Formaldehyde regulates S-adenosylmethionine biosynthesis and one-carbon metabolism

Abstract

One-carbon metabolism is an essential branch of cellular metabolism that intersects with epigenetic regulation. In this work, we show how formaldehyde (FA), a one-carbon unit derived from both endogenous sources and environmental exposure, regulates one-carbon metabolism by inhibiting the biosynthesis of S-adenosylmethionine (SAM), the major methyl donor in cells. FA reacts with privileged, hyperreactive cysteine sites in the proteome, including Cys120 in S-adenosylmethionine synthase isoform type-1 (MAT1A). FA exposure inhibited MAT1A activity and decreased SAM production with MAT-isoform specificity. A genetic mouse model of chronic FA overload showed a decrease n SAM and in methylation on selected histones and genes. Epigenetic and transcriptional regulation of Mat1a and related genes function as compensatory mechanisms for FA-dependent SAM depletion, revealing a biochemical feedback cycle between FA and SAM one-carbon units.

Fig. 1.. isoTOP-ABPP identifies privileged, formaldehyde-sensitive cysteine sites across the proteome.

(A) Workflow for isotopic tandem orthogonal proteolysis–activity-based protein profiling (isoTOP-ABPP) analysis of formaldehyde (FA)-sensitive cysteine sites applied to whole proteomes. Mouse liver lysate was treated with vehicle or formaldehyde, followed by iodoacetamide (IA)-alkyne cysteine activity-based probe, labeling of isotopic tags with copper-catalyzed azide-alkyne cycloaddition (CuAAC), downstream digestion, and subsequent analysis of peptide fragments using LC-MS/MS. (B) Waterfall plot of light/heavy ratios of formaldehyde (FA)-sensitive cysteine sites from isoTOP-ABPP (yellow), revealing a pattern of privileged targets for this one-carbon unit. Important enzymes involved in one-carbon and formaldehyde metabolism are labeled with the cysteine residue and MAT1A ratios in parentheses. Gray dotted line represents log₂ ratio of 1.58, equivalent to a ratio of 3. Targets were filtered for appearing in at least two technical replicates. (C) Pie chart summarizing the KEGG pathways most abundant with enzymes with ratios greater than 3 for formaldehyde-sensitive cysteines. Pathways were separated into two pie charts by metabolism and cellular processes (308 proteins, left) and organismal systems and diseases (172 proteins, right).

Fig. 2.. Formaldehyde forms site-specific covalent cysteine modifications on MAT1A.

(A) Workflow for the identification of formaldehyde (FA)-dependent covalent modifications on purified MAT1A protein in vitro. (B to E) Representative MS/MS spectra of formaldehyde modification of (B) Cys104, (C) Cys120, (D) Cys149, and (E) Cys376 upon fragmentation and sequencing to generate b (blue) and y (red) ions to indicate a (B) thiazolidine (Δm/z = +12.00) and (C to E) hemithioacetal (Δm/z = +30.01) modifications, respectively. (F) Crystal structure showing the position of all four identified cysteine residues relative to the SAM-bound active site of MAT1A. The angstrom distance between each cysteine residue and SAM are noted. SAM and imidotriphosphate (PPNP), substitute for ATP, are labeled.

Fig. 3.. Formaldehyde inhibits SAM biosynthesis of MAT1A at Cys120 on purified protein in a dose- and isoform-dependent manner, lowering levels of SAM in cells.

(A) Standard addition curve of formaldehyde in HepG2 by RFAP-1 fluorescent probe. Error bars are SD (n = 4) of technical cell replicates for each concentration. (B) Relative activity of MAT1A WT, MAT2A WT, MAT1A C120S, and MAT1A C376S enzymes in response to 0 (yellow), 100 (orange), or 500 μM (purple) formaldehyde (FA). Error bars denote SD (n = 3) of independent protein aliquots. (C) k_cat of MAT1A WT untreated or treated with 10 μM, 25 μM, 100 μM of formaldehyde, and 100 μM of reactive species (H₂O₂ or NO). Michaelis-Menten kinetic analysis and K_M values are reported in table S3. Error bars are SD (n = 3 or 4) of independent protein aliquots. (D) Measurements of SAE levels in CRISPR-generated HepG2 cell models showing that MAT1A-positive MAT2A KO cells are sensitive to formaldehyde and lead to SAE depletion whereas SAE levels in MAT2A-positive MAT1A KO cells are unaffected by formaldehyde exposure. Error bars are SD (n = 3) of technical cell replicates. Statistical significance (B to D) was determined with one-way ANOVA and P-values are from Tukey’s HSD post hoc analyses.

Fig. 4.. Genetic mouse model of chronic formaldehyde overload has reduced methylation potential that specifically targets histone methyl sinks.

(A) Schematic of SAM and its downstream methylation that were measured in WT and Adh5^−/− mice liver. (B) SAM and SAH measured by mass spectrometry in WT and Adh5^−/− liver. (C) Global DNA methylation measured by 5-methyldeoxycytidine (5mC) and normalized by deoxycytidine (dC). (D) Global RNA methylation measured by N⁶-methyladenosine (m6A) and normalized by adenosine (A). (E) Histone H3 methylation measured by immunoblotting for K4 and K79 mono- (me1), di- (me2), and trimethylation (me3). (F) Histone H3 methylation measured by immunoblotting for K9, K27, and K36 mono-, di-, and trimethylation and K27 acetylation (ac). All histone blots were normalized by total histone H3. WT (n = 5, yellow) and Adh5^−/− (n = 6, orange) of biological replicates. Error bars represent SD for all graphs. Statistical significance was determined with two-tailed t-test.

Fig. 5.. Chronic elevations in formaldehyde induce compensatory increases in MAT1A expression by decreasing promoter methylation.

(A) Schematic and summary of results of MAT1A regulation in response to elevated formaldehyde (FA) in Adh5^−/− mice liver. (B) MAT1A and MAT2A protein expression measured by immunoblotting for WT (n = 5, yellow) and Adh5^−/− liver (n = 6, orange) of biological replicates. (C) Mat1a and Mat2a mRNA transcript levels measured by RT-qPCR. (D to E) Difference in percent methylation of (D) Mat1a and (E) Mat2a DNA CpG sites between Adh5^−/− and WT liver. X-axis shows schematic of Mat1a and Mat2a promoters for CpG sites with greater than 2% methylation. Sites are numbered sequentially relative to the ATG site. P-values are determined with two-way t-test. (F) Heatmap of CpG beta values for MAT1A sites analyzed in the genome-wide CpG analysis. Beta values represent percent methylation from 0 (fully unmethylated) to 1 (fully methylated). (G) Volcano plot of the delta between Adh5^−/− compared to WT for genome-wide CpG beta values. One-carbon and fatty acid metabolism gene hits of interest are labeled. (H) Pathway enrichment analysis of methylation loss in Adh5^−/−. (I) Pathway enrichment analysis of methylation gain in Adh5^−/−. (J) Schematic of human MAT1A and mouse Mat1a promoter with transcription factors (TFs) studied in this work labeled. Transcription factors that were deemed as important for MAT1A promoter sensitivity for formaldehyde are in orange. (K) Luciferase assay of HepG2 human MAT1A methylated and unmethylated promoters with 0 or 200 μM formaldehyde and 5 mM SAM treatments. Data is normalized to untreated control. 0 and 200 μM formaldehyde treatment (n = 14) technical cell replicates from four experiments, formaldehyde with SAM treatment (n = 7) technical cell replicates from two experiments, unmethylated promoter (n = 13) technical cell replicates from four experiments. (L) Luciferase assay of HepG2 siRNA-mediated transcription factor knockdown with human MAT1A methylated promoter with 0 or 200 μM formaldehyde. Data is normalized to each untreated control. HepG2 (n = 7 to 9) of technical cell replicates from two experiments. (M) Expression of several MAT1A-associated transcription factors in WT (n = 8) versus Adh5^−/− (n = 7) of biological replicates. Error bars represent SD for all graphs. Statistical significance was determined with two-tailed t-test. (N) Proposed model of normal one-carbon metabolism in cells through MAT1A-catalyzed production of SAM and demethylation reactions, preserving the carbon unit through folate-mediated synthesis of methionine. (O) Proposed model of formaldehyde (FA)-dependent regulation of one-carbon metabolism in cells under situations of formaldehyde overload, showing decrease of SAM biosynthesis from isoform-specific formaldehyde-inhibition of MAT1A (gray arrows), disruption of the folate cycle due to hypermethioninemia, and further formaldehyde elevation from spontaneous folate degradation (dashed red arrows).