Journal: Theranostics

Article Title: Discovery of a novel Nrf2 activator that modulates mitochondrial function in neurons by regulating DHRS3-Nrf2 interaction after ischemic stroke

doi: 10.7150/thno.128602

Figure Lengend Snippet: DHRS3 bound to Nrf2 and inhibited its binding to the ARE element, which was inhibited by Cpd.51. (A) Co-staining with DHRS3 and DAPI in SH-SY5Y cells demonstrated that OGD/R increased nuclear localization of DHRS3, which Cpd.51 effectively inhibited. (B) Three-dimensional structural models of DHRS3 and Nrf2 proteins, as well as the specific binding interfaces mediating their direct interaction, were generated using molecular docking approaches. (C, D) Immunoprecipitation assay of the interaction in whole cell lysate between Nrf2 and DHRS3 in physiological or OGD/R conditions, and SH-SY5Y cells treated with Cpd.51 (100 nM) or Vehicle (DMSO). n = 3. (E) Cpd.51 inhibited the interaction between Nrf2 and DHRS3 detected by GST pulldown. (F) DHRS3-overexpression inhibited the activation effect of Cpd.51 on the ARE luciferase reporter gene. n = 6. (G, H) DHRS3-overexpression suppressed the expression of downstream target gene and protein of Nrf2. n = 3 or 4. (I) The GST pull-down assay demonstrated that Nrf2 specifically bound to the region of DHRS3 encompassing amino acids 100 to 190. (J, K) Overexpression of DHRS3 inhibited the expression of downstream target genes and proteins of Nrf2, except for DHRS3 (1-100 aa). n = 3. (L) Three-dimensional and two-dimensional structural models of Cpd.51 and DHRS3 proteins, as well as the specific binding interfaces mediating their direct interaction, were generated using molecular docking approaches. (M) RMSD of complexes, proteins and small molecule ligands. (N) The distance between the binding sites of proteins and small molecules. (O) Free Energy Landscape. (P) SPR was employed to determinate of the binding affinity between Cpd.51 and DHRS3. (Q) CETSA analysis of intracellular binding between Cpd.51 (100 nM) and DHRS3 at different temperature. n = 3. Results are expressed as mean ± SD. J, ### P < 0.001 vs. NC-OE group. * P < 0.05, *** P < 0.001 vs. DHRS3-OE group. Statistical differences among groups were analyzed by using one-way ANOVA followed by Tukey's post-hoc test. G, ## P < 0.01, ### P < 0.001. * P < 0.05, ** P < 0.01, *** P < 0.001. Statistical differences among groups were analyzed by using two-way ANOVA followed by Tukey's post-hoc test.

Article Snippet: GST-Nrf2 (YB710012, Ybio, China) or GST (Ag0040, Proteintech, China, as control) proteins were mixed with GST Resin (EA-IP-K008, Elabscience Biotechnology, China) for 2 h incubation on the shaker at 4 °C.

Techniques: Binding Assay, Staining, Generated, Immunoprecipitation, Over Expression, Activation Assay, Luciferase, Expressing, Pull Down Assay