Figure 1.
Characterization of SO for chemical proteomic studies.
(A) The chemical structures of the SO, CDDO-Im (TP-235), TP-154 (inactive) and TP-304 (active). (B) TP-304 but not TP-154 inhibits the proliferation of HE293 cells. HEK293 cells were treated with either DMSO (control), TP-304 or TP-154 at the concentrations indicated for 48 h. Proliferation was assessed by MTT assay. (C) CDDO-Im and TP-304 but not TP-154 prevent the TNFα-induced degradation of IκBα. HEK293 cells were treated with DMSO (control), CDDO-Im, TP-304 or TP-154 at the concentrations indicated for 1 h followed by treatment with 10 ng/ml TNFα. Lysates were immunoblotted with antibodies against IκBα or tubulin (loading control).
Figure 2.
Chemical proteomics studies for identification of SO targets.
(A) Schematic representation. HEK293 cells were treated with biotinylated SO compounds for 1 h in culture. Following cell lysis, target proteins were affinity purified with paramagnetic beads. Beads were washed extensively 5× and remaining proteins were eluted by boiling in Laemmli buffer. Proteins were separated by SDS-PAGE on 4–12% gradient gels. Gels were stained with Coomassie stain overnight followed by destaining until background staining was clear. Corresponding regions from both TP-154 and TP-304 gels were excised, trypsinised and protein identified by LC-MS/MS. (B) Image of Coomassie-stained gel submitted for mass-spectroscopic analysis. (C) Proteins identified in the TP-154 samples (control) were subtracted from the TP-304 treatment groups.
Figure 3.
Bioinformatic analysis of putative SO target proteins.
Gene ontology analysis of the candidate SO targets. GO_slim, gene ontology analysis of the candidate substrates was done with the CateGOrizer program. (A) SO target proteins, were annotated by molecular function. Categories with more than 20 assigned proteins are shown. (B) The 577 putative SO target proteins, were assigned to biological processes. Proteins for which no biological process could be assigned were omitted from this display. Categories with more than 20 assigned proteins are shown. (C) Overall connectivity of identified proteins was determined using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). (D) Highly connected sub-networks within the STRING network. Different line colors represent the types of evidence for each association, blue lines: direct binding, pink lines: post-translational modification. (E) For canonical pathway analysis, UniProt IDs were analyzed with Ingenuity pathway software. The top 40 most significant canonical signaling pathways from the Ingenuity canonical pathway library mapped to the SO target dataset are displayed. Threshold bar shows cut-off point of significance P<0.05, −log(P-value) of 1.3 as determined using a right-tailed Fisher's exact test. High resolution images of the STRING network and sub-network can be found in the supplementary information.
Figure 4.
Validation of putative SO targets identified by LC-MS/MS.
HE293 cells were treated with either DMSO (control) or 4 µM TP-304 for 1 h, as indicated. TP-304 binding proteins were affinity purified with NeutrAvidin resin. Proteins were separated by SDS-PAGE and immunoblots performed with various antibodies targeted toward a subset of proteins identified in the prior LC-MS/MS studies. Samples from whole cell lysate for both treatment conditions were also immunoblotted (lanes 1 and 2) to show that treatments had no effect on total protein levels.
Figure 5.
CDDO-Im inhibits the autokinase activity of mTOR.
CDDO-Im inhibits the mTOR pathway in HEK293 cells. (A) HEK293 cells were treated with rapamycin (R, 20 nM) CDDO-Im or TP-304 as indicated for 1 h. Then, cells were treated with 100 nM Insulin for 30 min. Cells were lysed in RIPA buffer and Western immunoblotting was performed with the antibodies listed. (B) CDDO-Im inhibits the auto-kinase activity of mTOR. HEK293 cells were treated with DMSO, rapamycin (20 nM) or CDDO-Im (500 nM) for 2 h. Cells were then stimulated with 100 nM insulin for 15 min. Cells were lysed, and lysates were immunoprecipitated with anti-RAPTOR antibodies and protein A beads. Immunoprecipitates were separated by SDS-PAGE and membranes blotted with anti-phospho-mTOR serine 2481 antibodies (top panel). Membranes were stripped and re-probed with antibodies for total-mTOR (second panel). Whole cell lysates were Western blotted with antibodies against phospho-p70, threonine 389 (third panel) and alpha tubulin (bottom panel). (C) CDDO-Im inhibits the mTOR pathway in cancer cells. PC-3 cells were treated with 10 µM of the PI3K inhibitor, LY 294002 (LY) or, CDDO-Im (500 nM) for 1 h. Cells were lysed in RIPA buffer and Western immunoblotting was performed with the antibodies listed.