Fig 2: NISCH C185 S-glutathionylation regulates cell migration and invasion.(a-d) Migration analysis of MCF7 cells expressing NISCH WT or C185S. (a) Analysis of NISCH knockout (KO) in MCF7 cells (left, n = 3), and cell migration analysis (right, n = 3) after CRISPR/Cas9-based NISCH KO. (b) The wound-healing migration analysis. MCF7-NISCH KO (MCF7NIS−KO) cells re-expressing NISCH WT or C185S (MCF7-NISCH WT or C185S) were incubated in high glucose (HG, 25 mM) or low glucose (LG, 5 mM) (n = 6). The yellow color indicates the area without cells. (c) Single-cell migration analysis. MCF7-NISCH WT or C185S cells in LG or HG were monitored by confocal microscopy every 10 min (0–180 min). The images were combined to analyze the migration of individual cells (n = 8–10). (d) Transwell migration analysis. MCF7-NISCH WT or C185S cells were incubated in LG or HG for 24 h (n = 3). (e) Spheroid invasion analysis of MCF7-NISCH WT or C185S. Individual spheroids were incubated in HG or LG and monitored daily for 96 h (n = 10). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test (a, c) or two-way ANOVA and Sidak’s post-hoc test (b, d-e), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Fig 3: NISCH S-glutathionylation activates the PAK1-LIMK1-cofilin pathway.MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG) for 16 h. (a) Phosphorylation levels of PAK1, ERK1/2, and MEK1/2 (n = 3). (b) Phosphorylation levels of LIMK1 and cofilin (n = 3). (c) Paxillin phosphorylation level (n = 3). (d) A model for cell migration induced by NISCH glutathionylation. In a non-stressed condition, NISCH binds to its protein partners, including Rac1 and PAK1, thus suppressing Rac1-and PAK1-mediated cell migration. NISCH may bind and retain Rac1 and integrin-α5 in the cytosol or endosome. However, upon ROS production, NISCH is glutathionylated at C185, dissociating Rac1 and PAK1, but not integrin-α5. The activated PAK1 activates its downstream LIMK1 and inhibits cofilin through phosphorylation, thereby increasing actin dynamics and polymerization. The dissociated Rac1 increases its localization to the membrane, where it activates actin filament branching, induces lamellipodia and membrane ruffles, and increases cell migration. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test (a–c), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Fig 4: NISCH S-glutathionylation disrupts protein-protein interactions, activating the Rac1 pathway.(a) NISCH domain and its interacting partners. NISCH domains interacting with protein partners were drawn in lines in different colors. (b-d) Analysis of NISCH-protein interactions and Rac1 downstream pathway in MDA-MB-231 cells. MDA-MB-231 cells expressing NISCH WT or C185S were incubated in low or high glucose (LG or HG). (b) NISCH glutathionylation dissociates NISCH from Rac1 and PAK1. After incubating cells in LG or HG for 16 h, the co-immunoprecipitation was used to examine the binding of Rac1, PAK1, or integrin α5 (n = 2–3). (c) Rac1 activation analysis after NISCH C185 glutathionylation. After incubating cells for 16 h, the GST-fused p21-binding domain (PBD) derived from PAK1 (GST-PAK1-PBD) was used to pull down the Rac1-GTP form, which was then analyzed by Western blot (n = 2). (d) The co-localization analysis of NISCH and Rac1. After 16 h, cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n = 10 images). The merged images were analyzed to examine the relative displacement of NISCH and Rac1 in the cell periphery at higher magnification (box). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test (b-d), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Fig 5: Engineered NISCH mutant suppresses cancer cell migration and invasion.(a) A scheme of the G-PROV approach that enables functional analysis of glutathionylation on the protein of interest (POI): dehydroglutathione (dhG) is used to introduce a non-reducible glutathione modification on the purified engineered NISCH construct (enNISCH). Subsequently, the glutathione-modified NISCH construct is delivered to cells via fusogenic liposome, where the effect of glutathionylation on NISCH can be analyzed. (b) dhG modification on purified enNISCH. Purified enNISCH WT and C185S were incubated with dhG. The dhG-mediated glutathione modification was analyzed by glutathione antibody (n = 2). (c) Analysis of enNISCH delivered to MDA-MB-231 cells via fusogenic liposome. After incubating fusogenic liposomes containing His-enNISCH constructs (modified or non-modified by dhG) for 2 h, lysates were analyzed by Western blot (n = 2). (d-e) The migration and invasion assays. After incubating fusogenic liposomes for 2 h, MDA-MB-231 cells were analyzed by the wound-healing migration (d, n = 6) and transwell invasion (e, n = 2) assays. (f) The colony formation by enNISCH constructs. Fusogenic liposomes containing enNISCH WT or C185S were incubated with MDA-MB-231 cells on plates, and the number of colonies formed was counted after 15 days. Cells were incubated in DMEM containing 25 mM Glc (d–e) or 5 mM Glc (f). The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test (b–e) or two-way ANOVA and Sidak’s post-hoc test (f), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.