A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites.

David W Cobb,Michelle Krakowiak,Heather M Kudyba,Baylee Bruton,Rodrigo P Baptista,Michael R Hoopmann,Robert L Moritz,Vasant Muralidharan,Alejandra Villegas,Michael J Blackman

doi:10.1371/journal.ppat.1009293

David W Cobb, Michelle Krakowiak + Show 8 more

Open Access

https://doi.org/10.1371/journal.ppat.1009293

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Abstract

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

Highlights

Today, the majority of the world’s population lives at risk for contracting malaria, a disease caused by eukaryotic parasites of the genus Plasmodium, with P. falciparum causing the most severe forms of the disease [1]
We know almost nothing about the function and essentiality of Trx-domain proteins that localize to the endoplasmic reticulum, the origin of the secretory pathway, within P. falciparum
We demonstrate that PfPDI8 is essential for the asexual lifecycle, and our data suggests that PfJ2 and PfPDI8 may work together with the Hsp70 PfBiP to promote protein folding in the endoplasmic reticulum (ER)

Summary

Introduction

The majority of the world’s population lives at risk for contracting malaria, a disease caused by eukaryotic parasites of the genus Plasmodium, with P. falciparum causing the most severe forms of the disease [1]. In 2018, the world saw approximately 228 million cases of malaria resulting in more than 400,000 deaths. The thioredoxin system and members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexan parasites, including in Plasmodium [2,3,4,5]. Members of the Trx superfamily typically contain at least one Trx domain with a “CXXC” active site. As modulators of protein redox states, members of the Trx superfamily regulate diverse aspects of cellular biology. Trx Reductase reduces Trx, which in turn serves as a reductase for other proteins potentially involved in protein synthesis and folding, anti-oxidant stress response, carbohydrate and lipid metabolism, and several other processes [8,9]

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