An amebic protein disulfide isomerase (PDI) complements the yeast PDI1 mutation but is unable to support cell viability under ER or thermal stress

Abstract

In eukaryotic cells, protein disulfide isomerases (PDI) are oxidoreductases that catalyze the proper disulfide bond formation during protein folding. The pathobiology of the protozoan parasite Entamoeba histolytica, the causative agent of human amebiasis, depends on secretion of several virulence factors, such as pore‐forming peptides and cysteine proteinases. Although the native conformation of these factors is stabilized by disulfide bonds, there is little information regarding the molecular machinery involved in the oxidative folding of amebic proteins. Whereas testing gene function in their physiological background would be the most suitable approach, we have taken advantage of the cellular benefits offered by the yeast Saccharomyces cerevisiae (as a model of eukaryotic cell) to examine the functional role of an amebic PDI (EhPDI). As the yeast PDI homolog is essential for cell viability, a functional complementation assay was carried out to test the ability of EhPDI to circumvent the lethal phenotype of a yeast PDI1 mutant. Also, its proficiency under stressful conditions was explored by examining the survival outcome following endoplasmic reticulum (ER) stress induced by a reductant agent (DTT) or thermal stress promoted by a nonpermissive temperature (37 °C). Our results indicate that EhPDI is functionally active when physiological conditions are stable. Nonetheless, when conditions are stressful (e.g., by the accumulation of misfolded proteins in the ER compartment), its functionality is exceeded, suggesting an inability to prevent unfolding, suppress aggregation, or assist refolding of proteins. Despite the latter, our findings constitute the initial step toward determining the participation of EhPDI in cellular mechanisms related to protein homeostasis.

N-glycosylation of the protein disulfide isomerase Pdi1 ensures full Ustilago maydis virulence

“…Moreover, Pdi1 ΔN-gly :GFP maintained wild-type Pdi1:GFP subcellular localization to cells membranes and the ER (Fig 7E). Consistent with defective Pdi1 function during plant infection when N-glycosylation target residues are removed, we observed that Pdi1 ΔN-gly does not complement the DTT sensitivity shown by the Pdi1 deletion mutant (Fig 7F) which has been previously associated to the lack of Pdi1 activity [37].…”

“…Modification of the two putative N-glycosylation motifs identified in the Pdi1 ORF affected its ability to recover the infective capacity of a pdi1 mutant. Because Pdi1 activity, in terms of cell growth capacity under DTT-induced reductive ER stress [37] it suggests that N-glycosylation of Pdi1 may be required for its in vivo function. Consistent with this idea, the modification of the putative N-glycosylation motifs affected Pdi1 electrophoretic mobility, without affecting Pdi1 cellular location or stability, making it unlikely that the Pdi1 ΔN-gly point mutations have grossly affected Pdi1 synthesis or conformation.…”

Transcriptomic Analysis of Pichia pastoris (Komagataella phaffii) GS115 During Heterologous Protein Production Using a High-Cell-Density Fed-Batch Cultivation Strategy

“…Likewise, Kar2 (BiP), which can activate Ire1 (Schroder et al, 2003), was not upregulated in this study. In accordance with the results of a study performed by Li et al (2019), protein disulfide isomerase (PDI, PAS_chr4_0844), which catalyzes appropriate disulfide bond formation during protein folding, is only significantly upregulated (Mares and Ramos, 2018) prior to and at the end of methanol induction (p-Value < 0.05 and log2 fold changes > 0.75) ( Figure 6 and Supplementary Table FIGURE 6 | Changes in proteins involved in the UPR and ERAD pathways. S3), while another PDI (PAS_chr1−3_0125) is significantly down-regulated during GM and the methanol induction stage (p-Value < 0.05 and log2 fold changes < −0.60) ( Figure 6 and Supplementary Table S3).…”

Entamoeba histolytica: Membrane and Non-Membrane Protein Structure, Function, Immune Response Interaction, and Vaccine Development

“…The recombinant type of EhPDI shows the specific properties of PDI enzymes, such as performing oxidase and reductase activities [59]. This protein also participates in the cellular mechanism related to protein homeostasis [61]. For example, during stressful conditions, this enzyme is involved in holding, refolding, and degrading unfolded or misfolded proteins [58].…”

“…Despite the lack of study on this protein, it merits further investigation as a drug target for anti-amoebic therapy and as a vaccine candidate. cellular mechanism related to protein homeostasis [61]. For example, during stressful conditions, this enzyme is involved in holding, refolding, and degrading unfolded or misfolded proteins [58].…”