Analysis of the isomerase and chaperone-like activities of an amebic PDI (EhPDI).

Rosa E Mares,Marco A Ramos,Samuel G Meléndez-López,Alexis Z Minchaca,Salvador Villagrana

doi:10.1155/2015/286972

Rosa E Mares, Marco A Ramos + Show 3 more

Open Access

https://doi.org/10.1155/2015/286972

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Abstract

Protein disulfide isomerases (PDI) are eukaryotic oxidoreductases that catalyze the formation and rearrangement of disulfide bonds during folding of substrate proteins. Structurally, PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. PDI enzymes are also involved in holding, refolding, and degradation of unfolded or misfolded proteins during stressful conditions. The EhPDI enzyme (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to gain insights into protein folding and disulfide bond formation in E. histolytica. Here, we performed a functional complementation assay, using a ΔdsbC mutant of E. coli, to test whether EhPDI exhibits isomerase activity in vivo. Our preliminary results showed that EhPDI exhibits isomerase activity; however, further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain. Additional studies confirmed that EhPDI protects heat-labile enzymes against thermal inactivation, extending our knowledge about its chaperone-like activity. The characterization of EhPDI, as an oxidative folding catalyst with chaperone-like function, represents the initial step to dissect the molecular mechanisms involved in protein folding in E. histolytica.

Highlights

In eukaryotic cells, folding and posttranslational modifications of proteins are the primary function of the endoplasmic reticulum (ER) [1]
Protein disulfide isomerases (PDI) enzymes share as a common feature the presence of at least one active thioredoxin-like domain
Our preliminary results showed that EhPDI exhibits isomerase activity; further mutagenic analysis revealed significant differences in the functional role of each thioredoxin-like domain

Summary

Introduction

In eukaryotic cells, folding and posttranslational modifications of proteins are the primary function of the endoplasmic reticulum (ER) [1]. PDI enzymes share as a common feature the presence of at least one active thioredoxin-like domain. Some organisms, such as yeast and mammals, have a family of PDI homologues that exhibit distinct domain organization and function [6,7,8]. The enzyme named EhPDI (a 38 kDa polypeptide with two active thioredoxin-like domains) has been used as a model to study protein folding and disulfide bond formation in E. histolytica. To test whether EhPDI exhibits isomerase activity in vivo, we performed a functional complementation assay using the ΔdsbC mutant of E. coli as a model and the defective expression of the periplasmic protein AppA as the phenotype. Additional studies confirmed that EhPDI protects two heatlabile enzymes, α-glucosidase and NdeI endonuclease, against thermal inactivation, extending our knowledge about its chaperone-like activity

Materials and Methods

DsbC Complementation and AppA Activity Assay

Results and Discussion

Linearized