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Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

ABSTRACT: MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cognate ligand CXCL12. By using peptide spot-array technology, site-directed mutagenesis, structure-activity-relationships, and molecular docking, we identified the Arg-Leu-Arg (RLR) sequence-region 87-89 that - in three-dimensional space - 'extends' the N-like-loop to control site-1-binding to CXCR4. Contrary to wildtype MIF, mutant R87A-L88A-R89A-MIF fails to bind to the N-terminal of CXCR4 and the contribution of RLR to the MIF/CXCR4-interaction is underpinned by an ablation of MIF/CXCR4-specific signaling and reduction in CXCR4-dependent chemotactic leukocyte migration of the RLR-mutant of MIF. Alanine-scanning, functional competition by RLR-containing peptides, and molecular docking indicate that the RLR residues directly participate in contacts between MIF and CXCR4 and highlight the importance of charge-interactions at this interface. Identification of the RLR region adds important structural information to the MIF/CXCR4 binding-site that distinguishes this interface from CXCR4/CXCL12 and will help to design MIF-specific drug-targeting approaches.

SUBMITTER: Lacy M

PROVIDER: S-EPMC5979958 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

Lacy Michael M Kontos Christos C Brandhofer Markus M Hille Kathleen K Gröning Sabine S Sinitski Dzmitry D Bourilhon Priscila P Rosenberg Eric E Krammer Christine C Thavayogarajah Tharshika T Pantouris Georgios G Bakou Maria M Weber Christian C Lolis Elias E Bernhagen Jürgen J Kapurniotu Aphrodite A

Scientific reports 20180326 1

MIF is a chemokine-like cytokine that plays a role in the pathogenesis of inflammatory and cardiovascular disorders. It binds to the chemokine-receptors CXCR2/CXCR4 to trigger atherogenic leukocyte migration albeit lacking canonical chemokine structures. We recently characterized an N-like-loop and the Pro-2-residue of MIF as critical molecular determinants of the CXCR4/MIF binding-site and identified allosteric agonism as a mechanism that distinguishes CXCR4-binding to MIF from that to the cogn ...[more]

PMID: 29581527

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Project description:Background The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets. Methods Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo. Results We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model. Conclusions Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-022-09725-8.

Dataset Information

Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

Publications

Identification of an Arg-Leu-Arg tripeptide that contributes to the binding interface between the cytokine MIF and the chemokine receptor CXCR4.

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