Marcel van Lith

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.

Tissue-specific expression and dimerization of the endoplasmic reticulum oxidoreductase Ero1β

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, Ero1α and Ero1β. Ero1β is incompletely characterized but is of physiological interest because it is induced by the unfolded protein response. Here, we show that Ero1β can form homodimers and mixed heterodimers with Ero1α, 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 Ero1β protein is constitutively strongly expressed in the stomach and the pancreas, but in a cell-specific fashion. In the stomach, selective expression of Ero1β occurs in the enzyme-producing chief cells. In pancreatic islets, Ero1β 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.

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 Ero1α, 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.

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.

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.

Activation of the unfolded protein response and alternative splicing of ATF6α 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 ATF6α 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, ATF6α mRNA is alternatively spliced during reducing stress in lymphocytes. This shorter ATF6α message lacks exon 7 and may have a regulatory role in the UPR.

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.

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.

The DMα and DMβ 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 DMα and β-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, DMαβ ER exit. However, glycosylation of DMα N15 is required for oxidation of the α-chain. The DMα and β-chains direct each others fate: single DMα chains cannot fully oxidize without DMβ, while DMβ forms disulfide-linked homodimers without DMα. Correct oxidation and subsequent ER egress depend on the unique DMβ 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.

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.