A crosslinker-based identification of redox relay targets

Araki, Kazutaka; Ushioda, Ryo; Kusano, Hidewo; Tanaka, Riko; Hatta, Tomohisa; Fukui, Kazuhiko; Nagata, Kazuhiro; Natsume, Tohru

doi:10.1016/j.ab.2016.12.025

Cited by 9 publications

(11 citation statements)

References 29 publications

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“…Furthermore, we showed that DVSF traps PfPDI11 to its substrates, but cysteine-to-alanine mutations in the PfPDI11 Trx domain active sites abolish this crosslinking even though this mutant has two cysteine residues that are not in the active site (S5 Fig). These data are consistent with observations in yeast and mammalian cells, in which DVSF has been validated to specifically trap Trx-domain proteins to the substrates with which they exchange disulfide bonds [28][29][30].…”

Section: Plos Pathogenssupporting

confidence: 91%

“…One major PfJ2 redox substrate-PfPDI8-was identified using a chemical biology approach. DVSF is a redox-specific crosslinker that has been used to identify redox partnerships between Thioredoxin proteins in the cytoplasm of model organisms [28][29][30]. To our knowledge, this compound had not yet been used to trap and define redox partnerships in Plasmodium, nor in the ER of any organism.…”

Section: Discussionmentioning

confidence: 99%

“…To do this, we attempted to generate parasites overexpressing either wild-type copies of PfJ2, PfPDI8, PfPDI11, or overexpressing these proteins with cysteine-to-alanine mutations in the Trx domain active sites. These types of mutations abolish DVSF-crosslinking in Trx proteins of model organisms [28,30]. We were unable to generate parasites overexpressing wild-type or mutant copies of PfJ2.…”

Section: Plos Pathogensmentioning

confidence: 96%

“…In Saccharomyces cerevisiae, DVSF was used to trap cytosolic Thioredoxin to two proteins known to exchange electrons with the CXXC active site, validating the compound's ability to covalently and irreversibly trap Trx domains to their redox substrates [28]. DVSF was subsequently used to identify substrates of other redox-active proteins containing hyper-reactive cysteines in S. cerevisiae and human cells [29,30]. To determine whether DVSF was capable of trapping redox partnerships in P. falciparum, we treated PfJ2 apt parasite cultures with DVSF and isolated proteins for western blot analysis.…”

Section: Pfj2 Is a Redox-active Protein In The P Falciparum Ermentioning

confidence: 99%

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

et al. 2021

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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.

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Section: Plos Pathogenssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Plos Pathogensmentioning

confidence: 96%

Section: Pfj2 Is a Redox-active Protein In The P Falciparum Ermentioning

confidence: 99%

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

et al. 2021

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“…Because such transient mixed disulfides tend to be unstable, the identification of substrates for oxidoreductases has proven to be technically difficult. Several experimental approaches have been developed to stabilize reaction intermediates ( 4 , 17 , 21 , 22 , 28 , 83 ), and these strategies can facilitate the identification of specific substrates of thioredoxin superfamily members ( Fig. 8A ).…”

Section: Identification Of Tmx Substratesmentioning

confidence: 99%

Introducing Thioredoxin-Related Transmembrane Proteins: Emerging Roles of Human TMX and Clinical Implications

Matsuo

2022

Antioxidants & Redox Signaling

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Significance:The presence of a large number of thioredoxin superfamily members suggests a complex mechanism of redox-based regulation in mammalian cells. However, whether these members are functionally redundant or play separate and distinct roles in each cellular compartment remains to be elucidated. Recent advances:In the mammalian endoplasmic reticulum (ER), approximately 20 thioredoxin-like proteins have been identified. Most ER oxidoreductases are soluble proteins located in the luminal compartment, while a small family of five thioredoxinrelated transmembrane proteins (TMX) also reside in the ER membrane and play crucial roles with specialized functions. Critical issues:In addition to the predicted function of ER protein quality control, several independent studies have suggested the diverse roles of TMX family proteins in the regulation of cellular processes, including calcium homeostasis, bioenergetics, and thioldisulfide exchange in the extracellular space. Moreover, recent studies have provided evidence of their involvement in the pathogenesis of various diseases. Future directions:Extensive research is required to unravel the physiological roles of TMX family proteins. Given that membrane-associated proteins are prime targets for drug discovery in a variety of human diseases, expanding our knowledge on the mechanistic details of TMX action on the cell membrane will provide molecular basis for developing novel diagnostic and therapeutic approaches as a potent molecular target in a clinical setting.

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The oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER

et al. 2023

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A crosslinker-based identification of redox relay targets

Cited by 9 publications

References 29 publications

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

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

Introducing Thioredoxin-Related Transmembrane Proteins: Emerging Roles of Human TMX and Clinical Implications

The oxidative folding of nascent polypeptides provides electrons for reductive reactions in the ER

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