A new nuclear function of the Entamoeba histolytica glycolytic enzyme enolase: the metabolic regulation of cytosine-5 methyltransferase 2 (Dnmt2) activity

Abstract

Cytosine-5 methyltransferases of the Dnmt2 family function as DNA and tRNA methyltransferases. Insight into the role and biological significance of Dnmt2 is greatly hampered by a lack of knowledge about its protein interactions. In this report, we address the subject of protein interaction by identifying enolase through a yeast two-hybrid screen as a Dnmt2-binding protein. Enolase, which is known to catalyze the conversion of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), was shown to have both a cytoplasmatic and a nuclear localization in the parasite Entamoeba histolytica. We discovered that enolase acts as a Dnmt2 inhibitor. This unexpected inhibitory activity was antagonized by 2-PG, which suggests that glucose metabolism controls the non-glycolytic function of enolase. Interestingly, glucose starvation drives enolase to accumulate within the nucleus, which in turn leads to the formation of additional enolase-E.histolytica DNMT2 homolog (Ehmeth) complex, and to a significant reduction of the tRNA(Asp) methylation in the parasite. The crucial role of enolase as a Dnmt2 inhibitor was also demonstrated in E.histolytica expressing a nuclear localization signal (NLS)-fused-enolase. These results establish enolase as the first Dnmt2 interacting protein, and highlight an unexpected role of a glycolytic enzyme in the modulation of Dnmt2 activity.

Figure 1. In vitro interaction between Ehmeth, dDnmt2, hDnmt2 and enolase.

³⁵S labeled proteins Ehmeth (TNT-Ehmeth), dDnmt2 (TNT-dDnmt2) and hDnmt2 (TNT-hDnmt2) were incubated respectively with glutathione beads coated with GST or GST- Enolase and the interacting proteins were analyzed by SDS-Page as described in the Materials and Methods . Left panel: Coomassie staining of GST and GST-Enolase fusion protein used in the pull-down procedure. Right panel: Pull down products TNT-Ehmeth, TNT-dDnmt2 and TNT-hDnmt2 were detected by exposure of the membrane to an x ray film.

Figure 2. Enolase is present in the cytoplasmatic and nuclear fraction of E.histolytica.

A. Cytoplasmatic (C) and nuclear (N) protein fractions of E.histolytica HM-1∶MSS and pJST4-Ehmeth trophozoites were separated on 12% SDS-PAGE and analyzed by western blot with an anti HA antibody, an anti enolase antibody, an anti EhMLBP antibody or an anti Myosin II antibody. B. Cellular localization of Ehenolase in E.histolytica trophozoites. Ehenolase was detected by immunofluorescence microscopy using anti-enolase antibody. Ehenolase distribution is shown in red using a primary anti-enolase antibody and a secondary antibody conjugated with Cy3. Nuclei (blue) were stained by DAPI. Computer-assisted image overlay analysis of the signal given by enolase antibody and by DAPI, shows that Ehenolase is ubiquitously present in trophozoites including in the nucleus. C. Cytoplasmatic and nuclear protein fractions of E.histolytica HM-1∶MSS, trophozoites expressing a NLS-fused-scramble peptide (NLS-Con) (30) and trophozoites expressing a NLS-fused enolase (NLS-Eno) were separated on 12% SDS-PAGE and analyzed by western blot with an anti enolase antibody, an anti actin antibody or an anti EhMLBP antibody.

Figure 3. In vivo interaction of Ehmeth with enolase.

Immunoprecipitation with an anti-HA antibody from a nuclear lysate of E. histolytica trophozoites that express Ehmeth as a CHH-tagged protein (pJST4-Ehmeth) grown in regular media (control) and from trophozoites grown in a glucose starvation media (glucose starvation). Detection of immunoprecipitated proteins was done by western blot with an anti-enolase antibody. To validate that the same amounts of Ehmeth were used in the assay, immunoprecipated proteins were analyzed with an anti His antibody which detects the CHH tagged Ehmeth. As a negative control, immunoprecipitation with an anti-HA antibody from a nuclear lysate of E. histolytica trophozoites that express CHH-klp5 was used (right panel). The physical interaction between enolase and Ehmeth is demonstrated only after immunoprecipitation from Ehmeth tagged trophozoites and this complex is enhanced following glucose starvation (3 fold according to Tina densitometry analysis).

Figure 4. Mapping of the enolase binding region of Ehmeth.

Upper panel: Scheme of the different Ehmeth mutants. Lower panel: Pull down experiment of different Ehmeth fragments with recombinant enolase. Whereas Ehmeth full length, Ehmeth (from amino acid 1 to103) and Ehmeth (from amino acid 88 to 322) where efficiently pull-down by enolase, Ehmeth that has its domain IV truncated interacts poorly with enolase.

Figure 5. Enolase inhibits Ehmeth and hDnmt2 functions.

A. The binding of γ−ATP labeled EhMRS2 DNA (EhMRS2*−0.33 µg) to Ehmeth or hDnmt2 was detected as a DNA-protein complex. No complex was observed between GST and GST-Enolase incubated with EhMRS2 DNA. Enolase inhibits the binding of Ehmeth and hDnmt2 to EhMRS2 DNA in a dose dependent manner. B. Effect of enolase on the Ehmeth (left panel) and hDnmt2 (right panel) tRNA methyltransferase activity. The results represent the mean and standard deviation of three independent experiments (P_value<0.05). U = one unit corresponds to 1 pmol of H³-Adomet incorporated/hour/nmol of enzyme.

Figure 6. The influence of 2-PG on enolase inhibitory effect over Dnmt2 tRNA MT activity.

A. Measure of the hDnmt2 tRNA methyltransferase activity in presence of enolase and increasing concentrations of 2 phosphoglycerate (2-PG). The activity of hDnmt2 measured in the presence of 7 mM 2-PG was regarded as 100%. As already reported enolase strongly inhibits hDnmt2 in absence of 2-PG. The activity of hDnmt2 in presence of enolase is restored by 2-PG in a dose dependent manner. The results represent the mean and standard deviation of three independent experiments (P_value<0.05). B. In vitro interaction between hDnmt2 and enolase in the presence of 7 mM 2-PG. ³⁵S labeled enolase (TNT-Eno) was incubated respectively with glutathione beads coated with GST or GST- hDnmt2 in presence or absence of 2-PG (7 mM). The pull down products was detected by exposure of the membrane to an x ray film. According to Tina densitometry analysis around 4 times less Enolase was pull down by hDnmt2 when 2-PG was present in the reaction.

Figure 7. The effect of enolase accumulation in the nucleus over Dnmt2 tRNA and DNA MT activity.

A. Western blot analysis of cytoplasmatic and nuclear protein fractions prepared from E.histolytica pJST4-Ehmeth trophozoites grown without glucose for (0, 6, 9 and 12 hours or for 12 hours of starvation followed by 12 hours of growth in presence of 1% glucose). Proteins were separated on 12% SDS-PAGE and analyzed by western blot with an anti HA antibody, an anti enolase antibody, or an anti Myosin II antibody. This figure is representative of at least three independent experiments. B. Effect of glucose starvation and continuous forced expression of enolase in the nucleus on the level of tRNA methylation in the parasite. RNA samples from trophozoites grown in regular (control), glucose starvation media (glucose starvation), NLS-Con trophozoites and NLS-Eno trophozoites were used as substrates for in vitro tRNA methylation assay performed with hDnmt2 (see materials and methods). The amount of methyl group incorporate in control RNA was taken as 100%. The significant higher amount of methyl group incorporated in RNA prepared from glucose starved trophozoites (38% increases) and NLS-Eno trophozoites (250% increases) indicates the tRNA present in this sample were less methylated. The results represent the mean and standard deviation of three independent experiments (P_value<0.05). C. RT PCR analysis of the tRNA^asp amount in trophozoites grown in regular (control) and glucose starvation media (glucose starvation). The amount of rDNA was used for the normalization of the data. D. Effect of glucose starvation and continuous forced expression of enolase in the nucleus on the level of m5C methylation in the parasite. Genomic DNA was prepared from trophozoites grown in regular (control), glucose starvation media (glucose starvation), NLS-Con trophozoites and NLS-Eno trophozoites and dot blotted on nitrocellulose membrane in the indicated amounts. Genomic DNA from calf thymus (CT) or PCR product (PCRP) were used as positive and negative controls respectively. DNA methylation was detected with an antibody directed against 5-methylcytosine (α5mc) and the total amount of DNA was estimated by hybridization with a radioactive probe against rDNA.