Stress granule formation inEntamoeba histolytica: cross-talk between EhMLBP, EhRLE3 reverse transcriptase and polyubiquitinated proteins

Abstract

SummaryThe Entamoeba histolytica-methylated LINEbinding protein (EhMLBP) binds to methylated repetitive DNA and is a positive regulator of a reverse transcriptase of a long interspersed nucleotide element (LINE). This protein protects trophozoites against heat shock by reducing protein aggregation. The presence of EhMLBP and polyubiquitinated proteins in heat shockinduced protein aggregates raised the question whether these proteins interact. This assumption was confirmed by co-immunoprecipitation experiments: ubiquitinated proteins were detected in the perinuclear region of non-stressed E. histolytica trophozoites, whereas ubiquitinated proteins were detected in the perinuclear region and colocalized with EhMLBP in cytoplasmic granules in heat-shocked trophozoites. We also observed that overexpression of the reverse transcriptase of EhRLE3 induced the upregulation of EhMLBP expression and the formation of these EhMLBP-containing granules. Since (i) these EhMLBP-containing granules in the cytoplasm of heat-shocked E. histolytica trophozoites also contain polyubiquitinated proteins and poly(A) + mRNA and (ii) their formation is promoted by sodium arsenate, puromycin, and pateamine A and is inhibited by cycloheximide, we propose that these cytoplasmic EhMLBP-containing granules are stress granules. Our data also suggest that the formation of these granules is dependent upon EhMLBP and LINE.

Functional Characterization of Entamoeba histolytica Argonaute Proteins Reveals a Repetitive DR-Rich Motif Region That Controls Nuclear Localization

“…2C). There are also some distinct punctate dots with induction of oxidative stress, similar to the stress granules reported earlier for E. histolytica (33). However, due to the lack of a marker for these granules, we cannot definitively say if the Eh Ago staining overlaps.…”

“…In human cells, hAgo2 is localized mainly in the cytoplasm and concentrated in P-bodies and is rapidly distributed to stress granules upon stresses (32), indicating a dynamic intracellular localization change of Ago with stresses. In E. histolytica , both cytoplasmic granules under stress condition and P-body-like structures have been reported (33, 34), and our previous transcriptional profiling showed high-level gene expression changes in E. histolytica after heat shock and oxidative stress (35, 36).…”

The Tudor Staphylococcal Nuclease Protein of Entamoeba histolytica Participates in Transcription Regulation and Stress Response

“…By immunofluorescence, we observed a low expression of EhHSP70 in trophozoites grown under normal conditions, and consequently a poor co-localization of this protein with EhTSN was detected (Figure 7A ). When trophozoites were submitted to heat shock, parasites showed a high expression of EhHSP70, which was detected in cytoplasmic structures similar to those reported as stress granules (SG) (Katz et al, 2014 ). Interestingly, in heat shock-treated trophozoites, EhTSN was concentered in SG-like structures, some of them showing the co-localizing with EhHSP70 (Figure 7B ).…”

“…In this work, we identify to EhHSP70 as one of the possible partners of EhTSN. Interestingly, the nuclear localization of EhTSN diminished in trophozoites submitted to heat shock and it was accumulated along with EhHSP70 in structures similar to that reported as stress granules (SGs) (Katz et al, 2014 ). In concordance, TSN of Arabidopsis thaliana has been detected in mRNA-processing SG (Yan et al, 2014 ).…”

Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase

“…In yeast, stress such as glucose deprivation, osmotic stress, and UV irradiation, also promotes P-bodies assembly [63]. The existence of stress granules has been described in eukaryotic cells [64,65] and more recently in this parasite [66]. Although several proteins are found in both stress granules and P-bodies, CAF1 is a specific marker of P-bodies.…”