Identification of a heritable deficiency of the folate-dependent enzyme 10-formyltetrahydrofolate dehydrogenase in mice.

Abstract

During the analysis of liver protein expression in the offspring of male mice Irradiated with fissionspectrum neutrons, one ofprin displayed a heritable 50% decrease in the abundance of two proteins. Homozygous mice lacing detectable quantities of these proteins were obtained through breeding. Characterization of this protein deficiency has identified these liver proteins as forms of the enzyme 10-formyltetrahydrofolate dehydrogenase (10- Although the physical properties and substrate and product binding characteristics of 10-formyl-THF DH have been described, the physiological role of this enzyme in mammalian metabolism has not been directly addressed. Certain characteristics of mice lacking 10-formyl-THF DH indicate that disruption of the structural gene has significant physiological consequences. While homozygous mice appear completely normal despite this enzyme deficiency, breeding pairs of homozygotes show a dramatically decreased reproductive efficiency (6). Analysis of pool sizes of hepatic folate derivatives revealed shifts in the steady-state abundance of some forms of THF in mice lacking 10-formyl-THF DH. EXPERVIMENTAL PROCEDURESLiver Protein Sample Preparation. Liver protein samples were prepared as described (7) and were either frozen at -700C and later ground in liquid nitrogen or homogenized in 8 volumes of a solution containing 9 M urea, 2% 2-mercaptoethanol, 2% ampholytes (Pharmacia LKB, pH 9-11), and 4% Nonidet P-40. The homogenates were centrifuged at 435,000 x g for 5 min in a Beckman TL100 ultracentrifuge. The resulting supernatants containing denatured liver proteins were collected and stored frozen at -700C until analysis by two-dimensional electrophoresis (2DE).Preparation of Clarified Mouse Liver Cytosol. Mouse liver was minced with scissors and homogenized in 5 volumes (4-5 ml) of 0.25 M sucrose buffered with 10 mM Hepes using a motor-driven pestle at 3000 rpm for 30 strokes on ice. Aliquots (1 ml) of homogenate were then centrifuged for 15 min at room temperature in a Beckman TL100 ultracentrifuge. The supernatants were collected, combined, and dialyzed at 40C against 0.02 M phosphate buffer (pH 7.1), which was changed three times at hourly intervals.2DE. Aliquots (10 pIl) of mouse liver samples were separated in the first dimension by isoelectric focusing in 10-inch rod gels (8). A mixture of 25% Biolyte (Bio-Rad) pH 3-10/ 25% Servalyte pH 3-10/50%6 Biolyte pH 5-7 was used to generate the pH gradient. The second-dimension separation was performed as described by O'Farrell (9) on 10-17% linear gradient slab gels prepared as described (10). Gels were fixed

Regulation of Folate-mediated One-carbon Metabolism by 10-Formyltetrahydrofolate Dehydrogenase

“…First, Krebs et al (19) suggested that FDH serves to remove excess one-carbon units from folate metabolism by depleting pools of folate-activated one-carbon units. In support of this hypothesis, mice with a heritable FDH deficiency exhibit 4-fold lower relative hepatic THF concentrations and 2.6-fold higher relative 10-formyl-THF concentrations compared with the livers of control mice (16). Second, FDH has been suggested to sequester and store cellular folate in the form of THF (2,20).…”

“…FDH catalyzes the NADP ϩ -dependent conversion of 10-formyl-THF to THF and CO 2 (1,15). Mice lacking FDH activity are viable and fertile, indicating that one or more FDH functions are not essential in the laboratory environment (16); FDH may function to regulate the folate-medi-* This work was supported by U.S. Public Health Services Grants HD35687 (to P. J. S.) and DK07158-28 (to M. C. A.).…”

“…Because intracellular FDH concentrations exceed THF concentrations, all cytoplasmic THF is expected to be bound by FDH in vivo (21). Mice lacking FDH exhibit a 35% reduction in total folate concentrations despite being maintained on folate sufficient diets, indicating that FDH may help to maintain cellular folate concentrations by sequestering cellular THF (16). Third, FDH has been proposed to inhibit purine biosynthesis by depleting cellular 10-formyl-THF pools (23).…”

Cooperation between JNK1 and JNK2 in activation of p53 apoptotic pathway

“…Although the enzymatic function of FDH, that is the conversion of 10-formyltetrahydrofolate to tetrahydrofolate, was known for decades, the main physiological role of the enzyme is still in question. Proposed FDH functions include regulation of reduced folate pools (Champion et al, 1994), storage of the intracellular folate (Min et al, 1988), regulation of de novo purine biosynthesis (Krupenko and Oleinik, 2002) and regulation of cellular methylation (Anguera et al, 2006).…”

906 variations among 27 genes encoding cytochrome P450 (CYP) enzymes and aldehyde dehydrogenases (ALDHs) in the Japanese population

“…ALDH1A3 is expressed in salivary gland, stomach, and kidney (Hsu et al 1994). The ALDH1L1 gene encodes a 10-formyltetrahydrofolate dehydrogenase (Champion et al 1994).…”