Characterization of the endoplasmic reticulum-resident peroxidases GPx7 and GPx8 shows the higher oxidative activity of GPx7 and its linkage to oxidative protein folding.

Oxidative protein folding occurs primarily in the mammalian endoplasmic reticulum, enabled by a diverse network comprising more than 20 members of the protein disulfide isomerase (PDI) family and more than five PDI oxidases. Although the canonical disulfide bond formation pathway involving Ero1α and PDI has been well-studied so far, the physiological roles of the newly identified PDI oxidases, glutathione peroxidase-7 (GPx7) and -8 (GPx8), are only poorly understood. We here demonstrated that human GPx7 has much higher reactivity with H2O2 and hence greater PDI oxidation activity than human GPx8. The high reactivity of GPx7 is due to the presence of a catalytic tetrad at the redox-active site, which stabilizes the sulfenylated species generated upon the reaction with H2O2 Although it was previously postulated that GPx7 catalysis involved a highly reactive peroxidatic cysteine that can be sulfenylated by H2O2, we revealed that a resolving cysteine instead regulates the PDI oxidation activity of GPx7. We also determined that GPx7 formed complexes preferentially with PDI and P5 in H2O2-treated cells. Altogether, these results suggest that human GPx7 functions as an H2O2-dependent PDI oxidase in cells, whereas PDI oxidation may not be the central physiological role of human GPx8.

Protein secondary structure determines the temporal relationship between folding and disulfide formation.

How and when disulfide bonds form in proteins relative to the stage of their folding is a fundamental question in cell biology. Two models describe this relationship: the folded precursor model, in which a nascent structure forms before disulfides do, and the quasi-stochastic model, where disulfides form prior to folding. Here we investigated oxidative folding of three structurally diverse substrates, ß2-microglobulin, prolactin, and the disintegrin domain of ADAM metallopeptidase domain 10 (ADAM10), to understand how these mechanisms apply in a cellular context. We used a eukaryotic cell-free translation system in which we could identify disulfide isomers in stalled translation intermediates to characterize the timing of disulfide formation relative to translocation into the endoplasmic reticulum and the presence of non-native disulfides. Our results indicate that in a domain lacking secondary structure, disulfides form before conformational folding through a process prone to nonnative disulfide formation, whereas in proteins with defined secondary structure, native disulfide formation occurs after partial folding. These findings reveal that the nascent protein structure promotes correct disulfide formation during cotranslational folding.

Dynamic assembly of protein disulfide isomerase in catalysis of oxidative folding.

Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.

Ca2+ Regulates ERp57-Calnexin Complex Formation.

ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.

Tumor-associated macrophage-derived inflammatory cytokine enhances malignant potential of malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is an asbestos-related aggressive malignant neoplasm. Due to the difficulty of achieving curative surgical resection in most patients with MPM, a combination chemotherapy of cisplatin and pemetrexed has been the only approved regimen proven to improve the prognosis of MPM. However, the median overall survival time is at most 12 mo even with this regimen. There has been therefore a pressing need to develop a novel chemotherapeutic strategy to bring about a better outcome for MPM. We found that expression of interleukin-1 receptor (IL-1R) was upregulated in MPM cells compared with normal mesothelial cells. We also investigated the biological significance of the interaction between pro-inflammatory cytokine IL-1ß and the IL-1R in MPM cells. Stimulation by IL-1ß promoted MPM cells to form spheroids along with upregulating a cancer stem cell marker CD26. We also identified tumor-associated macrophages (TAMs) as the major source of IL-1ß in the MPM microenvironment. Both high mobility group box 1 derived from MPM cells and the asbestos-activated inflammasome in TAMs induced the production of IL-1ß, which resulted in enhancement of the malignant potential of MPM. We further performed immunohistochemical analysis using clinical MPM samples obtained from patients who were treated with the combination of platinum plus pemetrexed, and found that the overexpression of IL-1R tended to correlate with poor overall survival. In conclusion, the interaction between MPM cells and TAMs through a IL-1ß/IL-1R signal could be a promising candidate as the target for novel treatment of MPM (Hyogo College of Medicine clinical trial registration number: 2973).

Structural basis of pH-dependent client binding by ERp44, a key regulator of protein secretion at the ER-Golgi interface.

ERp44 retrieves some endoplasmic reticulum (ER)-resident enzymes and immature oligomers of secretory proteins from the Golgi. Association of ERp44 with its clients is regulated by pH-dependent mechanisms, but the molecular details are not fully understood. Here we report high-resolution crystal structures of human ERp44 at neutral and weakly acidic pH. These structures reveal key regions in the C-terminal tail (C tail) missing in the original crystal structure, including a regulatory histidine-rich region and a subsequent extended loop. The former region forms a short α-helix (α16), generating a histidine-clustered site (His cluster). At low pH, the three Trx-like domains of ERp44 ("a," "b," and "b'") undergo significant rearrangements, likely induced by protonation of His157 located at the interface between the a and b domains. The α16-helix is partially unwound and the extended loop is disordered in weakly acidic conditions, probably due to electrostatic repulsion between the protonated histidines in the His cluster. Molecular dynamics simulations indicated that helix unwinding enhances the flexibility of the C tail, disrupting its normal hydrogen-bonding pattern. The observed pH-dependent conformational changes significantly enlarge the positively charged regions around the client-binding site of ERp44 at low pH. Mutational analyses showed that ERp44 forms mixed disulfides with specific cysteines residing on negatively charged loop regions of Ero1α. We propose that the protonation states of the essential histidines regulate the ERp44-client interaction by altering the C-tail dynamics and surface electrostatic potential of ERp44.

PDI Family Members as Guides for Client Folding and Assembly.

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest.

Small-cell lung cancer (SCLC) is characterized by one of neuroendocrine tumors, and is a clinically aggressive cancer due to its rapid growth, early dissemination, and rapid acquisition of multidrug resistance to chemotherapy. Moreover, the standard chemotherapeutic regimen in SCLC has not changed for three decades despite of the dramatic therapeutic improvement in non-SCLC. The development of a novel therapeutic strategy for SCLC has become a pressing issue. We found that expression of Eph receptor A2 (EphA2) is upregulated in three of 13 SCLC cell lines and five of 76 SCLC tumor samples. Genetic inhibition using siRNA of EphA2 significantly suppressed the cellular proliferation via induction of cell cycle arrest in SBC-5 cells. Furthermore, small molecule inhibitors of EphA2 (ALW-II-41-27 and dasatinib) also exclusively inhibited proliferation of EphA2-positive SCLC cells by the same mechanism. Collectively, EphA2 could be a promising candidate as a therapeutic target for SCLC.

Ero1-Mediated Reoxidation of Protein Disulfide Isomerase Accelerates the Folding of Cone Snail Toxins.

Disulfide-rich peptides are highly abundant in nature and their study has provided fascinating insight into protein folding, structure and function. Venomous cone snails belong to a group of organisms that express one of the largest sets of disulfide-rich peptides (conotoxins) found in nature. The diversity of structural scaffolds found for conotoxins suggests that specialized molecular adaptations have evolved to ensure their efficient folding and secretion. We recently showed that canonical protein disulfide isomerase (PDI) and a conotoxin-specific PDI (csPDI) are ubiquitously expressed in the venom gland of cone snails and play a major role in conotoxin folding. Here, we identify cone snail endoplasmic reticulum oxidoreductin-1 (Conus Ero1) and investigate its role in the oxidative folding of conotoxins through reoxidation of cone snail PDI and csPDI. We show that Conus Ero1 preferentially reoxidizes PDI over csPDI, suggesting that the reoxidation of csPDI may rely on an Ero1-independent molecular pathway. Despite the preferential reoxidation of PDI over csPDI, the combinatorial effect of Ero1 and csPDI provides higher folding yields than Ero1 and PDI. We further demonstrate that the highest in vitro folding rates of two model conotoxins are achieved when all three enzymes are present, indicating that these enzymes may act synergistically. Our findings provide new insight into the generation of one of the most diverse classes of disulfide-rich peptides and may improve current in vitro approaches for the production of venom peptides for pharmacological studies.

Human ER Oxidoreductin-1α (Ero1α) Undergoes Dual Regulation through Complementary Redox Interactions with Protein-Disulfide Isomerase.

In the mammalian endoplasmic reticulum, oxidoreductin-1α (Ero1α) generates protein disulfide bonds and transfers them specifically to canonical protein-disulfide isomerase (PDI) to sustain oxidative protein folding. This oxidative process is coupled to the reduction of O2 to H2O2 on the bound flavin adenine dinucleotide cofactor. Because excessive thiol oxidation and H2O2 generation cause cell death, Ero1α activity must be properly regulated. In addition to the four catalytic cysteines (Cys94, Cys99, Cys104, and Cys131) that are located in the flexible active site region, the Cys208-Cys241 pair located at the base of another flexible loop is necessary for Ero1α regulation, although the mechanistic basis is not fully understood. The present study revealed that the Cys208-Cys241 disulfide was reduced by PDI and other PDI family members during PDI oxidation. Differential scanning calorimetry and small angle X-ray scattering showed that mutation of Cys208 and Cys241 did not grossly affect the thermal stability or overall shape of Ero1α, suggesting that redox regulation of this cysteine pair serves a functional role. Moreover, the flexible loop flanked by Cys208 and Cys241 provides a platform for functional interaction with PDI, which in turn enhances the oxidative activity of Ero1α through reduction of the Cys208-Cys241 disulfide. We propose a mechanism of dual Ero1α regulation by dynamic redox interactions between PDI and the two Ero1α flexible loops that harbor the regulatory cysteines.

Inhibition of the functional interplay between endoplasmic reticulum (ER) oxidoreduclin-1α (Ero1α) and protein-disulfide isomerase (PDI) by the endocrine disruptor bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that may have adverse effects on human health. We recently isolated protein-disulfide isomerase (PDI) as a BPA-binding protein from rat brain homogenates and found that BPA markedly inhibited PDI activity. To elucidate mechanisms of this inhibition, detailed structural, biophysical, and functional analyses of PDI were performed in the presence of BPA. BPA binding to PDI induced significant rearrangement of the N-terminal thioredoxin domain of PDI, resulting in more compact overall structure. This conformational change led to closure of the substrate-binding pocket in b' domain, preventing PDI from binding to unfolded proteins. The b' domain also plays an essential role in the interplay between PDI and ER oxidoreduclin 1α (Ero1α), a flavoenzyme responsible for reoxidation of PDI. We show that BPA inhibited Ero1α-catalyzed PDI oxidation presumably by inhibiting the interaction between the b' domain of PDI and Ero1α; the phenol groups of BPA probably compete with a highly conserved tryptophan residue, located in the protruding ß-hairpin of Ero1α, for binding to PDI. Consistently, BPA slowed down the reoxidation of PDI and caused the reduction of PDI in HeLa cells, indicating that BPA has a great impact on the redox homeostasis of PDI within cells. However, BPA had no effect on the interaction between PDI and peroxiredoxin-4 (Prx4), another PDI family oxidase, suggesting that the interaction between Prx4 and PDI is different from that of Ero1α and PDI. These results indicate that BPA, a widely distributed and potentially harmful chemical, inhibits Ero1-PDI-mediated disulfide bond formation.

Phosphatidylinositol Induces Caspase-Independent Apoptosis of Malignant Pleural Mesothelioma Cells by Accumulating AIF in the Nucleus.

BACKGROUND/AIMS: Phosphatidylinositol (PI) regulates a variety of cell processes. The present study investigated the antitumor action of 1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DOPI) and 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol)(DPPI) on human malignant pleural mesothelioma (MPM) cell lines such as NCI-H28, NCI-H2052, NCI-H2452, and MSTO-211H cells. METHODS: MTT assay, TUNEL staining, flow cytometry using propidium iodide (PI) and annexin V (AV), enzymatic caspase assay, and nuclear staining using DAPI were carried out, and mitochondrial membrane potentials and intracellular distribution of apoptosis-inducing factor (AIF) were monitored in cells with and without the siRNA silencing the Bid-targeted gene. RESULTS: Both DOPI and DPPI reduced cell viability for all the investigated MPM cell lines in a concentration (0.01-100 µM)-dependent manner. DOPI and DPPI significantly increased TUNEL-positive cells and the population of PI-negative/AV-positive and PI-positive/AV-positive cells, corresponding to early apoptosis and late apoptosis/secondary necrosis, respectively. DOPI and DPPI perturbed mitochondrial membrane potentials in MSTO-211H cells, but no significant activation of caspase-3, -4, -8, and -9 was obtained. DOPI and DPPI upregulated expression of Bid in MSTO-211H cells. DOPI and DPPI significantly increased nuclear localization of AIF without affecting expression of the mRNAs and proteins in MSTO-211H cells, which was inhibited by knocking-down Bid. In the DAPI staining, nuclear fragmentation and condensation were found. CONCLUSION: The results of the present study indicate that DOPI and DPPI facilitate Bid-mediated AIF release from the mitochondria, to accumulate AIF in the nucleus and induce caspase-independent apoptosis of MPM cells.

Boosting the enzymatic activity of CxxC motif-containing PDI family members.

Compounds harboring high acidity and oxidizability of thiol groups permit tuning the redox equilibrium constants of CxxC sites of members of the protein disulphide isomerase (PDI) family and thus can be used to accelerate folding processes and increase the production of native proteins by minimal loading in comparison to glutathione.

Serum HMGB1 as a prognostic marker for malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive malignant tumor of mesothelial origin that shows a limited response to conventional chemotherapy and radiotherapy. Therefore, diagnosing MPM early is very important. Some researchers have previously reported that high-mobility group box 1 (HMGB1) was correlated with pulmonary fibrosis. MPM involves the malignant transformation of mesothelial cells, which originate from mesenchymal cells similar to lung fibroblasts. Here, we investigated serum levels of HMGB1 in patients with MPM and compared them with those of a population that had been exposed to asbestos without developing MPM. METHODS: HMGB1 production from MPM cell lines was measured using ELISA. Serum HMGB1 levels were also examined in 61 MPM patients and 45 individuals with benign asbestos-related diseases. RESULTS: HMGB1 concentrations of 2 out of 4 MPM cell lines were higher than that of normal mesothelial cell line, Met-5A. We demonstrated that patients with MPM had significantly higher serum levels of HMGB1 than the population who had been exposed to asbestos but had not developed MPM. The difference in overall survival between groups with serum HMGB1 levels that were lower and higher than assumed cut-off values was significant. CONCLUSIONS: Our data suggest that serum HMGB1 concentration is a useful prognostic factor for MPM.

Serum HMGB1 as a diagnostic marker for malignant peritoneal mesothelioma.

BACKGROUND: Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive malignant tumor of mesothelial origin that shows a limited response to cytoreductive surgery along with intraperitoneal chemotherapy. Therefore, early diagnosis of DMPM is very important. Some researchers have previously reported that high-mobility group box 1 (HMGB1) was correlated with pulmonary fibrosis. DMPM involves the malignant transformation of mesothelial cells, which originate from mesenchymal cells, similar to lung fibroblasts. Here, we investigated serum levels of HMGB1 in patients with MPM and compared them with those of a population that had been exposed to asbestos without developing MPM. STUDY: The serum concentrations of HMGB1 were measured in 13 DMPM patients and 45 individuals with benign asbestos-related diseases. RESULT: We demonstrated that the patients with DMPM had significantly higher serum levels of HMGB1 compared with the population who had been exposed to asbestos but did not develop DMPM. CONCLUSION: Our data suggest that serum HMGB1 concentration is a useful serum marker for DMPM.

Semi-enzymatic acceleration of oxidative protein folding by N-methylated heteroaromatic thiols.

We report the first example of a synthetic thiol-based compound that promotes oxidative protein folding upon 1-equivalent loading to the disulfide bonds in the client protein to afford the native form in over 70% yield. N-Methylation is a central post-translational processing of proteins in vivo for regulating functions including chaperone activities. Despite the universally observed biochemical reactions in nature, N-methylation has hardly been utilized in the design, functionalization, and switching of synthetic bioregulatory agents, particularly folding promotors. As a biomimetic approach, we developed pyridinylmethanethiols to investigate the effects of N-methylation on the promotion of oxidative protein folding. For a comprehensive study on the geometrical effects, constitutional isomers of pyridinylmethanethiols with ortho-, meta-, and para-substitutions have been synthesized. Among the constitutional isomers, para-substituted pyridinylmethanethiol showed the fastest disulfide-bond formation of the client proteins to afford the native forms most efficiently. N-Methylation drastically increased the acidity and enhanced the oxidizability of the thiol groups in the pyridinylmethanethiols to enhance the folding promotion efficiencies. Among the isomers, para-substituted N-methylated pyridinylmethanethiol accelerated the oxidative protein folding reactions with the highest efficiency, allowing for protein folding promotion by 1-equivalent loading as a semi-enzymatic activity. This study will offer a novel bioinspired molecular design of synthetic biofunctional agents that are semi-enzymatically effective for the promotion of oxidative protein folding including biopharmaceuticals such as insulin in vitro by minimum loading.

A unique leucine-valine adhesive motif supports structure and function of protein disulfide isomerase P5 via dimerization.

P5, also known as PDIA6, is a PDI family member involved in the ER quality control. Here, we revealed that P5 dimerizes via a unique adhesive motif contained in the N-terminal thioredoxin-like domain. Unlike conventional leucine zipper motifs with leucine residues every two helical turns on ∼30-residue parallel α helices, this adhesive motif includes periodic repeats of leucine/valine residues at the third or fourth position spanning five helical turns on 15-residue anti-parallel α helices. The P5 dimerization interface is further stabilized by several reciprocal salt bridges and C-capping interactions between protomers. A monomeric P5 mutant with the impaired adhesive motif showed structural instability and local unfolding, and behaved as aberrant proteins that induce the ER stress response. Disassembly of P5 to monomers compromised its ability to inactivate IRE1α via intermolecular disulfide bond reduction and its Ca2+-dependent regulation of chaperone function in vitro. Thus, the leucine-valine adhesive motif supports structure and function of P5.

Functional Interplay between P5 and PDI/ERp72 to Drive Protein Folding.

P5 is one of protein disulfide isomerase family proteins (PDIs) involved in endoplasmic reticulum (ER) protein quality control that assists oxidative folding, inhibits protein aggregation, and regulates the unfolded protein response. P5 reportedly interacts with other PDIs via intermolecular disulfide bonds in cultured cells, but it remains unclear whether complex formation between P5 and other PDIs is involved in regulating enzymatic and chaperone functions. Herein, we established the far-western blot method to detect non-covalent interactions between P5 and other PDIs and found that PDI and ERp72 are partner proteins of P5. The enzymatic activity of P5-mediated oxidative folding is up-regulated by PDI, while the chaperone activity of P5 is stimulated by ERp72. These findings shed light on the mechanism by which the complex formations among PDIs drive to synergistically accelerate protein folding and prevents aggregation. This knowledge has implications for understanding misfolding-related pathology.

The Protein Disulfide Isomerase Family: from proteostasis to pathogenesis.

In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by approximately 20 members of the protein disulfide isomerase family (PDIs). PDIs consist of multiple thioredoxin-like domains and recognize a wide variety of proteins via highly conserved interdomain flexibility. Although PDIs have been studied intensely for almost 50 years, exactly how they maintain protein homeostasis in the ER remains unknown, and is important not only for fundamental biological understanding but also for protein misfolding- and aggregation-related pathophysiology. Herein, we review recent advances in structural biology and biophysical approaches that explore the underlying mechanism by which PDIs fulfil their distinct functions to promote productive protein folding and scavenge misfolded proteins in the ER, the primary factory for efficient production of the secretome.

Antipsychotic olanzapine-induced misfolding of proinsulin in the endoplasmic reticulum accounts for atypical development of diabetes.

Second-generation antipsychotics are widely used to medicate patients with schizophrenia, but may cause metabolic side effects such as diabetes, which has been considered to result from obesity-associated insulin resistance. Olanzapine is particularly well known for this effect. However, clinical studies have suggested that olanzapine-induced hyperglycemia in certain patients cannot be explained by such a generalized mechanism. Here, we focused on the effects of olanzapine on insulin biosynthesis and secretion by mouse insulinoma MIN6 cells. Olanzapine reduced maturation of proinsulin, and thereby inhibited secretion of insulin; and specifically shifted the primary localization of proinsulin from insulin granules to the endoplasmic reticulum. This was due to olanzapine's impairment of proper disulfide bond formation in proinsulin, although direct targets of olanzapine remain undetermined. Olanzapine-induced proinsulin misfolding and subsequent decrease also occurred at the mouse level. This mechanism of olanzapine-induced ß-cell dysfunction should be considered, together with weight gain, when patients are administered olanzapine.