Article Title: Chemoproteomics Maps Glycolytic Targetome in Cancer Cells
Figure Lengend Snippet: G3P is an intrinsic proliferation-suppressive metabolite via targeting ENO1 and MTHFD1. (a) Intracellular concentrations of G3P in HCT116 cells treated with solvent control, NaF (3 mM) and GNE140 (10 μM) for 24 hr determined by targeted LC/MS-MS (n = 3). (b) Accumulative G3P upon direct supplementation of 0.5 mM G3P for 4 hr in HCT116 cells (n = 3). (c) Growth curve of HCT116 cells under G3P administration (n = 5). (d) Colony formation assay validates G3P-induced suppressive proliferation in HCT116 cells. (e) S-plot of TRPs shows top-ranked target proteins of G3P that displayed marked accessibility changes following G3P administration. (f) Thermal shift assay validated the ENO1-G3P engagement in HCT116 cell lysates. (g) Volcano plot of the TRAP-identified G3P TRPs in recombinant ENO1. Accessibility of the TRP carrying Lys330/Lys335 exhibited the most dramatic change after G3P incubation. (h) The ENO1 TRPs of G3P identified by TRAP are highlighted (PDB: 3B97). TRPs spanning residue 328-343, 407-426 and 254-269 are color-coded in blue, yellow and purple. (i) Binding affinities of G3P to the wide type (WT) and the K330E mutant ENO1 measured by SPR. (j) Stability changes of the WT and K330E mutant ENO1 induced by G3P incubation examined by nanoDSF. (k) Conservation analysis of K330 of ENO1 among several species. (l) Enzyme activity assay showed G3P inhibited the activity of WT ENO1 yet weakly to the K335E mutant ENO1 (n = 3). (m) Proliferation of the control and ENO1 knockdown cells under G3P treatment (n = 5). (n) Homology modeling of MTHFD1 indicates the TRP of G3P is located in proximity to the ATP binding site and thus the interaction likely interferes with the 10-formyl-THF synthetase reaction. (o) Multi-target regulatory network of G3P uncovered by TRAP. (p) DrugBank and non-DrugBank fraction of the identified glycolytic targetome. (q) NAMPT enzymatic assay suggests synergistic inhibition by FBP with FK866. (r) TRAP analysis reveals the TRP (blue, cartoon) of FBP located in proximity to FK866 (orange, stick) displayed reduced accessibility in response to FBP (n = 3), suggesting a FBP binding-promoted interaction between FK866 and NAMPT (PDB: 2GVJ). For (a)-(c) and (l)-(m), data represent mean ± SEM. For (a, b, m), *p < 0.05, **p < 0.01, ***p < 0.001, n.s. non-significant, Student’s t-test.
Article Snippet: Scrambled small interfering RNA (siRNA) targeting TRIM28 (sc-38550), ENO1 (sc-37007), MTHFD1 (sc-61082), GSTP1 (sc-72091) and control siRNA (scrambled siRNA, sc-37007) were purchased from Santa Cruz Biotechnology (USA).
Techniques: Liquid Chromatography with Mass Spectroscopy, Colony Assay, Thermal Shift Assay, Recombinant, Incubation, Binding Assay, Mutagenesis, Nano Differential Scanning Fluorimetry, Enzyme Activity Assay, Activity Assay, Enzymatic Assay, Inhibition