Biochemical Basis of Oxidative Protein Folding in the Endoplasmic Reticulum

Tu, Benjamin P.; Ho-Schleyer, Siew C.; Travers, Kevin; Weissman, Jonathan S.

doi:10.1126/science.290.5496.1571

Cited by 404 publications

(369 citation statements)

References 24 publications

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“…Disulfide bond formation has been established to consume oxygen and produce ROS (and thereby consume cellular resources to protect against the ROS) in stoichiometric quantities with the number of disulfide bonds formed [9]. When non-native disulfide linkages are formed, these linkages must be rearranged to the correct disulfide pairings for the native protein to be folded, a process called disulfide isomerization [30].…”

Section: Resultsmentioning

confidence: 99%

“…In Figure 4a, we assume that only PDIs interact with the folding protein. This appears the case, as kinetic rates for direct glutathione oxidation/reduction are too slow to be physiologically relevant [9]. Electron affinity (and therefore redox potential) is broadly determined by the proximity of the two cysteines, with the proximity determined by the current structure of the protein [46].…”

Section: Discussionmentioning

confidence: 99%

“…Proteins that are slow to fold or terminally misfolded proteins are removed from the ER via the ER-associated degradation (ERAD) pathway [8]. Disulfide bond formation requires the removal of electrons from cysteine thiols via protein disulfide isomerase (PDI) and Ero1p to the final electron acceptor, typically oxygen [9,10]. This process produces reactive oxygen species (ROS) in stoichiometric amounts to the number of disulfide bonds formed [11].…”

Section: Introductionmentioning

confidence: 99%

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Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

et al. 2012

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BackgroundThe protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor) or a larger protein (α-amylase).ResultsHAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a) degradation of protein/recycling amino acids, (b) overall transcription/translation repression, and (c) oxidative stress were broadly associated with secretory stress.ConclusionsApparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

et al. 2012

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“…The classical protein disulfide isomerase (PDI, EC 5.3.4.1) is an oxidoreductase that catalyzes the formation, reduction, and isomerization of disulfide bonds in nascent secretory proteins in the lumen of the endoplasmic reticulum (ER) (Frand and Kaiser, 1998;Tu et al, 2000). PDI properly folds its target proteins into conformations necessary for stability, catalytic activity, trafficking and interaction with other proteins (Aslund and Beckwith, 1999;Gruber et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Cho

Yuen

Kang

et al. 2011

Molecules and Cells

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Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXCcontaining thioredoxin catalytic sites (a,a′), two noncatalytic thioredoxin fold domains (b,b′), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein-folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2-GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP-and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus.

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“…This posttranslational modification is possible due to the high oxidizing ER lumen relative to the cytoplasm. The oxidizing potential of the ER lumen is controlled primarily by Ero1 in yeast [94, 95] and ERO1-β and peroxiredoxin IV in metazoans [96, 97]. In turn, members of the PDI family regulate formation of the correct disulfide bonds in a protein [98].…”

Section: Approaches For Imaging Er Stress and Upr Activity In Livinmentioning

confidence: 99%

Approaches to imaging unfolded secretory protein stress in living cells

Lajoie

Fazio

Snapp

2014

Endoplasmic Reticulum Stress in Diseases

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The endoplasmic reticulum (ER) is the point of entry of proteins into the secretory pathway. Nascent peptides interact with the ER quality control machinery that ensures correct folding of the nascent proteins. Failure to properly fold proteins can lead to loss of protein function and cytotoxic aggregation of misfolded proteins that can lead to cell death. To cope with increases in the ER unfolded secretory protein burden, cells have evolved the Unfolded Protein Response (UPR). The UPR is the primary signaling pathway that monitors the state of the ER folding environment. When the unfolded protein burden overwhelms the capacity of the ER quality control machinery, a state termed ER stress, sensor proteins detect accumulation of misfolded peptides and trigger the UPR transcriptional response. The UPR, which is conserved from yeast to mammals, consists of an ensemble of complex signaling pathways that aims at adapting the ER to the new misfolded protein load. To determine how different factors impact the ER folding environment, various tools and assays have been developed. In this review, we discuss recent advances in live cell imaging reporters and model systems that enable researchers to monitor changes in the unfolded secretory protein burden and activation of the UPR and its associated signaling pathways.

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Biochemical Basis of Oxidative Protein Folding in the Endoplasmic Reticulum

Cited by 404 publications

References 24 publications

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

Approaches to imaging unfolded secretory protein stress in living cells

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