Abstract
The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.
Highlights
Tetrahydrofolates (THF)3 are present in cells as a family of metabolic cofactors that carry and chemically activate single carbons for a network of biosynthetic pathways referred to as folate-mediated one-carbon metabolism (Fig. 1) [1, 2]
serine hydroxymethyltransferase (SHMT) is present in both the cytoplasm and mitochondria, and the SHMT isozymes are encoded on distinct genes, Shmt1 and Shmt2, respectively [7, 8]
This study confirms the metabolic role of cSHMT in a mammalian model system, and it establishes the essentiality of the cSHMT enzyme in mammalian systems
Summary
SalI fragment from the vector pGT1.8IresBgeo [10], which included the IRESgeo cDNA, was flanked with AscI linkers cloned into the AscI site of pKO. Previous studies in cell cultures have indicated that in intron 6 that is upstream of the sequence included in the cSHMT generates methylene-THF for dTMP synthesis targeting vector. The 3Ј homologous arm corre- ruption of the Shmt gene in the germ line was achieved by sponding to 5.0 kb of sequence, including most of intron 7, exon crossing Shmt1flox/ϩ mice with BALB/c-Tg(CMV-Cre)1Cgn/J. For AdoMet and S-adenosylhomocysteine (AdoHcy) analysis in cells, confluent MEFs were washed twice with PBS and cultured for 4 h in defined minimal essential medium (Hyclone Laboratories) that lacked methionine, vitamin B6, and folic acid but supplemented with 10% fetal calf serum (dialyzed and charcoal treated), 25 nM leucovorin, and 1 g/ml pyridoxal phosphate (PLP). Frozen tissues or cell pellets were sonicated in 500 l of 0.1 M NaAcO buffer (pH 6), and protein was precipitated by adding 312 l of 10% perchloric acid to each sample. All statistics were performed using JMP IN software, release 5.1.2
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