Another Mysterious Knife For the GSK-3 inhibitor

Using stable isotope labelling of amino acids in cell culture (SILAC), we identified the acetyltransferase p300 as a target of NleC and subsequently determined that the TAZ1 domain of p300 is required for this interaction. We also determined that nuclear p300 levels are decreased in the presence of NleC and that the recently characterized metalloprotease activity of NleC was responsible for p300 degradation. Furthermore, overexpression of p300 in Caco-2 cells dampens EPEC-mediated repression of IL-8 secretion during infection and siRNA knock-down of p300 causes decreased IL-8 secretion by EPEC ��nleC-infected cells. We therefore provide http://www.selleckchem.com/GSK-3.html the first example of a bacterial virulence factor targeting host p300, and a complementary mechanism to p65 degradation to influence IL-8 levels. Identification of host proteins targeted by bacterial virulence factors can provide insight into their function during infection. We used the A/E type III secreted effector NleC in a quantitative proteomic screen using SILAC to identify putative host protein targets. Two constructs, NleC fused to GST and a triple HA tag (GST-HA3-NleC) or GST-HA3 alone, either were expressed directly in HEK 293 cells as previously described (Rogers et?al., 2008; Shames et?al., 2010) or were produced in E.?coli BL21 (DE3) and purified on glutathione-agarose resin. HEK 293T cells were grown http://www.selleckchem.com/screening/tyrosine-kinase-inhibitor-library.html in media lacking l-lysine and l-arginine and supplemented with either light (normal) or heavy (2H4-lysine and 13C6-arginine) amino acids. Exogenously expressed constructs were immunoprecipitated from cell lysates and, in parallel, equal amounts of light and heavy 293T cell lysates were incubated with glutathione-agarose beads bound to GST-HA3-NleC and GST-HA3 respectively. Non-specific proteins were removed via sequential washes and proteins remaining on the column were eluted using 10?mM free glutathione and then eluates were http://en.wikipedia.org/wiki/VAV2 mixed prior to tryptic digestion. Peptides were then analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this scheme, non-specifically interacting proteins should present a ratio near 1.0 and, indeed, peptides from peroxiredoxin 2 had a heavy?:?light ratio of ?1.1 (Fig.?1A, top panel). However, 22 unique peptides of the E1A-associated protein p300 (herein called p300; Table?S1) all had low heavy: light ratios, indicating specific binding to NleC (Fig.?1A, bottom panel). All proteins identified in this SILAC experiment with heavy: light isotope ratios