Dysregulation of Folate-Dependent Mitochondrial de novo Thymidylate Biosynthesis Affects Mitohcondrial DNA Integrity
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Alonzo, Judith Raquel
Mitochondrial DNA (mtDNA) Depletion Syndrome (MDS) is a genetic disorder caused by mutations in nuclear-encoded mitochondrial proteins involved primarily in nucleotide or mtDNA synthesis. Folate-dependent one-carbon metabolism (FOCM) is a metabolic network compartmentalized in mitochondria, nucleus, and cytosol, where one-carbon units participate in the de novo synthesis of purines, thymidylate, and methionine metabolism. Either increase or decrease in mitochondrial dTTP pools reduces mtDNA content. However, FOCM has never been investigated as a pathway leading to loss of mtDNA integrity in MDS patients. Folate deficiency reduces de novo dTMP synthesis and increases uracil levels in nuclear DNA (nuDNA). Uracil misincorporation is not mutagenic but affects DNA integrity by inducing chromosome fragmentation and eventually apoptosis. Our knowledge of factors that contribute to uracil accumulation in mtDNA is limited but is expected to mirror uracil misincorporation in nuDNA. Enzymatic disruption of nuclear de novo dTMP synthesis due to lack of SHMT in mice and lack of mitochondrial SHMT2 in CHO cells results in reduced dTMP synthesis capacity. Similarly, mice deficient in MPV17, a protein of unknown function associated with hepatocerebral MDS, have reduced mitochondrial dTMP pools. This study investigates the role of genetic (MPV17 and SHMT2) and non-genetic factors (folate, glycine, and serine) on mtDNA integrity, in the context of mitochondrial de novo dTMP synthesis. These results reveal that mtDNA integrity is compromised in HeLa cells deficient in either MPV17 or SHMT2. MPV17-deficient cells exhibited reduced mitochondrial folate levels, suggesting it interacts with mitochondrial FOCM. Both, cells cultured in folate-depleted medium and MPV17-deficient cells, exhibit elevated uracil accumulation in mtDNA by at least 3-fold. Mitochondrial dTMP synthesis capacity and its incorporation into mtDNA were not affected in MPV17-deficient cells, but the elevated uracil levels imply that availability of dTMP for mtDNA synthesis is compromised. I propose that MPV17 is a mitochondrial dTMP transporter. SHMT2-deficient cells exhibited 164% more mtDNA content than control cells; glycine and serine in the culture medium did not have an effect, indicating that SHMT2 catalytic activity is fundamental for mitochondrial function. These data suggest that FOCM is involved in maintaining mtDNA integrity, which is highly susceptible to uracil misincorporation.
Nutrition; dTMP synthesis; Folate metabolism; Mitochondrial DNA integrity; Mitochondrial inner membrane protein Mpv17; serine hydroxymethyltransferase 2 SHMT2; uracil; Biochemistry; Molecular biology
Stover, Patrick J.
Alani, Eric E.; Weiss, Robert S.
Biochemistry, Molecular and Cell Biology
Ph. D., Biochemistry, Molecular and Cell Biology
Doctor of Philosophy
dissertation or thesis