Fig. 3: PPIA directly interacts with NRF2 and P174 of NRF2 is essential for PPIA binding.

A Co-immunoprecipitation analysis of endogenous proteins of PPIA and NRF2 using A549 cell lysates. B Pull-down assay of PPIA WT/NRF2 and PPIA^R55A&F60A/NRF2. A549 cell lysate was incubated with PPIA WT or PPIA^R55A&F60A pre-loaded beads, and NRF2 protein retained on the beads were detected by immunoblot. C The influence of CsA on the interaction between PPIA and NRF2. A549 cell lysate were incubated with PPIA pre-loaded beads in the presence of CsA (0, 3, 10 μM) and then subjected to pull-down assay. D Schematic diagram of various NRF2 truncation. E The binding of full-length and truncated NRF2 to PPIA as determined by pull-down assay. Various NRF2 truncations were overexpressed in HEK293T cells (as described in D). F CHX chase assay of NRF2 (WT or mutant P174A) protein stability. Cells were treated with CHX (100 μg/mL) at the indicated time points and then subjected to immunoblot (upper panel). Quantitative data was provided in the lower panel. G Crystal structure of PPIA in complex with NRF2 fragment PPIA-Binding Motif (PBM) (¹⁶⁹VAQVAPVD¹⁷⁶). PBM peptide is deeply embedded into catalytic pocket of PPIA presented as gray surface. ¹⁷²VAPV¹⁷⁵ residues of NRF2 peptide are displayed as slate stick. H Expansion of the catalytic pocket of PPIA in complex with NRF2 fragment PBM. PPIA is shown in gray cartoon and the interacted residues are displayed as salmon sticks. NRF2 PBM fragment is presented as slate sticks. The black dashed line denotes the hydrogen contact. The results of panels (A–C, E, F) are representative of three independent experiments. F represents mean ± SD of three independent experiments. P values were analyzed using Two-tailed unpaired Student’s t-test, P < 0.05 was considered statistically significant. Source data are provided as a Source Data file.