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. 2016 Aug 9;113(34):E4966–E4975. doi: 10.1073/pnas.1603244113

Fig. 1.

Fig. 1.

Characterization of RSL3 binding to GPX4. (A) Structure of RSL3 affinity probe used (Upper) and schematic drawing of the affinity complex formed during the pull-down assay (Lower). (B) A pull-down analysis showing that RSL3 does not bind to a mutant GPX4 where the active-site selenocysteine and all other cysteines were replaced with either alanine or serine. The schematic structure of GFP-GPX4 protein is shown (Upper). Cells stably expressing either WT or mutant [AllCys(-)] GFP-GPX4 protein were treated with (1S, 3R)-RSL3-Fcn, then the cell lysates were subjected to affinity purification using anti–Fcn-conjugated Sepharose beads. The binding between GFP-GPX4 protein and (1S, 3R)-RSL3-Fcn was determined by detecting the presence of GFP-GPX4 protein in the eluent using Western blot with anti-GFP antibody. Arrowheads indicate GFP-GPX4 protein; asterisks indicate nonspecific bands. W.C.L., whole-cell lysate before pulldown. (C) Selenocysteine is much more reactive to RSL3 than cysteine at the active site. Cells stably expressing A46U or A46C protein (see diagram in the figure) were treated with (1S, 3R)-RSL3-Fcn and subjected to the same analysis as in B. (1S, 3R)-RSL3-Fcn interacted with A46U protein but not with A46C protein. Arrowheads indicate GFP-GPX4 protein; asterisks indicate nonspecific bands. (D) RSL3 interacts weakly with other cysteines on GPX4. Cells stably expressing indicated mutant protein (see text for detailed information) were treated with (1S, 3R)-RSL3-Fcn and subjected to the same analysis as in B. Some mutant proteins such as A10C and S37C interacted with (1S, 3R)-RSL3-Fcn whereas others did not. The interaction was the strongest when WT GPX4 was expressed (thickest band in WT sample). Arrowheads indicate GFP-GPX4 protein; asterisks indicate nonspecific bands. (E) Some ferroptosis-inducing compounds competed off RSL3 binding to GPX4. HT-1080 cells were treated with (1S, 3R)-RSL3-Fcn in the presence or absence of indicated competitors, and cell lysates were subjected to pull-down experiment with anti-Fcn antibody. The binding between (1S, 3R)-RSL3-Fcn and endogenous GPX4 protein was determined by Western blotting with anti-GPX4 antibody. The (1S, 3R)-RSL3-Fcn binding to GPX4 was suppressed in samples where free (1S, 3R)-RSL3 was used as a competitor. Different GPX4 inhibitors competed off (1S, 3R)-RSL3-Fcn binding to GPX4 protein with varying degrees. (F) The amount of endogenous GPX4 protein bound to the affinity probe in E was quantified using Odyssey software (LI-COR Biosciences). Data represent mean ± SD calculated from technical triplicates. (G) Covalent interaction between GPX4 and RSL3 was confirmed by immunoprecipitating GFP-GPX4/(1S, 3R)-RSL3-Fcn complex with anti-GPX4 antibody and performing Western blot with anti-GFP or anti-Fcn antibodies. Arrow indicates GFP-GPX4 protein band in the Western blot analysis. (H) The (1S, 3R)-RSL3 covalently interacts with purified GPX4U46C protein. Purified GPX4U46C protein was mixed with (1S, 3R)-RSL3-Fcn, then the mixture was resolved on a denaturing gel and transferred to a membrane. Western blotting with anti-Fcn antibody detected (1S, 3R)-RSL3-Fcn in a position corresponding to the size of GPX4U46C protein (∼21 kDa), suggesting that (1S, 3R)-RSL3-Fcn covalently attached to GPX4U46C protein and migrated together in this denaturing condition. The Western blot band became thinner when a lesser amount of (1S, 3R)-RSL3-Fcn was added to the mixture (Upper). The band size remained the same across the sample when anti-GPX4 antibody was used because equal amount of GPX4U46C protein was added across the samples (Lower). (I) Inhibition of GPX4 enzyme activity by (1S, 3R)-RSL3 or (1R, 3R)-RSL3. GPX4 enzyme activity was assayed by mixing cell lysates with PC hydroperoxides (PC-OOH), a GPX4-specific substrate, and by determining the amount of substrate left in the reaction mixture using an LC-MS instrument. The arrow indicates the LC-MS peak corresponds to PC-OOH. (J) Area under the curve was determined from each mass chromatogram in I (retention time from 8.2 to 8.8 min; dotted lines in the chromatogram) and used to draw a concentration-dependent curve of GPX4 inhibition by (1S, 3R)-RSL3.