CONICET | Buscador de Institutos y Recursos Humanos

Background: Human echinococcosis is a zoonotic disease caused by the larval stage of tapeworms of the genus Echinococcus. Perturbations in protein folding and assembly can induce the UPRER, a genic transcriptional regulation program designed to expand the ER-processing capacity and alleviate cell damage in eukaryotic organisms. IRE1 is the most evolutionarily conserved ER stress transducer, which upon activation, undergoes dimerization-dependent autophosphorylation and allosterically induces its cytosolic endoribonuclease activity. Subsequently, IRE leads to unconventional splicing of the XBP1 mRNA enabling the translation of XBP1, which results in the transcriptional induction of genes expressing chaperones, ERAD components and autophagy regulators. Previously, we have identified orthologs of IRE, XBP1, and ATF6 in the genome of E. granulosus, but the orthologs of PERK/ATF4 were not found. In this work, we address the nature of the gene expression networks in the UPRER and autophagy under pharmacological UPR inducers in E. multilocularis, an endemic specie of Western and Central Europe. Methods: Using both computational and experimental tools, we functionally characterized Em-IRE and Em-XBP in parasites pharmacologically treated with tunicamycin, thapsigargin, bortezomib or sodium arsenite and in non-treated parasites. Results: Levels of total Em-xbp1 transcript in protoscoleces (proper larva) increased 30-fold after 4 h of tunicamycin treatment and consequently led to an increase of Em-Grp78 mRNA levels which indicates the beginning of ER stress in the parasite. Splicing of the Em-xbp1 mRNA was analyzed to assess Em-IRE activation. Increased Em-IRE activity in protoscoleces treated with tunicamycin, thapsigargin and sodium arsenite improves the efficiency of XBP1 mRNA splicing examined by RT-PCR. We identified Em-xbp1 mRNA variants (Em-xbp-unspliced and Em-xbp-spliced) by sequencing. Basic (N-x7-R/K motif) and leucine zipper (five heptad leucine-repeats) domains in N-terminal and a transactivation domain in the C-terminal end of Em-XBP1s were identified. In addition, a hydrophobic region (HR, which could target the translated Em-XBP1u mRNA to the ER membrane) in the XBP1u was recognized. Since Em-xbp1 mRNA appears spliced under physiological conditions, both Em-IRE and Em-XBP1 should act on certain target genes with physiological relevance in the parasite. On the other hand, secondary structure of the Em-xbp1 mRNA around the non-canonical intron has two short hairpins with splice sites located in the loop regions, common with other metazoan species. Also, Em-XBP1 orthologs with identities greater than 70% were identified in the genomes of main human zoonotic helminths. Em-XBP1 encoded by the spliced mRNA, is a conserved bZIP transcription factor, which constitutes one of the key transcriptional regulators of ER folding capacity in this cestode. Em-IRE showed a considerable similarity in secondary and tertiary structures to human IRE (3p23.1A homodimer, 35% identity and 0.49 QMEANDisCo). We analyzed possible exogenous ligands that may modulate Em-IRE activity, identifying it as a target for the employed of UPR inhibitors for Echinococcus. Moreover, in sequences of IRE of human helminths, we identified an additional C-terminal peptide (around 150 residues with coil-coil structures) that was previously unobserved. This region may be druggable, providing new opportunities for the development of IRE specific inhibitors in these parasites. Also, we verified that IRE/XBP1 activation induces autophagy, with overexpression of Em-atg8. Therefore, even in absence of PERK, we describe Em-TFEB transcriptional induction, which would promote the autophagy reducing ER stress?induced apoptosis. Conclusions: These interplay effects of the IRE/XBP1s branch on autophagy highlight the importance of both mechanisms in these pathogen parasites and encourage to consider further research targeting UPRER-autophagy as a potential therapeutic intervention.

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