Serge Ankri

A New Nuclear Function of the Entamoeba histolytica Glycolytic Enzyme Enolase: The Metabolic Regulation of Cytosine-5 Methyltransferase 2 (Dnmt2) Activity

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 tRNAAsp 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.

Consumption of L-arginine mediated by Entamoeba histolytica L-arginase (EhArg) inhibits amoebicidal activity and nitric oxide production by activated macrophages.

In this study we discuss the cloning and expression of Entamoeba histolytica arginase (EhArg), an enzyme that catalyses the hydrolysis of l‐arginine to l‐ornithine and urea. l‐norvaline, a competitive inhibitor of E. histolytica l‐arginase, inhibits the growth of the parasite, which suggests that the catabolism of l‐arginine mediated by EhArg is essential. Nitric oxide (NO) is an antimicrobial agent that inhibits some key enzymes in the metabolism of Entamoeba histolytica. NO is synthesized by activated macrophages from l‐arginine, the substrate of NO synthase (NOS‐II). We show that E. histolytica inhibits NO mediated amoebicidal activity of activated macrophages by consuming l‐arginine present in the medium.

Epigenetic and classical activation of Entamoeba histolytica heat shock protein 100 (EHsp100) expression.

The protozoan parasite Entamoeba histolytica expresses a cytosine‐5 DNA methyltransferase (Ehmeth) that belongs to the DNMT2 protein family. The biological function of members of this DNMT2 family is unknown. In the present study, the 5′ region of E. histolytica heat shock protein 100 (5′EHsp100) was isolated by affinity chromatography with 5‐methylcytosine antibodies as ligand. The methylation status of 5′EHsp100 was confirmed by sodium bisulfite sequencing. We showed that the expression of EHsp100 was induced by heat shock, 5‐azacytidine (5‐AzaC), an inhibitor of DNA methyltransferase and Trichostatin A (TSA), an inhibitor of histone deacetylase. The effect of TSA on EHsp100 expression was rapidly reversed by removing the drug from the culture. In contrast, EHsp100 expression was still detectable one month after removing 5‐AzaC from the media. Whereas 5‐AzaC and TSA caused demethylation in the promoter region of EHsp100, no demethylation was observed following heat shock. Remarkably, DNA that includes three putative heat shock elements identified in the promoter region of EHsp100 bound to a protein of 37 kDa present in the nuclear fraction of heat‐shocked trophozoites but absent in the nuclear fraction of 5‐AzaC and TSA treated trophozoites. Our data suggest that EHsp100 expression can be regulated by both a classical and an epigenetic mechanism.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation

ABSTRACT A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

Glucose starvation boosts Entamoeba histolytica virulence.

The unicellular parasite, Entamoeba histolytica, is exposed to numerous adverse conditions, such as nutrient deprivation, during its life cycle stages in the human host. In the present study, we examined whether the parasite virulence could be influenced by glucose starvation (GS). The migratory behaviour of the parasite and its capability to kill mammalian cells and to lyse erythrocytes is strongly enhanced following GS. In order to gain insights into the mechanism underlying the GS boosting effects on virulence, we analyzed differences in protein expression levels in control and glucose-starved trophozoites, by quantitative proteomic analysis. We observed that upstream regulatory element 3-binding protein (URE3-BP), a transcription factor that modulates E.histolytica virulence, and the lysine-rich protein 1 (KRiP1) which is induced during liver abscess development, are upregulated by GS. We also analyzed E. histolytica membrane fractions and noticed that the Gal/GalNAc lectin light subunit LgL1 is up-regulated by GS. Surprisingly, amoebapore A (Ap-A) and cysteine proteinase A5 (CP-A5), two important E. histolytica virulence factors, were strongly down-regulated by GS. While the boosting effect of GS on E. histolytica virulence was conserved in strains silenced for Ap-A and CP-A5, it was lost in LgL1 and in KRiP1 down-regulated strains. These data emphasize the unexpected role of GS in the modulation of E.histolytica virulence and the involvement of KRiP1 and Lgl1 in this phenomenon.

Sensing DNA methylation in the protozoan parasite Entamoeba histolytica

In the protozoan parasite Entamoeba histolytica, 5‐methylcytosine (m5C) was found predominantly in repetitive elements. Its formation is catalysed by Ehmeth, a DNA methyltransferase that belongs to the Dnmt2 subfamily. Here we describe a 32 kDa nuclear protein that binds in vitro with higher affinity to the methylated form of a DNA encoding a reverse transcriptase of an autonomous non‐long‐terminal repeat retrotransposon (RT LINE) compared with the non‐methylated RT LINE. This protein, named E. histolytica‐methylated LINE binding protein (EhMLBP), was purified from E. histolytica nuclear lysate, identified by mass spectrometry, and its corresponding gene was cloned. EhMLBP corresponds to a gene of unknown function that shares strong homology with putative proteins present in Entamoeba dispar and Entamoeba invadens. In contrast, the homology dropped dramatically when non‐Entamoebidae sequences were considered and only a weak sequence identity was found with Trypanosoma and several prokaryotic histone H1. Recombinant EhMLBP showed the same binding preference for methylated RT LINE as the endogenous EhMLBP. Deletion mapping analysis localized the DNA binding region at the C‐terminal part of the protein. This region is sufficient to assure the binding to methylated RT LINE with high affinity. Western blot and immunofluorescence microscopy, using an antibody raised against EhMLBP, showed that it has a nuclear localization. Chromatin immunoprecipitation (ChIP) confirmed that EhMLBP interacts with RT LINE in vivo. Finally, we showed that EhMLBP can also bind rDNA episome, a DNA that is methylated in the parasite. This suggests that EhMLBP may serve as a sensor of methylated repetitive DNA. This is the first report of a DNA‐methylated binding activity in protozoa.

Trichostatin A regulates peroxiredoxin expression and virulence of the parasite Entamoeba histolytica

Histone deacetylation is associated with a repressed chromatin state, and histone acetylase and deacetylase activities have been previously described in Entamoeba histolytica. To investigate their roles in the control of Entamoeba gene expression, the parasite was grown in 50 nM trichostatin A (TSA), an inhibitor of histone deacetylase. TSA enhanced the cytopathic and hemolytic activity of the parasite and its resistance to oxidative stress. We first focused our attention on peroxiredoxin, a protein previously associated with E. histolytica virulence and resistance to oxidative stress. We found that the expression of peroxiredoxin was increased after TSA treatment, but were unable to confirm that this was a direct consequence of histone modification at the promoter. By microarray analysis, we found that some other mRNAs encoding some other virulence factors, such as the galactose-inhibitable lectin small subunits, were also increased. The pattern of gene expression was surprisingly different from that previously described after treatment with 150 nM TSA.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress

ABSTRACT Nitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasite Entamoeba histolytica through S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites to S-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNAAsp methylation, the high levels suggested that Ehmeth-mediated tRNAAsp methylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treated E. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.

Identification of dihydropyrimidine dehydrogenase as a virulence factor essential for the survival of Entamoeba histolytica in glucose‐poor environments

Adaptation to nutritional changes is a key feature for successful survival of a pathogen within its host. The protozoan parasite Entamoeba histolytica normally colonizes the human colon and in rare occasions, this parasite spread to distant organs, such as the liver. E. histolytica obtains most of its energy from the fermentation of glucose into ethanol. In this study, we were intrigued to know how this parasite reacts to changes in glucose availability and we addressed this issue by performing a DNA microarray analysis of gene expression. Results show that parasites that were adapted to growth in absence of glucose increased their virulence and altered the transcription of several genes. One of these genes is the dihydropyrimidine dehydrogenase (DPD), which is involved in degradation of pyrimidines. We showed that this gene is crucial for the parasites growth when the availability of glucose is limited. These data contribute to our understanding of the parasites ability to survive in glucose‐poor environments and reveal a new role for the DPD enzyme.