Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a ) ARHGEF7 (human isoform-a, 646 residues, NM_001113513) domains, structure, and selected residues. The structure is an AlphaFold model (AF-Q14155-1-F1-model_v6_isoform_a) of ARHGEF7 showing the structured SH3, DH, and PH domains with cysteines and nearby residues. ( b ) Accessible surface areas (ASA) of cysteines in ARHGEF7. ( c ) Sequence alignments around C312 in ARHGEF7 orthologs. ( d ) Clickable glutathione approach. Cells expressing a glutathione synthetase mutant (GS M4) synthesize and produce clickable glutathione (N 3 -GSH) using endogenous γGlu-Cys and exogenous azido-Ala. Clickable glutathione forms S-glutathionylation with proteins, which are analyzed after click reactions. ( e - f ) Glutathionylation of ARHGEF7 WT and cysteine mutants in MDA-MB-231 cells in response to high glucose (HG, 25 mM) or low glucose (LG, 5 mM) conditions: cells were incubated for 20 h (n=3). ( g - h ) Glutathionylation of ARHGEF7 constructs in MDA-MB-231 cells upon adding EGF (n=3). EGF was incubated for 20 h. The lysates were used for click reactions with biotin-alkyne and analyzed by western blot with FLAG antibody before (input) and after (eluted) pull-down with streptavidin-agarose. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( e - h ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Sequencing, Expressing, Mutagenesis, Incubation, Construct, Western Blot

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a ) A potential mode of ARHGEF7 activation by S-glutathionylation. In the absence of ROS, the DH-PH active site is relatively closed, and the SH3 domain stays bound to the DH-PH or its binding partners, Rac1 or PAK1, through the C-terminal proline-rich sequence (PRS) in Rac1 and PAK1 (left). Upon ROS generation, ARHGEF7 is glutathionylated at C312 at the PH domain, which opens the active site in the DH-PH domains to which SH3-bound Rac1-GDP binds for GDP-GTP exchange (middle). Rac1 activation follows, resulting in PAK1 phosphorylation and activation (right). ( b ) A model for ARHGEF7 activation in cells. In the absence of ROS, ARHGEF7, in complex with GIT1, may be associated with none or PAK1 in cells, without significant Rac1 activation. In the presence of ROS, glutathionylated ARHGEF7/GIT1/PAK1 complex is recruited to the nascent focal adhesion at the protrusion, where PAK1 is dissociated, and glutathionylated ARHGEF7 increases the binding to Rac1 for Rac1 activation. Active Rac1 increases phosphorylation and activation of PAK1. The active Rac1 and PAK1 activate their downstream signaling, including Arp2/3, LIMK-cofilin, and MEK1/2-ERK1, which increases branching and polymerization of actin filaments, lamellipodia formation, and cell migration.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Activation Assay, Binding Assay, Sequencing, Phospho-proteomics, Migration

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a - b ) In-vitro scratch migration analysis of MDA-MB-231 cells expressing ARHGEF7 construct in high glucose (HG, 25 mM) or low glucose (LG, 5 mM) conditions (n=4) ( a ) or in response to EGF (n=9) ( b ). Yellow indicates areas without cells. ( c - d ) Transwell invasion analysis of MDA-MB-231 cells expressing ARHGEF7 constructs in HL or LG (n=3) ( c ) or upon incubating EGF (n=3) ( d ). Cells were incubated in HG or LG ( a , c ) or with EGF for 20 h ( b , d ). A scale bar = 500 μm. Data represent the mean ± SD. The statistical difference was analyzed by two-way ANOVA and Tukey’s post-hoc test ( a - d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: In Vitro, Migration, Expressing, Construct, Incubation

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: Analysis of ARHGEF7 protein interactions and Rac1-PAK1 downstream signaling. MDA-MB-231 cells expressing ARHGEF7 WT or C312S were incubated in high glucose (HG, 25 mM) or low glucose (LG, 5 mM) for 20 h. ( a ) ARHGEF7 S-glutathionylation increases its binding to Rac1. ARHGEF7 co-immunoprecipitation (co-IP) with Rac1 and PAK1 (n=3). ( b ) Rac1 is activated upon ARHGEF7 S-glutathionylation. Active Rac1 was enriched by purified GST-PBD (p21-binding domain derived from PAK1) and analyzed by western blots (n=3). ( c ) ARHGEF7 S-glutathionylation activates PAK1 and its downstream signaling. Phosphorylation levels of PAK1, LIMK1, and MEK1/2 were analyzed by western blot (n=3). ( d ) ARHGEF7 S-glutathionylation increases Rac1 localization at the cell periphery. The co-localization images of ARHGEF7 and Rac1 (left). Cells were fixed and analyzed by antibodies to FLAG (green) and Rac1/Cdc42 (red) (n=10 images). White arrows indicate the intense co-localization of ARHGEF7 and Rac1. Pearson’s correlation coefficients (right) were calculated after Intermodes thresholding to select for colocalization of ARHGEF7 and Rac1 at the cell periphery (see also Figure S6). A scale bar = 10 μm. Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a-d ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: Expressing, Incubation, Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Purification, Derivative Assay, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: ARHGEF7 S-glutathionylation promotes cancer cell migration through Rac1 activation

doi: 10.64898/2026.05.01.722049

Figure Lengend Snippet: ( a - c ) In vitro binding analysis of ARHGEF7 constructs (DP or SDP) with GST-Rac1. Purified ARHGEF7 constructs (DP and SDP) were incubated with oxidized glutathione (GSSG) to induce S-glutathionylation. Following dialysis, ARHGEF7 constructs were incubated with GST-Rac1 immobilized on glutathione-Sepharose. GST-Rac1 and ARHGEF7 constructs were analyzed by Western blot before and after GST pull-down. ( a ) Comparison of Rac1 binding to ARHGEF7 DP WT and ARHGEF7 SDP WT (n=3). ( b ) Comparison of Rac1 binding to ARHGEF7 SDP WT and ARHGEF7 SDP C312S (n=3). ( c ). Comparison of ARHGEF7 SDP WT binding to Rac1 with different active site states (none, GDP, GTPγS) (n=3). ( d ) In vitro binding analysis of ARHGEF7 with PI(3,5)P 2 . Purified ARHGEF7 SDP treated without and with GSSG were incubated with biotinylated PI(3,5)P 2 on micelle. Bound ARHGEF7 SDP constructs were analyzed by Coomassie stain (CM) (n=3). ( e - f ) ARHGEF7 increases its GDP-GTP exchange rate upon S-glutathionylation. Different purified ARHGEF7 constructs were incubated with GSSG and used for enzyme kinetics. Rates were determined by a loss of fluorescence when Bodipy-GDP bound to Rac1 dissociates from Rac1. ( e ) Rate measurement by purified ARHGEF7 DP or SDP constructs without or with incubation of GSSG. Rates were determined by increasing enzyme concentration (n=3) (left) and substrate concentration (Michaelis-Menten) (n=3) (right). ( f ) Rate measurement by a full-length ARHGEF7 construct without or with glutathionylation. FLAG-ARHGEF7 purified by affinity purification was incubated with GSSG and used to measure rates (n=4). Data represent the mean ± SD. The statistical difference was analyzed by one-way ANOVA and Tukey’s post-hoc test ( a-d ) or unpaired t-test ( f ), where *p < 0.03, **p < 0.002, ***p < 0.0002, ****p < 0.0001.

Article Snippet: For binding analysis with PI(3,5)P 2 , dialyzed glutathionylated ARHGEF7 SDP (WT and C312S) was then incubated with biotinylated PI(3,5)P 2 PolyPIPosomes (Echelon Biosciences, Cat# Y-P035) at room temperature for 30 min.

Techniques: In Vitro, Binding Assay, Construct, Purification, Incubation, Western Blot, Comparison, Staining, Fluorescence, Concentration Assay, Affinity Purification