ERp18 regulates activation of ATF6α during unfolded protein response.

Activation of the ATF6α signaling pathway is initiated by trafficking of ATF6α from the ER to the Golgi apparatus. Its subsequent proteolysis releases a transcription factor that translocates to the nucleus causing downstream gene activation. How ER retention, Golgi trafficking, and proteolysis of ATF6α are regulated and whether additional protein partners are required for its localization and processing remain unresolved. Here, we show that ER-resident oxidoreductase ERp18 associates with ATF6α following ER stress and plays a key role in both trafficking and activation of ATF6α. We find that ERp18 depletion attenuates the ATF6α stress response. Paradoxically, ER stress accelerates trafficking of ATF6α to the Golgi in ERp18-depleted cells. However, the translocated ATF6α becomes aberrantly processed preventing release of the soluble transcription factor. Hence, we demonstrate that ERp18 monitors ATF6α ER quality control to ensure optimal processing following trafficking to the Golgi.

A cytosolic reductase pathway is required for efficient N-glycosylation of an STT3B-dependent acceptor site.

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here, we used an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could be mimicked by the addition of a membrane-impermeable reducing agent. We identified a hypoglycosylated acceptor site that is adjacent to a cysteine involved in a short-range disulfide. We show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A- and STT3B-dependent glycosylation. Our results provide further insight into the important role of the endoplasmic reticulum redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins.

Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfides. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is crucial for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER. These results provide compelling evidence for the crucial role of the cytosol in regulating ER redox homeostasis, ensuring correct protein folding and facilitating the degradation of misfolded ER proteins.

Cytosolic thioredoxin reductase 1 is required for correct disulfide formation in the ER.

Folding of proteins entering the secretory pathway in mammalian cells frequently requires the insertion of disulfide bonds. Disulfide insertion can result in covalent linkages found in the native structure as well as those that are not, so-called non-native disulfides. The pathways for disulfide formation are well characterized, but our understanding of how non-native disulfides are reduced so that the correct or native disulfides can form is poor. Here, we use a novel assay to demonstrate that the reduction in non-native disulfides requires NADPH as the ultimate electron donor, and a robust cytosolic thioredoxin system, driven by thioredoxin reductase 1 (TrxR1 or TXNRD1). Inhibition of this reductive pathway prevents the correct folding and secretion of proteins that are known to form non-native disulfides during their folding. Hence, we have shown for the first time that mammalian cells have a pathway for transferring reducing equivalents from the cytosol to the ER, which is required to ensure correct disulfide formation in proteins entering the secretory pathway.

Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.

The membrane topology of vitamin K epoxide reductase is conserved between human isoforms and the bacterial enzyme.

The membrane topology of vitamin K epoxide reductase (VKOR) is controversial with data supporting both a three transmembrane and a four transmembrane model. The positioning of the transmembrane domains and the loops between these domains is critical if we are to understand the mechanism of vitamin K oxidation and its recycling by members of the thioredoxin family of proteins and the mechanism of action of warfarin, an inhibitor of VKOR. Here we show that both mammalian VKOR isoforms adopt the same topology, with the large loop between transmembrane one and two facing the lumen of the endoplasmic reticulum (ER). We used a redox sensitive green fluorescent protein (GFP) fused to the N- or C-terminus to show that these regions face the cytosol, and introduction of glycosylation sites along with mixed disulfide formation with thioredoxin-like transmembrane protein (TMX) to demonstrate ER localization of the major loop. The topology is identical with the bacterial homologue from Synechococcussp., for which the structure and mechanism of recycling has been characterized. Our results provide a resolution to the membrane topology controversy and support previous results suggesting a role for members of the ER protein disulfide isomerase (PDI) family in recycling VKOR.

Redox regulation in the endoplasmic reticulum.

The efficient folding, assembly and secretion of proteins from mammalian cells is a critically important process for normal cell physiology. Breakdown of the ability of cells to secrete functional proteins leads to disease pathologies caused by a lack of protein function or by cell death resulting from an aggravated stress response. Central to the folding of secreted proteins is the formation of disulfides which both aid folding and provide stability to the protein structure. For disulfides to form correctly necessitates the appropriate redox environment within the endoplasmic reticulum: too reducing and disulfides will not form, too oxidizing and non-native disulfides will not be resolved. How the endoplasmic reticulum maintains the correct redox balance is unknown. Although we have a good appreciation of the processes leading to a more oxidizing environment, our understanding of how any counterbalancing reductive pathway operates is limited. The present review looks at potential mechanisms for introducing reducing equivalents into the endoplasmic reticulum and discusses an approach to test these hypotheses.

Real-time monitoring of redox changes in the mammalian endoplasmic reticulum.

Redox-sensitive GFPs with engineered disulphide bonds have been used previously to monitor redox status in the cytosol and mitochondria of living cells. The usefulness of these redox probes depends on the reduction potential of the disulphide, with low values suiting the cytosol and mitochondrion, and higher values suiting the more oxidising environment of the endoplasmic reticulum (ER). Here, we targeted a modified redox-sensitive GFP (roGFP1-iL), with a relatively high reduction potential, to the ER of mammalian cells. We showed that the disulphide is partially oxidised, allowing roGFP1-iL to monitor changes in ER redox status. When cells were treated with puromycin, the redox balance became more reducing, suggesting that the release of nascent chains from ribosomes alters the ER redox balance. In addition, downregulating Ero1α prevented normal rapid recovery from dithiothreitol (DTT), whereas downregulating peroxiredoxin IV had no such effect. This result illustrates the contribution of the Ero1α oxidative pathway to ER redox balance. This first report of the use of roGFP to study the ER of mammalian cells demonstrates that roGFP1-iL can be used to monitor real-time changes to the redox status in individual living cells.

HLA-DP, HLA-DQ, and HLA-DR have different requirements for invariant chain and HLA-DM.

The MHC is central to the adaptive immune response. The human MHC class II is encoded by three different isotypes, HLA-DR, -DQ, and -DP, each being highly polymorphic. In contrast to HLA-DR, the intracellular assembly and trafficking of HLA-DP molecules have not been studied extensively. However, different HLA-DP variants can be either protective or risk factors for infectious diseases (e.g. hepatitis B), immune dysfunction (e.g. berylliosis), and autoimmunity (e.g. myasthenia gravis). Here, we establish a system to analyze the chaperone requirements for HLA-DP and to compare the assembly and trafficking of HLA-DP, -DQ, and -DR directly. Unlike HLA-DR1, HLA-DQ5 and HLA-DP4 can form SDS-stable dimers supported by invariant chain (Ii) in the absence of HLA-DM. Uniquely, HLA-DP also forms dimers in the presence of HLA-DM alone. In model antigen-presenting cells, SDS-stable HLA-DP complexes are resistant to treatments that prevent formation of SDS-stable HLA-DR complexes. The unexpected properties of HLA-DP molecules may help explain why they bind to a more restricted range of peptides than other human MHC class II proteins and frequently present viral peptides.

A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells.

Glycoprotein folding is mediated by lectin-like chaperones and protein disulfide isomerases (PDIs) in the endoplasmic reticulum. Calnexin and the PDI homologue ERp57 work together to help fold nascent polypeptides with glycans located toward the N-terminus of a protein, whereas PDI and BiP may engage proteins that lack glycans or have sugars toward the C-terminus. In this study, we show that the PDI homologue PDILT is expressed exclusively in postmeiotic male germ cells, in contrast to the ubiquitous expression of many other PDI family members in the testis. PDILT is induced during puberty and represents the first example of a PDI family member under developmental control. We find that PDILT is not active as an oxido-reductase, but interacts with the model peptide Delta-somatostatin and nonnative bovine pancreatic trypsin inhibitor in vitro, indicative of chaperone activity. In vivo, PDILT forms a tissue-specific chaperone complex with the calnexin homologue calmegin. The identification of a redox-inactive chaperone partnership defines a new system of testis-specific protein folding with implications for male fertility.

Activation of the unfolded protein response and alternative splicing of ATF6alpha in HLA-B27 positive lymphocytes.

Misfolding of major histocompatibility complex (MHC) class I molecules has been implicated in the rheumatic autoimmune disease ankylosing spondylitis (AS), and has been linked to the unfolded protein response (UPR) in rodent AS models. XBP1 and ATF6alpha are two important transcription factors that initiate and co-ordinate the UPR. Here we show that misoxidised MHC class I heavy chains activate XBP1 processing in a similar manner to tunicamycin, with tunicamycin and dithiothreitol (DTT) inducing differential XBP1 processing. Unexpectedly, ATF6alpha mRNA is alternatively spliced during reducing stress in lymphocytes. This shorter ATF6alpha message lacks exon 7 and may have a regulatory role in the UPR.

Inhibition of IRE1α-mediated XBP1 mRNA cleavage by XBP1 reveals a novel regulatory process during the unfolded protein response.

Background: The mammalian endoplasmic reticulum (ER) continuously adapts to the cellular secretory load by the activation of an unfolded protein response (UPR).  This stress response results in expansion of the ER, upregulation of proteins involved in protein folding and degradation, and attenuation of protein synthesis.  The response is orchestrated by three signalling pathways each activated by a specific signal transducer, either inositol requiring enzyme α (IRE1α), double-stranded RNA-activated protein kinase-like ER kinase (PERK) or activating transcription factor 6 (ATF6).  Activation of IRE1α results in its oligomerisation, autophosphorylation and stimulation of its ribonuclease activity.  The ribonuclease initiates the splicing of an intron from mRNA encoding the transcription factor, X-box binding protein 1 (XBP1), as well as degradation of specific mRNAs and microRNAs. Methods: To investigate the consequence of expression of exogenous XBP1, we generated a stable cell-line expressing spliced XBP1 mRNA under the control of an inducible promotor. Results: Following induction of expression, high levels of XBP1 protein were detected, which allowed upregulation of target genes in the absence of induction of the UPR.  Remarkably under stress conditions, the expression of exogenous XBP1 repressed splicing of endogenous XBP1 mRNA without repressing the activation of PERK. Conclusions: These results illustrate that a feedback mechanism exists to attenuate Ire1α ribonuclease activity in the presence of XBP1.

Cloning and initial characterization of the Arabidopsis thaliana endoplasmic reticulum oxidoreductins.

The oxidation and isomerization of disulfide bonds is necessary for the growth of all organisms. In yeast, the oxidative folding of secretory pathway proteins is catalyzed by protein disulfide isomerase (PDI), which requires Ero1p (endoplasmic reticulum oxidoreductin) for its own oxidation. In Homo sapiens, two homologues of Ero1p, Ero1-Lalpha and Ero1-Lbeta, have been cloned. Both Ero1-Lalpha and Ero1-Lbeta interact via disulfide bonds with PDI and support the oxidation of immunoglobulin light chains. However, the function of Ero proteins in plants has not yet been analyzed. In this article, we report the cloning of the two Ero1p homologues present in Arabidopsis thaliana, demonstrating that one of the cDNAs has a shorter terminal exon than predicted and differs from the annotated sequence found in the genome database. Sequence analysis of the Arabidopsis endoplasmic reticulum oxidoreductins (AEROs) reveals that both AERO1 and AERO2 are more closely related to each other than to either of the human Eros. Both in vitro translated AERO proteins are targeted to the endoplasmic reticulum and glycosylated. The ability to use a genetically tractable multicellular organism in combination with biochemical approaches should further our understanding of redox networks and Ero function in both plants and animals.

Ero1-PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum.

The protein folding machinery of the endoplasmic reticulum (ER) ensures that proteins entering the eukaryotic secretory pathway acquire appropriate post-translational modifications and reach a stably folded state. An important component of this protein folding process is the supply of disulfide bonds. These are introduced into client proteins by ER resident oxidoreductases, including ER oxidoreductin 1 (Ero1). Ero1 is usually considered to function in a linear pathway, by 'donating' a disulfide bond to protein disulfide isomerase (PDI) and receiving electrons that are passed on to the terminal electron acceptor molecular oxygen. PDI engages with a range of clients as the direct catalyst of disulfide bond formation, isomerization or reduction. In this paper, we will consider the interactions of Ero1 with PDI family proteins and chaperones, highlighting the effect that redox flux has on Ero1 partnerships. In addition, we will discuss whether higher order protein complexes play a role in Ero1 function.

The DMalpha and DMbeta chain cooperate in the oxidation and folding of HLA-DM.

HLA-DM (DM) is a heterodimeric MHC molecule that catalyzes the peptide loading of classical MHC class II molecules in the endosomal/lysosomal compartments of APCs. Although the function of DM is well-established, little is known about how DMalpha and beta-chains fold, oxidize, and form a complex in the endoplasmic reticulum (ER). In this study, we show that glycosylation promotes, but is not essential for, DMalphabeta ER exit. However, glycosylation of DMalpha N15 is required for oxidation of the alpha-chain. The DMalpha and beta-chains direct each others fate: single DMalpha chains cannot fully oxidize without DMbeta, while DMbeta forms disulfide-linked homodimers without DMalpha. Correct oxidation and subsequent ER egress depend on the unique DMbeta C25 and C35 residues. This suggests that the C25-C35 disulfide bond in the peptide-binding domain overcomes the need for stabilizing peptides required by other MHC molecules.

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.

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.

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1beta.

Endoplasmic reticulum oxidoreductases (Eros) are essential for the formation of disulfide bonds. Understanding disulfide bond catalysis in mammals is important because of the involvement of protein misfolding in conditions such as diabetes, arthritis, cancer, and aging. Mammals express two related Ero proteins, Ero1alpha and Ero1beta. Ero1beta is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1beta can form homodimers and mixed heterodimers with Ero1alpha, in addition to Ero-PDI dimers. Ero-Ero dimers require the Ero active site, occur in vivo, and can be modeled onto the Ero1p crystal structure. Our data indicate that the Ero1beta protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1beta occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1beta expression is high, but is inversely correlated with PDI and PDIp levels, demonstrating that cell-specific differences exist in the regulation of oxidative protein folding in vivo.

Spatial separation of HLA-DM/HLA-DR interactions within MIIC and phagosome-induced immune escape.

Major Histocompatibility Complex (MHC) class II molecules, including Human Leukocyte Antigen (HLA)-DR, present peptide fragments from proteins degraded in the endocytic pathway. HLA-DR is targeted to late-endocytic structures named MHC class II-containing Compartments (MIIC), where it interacts with HLA-DM. This chaperone stabilizes HLA-DR during peptide exchange and is critical for successful peptide loading. To follow this process in living cells, we have generated cells containing HLA-DR3/Cyan Fluorescent Protein (CFP), HLA-DM/Yellow Fluorescent Protein (YFP), and invariant chain. HLA-DR/DM interactions were observed by Fluorescence Resonance Energy Transfer (FRET). These interactions were pH insensitive, yet occurred only in internal structures and not at the limiting membrane of MIIC. In a cellular model of infection, phagosomes formed a limiting membrane surrounding internalized Salmonella. HLA-DR and HLA-DM did not interact in Salmonella-induced vacuoles, and HLA-DR was not loaded with antigens. The absence of HLA-DR/DM interactions at the limiting membrane prevents local loading of MHC class II molecules in phagosomes. This may allow these bacteria to successfully evade the immune system.

Stable expression of MHC class I heavy chain/HLA-DO complexes at the plasma membrane.

Major histocompatibility complex (MHC) class I and II molecules present antigenic fragments to the immune system. MHC-like chaperones, like HLA-DM, HLA-DO and tapasin support peptide loading. HLA class I heavy chains require association with beta 2-microglobulin and peptide for endoplasmic reticulum (ER) exit. Likewise, HLA-DO is only able to leave the ER by association to DM. Here we show that HLA-DO and free MHC class I heavy chains associate into a stable complex early during biosynthesis and are expressed at the plasma membrane as a complex. These DO/heavy chain complexes are found on DO-transfected cells and on low amounts on human B cells.