Fig. 6: Zn²⁺ tunes SENP1 redox sensitivity and SENP1-dependent β-cell exocytosis.

A Activity of SENP1 WT and C535S in the presence or absence of Ca²⁺, Co²⁺, Ni²⁺ and Zn²⁺ (n = 3 experiments, ^**p = 0.0071, ^***p = 0.00029). Data were normalized to C535S activity, as this is more resistant to oxidation. B Dose-response curve of SENP1 inhibition by ZnCl₂ (n = 3 experiments, ^***p = 1.9 × 10⁻¹⁴). C SENP1 inhibition by ZnCl₂ could be reversed with the chelating reagent EDTA (n = 4 experiments, ^***p = 1.6 × 10⁻⁸). D–F SENP1 activity, measured using a native-PAGE assay, inhibited by Zn²⁺-carrying (MT1X, panel D, ^***p = <1 × 10⁻¹⁵, 1.5 × 10⁻⁶, 1.5 × 10⁻¹²) but not Zn²⁺-depleted (T1X, panel E) metallothionine or by Zn²⁺-carrying MT1X in the presence of EDTA (panel F, ^***p = all are 1.9 × 10⁻¹⁴) (n = 6 experiments). G Effect of Zn²⁺ on exocytosis from β-cells of βSENP1-WT and -KO mice at 5 mM glucose (n = 46, 35, 46, 39, 23, 23 cells, ^@p = 0.023, ^@@p = 0.0035, 0.0041, 0.0012, ^@@@p = 7.4 × 10⁻⁷; from 6, 6, 3 pairs of mice, ^*p = 0.030, ^**p = 0.030). H Concentration-response curve of SENP1 activity, with enzyme prepared with or without ZnCl₂ during refolding, to inhibition by H₂O₂. Activity of SENP1 C603S is shown for comparison (n = 6 experiments). I Activity of SENP1 C603S, WT, and C535S in the presence of 1 µM Zn²⁺ and subsequent activation with 5 mM GSH and 10 µg/ml GRX1. 2-mercaptoethanol (2-ME, 1 mM) was used to fully activate enzymes (n = 4 experiments, ^*p = 0.014, ^***p = 3.5 × 10⁻⁶, 5.1 × 10⁻¹⁰, 5.1 × 10⁻¹¹, 2.6 × 10⁻⁶, 6.5 × 10⁻⁵). In panel G data are shown as individual cells (gray) or cells averaged by animal (dark). Data are mean ± SEM, compared with RM one-way ANOVA followed by Tukey post-test (panels C–F) or RM two-way ANOVA followed by Bonferroni post-test within groups (panels A, I) or across conditions (panels B, G). Source data are provided as a Source Data file.