in the CXCL12 dimer. Functional analysis of CXCR4 mutants validated the relative importance of individual CXCR4 sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12 binding and receptor activation. Biophysical measurements also revealed a cooperative relationship between sulfopeptide binding at the Tyr21 site and CXCL12 dimerization, the first example of allosteric behavior in a chemokine. Future ligands that occupy the sTyr21 recognition site may act as both competitive inhibitors of receptor binding and allosteric modulators of chemokine function. Together, our data suggests that sulfation does not ubiquitously enhance complex affinity and that distinct patterns of tyrosine sulfation could encode oligomer selectivity, implying another layer of regulation for chemokine signaling
Sulfopeptide Probes of the CXCR4/CXCL12 Interface Reveal Oligomer-Specific Contacts and Chemokine Allostery
Tyrosine
sulfation is a post-translational modification that enhances
proteinâprotein interactions and may identify druggable sites
in the extracellular space. The G protein-coupled receptor CXCR4 is
a prototypical example with three potential sulfation sites at positions
7, 12, and 21. Each receptor sulfotyrosine participates in specific
contacts with its chemokine ligand in the structure of a soluble,
dimeric CXCL12:CXCR4(1â38) complex, but their relative importance
for CXCR4 binding and activation by the monomeric chemokine remains
undefined. NMR titrations with short sulfopeptides showed that the
tyrosine motifs of CXCR4 varied widely in their contributions to CXCL12
binding affinity and site specificity. Whereas the Tyr21 sulfopeptide
bound the same site as in previously solved structures, the Tyr7 and
Tyr12 sulfopeptides interacted nonspecifically. Surprisingly, the
unsulfated Tyr7 peptide occupied a hydrophobic site on the CXCL12
monomer that is inaccessible in the CXCL12 dimer. Functional analysis
of CXCR4 mutants validated the relative importance of individual CXCR4
sulfotyrosine modifications (Tyr21 > Tyr12 > Tyr7) for CXCL12
binding
and receptor activation. Biophysical measurements also revealed a
cooperative relationship between sulfopeptide binding at the Tyr21
site and CXCL12 dimerization, the first example of allosteric behavior
in a chemokine. Future ligands that occupy the sTyr21 recognition
site may act as both competitive inhibitors of receptor binding and
allosteric modulators of chemokine function. Together, our data suggests
that sulfation does not ubiquitously enhance complex affinity and
that distinct patterns of tyrosine sulfation could encode oligomer
selectivity, implying another layer of regulation for chemokine signaling
Targeting the CCR6/CCL20 axis in entheseal and cutaneous inflammation
Objectives:
To assess the involvement of the CCR6/CCL20 axis in psoriatic arthritis (PsA) and psoriasis (PsO) and to evaluate its potential as a therapeutic target.
Methods:
First, we quantified CCL20 levels in peripheral blood and synovial fluid of PsA patients and the presence of CCR6+ cells in synovial and tendon tissue. Utilizing an IL-23 minicircle DNA (MC) mouse model exhibiting key features of both PsO and PsA, we investigated CCR6 and CCL20 expression and the preventive and therapeutical effect of CCL20 blockade. Healthy tendon stromal cells were stimulated in vitro with IL-1β to assess the production of CCL20 by qPCR and ELISA. The effect of conditioned media from stimulated tenocytes in inducing T cell migration was interrogated with a transwell system.
Results:
We observed an upregulation of both CCR6 and CCL20 in the enthesis of IL-23 MC-treated mice, which was confirmed in human biopsies. Specific targeting of the CCR6/CCL20 axis with a CCL20 locked dimer (CCL20LD) blocked entheseal inflammation, leading to profound reductions in clinical and proinflammatory markers in the joints and skin of IL-23 MC-treated mice. The stromal compartment in the tendon was the main source of CCL20 in this model and accordingly, in vitro activated human tendon cells were able to produce this chemokine and to induce CCR6+ T cell migration, the latter of which could be blocked by CCL20LD.
Conclusions:
Our studies highlight the pathogenic role of CCR6-CCL20 axis in enthesitis and raise the prospect of a novel therapeutic approach for treating patients with PsO and PsA