Inflammation and Alzheimer’s Disease (AD) Figure 6. Electrophoresis and gas-phase electrophoretic mobility molecular analysis (GEMMA) of S100A9 and S100A9/Ab1–40 mixture. (a) SDS PAGE of S100A9 with (1) 0.002 mM; (2) 0.01 mM; (3) 0.02 mM; (4) 0.1 mM Ab1–40 and native PAGE of (5) 0.2 mM S100A9 with 0.01 mM Ab1–40; (6) 0.2 mM S100A9 with 0.1 mM Ab1–40; (7) 0.1 mM Ab1–40; (8) 0.01 mM Ab1–40. The final concentration of S100A9 is 0.2 mM. The data was obtained by several experiments, and integrated together according to the ladders. (b) Size distribution of S100A9 monomer and dimer at pH 7.4 and 3.0 examined by gas-phase electrophoretic mobility molecular analysis (GEMMA). The red columns represent S100A9 at pH 7.4. The blue ones represent S100A9 at pH3.0. doi:10.1371/journal.pone.0032953.g006 two monomers make contact with each other, and form an antiparallel-packed hydrophobic core [19]. Because the electron density of 28 residues in C-terminal tail is low and the residues are mostly hydrophilic, it is believed that the residues do not participate in electrostatic and hydrophobic protein-protein interactions [19]. As an exposed part of the intra-molecule hydrophobic cluster, the hinge region is identified as a target- binding site for proteins [20]. The interaction between Ab1-40 and S100A9 is possibly initiated by binding to S100A9 hinge region. According to our previous simulation results, the contact of the Ab12-24 hydrophobic core (L17V18F19F20A21) leads to the formation of b-sheet structures, which then act as a seed for further amyloid formation [21]. Besides aggregation, proper stacking (parallel or anti-parallel) is required, which demands a specific residue sequence. The hinge region of S100A9 consists of 11 residues (Leu45-Asn55) (Fig. 7). Similarly to Ab peptides, the hinge region has a hydrophobic core (L44Q45N46F47L48), following which K (50, 51, 54) residues are positively charged and E52 is negatively charged. By electrostatic interaction, E22D23 residues pair with K50K51 residues. Such stacking allows Ab peptide molecules to be aligned in a certain direction. As the anti-parallel stacking is energetically preferred for Ab amyloid formation at physiological pH, it is plausible that Ab amyloid formation is initiated by binding of Ab peptide molecules to different sites of S100A9 [20]. The S100A9 homodimer is formed by anti-parallel stacking of two monomers. Accordingly, the binding of Ab peptides to the hinge regions of two monomers provides the possibility of anti-parallel stacking. This is consistent with our assumption that the S100A9 homodimer interacts with Ab1–40 and our observation of the interactions between S100A9, Ab12– 24, and Ab1–42 peptide. Similar to Ab1–40, Ab12–24, and Ab1– 42 amyloid formation was greatly promoted by S100A9, which emphasizes the importance of the Ab hydrophobic core (Fig. S6). With no hydrophobic core (L17V18F19F20A21), Ab1-16 showed no obvious change upon addition of S100A9 in both AFM and Cytotoxicity experiments. Overall, the results of this study, combined with those in previous reports, suggest that Ab amyloid production is induced by the pro-inflammatory protein S100A9. The Ab-induced reduction of cytotoxicity indicates that the formation of AD amyloid plaques could be a beneficial response to inflammation. It ameliorates the potentially destructive effects of inflammatory molecules such as S100A9. S100A9-induced Ab amyloid formation also explains the presence of amyloid plaques in normal aged brain [22,23]. Further computer simulations will be conducted to elucidate the interaction between S100A9 and Ab peptides. Figure 7. Amino acid sequence alignment of S100A9 in the hinge region and Ab12–24 peptide. The hydrophobic cores of both proteins are marked in red. The charged residues are indicated with their approximate charges. All sequences were obtained from the SWISS-PORT protein database [26]. doi:10.1371/journal.pone.0032953.g007 PLoS ONE | www.plosone.org 5 March 2012 | Volume 7 | Issue 3 | e32953