Fig 1: Hypoglycemia and CRT0063465 regulate interaction of PGK1/DJ1 with TRF2 and telomeres. (a), CRT0063465 rescues a nuclear telomere-binding activity in hypoglycemia. HCT116 cells were cultured for 1 week in 5 mM (physiological) or 500 μM (hypoglycemic) glucose with or without 10 nM CRT0063465. Post-treatment, nuclear extracts were prepared for gel shift analysis. Mobility of labeled probe was visualized in the absence of nuclear extract (“probe” lanes), or in the presence of 5 μg nuclear extract from each treatment condition with (“NucEx + comp” lanes) or without (“NucEx” lanes) presence of excess cold-competitor. The experiment was performed twice. A representative image is shown. (b), PGK1 and DJ1 binding to TRF2 is modulated by hypoglycemia and CRT0063465. HCT116 cells were cultured for 1 week in 5 mM (physiological) or 500 μM (hypoglycemic) glucose with or without 10 nM CRT0063465. Co-immunoprecipitation of TRF2 was performed using Dynabeads. Immunoprecipitates were blotted for the presence of DJ1, PGK1, Ku80 and TRF2. The experiment was performed twice. A representative blot is shown. (c), PGK1 and DJ1 differentially bind to telomeric DNA under hypoglycemia and CRT0063465 treatment. HCT116 cells were cultured for 1 week in physiological or hypoglycemic conditions with or without 10 nM CRT0063465 prior to chromatin immunoprecipitation using the antibodies shown. Mean ± SEM of 3 experiments with qPCR performed in triplicate.
Fig 2: Identification of a complex between PGK1 and DJ1 modulated by CRT0063465. (a), Putative mode of binding between CRT0063465 and DJ1. (b) Close up of proposed binding mode, showing electron density of the oxidized C106 residue and adjacent residual electron density overlaid with the CRT0063465 carboxylate moiety. (c), Detection of a cellular PGK1/DJ1 complex. A2780 cells were treated with CRT0063465 or vehicle for 1 h and harvested for immunoprecipitation of either target. Immunoblots were performed to detect the presence of each protein in unprecipitated cell lysates or in immunoprecipitates. The experiment was performed twice. Representative blots are shown. (d), Close-up of candidate interfacial binding mode. Interaction between CRT0063465 and oxidized C106 in DJ1 leaves the PGK1 hydrophobic tunnel accessible to the bromoaryl portion of CRT0063465. (e), Molecular docking simulation of proposed 2:2:2 complex between PGK1, DJ1 and CRT0063465. The compound-bound PGK1 and DJ1 X-ray structures were aligned along the axis of CRT0063465 by overlaying the compound scaffold in either structure.
Supplier Page from Abcam for Recombinant Human PARK7/DJ1 protein