title: Alcohol Dehydrogenase 5 Is a Source of Formate for De Novo Purine Biosynthesis in HepG2 Cells.
abstract: Background:Formate provides one-carbon units for de novo purine and thymidylate (dTMP) synthesis and is produced via both folate-dependent and folate-independent pathways. Folate-independent pathways are mediated by cytosolic alcohol dehydrogenase 5 (ADH5) and mitochondrial aldehyde dehydrogenase 2 (ALDH2), which generate formate by oxidizing formaldehyde. Formate is a potential biomarker of B-vitamin-dependent one-carbon metabolism.Objective:This study investigated the contributions of ADH5 and ALDH2 to formate production and folate-dependent de novo purine and dTMP synthesis in HepG2 cells.Methods:ADH5knockout and ALDH2 knockdown HepG2 cells were cultured in folate-deficient [0 nM (6S) 5-formyltetrahydrofolate] or folate-sufficient [25 nM (6S) 5-formyltetrahydrofolate] medium. Purine biosynthesis was quantified as the ratio of [14C]-formate to [3H]-hypoxanthine incorporated into genomic DNA, which indicates the contribution of the de novo purine synthesis pathway relative to salvage synthesis. dTMP synthesis was quantified as the ratio of [14C]-deoxyuridine to [3H]-thymidine incorporation into genomic DNA, which indicates the capacity of de novo dTMP synthesis relative to salvage synthesis.Results:The [14C]-formate-to-[3H]-hypoxanthine ratio was greater inADH5knockout than in wild-type HepG2 cells, under conditions of both folate deficiency (+30%;P< 0.001) and folate sufficiency (+22%;P= 0.02). These data indicate that ADH5 deficiency increases the use of exogenous formate for de novo purine biosynthesis. The [14C]-deoxyuridine-to-[3H]-thymidine ratio did not differ betweenADH5knockout and wild-type cells, indicating that ADH5 deficiency does not affect de novo dTMP synthesis capacity relative to salvage synthesis. Under folate deficiency, ALDH2 knockdown cells exhibited a 37% lower ratio of [14C]-formate to [3H]-hypoxanthine (P< 0.001) compared with wild-type HepG2 cells, indicating decreased use of exogenous formate, or increased endogenous formate synthesis, for de novo purine biosynthesis.Conclusions:In HepG2 cells, ADH5 is a source of formate for de novo purine biosynthesis, especially during folate deficiency when folate-dependent formate production is limited. Formate is also shown to be limiting in the growth of HepG2 cells.
authors.affiliation: Division of Nutritional Sciences and.
authors.forename: Sajin
authors.initials: S
authors.lastname: Bae
authors.affiliation: Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY.
authors.forename: James
authors.initials: J
authors.lastname: Chon
authors.affiliation: Division of Nutritional Sciences and.
authors.forename: Martha S
authors.initials: MS
authors.lastname: Field
authors.affiliation: Division of Nutritional Sciences and pjs13@cornell.edu.|Graduate Field of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Stover
mesh_terms: D000071396:Aldehyde Dehydrogenase, Mitochondrial / Q000235:genetics / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D005561:Formates / Q000378:metabolism*; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D056945:Hep G2 Cells; D006801:Humans; D011687:Purines / Q000096:biosynthesis*; D013938:Thymidine Monophosphate / Q000096:biosynthesis
chemical_list: D005561:Formates; D011687:Purines; D013938:Thymidine Monophosphate; D000445:Aldehyde Oxidoreductases; C096127:ALDH2 protein, human; D000071396:Aldehyde Dehydrogenase, Mitochondrial; C087006:formaldehyde dehydrogenase, glutathione-independent
keywords: alcohol dehydrogenase 5; aldehyde dehydrogenase 2; folate; formaldehyde dehydrogenase; formate; purine synthesis; thymidylate synthesis