Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity

Hudson, Devin A.; Thorpe, Colin

doi:10.1016/j.abb.2015.05.005

Cited by 13 publications

(10 citation statements)

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“…Apparently, the thioredoxin-based oxidation system of the periplasm of the endosymbiont that served as the ancestor of mitochondria was replaced during evolution by the Mia40 system. This replacement during evolution was hardly driven to increase protein oxidation in the IMS since Mia40 is a poor catalyst: in its oxidizing power, it is weaker than DsbA, and in forming correct disulfides, it is slower than PDI ( Koch and Schmid, 2014b ; 2014a ; Hudson and Thorpe, 2015 ; Tienson et al, 2009 ). However, Mia40 binds much stronger and for much longer interaction times to its substrates than DsbA or PDIs ( Koch and Schmid, 2014b ; Mesecke et al, 2005 ; Naoé et al, 2004 ; Chacinska et al, 2004 ).…”

Section: Discussionmentioning

confidence: 99%

Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

Peleh

Cordat

Herrmann

2016

eLife

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Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a ‘holding trap’ rather than a ‘folding trap’ mechanism.DOI: http://dx.doi.org/10.7554/eLife.16177.001

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Section: Discussionmentioning

confidence: 99%

Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

Peleh

Cordat

Herrmann

2016

eLife

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“…Reduced RNase was prepared as described previously [46]. Solutions of increasing rRNase concentrations were added to 40 μM MNS in buffer and the absorbance at 378 nm was monitored over time, applying a 530–730 nm scatter correction.…”

Section: Methodsmentioning

confidence: 99%

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Foster

Thorpe

2017

Free Radical Biology and Medicine

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This paper addresses how to evaluate the efficacy of the growing inventory of thiol-reactive inhibitors of mammalian protein disulfide isomerase (PDI) enzymes under realistic concentrations of potentially competing thiol-containing peptides and proteins. For this purpose, we introduce a variant of the widely-used reductase assay by using a commercially-available cysteine derivative (BODIPY FL L-Cystine; BD-SS) that yields a 55-fold increase in fluorescence (excitation/emission; 490/513 nm) on scission of the disulfide bond. This plate reader-compatible method detects human PDI down to 5–10 nM, can utilize a range of thiol substrates (including 5 μM dithiothreitol, 10 μM reduced RNase thiols, and 5 mM glutathione; GSH), and can operate from pH 6–9.5 in a variety of buffers. PDI assays often employ low micromolar levels of substrates leading to ambiguities when thiol-directed inhibitors are evaluated. The present work utilizes 5 mM GSH for both pre-incubation and assay phases to more realistically reflect the high concentration of thiols that an inhibitor would encounter intracellularly. Extracellular PDI faces a much lower concentration of potentially competing thiols; to assess reductase activity under these conditions, the pre-reduced PDI is treated with inhibitor and then fluorescence increase upon reduction of BD-SS is followed in the absence of additional competing thiols. Both assay modes were tested with four mechanistically diverse PDI inhibitors. Two reversible reagents, 3,4-methylenedioxy-β-nitrostyrene (MNS) and the arsenical APAO, were found to be strong inhibitors of PDI in the absence of competing thiols, but were ineffective in the presence of 5 mM GSH. A further examination of the nitrostyrene showed that MNS not only forms facile Michael adducts with GSH, but also with the thiols of unfolded proteins (Kd values of 7 and <0.1 μM, respectively) suggesting the existence of multiple potential intracellular targets for this membrane-permeant reagent. The inhibition of PDI by the irreversible alkylating agent, the chloroacetamide 16F16, was found to be only modestly attenuated by 5 mM GSH. Finally, the thiol-independent flavonoid inhibitor quercetin-3-O-rutinoside was found to show equal efficacy in reoxidation and turnover assay types. This work provides a framework to evaluate inhibitors that may target the CxxC motifs of PDI and addresses some of the complexities in the interpretation of the behavior of thiol-directed reagents in vivo.

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“…Accordingly, Mia40 is a thiol-disulfide oxidoreductase in the intermembrane space of mitochondria that displays good oxidase activity, but poor disulfide isomerase activity. Furthermore, Mia40 is a monomeric, single domain thiol-disulfide oxidoreductase [202]. Disulfide formation/ isomerization is a vast field that we do not intend to cover here.…”

Section: Other Thiol-disulfide Oxidoreductasesmentioning

confidence: 99%

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions

Netto

Oliveira

Tairum

et al. 2016

Free Radical Research

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Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

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Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity

Cited by 13 publications

References 63 publications

Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding

Challenges in the evaluation of thiol-reactive inhibitors of human protein disulfide Isomerase

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions

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