Studies On The Interaction Of S100A8/A9with RAGE

Sepsis remains to be one of the chief causes of death in intensive care units at present, with the mortality is between30～70%. It has been found that with the stimulation of LPS, TNF or IL-1, S100A8/A9is actively secreted from monocytes and macrophages. S100A8/A9can also be passively released from necrotic cells. Once released, S100A8/A9is able to activate many other cells including monocytes, macrophages, endothelial cells and epithelium to produce proinflammatory cytokine and adhesion molecules by the interaction with the receptors on membrane. S100A8/A9causes inflammatory responses in various systems in vivo, including brain, lung, gastrointestinal tract, joints, bladder, prostate and heart, leading to the systemic inflammatory response even death. This implicates that S100A8/A9plays a key role in inflammatory reactions. The studies on the interaction of S100A8/A9and receptors on membrane will promote our clinic therapy.The S100protein family represents the largest subgroup within the Ca2+binding EF-hand superfamily. The name of the protein family has derived from the fact that the first identified S100proteins were obtained from the soluble (S) bovine brain fraction upon fractionation with saturated(100%) ammonium sulfate. The genes encoding the large majority of human S100proteins are organized in a gene cluster located in chromosomal region1q21. The S100protein family consists of21members, which are expressed in a controlled tissue-or cell-type-specific manner. S100proteins are low molecular weight (9-14kD) acid proteins with two EF domains that bind Ca2+ions selectively and with high to low affinity. Each S100monomer is composed of two EF-hand helix-loop-helix structural motifs arranged in a back to back manner and connected by a flexible linker. The C-terminal EF-hand contains the classical Ca2+-binding motif, common to all EF-hand proteins. The loop has a typical sequence signature of12amino acids flanked by helices HⅢ and HⅣ. The N-terminal EF-hand exhibits a slightly different architecture and contains a specific14amino acid motif flanked by helices HⅠ and HⅡ. This motif is characteristic for S100proteins and therefore it is often called’S100-specific’or’pseudo EF-hand’. Generally, the dimeric S100proteins bind four Ca2+ions per dimer with micromolar to hundreds micromolar binding constants and strong cooperativity. The S100protein dimer interface is formed by helices HⅠ and HⅣ from both monomers, building a compact four helix bundle.The conformational properties and function of S100proteins are modulated by metal ion binding. The binding of Ca2+to EF-hand type domains triggers conformational changes allowing interactions with other proteins and regulating various proteins involved in a large number of cellular functions such as calcium homeostasis, cell growth and differentiation, dynamic of cytoskeleton or energy metabolism. In many S100proteins, additional binding of Zn2+fine tunes protein folding and function. Intracellularly, S100proteins act as Ca2+sensors, translating intracellular Ca2+level increases into a cellular response. S100A8/A9can form heterodimers in the absence of calcium and heterotetramers (S100A8/A9)2in the presence of calcium. Elevated serum levels of these S100proteins are found in patients suffering from inflammatory diseases such as rheumatoid arthritis, cystic fibrosis or Crohn’s disease.High levels of S100A8/A9have also been found in the microglia of patients with Alzheimer’s disease or suffering from ischemic lesions and may also paly a role in several cancers such as gastric cancer, colorectal carcinoma or prostate cancer. To date, more and more studies focus on the target proteins, such as RAGE and TLR4, but only little is about the relationship of metal ions and the interaction of S100A8, S100A9with RAGE.RAGE is a member of the immunoglobulin protein family of cell surface molecules and shares structural homology with other immunoglobulin like receptors. Although RAGE is not essential to life, it plays important roles in certain human pathologies including diabetes, Alzheimer’s disease and cancer. Human RAGE (hRAGE) consists of404amino acid residues, including an extracellular part (321amino acid), a single transmembrane spanning helix (19amino acid) and a short cytosolic domain (41amino acid). The extracellular part of RAGE is also named soluble RAGE (sRAGE). sRAGE has two N-linked glycosylation sites locating near the anlino terminus (at amino acid positions25and81of the mature protein), which lie in a V-like domain. There are also two C-like domains in this region. These structures are important to the molecular stability and the function of identifying ligands. Besides, there is a transmembrance region of21hydrophobic amino acids followed by a highly charged intracellular domain of41amino acids. It has been demonstrated that RAGE is not a specifical receptor for advanced glycation end product(AGE) which has been recognized firstly as a ligand of RAGE. In fact, RAGE recognizes a ligand family whose member inchide advanced glycation end products, HMGB1, amyloid-β peptide and S100proteins (such as S100B, S100P, S100A4, S100A6, S100A8/A9, S100A11-13). The interaction of these ligands and RAGE plays important role in the pathogenesis of many diseases, such as diabetic mellitus and Alzheimer’s diseas (AD). With the development of studies on RAGE, we highly recognized the role of blocking the interaction of RAGE and its ligands in the pathogenesis of diseases.Many proteins function in association with partner proteins or as components of large multiprotein complexes. Understanding these protein interactions is critical to our understanding of biological pathways and cellular function. The yeast two-hybrid system is commonly used to determine protein-protein interactions but generates many false positives, requiring confirmation of results by another method. GST pull-down is becoming an important tool for validation of suspected protein-protein interactions or for discovering novel protein interactions. GST pull-down uses a GST-fusion protein (bait) bound to glutathione (GSH)-coupled particles to affinity purify any proteins (prey) that interact with the bait from a pool of proteins in solution. Bait and prey proteins can be obtained from multiple sources including cell lysates, purified proteins, expression systems and in vitro transcription/translation systems.Based on the understandings mentioned above, in our experiment, we constructed fusion protein expression vectors with GST purification tags. After the induction of expression in BL21(DE3) host bacteria and the purification with Ni2+-NTA and GST·BindTM, we got the corresponding fusion proteins. Then, used the GST pull-down method, we validate the relationship of metal ions with the interaction of S100A8, S100A9and RAGE, further to provide foundations for pathogenesis and gene therapy of diseases.Taken together the above data, we draw the following conclusions:First. We have successfully constructed the prokaryotic expressing fusion protein expression vectors:pGEX-4T-hS100A8and pGEX-4T-hS100A9; and successfully purifed the fusion proteins:GST-hS100A8protein, GST-hS100A8protein, GST protein and His-esRAGE.Second. We find that hS100A8and esRAGE can be interact, and the interaction of hS100A8and esRAGE dependent of metal divalent cations Ca2+, Zn2+Third. We find that hS100A9and esRAGE can be interact, and the interaction of hS100A9and esRAGE dependent of metal divalent cations Ca2+, Zn2+In general, based on the principal of protein-protein interactions, we validate the relationship of metal divalent cations with the interaction of S100A8, S100A9and esRAGE by GST pull-down technology. In addition, our research could help us further understand the meaning of S100A8/A9and its interaction with receptors, and also provide us new theoretical basis and possible therapy target of clinical trial of endoxic shock.