The ASPP2 Ank-SH3 – p53CD complex PDB ID: 1YCS [14].

<p>(A) Crystal structure of the complex between p53CD (black) and ASPP2 927–1119 (gray). The RT loop (residues 1067–1080, blue) and the n-Src loop (residues 1089–1096, purple) in the ASPP2 SH3 domain bind the L3 loop of p53 (residues 241–249, cyan), while the fourth ankyrin repeat (residues 1020–1027, red) binds the L2 loop (residues 178–183, cyan) of p53CD <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Gorina1" target="_blank">[14]</a>; (B) The structure of the four ankyrin repeats (light gray) and the SH3 domain (dark gray) of ASPP2. The p53CD binding sites in the ASPP2 SH3 domain of ASPP2 are shown with the RT loop in blue and the n-src loop in purple. (C) The binding sites of ASPP2 Ank-SH3 to p53CD (cyan), Bcl2 (orange) and NFκB (green) are all on the same face of the molecule but with minimal overlap between the binding sites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Benyamini1" target="_blank">[21]</a>; (D) The ASPP2 Pro (residues 693–918) binding sites in ASPP2 Ank-SH3 (magenta) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058470#pone.0058470-Rotem1" target="_blank">[13]</a>.</p

ASPP2 Ank-SH3 binds ASPP2 722–737 and is not displaced by Bcl-2 peptides.

<p>(A) ASPP2 Ank-SH3 binds Fl-ASPP2 722–737 with K<i>_d</i> = 0.82±0.08 µM (magenta); (B) Non-labeled Bcl-2 103–120 (orange square) or Bcl-2 7–24 (orange triangle) peptides were titrated into the fully formed complex between Fl- ASPP2 722–737 and ASPP2 Ank-SH3 but did not compete with ASPP2 Ank-SH3 on peptide binding.</p

p53CD and the Fl-ASPP2 Pro 722–737 peptide compete for binding ASPp2 Ank-SH3.

<p>(A) ASPP2 Ank-SH3 binds ASPP2 Pro 722–737 with <i>K</i>_d = 0.68±0.07 µM. (B) p53CD was titrated into the fully formed complex between ASPP2 Ank-SH3 and Fl-ASPP2 722–737 and displaced the Fl-ASPP2 722–737 peptide.</p

The ASPP2 Ank-SH3 binding peptides used in this study.

*<p>Trp was added at the N terminus of the peptide for UV spectroscopy.</p

Bcl-2 and NFκB peptides bind the ASPP2 n-src loop 1089–1097 peptide in a different site than p53CD.

<p>(A) p53CD binds Fl-ASPP2 1089–1097 ASPP2 SH3 derived peptide In fluorescence anisotropy experiments with <i>K</i>_d = 0.63±0.02 µM (B) Non-labeled Bcl-2 or NFκB peptides were titrated into the fully formed complex between Fl-ASPP2 1089–1097 and p53CD. None of the peptides displaced p53CD from the complex.</p

ASPP2 Ank-SH3 binds NFκB 303–332 and is not displaced by Bcl-2 peptides.

<p>(A) ASPP2 Ank-SH3 binds Fl-NFκB 303–332 with K<i>_d</i> = 0.13±0.01 µM; (B) Non-labeled Bcl-2 103–120 (orange square) or Bcl-2 7–24 (orange triangle) peptides were titrated into the fully formed complex between Fl-NFκB 303–332 and ASPP2 Ank-SH3 but did not compete with ASPP2 Ank-SH3 on peptide binding.</p

p53CD and ASPP2 Pro 723–737 compete for binding the Fl-ASPP2 n-src loop peptide residues 1089–1097: fluorescence anisotropy experiments.

<p>(A) A peptide derived from the proline rich domain of ASPP2 residues 722–737 binds the ASPP2 n-src peptide with <i>K</i>_d = 0.50±0.03 µM (magenta); Following titration of p53CD (cyan) into to the fully formed complex between Fl-ASPP2 1089–1097 and ASPP2 722–737, the anisotropy increased (B) p53CD residues 94–312 bind the ASPP2 n-src peptide with <i>K</i>_d = 0.63±0.02 µM (cyan). Unlabeled ASPP2 722–737 (magenta) displaced p53CD form its fully formed complex with ASPP2 1089–1097.</p

Effect of protein synthesis arrest on AGEs secretion.

<p>Bacteria were grown in MOPS minimal medium and samples were collected for AGEs determination as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017974#s4" target="_blank">materials and methods</a>. AGEs-specific fluorescence (Ex. 370, Em. 440) was determined and normalized to cells density. A) Intracellular and extracellular AGEs levels following exposure to chloramphenicol. B) Effect of chloramphenicol (open circles) and Arsenate (triangles) on AGEs secretion kinetics. The data represent three independent experiments.</p

Effect of extracellular AGEs on secretion of TNF-alpha by THP-1 cells.

<p>The levels of TNF-alpha secreted by THP-1 cells were measured using sandwich-ELISA (see methods). A) TNF-alpha levels following exposure to elevated concentration of AGEs-containing fraction. B) Relative TNF-alpha levels secreted from THP-1 cells following exposure to the extracellular fractions (1/80 stock dilutions in distilled water) secreted from wild type cells, peprazine-treated cells and Δ<i>tolc</i> mutant cells. The data represent three independent experiments.</p

Effect of metallo-protease inhibition on AGEs accumulation and secretion.

<p>Bacterial growth and sample collection were described in materials and methods. AGEs-specific fluorescence was determined and normalized to cells density. A) Extracellular fraction was further separated into proteins and low molecular weight compounds fractions. B) Effect of 100 µM and 1 mM of 1,10 phenantholine (Sigma) on intracellular AGEs accumulation. C) Effect of 100 µM of 1,10 phenantholine (Sigma) on AGEs secretion after 2 hours in a non-growing cultures.</p