Fig 2: MTHFD2 is acetylated at K88.a Western blot detection of acetylation levels of ectopically expressed MTHFD2(left) and endogenous MTHFD2 (right) after treated with 5 µm NAM for the duration indicated. Flag-MTHFD2 was immunoprecipitated from cell lysate and its acetylation was examined with a pan-acetylated lysine antibody (a-AcK). Endogenous MTHFD2 acetylation was examined with pan-acetylated lysine antibody (a-AcK) and site-specific K88 acetylation antibody (Ac-K88). Relative MTHFD2 acetylation was normalized by Flag protein or endogenous MTHFD2. b In vitro MTHFD2 acetylation assay. MTHFD2 proteins were incubated with different concentrations of acetyl-CoA as indicated. Protein acetylation level was analyzed, and relative MTHFD2 acetylation was normalized by His protein. c Identification of acetylated MTHFD2 peptides by tandem mass spectrum. Identified sites were shown in chat. d Acetylated MTHFD2 K88 was identified by a tandem mass spectrum. The identified peptide is shown. e K88 in MTHFD2 is evolutionarily conserved. The sequences around MTHFD2 K88 from different species were aligned. f K88 is the major acetylation residue of MTHFD2. Flag-tagged WT MTHFD2 or mutants (K44Q, K50Q, K88Q, and K104Q) were expressed in HEK293T cells, followed by treatments with or without 5 µm NAM. Flag-MTHFD2 was immunoprecipitated and its acetylation was examined with a-AcK. g Characterization of anti-acetyl-MTHFD2 (K88) (a-acK88) antibody. Specificity of antibody against acetylated K88 residue of MTHFD2 was determined by dot blot assay. Nitrocellulose membrane was spotted with different amounts of acetyl-K88 peptide or unmodified peptide and immunoblotted with anti-acetyl-MTHFD2 (K88) antibody. h Characterization of acetyl-MTHFD2 (K88) antibody. Acetylation level of MTHFD2-Flag, MTHFD2-K88R-Flag, or MTHFD2 K88Q-Flag ectopically expressed in HCT116 cells was measured by the site-specific K88 acetylation antibody (Ac-K88).

Fig 3: K88 acetylation of MTHFD2 is downregulated in CRC.a In total, 15 pairs of tumor tissues (T) and adjacent normal tissues (N) were lysed. Protein levels of MTHFD2-K88Ac, MTHFD2, and SIRT3 were determined by direct western blot. Relative protein levels were normalized by a-Tubulin. Shown are eight pairs of samples. b Quantification of relative MTHFD2, MTHFD2-K88Ac and SIRT3 protein levels in the 15 pairs of samples tested. The intensities of indicated proteins were quantified using the ImageJ software, followed by statistical analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. c SIRT3 protein levels show negative correlation with MTHFD2-K88Ac. Correlation between MTHFD2 protein levels and MTHFD2-K88Ac levels in the tested 15 pairs of samples. Statistical analyses were performed with F test. d Working model. Acetylation at K88 under cisplatin treatment decreases SIRT3 expression, increases MTHFD2 acetylation level and inhibits MTHFD2 enzyme activity. In CRC, high SIRT3 levels promotes deacetylation of MTHFD2 to maintain its high activity and restraining cellular redox balance.

Fig 4: SIRT3 is the major deacetylase for MTHFD2.a, b SIRT3 interacts MTHFD2. Interactions between MTHFD2 and SIRT3 were determined when proteins were co-expressed with each other in HEK293T cells. c, d endogenous SIRT3 interacts with MTHFD2 in vivo. Whole-cell lysates were immunoprecipitated with control IgG, anti-SIRT3, or anti-MTHFD2 antibody, and the precipitated proteins were detected with anti-MTHFD2 or anti-SIRT3 antibody, respectively. e SIRT3 deacetylates MTHFD2 in a dose-dependent manner. Acetylation levels were detected of ectopically expressed MTHFD2(left) and endogenous MTHFD2 (right) after transfected with plasmids that express increasing amounts of SIRT3. Whole-cell lysates were immunoprecipitated with Flag-beads(left) or anti-MTHFD2 antibody(right), and the precipitated proteins were detected by an acetylation assay. f SIRT3-knockout increases MTHFD2 acetylation level. SIRT3-knockout generated by CRISPR/Cas9 were analyzed by western blotting for SIRT3 expression. Whole-cell lysates of HCT116 WT cells or SIRT3-knockout cells were immunoprecipitated with anti-MTHFD2 antibody, and the precipitated proteins were detected by an acetylation assay. g Acetylation levels were detected of ectopically expressed(left) and endogenous MTHFD2 (right) after transfected with empty vector or with SIRT3(WT) or SIRT3-H248Y (H248Y). Whole-cell lysates were immunoprecipitated with Flag-beads(left) or anti-MTHFD2 antibody(right), and the precipitated proteins were detected by an acetylation assay. h SIRT3 deacetylates MTHFD2 in vitro. Recombinant human (rh) SIRT3 deacetylates the site-specific K88-acetylated MTHFD2 proteins incubated with NAD+ or NAM, and acetylation level was determined by Ac-K88 and pan-AcK. i Recombinant catalytically inactive (rh) SIRT3 mutant H248Y and the site-specific K88-acetylated MTHFD2 incubated with NAD+ or NAM, and acetylation level was determined by Ac-K88 and pan-AcK.

Fig 5: K88 acetylation reduces MTHFD2 enzymatic activity.a In vitro acetylation of MTHFD2 decreases its activity. MTHFD2 proteins were purified and incubated with or without 400 µm acetyl-CoA for 15 min at 30 °C before specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. b, c K88Q mutation decreases MTHFD2 activity. MTHFD2 proteins were purified by immunoprecipitation from HCT116 cells b or recombinant expressed in E. coli and purified by nickel affinity chromatography c, and the enzyme activity assays were performed. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. d The site-specific K88-acetylated MTHFD2 decreases its enzyme activity. MTHFD2 and K88Ac-MTHFD2 were recombinant expressed and purified by nickel affinity chromatography and performed activity assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. e Cisplatin represses MTHFD2 activity by acetylation. HCT116 cells were transfected with Flag-tagged MTHFD2 and treated with or without Cisplatin for 24 h. Immunoprecipitated MTHFD2 activity was detected by specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. f Cisplatin cannot repress MTHFD2 activity in vitro. MTHFD2 proteins were purified and incubated with or without 40 µm cisplatin for 15 min at 30 °C before specific enzymatic assay. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. g Molecular modeling of acetylation of K88 in MTHFD2 (left, WT; right, K88Ac) with K88 (PDB ID 5TC4) bounded with LY345899 (an inhibitor of MTHFD2 similar to substrate). The WT MTHFD2 is shown in green chains, K88Ac is shown in blue chains and LY345899 is shown in stick format. Bottom view is enlarged to show that LY345899 binds to K88 of MTHFD2.