Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Ana-Maria Filip,Jörg Klug,Sevil Cayli,Suada Fröhlich,Tamara Henke,Philipp Lacher,Regina Eickhoff,Patrick Bulau,Monika Linder,Christine Carlsson-Skwirut,Lin Leng,Richard Bucala,Sandra Kraemer,Jürgen Bernhagen,Andreas Meinhardt

doi:10.1074/jbc.m808620200

Abstract

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

Highlights

Despite its role as a key mediator in immune and inflammatory diseases, very little is known of how migration inhibitory factor (MIF) action is regulated and terminated
This 16-kDa protein was analyzed by tryptic in-gel digestion followed by Matrix-assisted Laser Desorption/ Ionization (MALDI)-time of flight (TOF) mass spectrometry and identified as ribosomal protein S19 (RPS19)
In the past 20 years significant progress has been made in our understanding of the role MIF plays in normal cellular physiology and in a variety of pathological conditions ranging from infection to autoimmunity and cancer [33, 34]

Summary

Introduction

Despite its role as a key mediator in immune and inflammatory diseases, very little is known of how MIF action is regulated and terminated. To completely remove free biotinylated MIF, beads were washed with PBS and incubated in 500 ␮l of lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% IGEPAL CA-630) with increasing amounts of GST-RPS19 (50, 100, and 200 ng) on a rotating wheel at 4 °C for 1 h.

Results

Conclusion