Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding

Moilanen, Antti; Korhonen, Kati; Saaranen, Mirva J.; Ruddock, Lloyd W.

doi:10.26508/lsa.201800090

Cited by 23 publications

(24 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The K m of Ero1, the ER resident sulfhydryl oxidase enzymes, was determined to be 4 μ m for yeast Ero1 and 5 μ m for mammalian Ero1‐Lα . Interestingly, a recent study showed that Ero1not only has a high affinity for molecular oxygen but also a high cooperativity of oxygen binding, whilst yeast Erv1 has no such cooperativity . Moreover, using GSH‐PDI (protein disulphide isomerase) as electron donor, a K m of 89 μ m and k cat of 0.41 s −1 for Erv1 oxidase activity was obtained, which are both clearly larger than that we obtained with GSH‐Mia40 system (89 vs 3 μ m ; 0.41 vs 0.05 s −1 ).…”

Section: Discussionmentioning

confidence: 99%

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

et al. 2019

View full text Add to dashboard Cite

Yeast (Saccharomyces cerevisiae) essential for respiration and viability 1 (Erv1; EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html), a member of the flavin adenine dinucleotide‐dependent Erv1/ALR disulphide bond generating enzyme family, works together with Mia40 to catalyse protein import and oxidative folding in the mitochondrial intermembrane space. Erv1/ALR functions either as an oxidase or cytochrome c reductase by passing electrons from a thiol substrate to molecular oxygen (O2) or cytochrome c, respectively. However, the substrate specificity for oxygen and cytochrome c is not fully understood. In this study, the oxidase and cytochrome c reductase kinetics of yeast Erv1 were investigated in detail, under aerobic and anaerobic conditions, using stopped‐flow absorption spectroscopy and oxygen consumption analysis. Using DTT as an electron donor, our results show that cytochrome c is ~ 7‐ to 15‐fold more efficient than O2 as electron acceptors for yeast Erv1, and that O2 is a competitive inhibitor of Erv1 cytochrome c reductase activity. In addition, Mia40, the physiological thiol substrate of Erv1, was used as an electron donor for Erv1 in a detailed enzyme kinetic study. Different enzyme kinetic kcat and Km values were obtained with Mia40 compared to DTT, suggesting that Mia40 modulates Erv1 enzyme kinetics. Taken together, this study shows that Erv1 is a moderately active enzyme with the ability to use both O2 and cytochrome c as the electron acceptors, indicating that Erv1 contributes to mitochondrial hydrogen peroxide production. Our results also suggest that Mia40‐Erv1 system may involve in regulation of the redox state of glutathione in the mitochondrial intermembrane space. Erv1 EC number http://www.chem.qmul.ac.uk/iubmb/enzyme/1/8/3/2.html.

show abstract

Section: Discussionmentioning

confidence: 99%

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Expression tests to screen for optimal conditions were carried out for the constructs in 24 deep well plates. The constructs were co-expressed together with CyDisCo components from pMJS205 [18] in up to four different E. coli strains (BL21(DE3) (from Stratagene), the Keio collection parental strain BW25113 [53], MDS42 [54] and BL21 (DE3) ∆trxA/trxC [55]), up to two different media (chemically defined [56] and terrific broth autoinduction media (Formedium) with trace elements and 0.8% glycerol and at two different temperatures (30 • C and 15 • C). All the constructs were found to be best expressed at 15 • C, in BL21(DE3) cells.…”

Section: Expressionmentioning

confidence: 99%

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories

Sohail

Gaikwad

Khadka

et al. 2020

IJMS

Self Cite

View full text Add to dashboard Cite

Escherichia coli is the most widely used protein production host in academia and a major host for industrial protein production. However, recombinant production of eukaryotic proteins in prokaryotes has challenges. One of these is post-translational modifications, including native disulfide bond formation. Proteins containing disulfide bonds have traditionally been made by targeting to the periplasm or by in vitro refolding of proteins made as inclusion bodies. More recently, systems for the production of disulfide-containing proteins in the cytoplasm have been introduced. However, it is unclear if these systems have the capacity for the production of disulfide-rich eukaryotic proteins. To address this question, we tested the capacity of one such system to produce domain constructs, containing up to 44 disulfide bonds, of the mammalian extracellular matrix proteins mucin 2, alpha tectorin, and perlecan. All were successfully produced with purified yields up to 6.5 mg/L. The proteins were further analyzed using a variety of biophysical techniques including circular dichroism spectrometry, thermal stability assay, and mass spectrometry. These analyses indicated that the purified proteins are most likely correctly folded to their native state. This greatly extends the use of E. coli for the production of eukaryotic proteins for structural and functional studies.

show abstract

“…Human Ero1α requires high concentrations of reduced substrate PDI for redox activation as well as for catalytic turnover (12). Both events are mediated by the same molecular determinant, specifically, the protruding β-hairpin of Ero1α interacting with the hydrophobic substrate-binding site of PDI in the b' domain (11,12). Non-native proteins, such as misfolded proteins or folding intermediates, contain exposed hydrophobic amino acids which are bound by the same substratebinding site in the b' domain of PDI (19) that interacts with Ero1α.…”

Section: Non-native Proteins Inhibit Ero1α Activity and Activationmentioning

confidence: 99%

“…As an additional control, we measured similar inhibition assays replacing Ero1α with another sulfhydryl oxidase, Erv1p, capable of oxidizing PDI despite lacking the hairpin structure of Ero1α (12,26). The oxygen consumption rate was not strongly inhibited by either 17β-E2 or BPA (Fig.…”

Section: Pdi Binds Small Hydrophobic Molecules Which Inhibit the Regmentioning

confidence: 99%

See 1 more Smart Citation

Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded

Moilanen

Ruddock

2020

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Protein maturation in the endoplasmic reticulum (ER) depends on a fine balance between oxidative protein folding and quality control mechanisms, which together ensure high-capacity export of properly folded proteins from the ER. Oxidative protein folding needs to be regulated to avoid hyperoxidation. The folding capacity of the ER is regulated by the unfolded protein response (UPR) and ER-associated degradation (ERAD). The UPR is triggered by unfolded protein stress and leads to up-regulation of cellular components such as chaperones and folding catalysts. These components relieve stress by increasing folding capacity and up-regulating ERAD components that remove non-native proteins. Although oxidative protein folding and the UPR/ERAD pathways each are well-understood, very little is known about any direct cross-talk between them. In this study, we carried out comprehensive in vitro activity and binding assays, indicating that the oxidative protein folding relay formed by ER oxidoreductin 1 (Ero1), and protein disulfide-isomerase can be inactivated by a feedback inhibition mechanism involving unfolded proteins and folding intermediates when their levels exceed the folding capacity of the system. This mechanism allows client proteins to remain mainly in the reduced state and thereby minimizes potential futile oxidation–reduction cycles and may also enhance ERAD, which requires reduced protein substrates. Relief from excess levels of non-native proteins by increasing the levels of folding factors removed the feedback inhibition. These results reveal regulatory cross-talk between the oxidative protein folding and UPR and ERAD pathways.

show abstract

Molecular analysis of human Ero1 reveals novel regulatory mechanisms for oxidative protein folding

Abstract: This study reveals novel regulatory mechanisms for human Ero1α and Ero1β, including cooperativity for oxygen binding and complex formation with PDI via a novel mixed disulfide linkage.

Cited by 23 publications

References 49 publications

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Kinetic characterisation of Erv1, a key component for protein import and folding in yeast mitochondria

Production of Extracellular Matrix Proteins in the Cytoplasm of E. coli: Making Giants in Tiny Factories

Non-native proteins inhibit the ER oxidoreductin 1 (Ero1)–protein disulfide-isomerase relay when protein folding capacity is exceeded

Contact Info

Product

Resources

About