Figure 1.
Disposition of GNMT in cellular metabolism.
GNMT converts SAM to SAH, methylating glycine to sarcosine. This reaction regulates SAM/SAH ratio and shuttles methyl groups, from activated methyl cycle back to the folate pool. Inhibitory effect of 5-CH3-THF (5-MTHF) on GNMT catalysis is indicated. Hcy, homocysteine; Sarc, sarcosine; THF, tetrahydrofolate.
Figure 2.
Presence of GNMT in human tissues.
A. Immunohistochemical staining of normal human tissues with GNMT-specific antibody or preimmune (PI) serum (control). B. Immunohistochemical staining of GNMT in normal tissues compared to malignant tumors of the same origin. C. Levels of GNMT mRNA in rat tissues; for pancreas, ten-fold less cDNA was used for PCR compared to other tissue; for retina, ten-fold more cDNA was used (in all cases the same amount of cDNA was used for GAPDH amplification). D. Levels of GNMT in normal and regenerating rat liver (Western blot of samples obtained 1-7 days after 70% hepatectomy; control sample represents levels of GNMT in resting liver. Numbers indicate ratio of the intensity of GNMT and actin bands.
Figure 3.
Effect of GNMT transient transfection on cellular proliferation.
Cell viability was assessed by MTT assay (absorbance at 570 nm reflects the number of live cells). Error bars represent ± S.D., n =3. Insets show levels of GNMT (Western blot) at different time points after transfection.
Figure 4.
Cellular responses to GNMT expression.
A. Distribution of GNMT-expressing cells (right panel) between cell cycle phases (propidium iodide staining) compared to control (left panel) GNMT-deficient cells. B. Assessment of DNA damage in GNMT expressing cells by the Comet assay. C. Apoptotic cells assessed by Annexin V/propidium iodide staining after GNMT expression (bottom right quadrant, early apoptotic cells; upper right quadrant, late apoptotic cells); only green cells (expressing GFP-GNMT) were evaluated. D. Calculation of apoptotic cells from C. E. Activation of ERK phosphorylation in response to GNMT expression. F. zVAD-fmk, but not ERK inhibitor PD98059, partially rescues cells from the antiproliferative effect of GNMT (data for A549 cells are shown).
Figure 5.
Catalytically inactive or folate-binding deficient GNMT mutants are capable of the antiproliferative effect.
A. Crystal structure of GNMT tetramer (RCSB Protein Data Bank 3ths; subunits are shown in different colors) with bound 5-MTHF monoglutamate (two molecules shown in spacefill mode are bound per tetramer). B. Positions of amino acid residues in the GNMT catalytic center (from RCSB Protein Data Bank 1XVA). Acetate (Ac) is the competitive inhibitor of Gly and presumably occupies the same position in the active center. Glu 15 (E15*) is from a different subunit. Dotted lines indicate hydrogen bonds. C and D. The enzyme activities and CD spectra of GNMT mutants, analyzed in this study. E. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or corresponding mutants. Error bars represent ± S.D., n =3. F. Folate binding site at the GNMT subunit interface (as shown in panel A); Selected for mutagenesis are residues within close distance to 5-MTHF molecule (these residues are from all four subunits, which are denoted in parentheses). G. Binding of 5-MTHF by GNMT mutants. Error bars represent ± S.D., n =2. H. The MTT proliferation assay of cells transfected with empty vector (control), wild type GNMT (WT), or folate-binding deficient mutants mutants. Error bars represent ± S.D., n =3. I. The supplementation with excessively high media folate or SAM does not rescue cells from the GNMT antiproliferative effect. Error bars represent ± S.D., n =3.
Figure 6.
A. Detection of GNMT in a panel of normal human tissues by immunohistochemical staining and confocal microscopy (left panel, GNMT (green) detected by immunostaining with anti-GNMT antibody and secondary antibody conjugated with Alexa Fluor 488 dye; middle panel, nuclei (red) stained with To-Pro-3 dye; right panel, overlay with yellow indicating co-localization). B. Nuclear localization of GNMT in fixed PC3 cells assessed by confocal microscopy: left panel, GNMT (green) detected by immunostaining with anti-GNMT antibody and secondary antibody conjugated with Alexa Fluor 488 dye; middle panel, nuclei (red) stained with To-Pro-3 dye; right panel, overlay with yellow indicating co-localization. C. Accumulation of GNMT in nuclei monitored by confocal microscopy in live cells: left panel, GFP-GNMT (green); middle panel, RFP-H2B (red); right panel, overlay with yellow indicating co-localization. D. Exclusion of GFP-GNMT from nuclei in HEK293 cells detected by fluorescence microscopy. E. Detection of GNMT in nuclear and cytosolic fractions of PC-3 and HEK239 cells 96 h post-transfection. Subcellular fractions were obtained by differential centrifugation. Control (-) or GNMT transfected (+) cell fractions are shown.
Figure 7.
Subcellular localization-specific effects of GNMT.
A. Sequences used to target GNMT to cytosol or nucleus. B. Distribution of GNMT fusion constructs between cytosol and the nucleus. All constructs included GFP tag at the C-terminus of GNMT. Respective subcellular targeting sequences were introduced at the C-terminus of the GFP tag. C. Levels of corresponding GNMT constructs after transient transfection (Western blot assay). D. MTT assay of cells transfected with GNMT constructs. Error bars represent ± S.D., n =2; *, p < 0.05.