Manipulation of oxidative protein folding and PDI redox state in mammalian cells.

In the endoplasmic reticulum (ER), disulfide bonds are simultaneously formed in nascent proteins and removed from incorrectly folded or assembled molecules. In this compartment, the redox state must be, therefore, precisely regulated. Here we show that both human Ero1-Lalpha and Ero1-Lbeta (hEROs) facilitate disulfide bond formation in immunoglobulin subunits by selectively oxidizing PDI. Disulfide bond formation is controlled by hEROs, which stand at a crucial point of an electron-flow starting from nascent secretory proteins and passing through PDI. The redox state of ERp57, another ER-resident oxidoreductase, is not affected by over-expression of Ero1-Lalpha, suggesting that parallel and specific pathways control oxidative protein folding in the ER. Mutants in the Ero1-Lalpha CXXCXXC motif act as dominant negatives by limiting immunoglobulin oxidation. PDI-dependent oxidative folding in living cells can thus be manipulated by using hERO variants.     

Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum.

Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p. Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI. PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant. Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides. Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY). Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo. Together, these results define a pathway for protein disulfide bond formation in the ER. The PDI homolog Mpd2p is also oxidized by Ero1p.     

Oxidative protein folding in the plant endoplasmic reticulum

For most of the proteins synthesized in the endoplasmic reticulum (ER), disulfide bond formation accompanies protein folding in a process called oxidative folding. Oxidative folding is catalyzed by a number of enzymes, including the family of protein disulfide isomerases (PDIs), as well as other proteins that supply oxidizing equivalents to PDI family proteins, like ER oxidoreductin 1 (Ero1). Oxidative protein folding in the ER is a basic vital function, and understanding its molecular mechanism is critical for the application of plants as protein production tools. Here, I review the recent research and progress related to the enzymes involved in oxidative folding in the plant ER. Firstly, nine groups of plant PDI family proteins are introduced. Next, the enzymatic properties of plant Ero1 are described. Finally, the cooperative folding by multiple PDI family proteins and Ero1 is described.     

ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

In human cells, Ero1-Lalpha and -Lbeta (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein--protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1-Lalpha. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport-competent Ig-K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short-lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1-Lalpha redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.     

Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.     

Glutathione limits Ero1-dependent oxidation in the endoplasmic reticulum

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1 alpha and Ero1 beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1 alpha, indicating that cytosolic GSH and lumenal Ero1 alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1 alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins.     

The FAD- and O(2)-dependent reaction cycle of Ero1-mediated oxidative protein folding in the endoplasmic reticulum

The endoplasmic reticulum (ER) supports disulfide formation through an essential protein relay involving Ero1p and protein disulfide isomerase (PDI). We find that in addition to having a tightly associated flavin adenine dinucleotide (FAD) moiety, yeast Ero1p is highly responsive to small changes in physiological levels of free FAD. This sensitivity underlies the dependence of oxidative protein folding on cellular FAD levels. FAD is synthesized in the cytosol but can readily enter the ER lumen and promote Ero1p-catalyzed oxidation. Ero1p then uses molecular oxygen as its preferred terminal electron acceptor. Thus Ero1p directly couples disulfide formation to the consumption of molecular oxygen, but its activity is modulated by free lumenal FAD levels, potentially linking disulfide formation to a cell's nutritional or metabolic status.     

The C-terminal domain of yeast Ero1p mediates membrane localization and is essential for function.

In eukaryotes, members of the Ero1 family control oxidative protein folding in the endoplasmic reticulum (ER). Yeast Ero1p is tightly associated with the ER membrane, despite cleavage of the leader peptide, the only hydrophobic sequence that could mediate lipid insertion. In contrast, human Ero1-Lalpha and a yeast mutant (Ero1pDeltaC) lacking the 127 C-terminal amino acids are soluble when expressed in yeast. Neither Ero1-Lalpha nor Ero1pDeltaC complements an ERO1 disrupted strain. Appending the yeast C-terminal tail to human Ero1-Lalpha restores membrane association and allows growth of ERO1 disrupted cells. Therefore, the tail of Ero1p mediates membrane association and is crucial for function.     

Oxidative protein folding in eukaryotes: mechanisms and consequences

The endoplasmic reticulum (ER) provides an environment that is highly optimized for oxidative protein folding. Rather than relying on small molecule oxidants like glutathione, it is now clear that disulfide formation is driven by a protein relay involving Ero1, a novel conserved FAD-dependent enzyme, and protein disulfide isomerase (PDI); Ero1 is oxidized by molecular oxygen and in turn acts as a specific oxidant of PDI, which then directly oxidizes disulfide bonds in folding proteins. While providing a robust driving force for disulfide formation, the use of molecular oxygen as the terminal electron acceptor can lead to oxidative stress through the production of reactive oxygen species and oxidized glutathione. How Ero1p distinguishes between the many different PDI-related proteins and how the cell minimizes the effects of oxidative damage from Ero1 remain important open questions.     

Two pairs of conserved cysteines are required for the oxidative activity of Ero1p in protein disulfide bond formation in the endoplasmic reticulum

In the major pathway for protein disulfide-bond formation in the endoplasmic reticulum (ER), oxidizing equivalents flow from the conserved ER-membrane protein Ero1p to secretory proteins via protein disulfide isomerase (PDI). Herein, a mutational analysis of the yeast ERO1 gene identifies two pairs of conserved cysteines likely to form redox-active disulfide bonds in Ero1p. Cys100, Cys105, Cys352, and Cys355 of Ero1p are important for oxidative protein folding and for cell viability, whereas Cys90, Cys208, and Cys349 are dispensable for these functions. Substitution of Cys100 with alanine impedes the capture of Ero1p-Pdi1p mixed-disulfide complexes from yeast, and also blocks oxidation of Pdi1p in vivo. Cys352 and Cys355 are required to maintain the fully oxidized redox state of Ero1p, and also play an auxiliary role in thiol-disulfide exchange with Pdi1p. These results suggest a model for the function of Ero1p wherein Cys100 and Cys105 form a redox-active disulfide bond that engages directly in thiol-disulfide exchange with ER oxidoreductases. The Cys352-Cys355 disulfide could then serve to reoxidize the Cys100-Cys105 cysteine pair, possibly through an intramolecular thiol-disulfide exchange reaction.     

Novel Roles of the Non-catalytic Elements of Yeast Protein-disulfide Isomerase in Its Interplay with Endoplasmic Reticulum Oxidoreductin 1

The formation of disulfide bonds in the endoplasmic reticulum (ER) of eukaryotic cells is catalyzed by the sulfhydryl oxidase, ER oxidoreductin 1 (Ero1), and protein-disulfide isomerase (PDI). PDI is oxidized by Ero1 to continuously introduce disulfides into substrates, and feedback regulates Ero1 activity by manipulating the regulatory disulfides of Ero1. In this study we find that yeast Ero1p is enzymatically active even with its regulatory disulfides intact, and further activation of Ero1p by reduction of the regulatory disulfides requires the reduction of non-catalytic Cys⁹⁰-Cys⁹⁷ disulfide in Pdi1p. The principal client-binding site in the Pdi1p b′ domain is necessary not only for the functional Ero1p-Pdi1p disulfide relay but also for the activation of Ero1p. We also demonstrate by complementary activation assays that the regulatory disulfides in Ero1p are much more stable than those in human Ero1α. These new findings on yeast Ero1p-Pdi1p interplay reveal significant differences from our previously identified mode of human Ero1α-PDI interplay and provide insights into the evolution of the eukaryotic oxidative protein folding pathway.     

Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

The toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show that upon oxidation of the COOH-terminal disulfide bond in PDI by the enzyme Ero1, the A1 chain is released. Both yeast Ero1 and the mammalian Ero1alpha isoform are active in this reaction. Ero1 has a preference for the PDI-toxin complex. We further show that the complex is transferred to a protein at the lumenal side of the ER membrane, where the unfolded toxin is released from PDI by the action of Ero1. Taken together, our results identify Ero1 as the enzyme mediating the release of unfolded CT from PDI and characterize an additional step in retrotranslocation of the toxin.     

Disulfide relays between and within proteins: the Ero1p structure

The essential flavoenzyme Ero1p both creates de novo disulfide bonds and transfers these disulfides to the folding catalyst protein disulfide isomerase (PDI). The recently solved crystal structure of Ero1p, in combination with previous biochemical, genetic and structural data, provides insight into the mechanism by which Ero1p accomplishes these tasks. A comparison of Ero1p with the smaller flavoenzyme Erv2p highlights important structural elements that are shared by these flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases and suggests some general themes that might be common to proteins that generate disulfide bonds.     

The Reduction Potential of the Active Site Disulfides of Human Protein Disulfide Isomerase Limits Oxidation of the Enzyme by Ero1��

Disulfide formation in newly synthesized proteins entering the mammalian endoplasmic reticulum is catalyzed by protein disulfide isomerase (PDI), which is itself thought to be directly oxidized by Ero1α. The activity of Ero1α is tightly regulated by the formation of noncatalytic disulfides, which need to be broken to activate the enzyme. Here, we have developed a novel PDI oxidation assay, which is able to simultaneously determine the redox status of the individual active sites of PDI. We have used this assay to confirm that when PDI is incubated with Ero1α, only one of the active sites of PDI becomes directly oxidized with a slow turnover rate. In contrast, a deregulated mutant of Ero1α was able to oxidize both PDI active sites at an equivalent rate to the wild type enzyme. When the active sites of PDI were mutated to decrease their reduction potential, both were now oxidized by wild type Ero1α with a 12-fold increase in activity. These results demonstrate that the specificity of Ero1α toward the active sites of PDI requires the presence of the regulatory disulfides. In addition, the rate of PDI oxidation is limited by the reduction potential of the PDI active site disulfide. The inability of Ero1α to oxidize PDI efficiently likely reflects the requirement for PDI to act as both an oxidase and an isomerase during the formation of native disulfides in proteins entering the secretory pathway.     

Dynamic Regulation of Ero1α and Peroxiredoxin 4 Localization in the Secretory Pathway

In the early secretory compartment (ESC), a network of chaperones and enzymes assists oxidative folding of nascent proteins. Ero1 flavoproteins oxidize protein disulfide isomerase (PDI), generating H₂O₂ as a byproduct. Peroxiredoxin 4 (Prx4) can utilize luminal H₂O₂ to oxidize PDI, thus favoring oxidative folding while limiting oxidative stress. Interestingly, neither ER oxidase contains known ER retention signal(s), raising the question of how cells prevent their secretion. Here we show that the two proteins share similar intracellular localization mechanisms. Their secretion is prevented by sequential interactions with PDI and ERp44, two resident proteins of the ESC-bearing KDEL-like motifs. PDI binds preferentially Ero1α, whereas ERp44 equally retains Ero1α and Prx4. The different binding properties of Ero1α and Prx4 increase the robustness of ER redox homeostasis.     

PDILT, a divergent testis-specific protein disulfide isomerase with a non-classical SXXC motif that engages in disulfide-dependent interactions in the endoplasmic reticulum

Protein disulfide isomerase (PDI) is the archetypal enzyme involved in the formation and reshuffling of disulfide bonds in the endoplasmic reticulum (ER). PDI achieves its redox function through two highly conserved thioredoxin domains, and PDI can also operate as an ER chaperone. The substrate specificities and the exact functions of most other PDI family proteins remain important unsolved questions in biology. Here, we characterize a new and striking member of the PDI family, which we have named protein disulfide isomerase-like protein of the testis (PDILT). PDILT is the first eukaryotic SXXC protein to be characterized in the ER. Our experiments have unveiled a novel, glycosylated PDI-like protein whose tissue-specific expression and unusual motifs have implications for the evolution, catalytic function, and substrate selection of thioredoxin family proteins. We show that PDILT is an ER resident glycoprotein that liaises with partner proteins in disulfide-dependent complexes within the testis. PDILT interacts with the oxidoreductase Ero1alpha, demonstrating that the N-terminal cysteine of the CXXC sequence is not required for binding of PDI family proteins to ER oxidoreductases. The expression of PDILT, in addition to PDI in the testis, suggests that PDILT performs a specialized chaperone function in testicular cells. PDILT is an unusual PDI relative that highlights the adaptability of chaperone and redox function in enzymes of the endoplasmic reticulum.     

Analysis of myotilin turnover provides mechanistic insight into the role of myotilinopathy-causing mutations

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the á domain of PDI. Our results reveal that Ero1β oxidase activity is regulated by long-range disulfide bonds and that Cys130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1β is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.     

Ero1-α and PDIs constitute a hierarchical electron transfer network of endoplasmic reticulum oxidoreductases

Ero1-α and endoplasmic reticulum (ER) oxidoreductases of the protein disulfide isomerase (PDI) family promote the efficient introduction of disulfide bonds into nascent polypeptides in the ER. However, the hierarchy of electron transfer among these oxidoreductases is poorly understood. In this paper, Ero1-α–associated oxidoreductases were identified by proteomic analysis and further confirmed by surface plasmon resonance. Ero1-α and PDI were found to constitute a regulatory hub, whereby PDI induced conformational flexibility in an Ero1-α shuttle cysteine (Cys99) facilitated intramolecular electron transfer to the active site. In isolation, Ero1-α also oxidized ERp46, ERp57, and P5; however, kinetic measurements and redox equilibrium analysis revealed that PDI preferentially oxidized other oxidoreductases. PDI accepted electrons from the other oxidoreductases via its a′ domain, bypassing the a domain, which serves as the electron acceptor from reduced glutathione. These observations provide an integrated picture of the hierarchy of cooperative redox interactions among ER oxidoreductases in mammalian cells.     

Mutations in the FAD binding domain cause stress-induced misoxidation of the endoplasmic reticulum oxidoreductase Ero1beta

Disulfide bond catalysis is an essential component of protein biogenesis in the secretory pathway, from yeast through to man. In the endoplasmic reticulum (ER), protein-disulfide isomerase (PDI) catalyzes the oxidation and isomerization of disulfide bonds and is re-oxidized by an endoplasmic reticulum oxidoreductase (ERO). The elucidation of ERO function was greatly aided by the genetic analysis of two ero mutants, whose impairment results from point mutations in the FAD binding domain of the Ero protein. The ero1-1 and ero1-2 yeast strains have conditional and dithiothreitol-sensitive phenotypes, but the effects of the mutations on the behavior of Ero proteins has not been reported. Here, we show that these Gly to Ser and His to Tyr mutations do not prevent the dimerization of Ero1beta or the non-covalent interaction of Ero1beta with PDI. However, the Gly to Ser mutation abolishes disulfide-dependent PDI-Ero1beta heterodimers. Both the Gly to Ser and His to Tyr mutations make Ero1beta susceptible to misoxidation and aggregation, particularly during a temperature or redox stress. We conclude that the Ero FAD binding domain is critical for conformational stability, allowing Ero proteins to withstand stress conditions that cause client proteins to misfold.     

Redox signaling loops in the unfolded protein response

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology.