Identification and functional characterization of lysine methyltransferases of Entamoeba histolytica

Abstract

Lysine methylation of histones, a posttranslational modification catalyzed by lysine methyltransferases (HKMTs), plays an important role in the epigenetic regulation of transcription. Lysine methylation of non-histone proteins also impacts the biological function of proteins. Previously it has been shown that lysine methylation of histones of Entamoeba histolytica, the protozoan parasite that infects 50 million people worldwide each year and causing up to 100,000 deaths annually, is implicated in the epigenetic machinery of this microorganism. However, the identification and characterization of HKMTs in this parasite had not yet been determined. In this work we identified four HKMTs in E. histolytica (EhHKMT1 to EhHKMT4) that are expressed by trophozoites. Enzymatic assays indicated that all of them are able to transfer methyl groups to commercial histones. EhHKMT1, EhHKMT2 and EhHKMT4 were detected in nucleus and cytoplasm of trophozoites. In addition EhHKMT2 and EhHKMT4 were located in vesicles containing ingested cells during phagocytosis, and they co-immunoprecipitated with EhADH, a protein involved in the phagocytosis of this parasite. Results suggest that E. histolytica uses its HKMTs to regulate transcription by epigenetic mechanisms, and at least two of them could also be implicated in methylation of proteins that participate in phagocytosis.

Are Metabolites From the Gut Microbiota Capable of Regulating Epigenetic Mechanisms in the Human Parasite Entamoeba histolytica?

“…Inactivation of genes expression was linked to the demethylation of lysine 4 of histone H3 and the dimethylation of lysine 27 of histone H3 ( Anbar et al, 2005 ) ( Foda and Singh, 2015 ). Four expressed and catalytically active E. histolytica histone lysine methyltransferases (EhHKMTases) have been characterized ( Borbolla-Vazquez et al, 2016 ). Two of these EhHKMTases, EhHKMT2 and EhHKMT4, may in addition to catalyze epigenetic marks, methylate proteins involved in phagocytosis.…”

Lysine Methyltransferase EhPKMT2 Is Involved in the In Vitro Virulence of Entamoeba histolytica

“…In addition, for erythrophagocytosis, trophozoites were incubated with fresh human red blood cells (RBCs) (ratio 1:25) for 10, 20 and 30 min at 37 °C. After the established times for each treatment, total extracts were obtained in the presence of proteases inhibitors (Complete Mini, Roche-Mannheim, Mannheim, Germany) and submitted to Western blot assays using the anti-EhPKMT2 antibody [ 12 ] (dilution 1: 500) and subsequently with a peroxidase-coupled secondary antibody (1: 10,000) (anti-rabbit IgG, GenTex, Carbondale, PA, USA, catalog no. GTX 213110-01).…”

“…The full-length Ehpkmt2 gene was amplified by PCR using specific oligonucleotide primers (sense: 5´-cccc gcggccgc atg gat ttt tcg tta aaa tat c-3´; antisense: 5′-cccc agatct ata ctc aac ata ttc agt ata-3′, underlined sequences correspond to the NotI and BglII recognition sites in sense and anti-sense primers, respectively) and Ehpkmt2/GST construct [ 12 ] as template. PCR reaction was performed in a 50 µL volume reaction containing 0.2 mM of each primer, dNTPs 0.2 mM, MgCl 2 3 mM and 1 U of Taq polymerase (Invitrogen).…”

“…Adherence to target cells, amoebapores- and proteases-mediated citotoxicity, phagocytosis and stress response are events necessary for its virulence and pathogenicity [ 11 ]; however, little is known about the molecules that regulate these activities. This microorganism has four PKMTs (EhPKMT1 to EhPKMT4), whose recombinant proteins showed the ability to transfer methyl groups to commercial histones [ 12 ]. Furthermore, antibodies against histone-methylated lysines showed that EhPKMT1, EhPKMT2 and EhPKMT4 catalyze epigenetic marks H4K20me2, H3K4me3 and H4K20me3, respectively [ 12 ], strongly suggesting that these PTMs occur in the pathogen and may participate in epigenetic regulation of transcription.…”

“…This microorganism has four PKMTs (EhPKMT1 to EhPKMT4), whose recombinant proteins showed the ability to transfer methyl groups to commercial histones [ 12 ]. Furthermore, antibodies against histone-methylated lysines showed that EhPKMT1, EhPKMT2 and EhPKMT4 catalyze epigenetic marks H4K20me2, H3K4me3 and H4K20me3, respectively [ 12 ], strongly suggesting that these PTMs occur in the pathogen and may participate in epigenetic regulation of transcription. On the other hand, during erythrophagocytosis, EhPKMT2 and EhPKMT4 were also detected in phagocytic cups and erythrocyte-containing vacuoles [ 12 ], proposing that these enzymes might somehow regulate this virulence-related property.…”