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

Abstract

Entamoeba histolytica is the protozoa parasite responsible of human amoebiasis, disease that causes from 40,000 to 100,000 deaths annually worldwide. However, few are known about the expression regulation of molecules involved in its pathogenicity. Transcription of some virulence-related genes is positively controlled by the cis-regulatory element named URE1. Previously we identified the transcription factor that binds to URE1, which displayed a nuclear and cytoplasmic localization. This protein belongs to the Tudor Staphyococcal nuclease (TSN) family, which in other systems participates in virtually all pathways of gene expression, suggesting that this amoebic transcription factor (EhTSN; former EhURE1BP) could also play multiple functions in E. histolytica. The aim of this study was to identify the possible cellular events where EhTSN is involved. Here, we found that EhTSN in nucleus is located in euchromatin and close to, but not into, heterochromatin. We also showed the association of EhTSN with proteins involved in transcription and that the knockdown of EhTSN provokes a diminishing in the mRNA level of the EhRabB gene, which in its promoter region contains the URE1 motif, confirming that EhTSN participates in transcription regulation. In cytoplasm, this protein was found linked to the membrane of small vesicles and to plasma membrane. Through pull-down assays and mass spectrometry we identity thirty two candidate proteins to interact with EhTSN. These proteins participate in transcription, metabolism, signaling, and stress response, among other cellular processes. Interaction of EhTSN with some candidate proteins involved in metabolism, and signaling was validated by co-immunoprecipitation or co-localization. Finally we showed the co-localization of EhTSN and HSP70 in putative stress granules during heat shock and that the knockdown of EhTSN increases the cell death during heat shock treatment, reinforcing the hypothesis that EhTSN has a role during stress response. All data support the proposal that EhTSN is a multifunctional protein of E. histolytica.

The state of the art of extracellular vesicle research in protozoan infection

“…Notably, TSN can bind to upstream regulatory element 1 (URE1), which is related to an amoeba virulence protein. It has been suggested that TSN plays a role in virulence regulation as a transcription factor (Calixto-Gálvez et al, 2011;Cázares-Apátiga et al, 2017). In addition, after heat shock treatment (42 °C for 2 h), TSN colocalized with HSP70 (another protein in Entamoeba-shed EVs).…”

Protein arginine methyltransferases in protozoan parasites

“…1), although the EhPRMTA recombinant protein displayed enzymatic activity on commercial histones (Medina-Gómez et al ., 2021). On the other hand, EhPRMTA interacts with the EhTSN protein, which is a transcription factor of E. histolytica (Cázares-Apátiga et al ., 2017) and its homologues in other protozoan parasites participate in RNA metabolism (Hossain et al ., 2008; Musiyenko et al ., 2012), suggesting that this EhPRMT is involved in transcription regulation and in RNA metabolism; moreover, the EhPRMTA-knockdown increased cell growth and phagocytosis, but decreased cell migration and survival of trophozoites submitted to heat shock (Medina-Gómez et al ., 2021), indicating that this PRMT participates in these events (Table 1), although the identification of its protein targets remains to be determined.…”

Tudor staphylococcal nuclease is a docking platform for stress granule components and is essential for SnRK1 activation in Arabidopsis

“…Two recent lines of evidence suggest that TSN might be important for assembly and/or function of SGs. First, TSN localizes in SGs in such distant lineages as protozoa, animals and plants (Zhu et al , 2013 ; Yan et al , 2014 ; Gao et al , 2015 ; Gutierrez‐Beltran et al , 2015b ; Cazares‐Apatiga et al , 2017 ). Second, TSN interacts with proteins constituting the core of SGs, such as PAB1, eIF4E and eIF5A in different organisms including mammalian and Bombyx mori cells (Weissbach & Scadden, 2012 ; Zhu et al , 2013 ; Gao et al , 2014 ).…”