Dissecting endoplasmic reticulum unfolded protein response in Echinococcus sp. through IRE-bZIP signaling

NICOLAO, MARÍA CELESTE; LEDO, CAMILA; DÍAZ,, MALENA; CHOP, MAIA; RODRIGUEZ RODRIGUES, CHRISTIAN; LANZA CASTRONUOVO, P.; VERA, D.M; CYBULSKI, L.E; CUMINO, ANDREA C.

Paris-Virtual

Conferencia; Endoplasmic Reticulum Conference ?Cell biology, development and evolution?; 2021

Background: Human echinococcosis is a zoonotic disease caused by 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, inducing allosterically its cytosolic endoribonuclease activity. Subsequently IRE leads to unconventional splicing of the mRNA encoding the XBP1 transcription factor, resulting in the transcriptional induction of genes expressing chaperones, ERAD components and autophagy regulators. Previously, we have identified one ortholog of IRE2, XBP1, and ATF6 in the genome of E. granulosus, but the ortholog of PERK/ATF4 was not found. In this work, we address the nature of the gene expression networks in the UPRER and autophagy under UPR-experimental activators 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 control and in vitro pharmacological-treated parasites with tunicamycin, thapsigargin, bortezomib and sodium arsenite. Results: Levels of total Em-xbp1 transcript in protoscoleces (proper larva) increased 30-fold following tunicamycin treatment at 4 h and consequently led increase of Em-Grp78 mRNA levels that indicated the beginning of ER stress in the parasite. Splicing of the Em-xbp1 mRNA was analyzed to assess Em-IRE1 activation. Increased Em-IRE1 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 RNAm 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, as well as, a hydrophobic region (HR, which could target the translated Em-XBP1u mRNA to the ER membrane, enhancing its processing by IRE) in the XBP1u. Since Em-xbp1 mRNA appear 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 metazoa species. Also, Em-XBP1 orthologs with greater that 70% identity were identified in the genomes of main human zoonotic helminths. Em-XBP1 encoded by the spliced mRNA, is a conserved bZIP transcription factor, which constitute one of the key transcriptional regulator 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 to modulate Em-IRE1 activity, identifying it as a target for the employed of UPR inhibitors for Echinococcus. Moreover, in sequences of IRE in 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 IRE1/XBP1 activation induces autophagy, with overexpression of Em-atg8. Therefore, even in absence of PERK, we describe Em-TFEB transcriptional induction, which would promote to the autophagy making more resistant to ER stress?induced apoptosis. Conclusions: These interplay effects of the IRE1/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.