Biophysical characterization of S100A8 and S100A9 in the absence and presence of bivalent cations

AbstractS100A8 and S100A9 are two proinflammatory molecules belonging to the S100 family of calcium-binding proteins. Common to all S100 proteins S100A8 and S100A9 form non-covalently associated complexes which have been shown to exhibit different functional properties. Besides dimerization, recent research is focused on the importance of higher oligomeric structures of S100 proteins induced by bivalent cations. While S100A8/S100A9-heterodimers are formed in the absence of calcium, tetramerization is strictly calcium-dependent. Heterodimer formation is not a simple process and our biophysical analyses (FRET, ESI-MS) demonstrate that simply mixing both subunits is not sufficient to induce complex formation. Steps of denaturation/renaturation are necessary for the recombinant complex to show identical biophysical properties as S100A8/S100A9 obtained from granulocytes. In addition to calcium both proteins are able to bind zinc with high affinity. Here we demonstrate for the first time by different biophysical methods (MALDI-MS, ESI-MS, fluorescence spectroscopy) that zinc-binding, like calcium, induces (S100A8/S100A9)2-tetramers. Using mass spectrometric investigations we demonstrate that zinc triggers the formation of (S100A8/S100A9)2-tetramers by zinc-specific binding sites rather than by interactions with calcium-specific EF-hands. The zinc-induced tetramer is structurally very similar to the calcium-induced tetramer. Thus, like calcium, zinc acts as a regulatory factor in S100A8/S100A9-dependent signaling pathways

Molekulare Charakterisierung verschiedener Komplexformen der Calcium-bindenden Proteine S100A8 und S100A9

Die Proteine S100A8 (MRP8) und S100A9 (MRP14), die spezifisch in Monozyten und Granulozyten exprimiert werden, zeichnen sich strukturell durch zwei Calcium-Bindungsstellen des EF-Hand Typs aus. In Abhängigkeit von der Calcium-Konzentration bilden sie nicht-kovalent assoziierte Komplexe aus, die neben den Monomeren als biologisch funktionelle Formen der Proteine angesehen werden. Im Rahmen der vorliegenden Arbeit konnten sowohl die humanen, heterodimeren S100A8/S100A9-Komplexe, als auch die höhermolekularen (S100A8/S100A9)2-Heterotetramerkomplexe als physiologisch relevant identifiziert werden. Dazu wurde einerseits die Komplexstruktur der S100A8/S100A9-Heterodimere durch den Austausch konservierter, hydrophober Aminosäuren mit Hilfe des Yeast Two-Hybrid Systems untersucht. Andererseits konnte mit Hilfe proteinbiochemischer Methoden nachgewiesen werden, dass die Calcium-abhängige Ausbildung höhermolekularer Tetramerkomplexe durch spezifische Mutationen innerhalb der C-terminalen EF-Hand des S100A9 Proteins gestört werden kann, wohingegen die Heterodimerisierung unbeeinträchtigt bleibt

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor-associated kinase-1 and nuclear factor-kappaB, resulting in elevated expression of tumor necrosis factor-alpha (TNF-alpha). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNFalpha-dependent effect