Extended Data Fig. 4: Characterization of functional effects of lactate and zinc on SENP family.

(a) Effect of ZnSO₄ on recombinant human SENP1 deSUMOylation activity. n = 6; 7.5 nM n = 5 independent samples. (b) Effect of lactate on zinc-mediated inhibition of recombinant SENP1 deSUMOylation activity. n = 6; 120 nM zinc n = 4 independent samples. (c) Effect of MgCl₂ and CaCl₂ on recombinant human SENP1 deSUMOylation activity. n = 6; 500 nM CaCl₂ n = 5 independent samples. (d) Effect of lactate on recombinant human SENP1 deSUMOylation activity. n = 6 independent samples. (e) Effect of ZnSO₄ and lactate on recombinant human SENP2, SENP3, SENP5, SENP6, and SENP7 deSUMOylation activity. n shown in panel and indicate independent samples. (f) Sequence alignment of human SENP family active site regions highlighting residues invoked in SENP1 co-ordination of lactate-zinc complex (C535 and N556). (g) SUMO2/3 (top) and APC4 (bottom) immunoblot of immunopurified APC4 following lactate treatment in cellular lysates in the presence of EDTA to chelate zinc, including supplementation with Mg²⁺ to control for effects of EDTA on Mg²⁺ chelation. Representative blot from three experiments shown. (h) SUMO2/3 (top) and APC4 (bottom) immunoblot of immunopurified APC4 following ZnSO₄ treatment in cellular lysates demonstrate that elevation of zinc stabilizes APC4 SUMOylation. Representative blot from three experiments shown. (i) SUMO2/3 (top) and APC4 (bottom) immunoblot of immunopurified APC4 following treatment with sub-optimal zinc and lactate in cellular lysates. Representative blot from four experiments shown. (j) Densitometry quantification immunoblot replicates from panel i of immunopurified APC4 following suboptimal zinc treatment in the presence or absence of lactate. n = 4 cell replicates. (k) Sequencing of SENP1 in HCT116 expressing lentiviral WT and mutant constructs containing WT sequence N556, and mutant A556. Blue region highlights silent PAM mutation, red region highlights N556A mutation. (l) Densitometry of immunopurified APC4 following lactate treatment in cellular lysates from SENP1 WT and N556A HCT116 cells, related to Fig. 3k. (m) Effect of lactate on zinc-mediated inhibition of recombinant human SENP1 deSUMOylation activity in the presence of 20 mg/ml albumin. n = 3 independent samples. (two-tailed Student’s t-test for pairwise comparisons in l, one-way ANOVA for multiple comparisons in a,b,j,m). ANOVA analyses were subjected to Bonferroni’s post hoc test. Data are mean ± s.e.m.