Redox-active chemical chaperones exhibiting promiscuous binding promote oxidative protein folding under condensed sub-millimolar conditions.

Proteins form native structures through folding processes, many of which proceed through intramolecular hydrophobic effect, hydrogen bond and disulfide-bond formation. In vivo, protein aggregation is prevented even in the highly condensed milieu of a cell through folding mediated by molecular chaperones and oxidative enzymes. Chemical approaches to date have not replicated such exquisite mediation. Oxidoreductases efficiently promote folding by the cooperative effects of oxidative reactivity for disulfide-bond formation in the client unfolded protein and chaperone activity to mitigate aggregation. Conventional synthetic folding promotors mimic the redox-reactivity of thiol/disulfide units but do not address client-recognition units for inhibiting aggregation. Herein, we report thiol/disulfide compounds containing client-recognition units, which act as synthetic oxidoreductase-mimics. For example, compound ßCDWSH/SS bears a thiol/disulfide unit at the wide rim of ß-cyclodextrin as a client recognition unit. ßCDWSH/SS shows promiscuous binding to client proteins, mitigates protein aggregation, and accelerates disulfide-bond formation. In contrast, positioning a thiol/disulfide unit at the narrow rim of ß-cyclodextrin promotes folding less effectively through preferential interactions at specific residues, resulting in aggregation. The combination of promiscuous client-binding and redox reactivity is effective for the design of synthetic folding promoters. ßCDWSH/SS accelerates oxidative protein folding at highly condensed sub-millimolar protein concentrations.

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.

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.

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.

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+.

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.

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.

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.

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.

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).

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.

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.

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.

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.

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.

Successful Treatment of Pulmonary Pleomorphic Carcinoma with Nivolumab: A Case Report.

Pulmonary pleomorphic carcinoma (PPC) has a poor prognosis due to the poor results of treatment with systemic chemotherapy. We report the case of a 73-year-old woman with PPC who showed a favorable response to nivolumab. As first-line treatment for postoperative recurrence, she received carboplatin and nanoparticle albumin-bound paclitaxel. However, 12 months later, a new metastatic lymph node appeared. Nivolumab was administered as second-line treatment, and the patient showed a favorable prolonged response. The effects of treatment of PPC with nivolumab seem promising. The results of a future prospective study are expected to identify indicators for the treatment of PPC.

Impact of membrane curvature on amyloid aggregation.

The misfolding, amyloid aggregation, and fibril formation of intrinsically disordered proteins/peptides (or amyloid proteins) have been shown to cause a number of disorders. The underlying mechanisms of amyloid fibrillation and structural properties of amyloidogenic precursors, intermediates, and amyloid fibrils have been elucidated in detail; however, in-depth examinations on physiologically relevant contributing factors that induce amyloidogenesis and lead to cell death remain challenging. A large number of studies have attempted to characterize the roles of biomembranes on protein aggregation and membrane-mediated cell death by designing various membrane components, such as gangliosides, cholesterol, and other lipid compositions, and by using various membrane mimetics, including liposomes, bicelles, and different types of lipid-nanodiscs. We herein review the dynamic effects of membrane curvature on amyloid generation and the inhibition of amyloidogenic proteins and peptides, and also discuss how amyloid formation affects membrane curvature and integrity, which are key for understanding relationships with cell death. Small unilamellar vesicles with high curvature and large unilamellar vesicles with low curvature have been demonstrated to exhibit different capabilities to induce the nucleation, amyloid formation, and inhibition of amyloid-ß peptides and α-synuclein. Polymorphic amyloidogenesis in small unilamellar vesicles was revealed and may be viewed as one of the generic properties of interprotein interaction-dominated amyloid formation. Several mechanical models and phase diagrams are comprehensively shown to better explain experimental findings. The negative membrane curvature-mediated mechanisms responsible for the toxicity of pancreatic ß cells by the amyloid aggregation of human islet amyloid polypeptide (IAPP) and binding of the precursors of the semen-derived enhancer of viral infection (SEVI) are also described. The curvature-dependent binding modes of several types of islet amyloid polypeptides with high-resolution NMR structures are also discussed.

First-line chemotherapy with pemetrexed plus cisplatin for malignant peritoneal mesothelioma.

BACKGROUND: Mesothelioma of peritoneal origin has wider variation in treatment outcomes than mesothelioma of pleural origin, likely because peritoneal mesothelioma comprises borderline malignant variants and aggressive malignant peritoneal mesothelioma (MPeM). This study retrospectively evaluates the efficacy of first-line systemic pemetrexed and cisplatin chemotherapy in MPeM. RESEARCH DESIGN AND METHODS: Twenty-four patients with histologically proven MPeM were treated with pemetrexed plus cisplatin as a first-line systemic chemotherapy. The response was evaluated radiologically according to standard Response Evaluation Criteria In Solid Tumors (RECIST) criteria. Twenty-two patients underwent 18F-fluorodeoxyglucose positron emission tomography/(FDG-PET)/computed tomography(CT) at baseline, and 13 were eligible for metabolic assessment. RESULTS: Two complete responses and 9 partial responses were achieved. Overall response rate and disease control rate were 45.8% and 91.7%, respectively. Median progression-free survival and median overall survival were 11.0 months and 15.8 months, respectively. Wet- type MPeM had significantly longer survival (40.9 months median) than other clinical types (15.5 months) (P = 0.045). The baseline maximum standardized uptake value in 22 patients was 8.93 (range, 2.5-16.77). CONCLUSIONS: Systemic pemetrexed plus cisplatin is active for MPeM. Disparity with the outcome of cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (CRS/HIPEC) needs to receive more emphasis, since peritoneal mesothelioma has a 5-year survival rate of 50%.

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.

Metabolic response assessment with 18F-FDG-PET/CT is superior to modified RECIST for the evaluation of response to platinum-based doublet chemotherapy in malignant pleural mesothelioma.

PURPOSE: Efficient monitoring of tumor responsiveness to chemotherapy is essential to mitigate high mortality risks and cytotoxic effects of chemotherapeutics. However, there is no consensus on the most suitable diagnostic technique/parameters for assessing response to chemotherapy in malignant pleural mesothelioma (MPM). We compared the tumor responsiveness of MPM patients as assessed using modified RECIST (mRECIST) criteria and integrated 18F-FDG-PET/CT. METHODS: Histologically confirmed MPM patients (N=82) who were treated with three cycles of cisplatin and pemetrexed, or carboplatin and pemetrexed, were included. mRECIST and integrated 18F-FDG-PET/CT were used to evaluate MPM tumor response to chemotherapy. Metabolic non-responders were defined as those with a 25% or greater increase in SUVmax compared with the previous value. Time to progression (TTP) and overall survival (OS) were compared between metabolic-responders and non-responders. RESULTS: After three cycles of chemotherapy, 62(75.6%) of the patients were classified as having SD, 15 (18%) with partial remission (PR), and 5 (6%) with progressive disease (PD), based on mRECIST criteria. The cumulative median OS was 728.0days (95% confidence interval [CI]: 545.9-910.1) and cumulative median TTP was 365.0days (95% CI: 296.9-433.1). For the 82 patients, the disease control rate was 93.9%, whereas the metabolic response rate was only 71.9% (p<0.001). All PD and PR patients were found to be metabolic responders on 18F-FDG-PET/CT; however, among the 62mRECIST SD patients, 18 (29%) were classified as metabolic non-responders. The median TTP for metabolic responders was 13.7 months, while it was 10.0 months for non-responders(p<0.001). Metabolic responders had a trend toward longer OS, although the difference did not reach statistical significance (metabolic responders:33.9 months; non-responders: 21.6 months; p>0.05). CONCLUSION: Several mRECIST-confirmed SD MPM patients may be classified as metabolic non-responders on18F-FDGPET/CT. Metabolic response is significantly correlated with the median TTP, suggesting it should be included in the evaluation of the response to chemotherapy in MPM patients classified as mRECIST SD, to identify non-responders.