Tuesday, August 16, 2011

Glycine N-methyltransferase

GNMT gene provides instructions for producing the enzyme glycine N-methyltransferase and is involved in a multistep process that breaks down the protein building block (amino acid) methionine . It starts a reaction that converts the compounds glycine and S-adenosylmethionine to N-methylglycine and S-adenosylhomocysteine. This helps to control the relative amounts of AdoMet and AdoHcy. S-adenosylmethionine also called as AdoMet and S-adenosylhomocysteine as  AdoHcy.

This control the relative amounts of AdoMet and AdoHcy. These ratio is important in many body processes, including the regulation of other genes by the addition of methyl groups .These are also involved in processing toxic compounds in the liver. Inherited variations in the GNMT gene have been associated with an increased risk of liver and prostate cancers .

Glycine N-methyltransferase is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine. This is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice.

Deletion of glycine N-methyltransferase (GNMT), the main gene involved in liver S-adenosylmethionine (SAM) catabolism, leads to the hepatic accumulation of this molecule and the development of fatty liver and fibrosis in mice. To demonstrate that the excess of hepatic SAM is the main agent contributing to liver disease in GNMT knockout (KO) mice, we treated 1.5-month-old GNMT-KO mice for 6 weeks with nicotinamide (NAM), a substrate of the enzyme NAM N-methyltransferase. NAM administration markedly reduced hepatic SAM content, prevented DNA hypermethylation, and normalized the expression of critical genes involved in fatty acid metabolism, oxidative stress, inflammation, cell proliferation, and apoptosis.  

Genotypic analysis revealed that the rates of loss heterozygosity at the GNMT locus in paired tumor and tumor-adjacent tissues from hepatocellular carcinoma (HCC) patients were approximately 36-47%. Previously, we generated a Gnmt-/- mouse model and showed that Gnmt-/- mice develop chronic hepatitis, glycogen storage disease, fatty nodules, and HCC. Subsequently, we showed that GNMT is consistently down-regulated in both human and mouse fatty liver tissues. Yeast two-hybrid screenings indicate that Niemann-Pick type C2 protein (NPC2) is a GNMT-binding protein. NPC2 can be released from lysosomes to the cytosol where it interacts with GNMT. GNMT over-expression doubled the half-lives of NPC2 isoforms and reduced cholesterol accumulation in cells. Compared to wild-type mice, Gnmt-/- mice suffered from more severe steatohepatitis and liver damage after being fed methionine/choline-deficient diets; Gnmt-transgenic mice expressed much milder symptoms. Our results suggest that GNMT-NPC2 interaction plays an important role in intracellular cholesterol trafficking and steatohepatitis pathogenesis. GNMT also expresses in neurons distributed in cortex, mid brain and substancia nigra.

Based on the identification of GNMT as a 4 S polyaromatic hydrocarbon-binding protein, we used liver cancer cell lines that expressed GNMT either transiently or stably in cDNA transfections to analyze the role of GNMT in the benzo(a)pyrene (BaP) detoxification pathway. Results from an indirect immunofluorescent antibody assay showed that GNMT was expressed in cell cytoplasm before BaP treatment and translocated to cell nuclei after BaP treatment. Compared with cells transfected with the vector plasmid, the number of BaP-7,8-diol 9,10-epoxide-DNA adducts that formed in GNMT-expressing cells was significantly reduced. Furthermore, the dose-dependent inhibition of BaP-7,8-diol 9,10-epoxide-DNA adduct formation by GNMT was observed in HepG2 cells infected with different multiplicities of infection of recombinant adenoviruses carrying GNMT cDNA. According to an aryl hydrocarbon hydroxylase enzyme activity assay, GNMT inhibited BaP-induced cytochrome P450 1A1 enzyme activity.  

Messenger RNA differential display was used to study liver-gene expression in paired tumor and non-tumor tissues from hepatocellular carcinoma (HCC) patients. mRNA differ- ential display and Northern-blot analyses showed that a 0.8-kb cDNA fragment was diminished or absent from the tumorous tissues of 7 HCC patients. The cDNA fragment was sequenced and found to have 98.7% nucleotide sequence homology with human glycine-N-methyltransferase cDNA (GNMT). In addition, there was no detectable level of GNMT expression in 4 human HCC cell lines, SK-Hep1, Hep 3B, HuH-7 and HA22T, examined by Northern-blot assay.