The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

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.     

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.     

Secretion of the adipocyte-specific secretory protein adiponectin critically depends on thiol-mediated protein retention

Adiponectin is a secretory protein abundantly secreted from adipocytes. It assembles into a number of different higher-order complexes. Adipocytes maintain tight control over circulating plasma levels, suggesting the existence of a complex, highly regulated biosynthetic pathway. However, the critical mediators of adiponectin maturation within the secretory pathway have not been elucidated. Previously, we found that a significant portion of de novo-synthesized adiponectin is not secreted and retained in adipocytes. Here, we show that there is an abundant pool of properly folded adiponectin in the secretory pathway that is retained through thiol-mediated retention, as judged by the release of adiponectin in response to treatment of adipocytes with reducing agents. Adiponectin is covalently bound to the ER chaperone ERp44. An adiponectin mutant lacking cysteine 39 fails to stably interact with ERp44, demonstrating that this residue is the primary site mediating the covalent interaction. Another ER chaperone, Ero1-Lalpha, plays a critical role in the release of adiponectin from ERp44. Levels of both of these proteins are highly regulated in adipocytes and are influenced by the metabolic state of the cell. While less critical for the secretion of trimers, these chaperones play a major role in the assembly of higher-order adiponectin complexes. Our data highlight the importance of posttranslational events controlling adiponectin levels and the release of adiponectin from adipocytes. One mechanism for increasing circulating levels of specific adiponectin complexes by peroxisome proliferator-activated receptor gamma agonists may be selective upregulation of rate-limiting chaperones.     

Post-translational modifications of adiponectin: mechanisms and functional implications

Adiponectin is an insulin-sensitizing adipokine with anti-diabetic, anti-atherogenic, anti-inflammatory and cardioprotective properties. This adipokine is secreted from adipocytes into the circulation as three oligomeric isoforms, including trimeric, hexameric and the HMW (high-molecular-mass) oligomeric complex consisting of at least 18 protomers. Each oligomeric isoform of adiponectin exerts distinct biological properties in its various target tissues. The HMW oligomer is the major active form mediating the insulin-sensitizing effects of adiponectin, whereas the central actions of this adipokine are attributed primarily to the hexameric and trimeric oligomers. In patients with Type 2 diabetes and coronary heart disease, circulating levels of HMW adiponectin are selectively decreased due to an impaired secretion of this oligomer from adipocytes. The biosynthesis of the adiponectin oligomers is a complex process involving extensive post-translational modifications. Hydroxylation and glycosylation of several conserved lysine residues in the collagenous domain of adiponectin are necessary for the intracellular assembly and stabilization of its high-order oligomeric structures. Secretion of the adiponectin oligomers is tightly controlled by a pair of molecular chaperones in the ER (endoplasmic reticulum), including ERp44 (ER protein of 44 kDa) and Ero1-Lalpha (ER oxidoreductase 1-Lalpha). ERp44 inhibits the secretion of adiponectin oligomers through a thiol-mediated retention. In contrast, Ero1-Lalpha releases HMW adiponectin trapped by ERp44. The PPARgamma (peroxisome-proliferator-activated receptor gamma) agonists thiazolidinediones selectively enhance the secretion of HMW adiponectin through up-regulation of Ero1-Lalpha. In the present review, we discuss the recent advances in our understanding of the structural and biological properties of the adiponectin oligomeric isoforms and highlight the role of post-translational modifications in regulating the biosynthesis of HMW adiponectin.     

Disulphide production by Ero1���CPDI relay is rapid and effectively regulated

The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady‐state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1‐deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed‐disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI‐family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1‐ and glutathione disulphide‐mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.     

Secretory kinase Fam20C tunes endoplasmic reticulum redox state via phosphorylation of Ero1α

Family with sequence similarity 20C (Fam20C), the physiological Golgi casein kinase, phosphorylates numerous secreted proteins that are involved in a wide variety of biological processes. However, the role of Fam20C in regulating proteins in the endoplasmic reticulum (ER) lumen is largely unknown. Here, we report that Fam20C interacts with various luminal proteins and that its depletion results in a more reduced ER lumen. We further show that ER oxidoreductin 1α (Ero1α), the pivotal sulfhydryl oxidase that catalyzes disulfide formation in the ER, is phosphorylated by Fam20C in the Golgi apparatus and retrograde‐transported to the ER mediated by ERp44. The phosphorylation of Ser145 greatly enhances Ero1α oxidase activity and is critical for maintaining ER redox homeostasis and promoting oxidative protein folding. Notably, phosphorylation of Ero1α is induced under hypoxia, reductive stress, and secretion‐demanding conditions such as mammalian lactation. Collectively, our findings open a door to uncover how oxidative protein folding is regulated by phosphorylation in the secretory pathway.     

A novel disulphide switch mechanism in Ero1alpha balances ER oxidation in human cells

Oxidative maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is powered by Ero1 oxidases. To prevent cellular hyperoxidation, Ero1 activity can be regulated by intramolecular disulphide switches. Here, we determine the redox-driven shutdown mechanism of Ero1alpha, the housekeeping Ero1 enzyme in human cells. We show that functional silencing of Ero1alpha in cells arises from the formation of a disulphide bond-identified by mass spectrometry--between the active-site Cys(94) (connected to Cys(99) in the active enzyme) and Cys(131). Competition between substrate thiols and Cys(131) creates a feedback loop where activation of Ero1alpha is linked to the availability of its substrate, reduced protein disulphide isomerase (PDI). Overexpression of Ero1alpha-Cys131Ala or the isoform Ero1beta, which does not have an equivalent disulphide switch, leads to augmented ER oxidation. These data reveal a novel regulatory feedback system where PDI emerges as a central regulator of ER redox homoeostasis.     

ERp44 mediates a thiol-independent retention of formylglycine-generating enzyme in the endoplasmic reticulum

Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.     

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.     

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.     

Two conserved cysteine triads in human Ero1alpha cooperate for efficient disulfide bond formation in the endoplasmic reticulum

Human Ero1alpha is an endoplasmic reticulum (ER)-resident protein responsible for protein disulfide isomerase (PDI) oxidation. To clarify the molecular mechanisms underlying its function, we generated a panel of cysteine replacement mutants and analyzed their capability of: 1) complementing a temperature-sensitive yeast Ero1 mutant, 2) favoring oxidative folding in mammalian cells, 3) forming mixed disulfides with PDI and ERp44, and 4) adopting characteristic redox-dependent conformations. Our results reveal that two essential cysteine triads (Cys85-Cys94-Cys99 and Cys391-Cys394-Cys397) cooperate in electron transfer, with Cys94 likely forming mixed disulfides with PDI. Dominant negative phenotypes arise when critical residues within the triads are mutated (Cys394, Cys397, and to a lesser extent Cys99). Replacing the first cysteine in either triad (Cys85 or Cys391) generates mutants with weaker activity. In addition, mutating either Cys85 or Cys391, but not Cys397, reverts the dominant negative phenotype of the C394A mutant. These findings suggest that interactions between the two triads, dependent on Cys85 and Cys391, are important for Ero1alpha function, possibly stabilizing a platform for efficient PDI oxidation.     

Pathogenesis of ER Storage Disorders: Modulating Russell Body Biogenesis by Altering Proximal and Distal Quality Control

In many protein storage diseases, detergent‐insoluble proteins accumulate in the early secretory compartment (ESC). Protein condensation reflects imbalances between entry into (synthesis/translocation) and exit from (secretion/degradation) ESC, and can be also a consequence of altered quality control (QC) mechanisms. Here we exploit the inducible formation of Russell bodies (RB), dilated ESC cisternae containing mutant Ig‐µ chains, as a model to mechanistically dissect protein condensation. Depending on the presence or absence of Ig‐L chains, mutant Ig‐µ chains lacking their first constant domain (Ch 1) accumulate in rough or smooth RB (rRB and sRB), dilations of the endoplasmic reticulum (ER) and ER‐Golgi intermediate compartment (ERGIC), respectively, reflecting the proximal and distal QC stations in the stepwise biogenesis of polymeric IgM. Either weakening ERp44‐dependent distal QC or facilitating ER‐associated degradation (ERAD) inhibits RB formation. Overexpression of PDI or ERp44 inhibits µΔCh 1 secretion. However, PDI inhibits while ERp44 promotes µΔCh 1 condensation. Both Ero1α silencing and overexpression prevent RB formation, demonstrating a strict redox dependency of the phenomenon. Altogether, our findings identify key controllers of protein condensation along the ESC as potential targets to handle certain storage disorders.     

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.     

Analysis of the retention signals of two resident luminal endoplasmic reticulum proteins by in vitro mutagenesis

Protein disulfide isomerase (PDI, ERp59), ERp72, and ERp61 are luminal proteins of the endoplasmic reticulum (ER) that are characterized by the presence of sequences corresponding to the active site regions of PDI. Each one of these proteins possesses a different COOH-terminal tetrapeptide ER retention signal. In order to investigate what other tetrapeptide sequences could serve as retention signals and to determine to what extent the function of the retention signal is modulated by the protein carrying the signal, we have constructed a set of mutants of two of these resident ER proteins, PDI and ERp72. In each of these proteins, the wild type tetrapeptide sequences were replaced by each member of the set of the 12 possible combinations consisting of (K,R,Q)-(D,E)-(D,E)-L. Analysis of the efficiency of retention of the variant proteins when each was transiently expressed in COS cells showed that the retention efficiencies vary with both the COOH-terminal sequence and with the protein that carries this sequence.     

Consequences of ERp57 deletion on oxidative folding of obligate and facultative clients of the calnexin cycle

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.     

Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.     

Ero1L, a thiol oxidase, is required for Notch signaling through cysteine bridge formation of the Lin12-Notch repeats in Drosophila melanogaster

Notch-mediated cell–cell communication regulates numerous developmental processes and cell fate decisions. Through a mosaic genetic screen in Drosophila melanogaster, we identified a role in Notch signaling for a conserved thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1–like (Ero1L). Although Ero1L is reported to play a widespread role in protein folding in yeast, in flies Ero1L mutant clones show specific defects in lateral inhibition and inductive signaling, two characteristic processes regulated by Notch signaling. Ero1L mutant cells accumulate high levels of Notch protein in the ER and induce the unfolded protein response, suggesting that Notch is misfolded and fails to be exported from the ER. Biochemical assays demonstrate that Ero1L is required for formation of disulfide bonds of three Lin12-Notch repeats (LNRs) present in the extracellular domain of Notch. These LNRs are unique to the Notch family of proteins. Therefore, we have uncovered an unexpected requirement for Ero1L in the maturation of the Notch receptor.     

Protein disulfide isomerase-like proteins play opposing roles during retrotranslocation

Misfolded proteins in the endoplasmic reticulum (ER) are retained in the organelle or retrotranslocated to the cytosol for proteasomal degradation. ER chaperones that guide these opposing processes are largely unknown. We developed a semipermeabilized cell system to study the retrotranslocation of cholera toxin (CT), a toxic agent that crosses the ER membrane to reach the cytosol during intoxication. We found that protein disulfide isomerase (PDI) facilitates CT retrotranslocation, whereas ERp72, a PDI-like protein, mediates its ER retention. In vitro analysis revealed that PDI and ERp72 alter CT's conformation in a manner consistent with their roles in retrotranslocation and ER retention. Moreover, we found that PDI's and ERp72's opposing functions operate on endogenous ER misfolded proteins. Thus, our data identify PDI family proteins that play opposing roles in ER quality control and establish an assay to further delineate the mechanism of CT retrotranslocation.