Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b'a' Domain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b'a' domain of protein disulfide isomerase (PDIb'a') greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb'a'-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC50 of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.

Defining the mechanism of PDI interaction with disulfide-free amyloidogenic proteins: Implications for exogenous protein expression and neurodegenerative disease.

Protein disulfide isomerase (PDI) is an important molecular chaperone capable of facilitating protein folding in addition to catalyzing the formation of a disulfide bond. To better understand the distinct substrate-screening principles of Pichia pastoris PDI (Protein disulfide isomerase) and the protective role of PDI in amyloidogenic diseases, we investigated the expression abundance and intracellular retention levels of three archetypal amyloidogenic disulfide bond-free proteins (Aß42, α-synuclein (α-Syn) and SAA1) in P. pastoris GS115 strain without and with the overexpression of PpPDI (P. pastoris PDI). Intriguingly, amyloidogenic Aß42 and α-Syn were detected only as intracellular proteins whereas amyloidogenic SAA1 was detected both as intracellular and extracellular proteins when these proteins were expressed in the PpPDI-overexpressing GS115 strain. The binding between PpPDI and each of the three amyloidogenic proteins was investigated by molecular docking and simulations. Three different patterns of PpPDI-substrate complexes were observed, suggesting that multiple modes of binding might exist for the binding between PpPDI and its amyloidogenic protein substrates, and this could represent different specificities and affinities of PpPDI toward its substrates. Further analysis of the proteomics data and functional annotations indicated that PpPDI could eliminate the need for misfolded proteins to be partitioned in ER-associated compartments.

Male predisposition to severe COVID-19: Review of evidence and potential therapeutic prospects.

The severe form of COVID-19 has significant sex disparities, with high fatalities commonly reported among males than females. The incidence of COVID-19 has also been higher in males compared with their female counterparts. This trend could be attributed to a better responsive and robust immune system in females. Cytokine storm is one of the pathophysiological features of severe COVID-19, and it occurs as a result of over-activation of immune cells leading to severe inflammation and tissue damage. Nevertheless, it is well modulated in females compared to their male counterparts. Severe inflammation in males is reported to facilitate progression of mild to severe COVID-19. The sex hormones, estrogens and androgens which exist in varying functional levels respectively in females and males are cited as the underlying cause for the differential immune response to COVID-19. Evidence abounds that estrogen modulate the immune system to protect females from severe inflammation and for that matter severe COVID-19. On the contrary, androgen has been implicated in over-activation of immune cells, cytokine storm and the attendant severe inflammation, which perhaps predispose males to severe COVID-19. In this review efforts are made to expand understanding and explain the possible roles of the immune system, the sex hormones and the angiotensin-converting enzyme (ACE) systems in male bias to severe COVID-19. Also, this review explores possible therapeutic avenues including androgen deprivation therapy (ADT), estrogen-based therapy, and ACE inhibitors for consideration in the fight against COVID-19.

Assessing the oncolytic potential of rotavirus on mouse myeloma cell line Sp2/0-Ag14. / Evaluación del potencial oncolítico del rotavirus en la línea celular Sp2/0-Ag14 de mieloma de ratón

INTRODUCTION: Cancer is the second leading cause of death in the United States, surpassed only by cardiovascular disease. However, cancer has now overtaken cardiovascular disease as the main cause of death in 12 countries in Western Europe. The burden of cancer is posing a major challenge to health care systems worldwide and demanding improvements in methods for cancer prevention, diagnosis, and treatment. Alternative and complementary strategies for orthodox surgery, radiotherapy, and chemotherapy need to be developed. OBJECTIVE: To determine the oncolytic potential of tumor cell-adapted rotavirus in terms of their ability to infect and lysate murine myeloma Sp2/0-Ag14 cells. MATERIALS AND METHODS: We inoculated rotaviruses Wt1-5, WWM, TRUYO, ECwt-O, and WTEW in Sp2/0-Ag14 cells and we examined their infectious effects by immunocytochemistry, immunofluorescence, flow cytometry, and DNA fragmentation assays. RESULTS: Rotavirus infection involved the participation of some heat shock proteins, of protein disulfide isomerase (PDI), and integrin ß3. We detected the accumulation of viral antigens within the virus-inoculated cells and in the culture medium in all the rotavirus isolates examined. The rotavirus-induced cell death mechanism in Sp2/0-Ag14 cells involved changes in cell membrane permeability, chromatin condensation, and DNA fragmentation, which were compatible with cytotoxicity and apoptosis. CONCLUSIONS: The ability of the rotavirus isolates Wt1-5, WWM, TRUYO, ECwt-O, and WTEW to infect and cause cell death of Sp2/0-Ag14 cells through mechanisms that are compatible with virus-induced apoptosis makes them potential candidates as oncolytic agents.

Introducción. El cáncer es la segunda causa de muerte en los Estados Unidos, solamente superado por la enfermedad cardiovascular. Sin embargo, el cáncer aventaja a la enfermedad cardiovascular como primera causa de muerte en doce países de Europa occidental. Se requieren mejores métodos de prevención, diagnóstico y tratamiento para afrontar el gran desafío que el cáncer representa mundialmente para los sistemas de salud, y se necesita desarrollar estrategias alternativas y complementarias a la cirugía, la radioterapia y la quimioterapia convencionales. Objetivo. Evaluar el potencial oncolítico de rotavirus adaptados a células tumorales por su capacidad para infectar y lisar células Sp2/0-Ag14 de mieloma de ratón. Materiales and métodos. Los aislamientos de rotavirus Wt1-5, WWM, TRUYO, ECwt-O y WTEW se inocularon en células Sp2/0-Ag14 y se examinaron sus efectos infecciosos mediante inmunocitoquímica, inmunofluorescencia, citometría de flujo y ensayos de fragmentación del ADN. Resultados. La infección con los rotavirus Wt1-5, WWM, TRUYO, ECwt-O y WTEW implicó la participación de algunas proteínas de choque térmico, la proteína disulfuro isomerasa y la integrina ß3. La acumulación de antígenos virales intracelulares y extracelulares se detectó en todos los virus utilizados. Los mecanismos de muerte inducidos por los rotavirus en células Sp2/0-Ag14 indujeron cambios en la permeabilidad de la membrana celular, la condensación de cromatina y la fragmentación de ADN, los cuales fueron compatibles con citotoxicidad y apoptosis. Conclusiones. La capacidad de los rotavirus estudiados para infectar y causar la muerte de células Sp2/0-Ag14 mediante mecanismos compatibles con la apoptosis inducida viralmente los convierte en candidatos potenciales para ser utilizados como agentes oncolíticos.

Urate hydroperoxide oxidizes endothelial cell surface protein disulfide isomerase-A1 and impairs adherence.

BACKGROUND: Extracellular surface protein disulfide isomerase-A1 (PDI) is involved in platelet aggregation, thrombus formation and vascular remodeling. PDI performs redox exchange with client proteins and, hence, its oxidation by extracellular molecules might alter protein function and cell response. In this study, we investigated PDI oxidation by urate hydroperoxide, a newly-described oxidant that is generated through uric acid oxidation by peroxidases, with a putative role in vascular inflammation. METHODS: Amino acids specificity and kinetics of PDI oxidation by urate hydroperoxide was evaluated by LC-MS/MS and by stopped-flow. Oxidation of cell surface PDI and other thiol-proteins from HUVECs was identified using impermeable alkylating reagents. Oxidation of intracellular GSH and GSSG was evaluated with specific LC-MS/MS techniques. Cell adherence, detachment and viability were assessed using crystal violet staining, cellular microscopy and LDH activity, respectively. RESULTS: Urate hydroperoxide specifically oxidized cysteine residues from catalytic sites of recombinant PDI with a rate constant of 6 × 103 M-1 s-1. Incubation of HUVECs with urate hydroperoxide led to oxidation of cell surface PDI and other unidentified cell surface thiol-proteins. Cell adherence to fibronectin coated plates was impaired by urate hydroperoxide, as well as by other oxidants, thiol alkylating agents and PDI inhibitors. Urate hydroperoxide did not affect cell viability but significantly decreased GSH/GSSG ratio. CONCLUSIONS: Our results demonstrated that urate hydroperoxide affects thiol-oxidation of PDI and other cell surface proteins, impairing cellular adherence. GENERAL SIGNIFICANCE: These findings could contribute to a better understanding of the mechanism by which uric acid affects endothelial cell function and vascular homeostasis.

Male-Specific Protein Disulphide Isomerase Function is Essential for Plasmodium Transmission and a Vulnerable Target for Intervention.

Inhibiting transmission of Plasmodium is an essential strategy in malaria eradication, and the biological process of gamete fusion during fertilization is a proven target for this approach. Lack of knowledge of the mechanisms underlying fertilization have been a hindrance in the development of transmission-blocking interventions. Here we describe a protein disulphide isomerase essential for malarial transmission (PDI-Trans/PBANKA_0820300) to the mosquito. We show that PDI-Trans activity is male-specific, surface-expressed, essential for fertilization/transmission, and exhibits disulphide isomerase activity which is up-regulated post-gamete activation. We demonstrate that PDI-Trans is a viable anti-malarial drug and vaccine target blocking malarial transmission with the use of PDI inhibitor bacitracin (98.21%/92.48% reduction in intensity/prevalence), and anti-PDI-Trans antibodies (66.22%/33.16% reduction in intensity/prevalence). To our knowledge, these results provide the first evidence that PDI function is essential for malarial transmission, and emphasize the potential of anti-PDI agents to act as anti-malarials, facilitating the future development of novel transmission-blocking interventions.

Peri/epicellular protein disulfide isomerase-A1 acts as an upstream organizer of cytoskeletal mechanoadaptation in vascular smooth muscle cells.

Although redox processes closely interplay with mechanoresponses to control vascular remodeling, redox pathways coupling mechanostimulation to cellular cytoskeletal organization remain unclear. The peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) supports postinjury vessel remodeling. Using distinct models, we investigated whether pecPDIA1 could work as a redox-dependent organizer of cytoskeletal mechanoresponses. In vascular smooth muscle cells (VSMCs), pecPDIA1 immunoneutralization impaired stress fiber assembly in response to equibiaxial stretch and, under uniaxial stretch, significantly perturbed cell repositioning perpendicularly to stretch orientation. During cyclic stretch, pecPDIA1 supported thiol oxidation of the known mechanosensor ß1-integrin and promoted polarized compartmentalization of sulfenylated proteins. Using traction force microscopy, we showed that pecPDIA1 organizes intracellular force distribution. The net contractile moment ratio of platelet-derived growth factor-exposed to basal VSMCs decreased from 0.90 ± 0.09 (IgG-exposed controls) to 0.70 ± 0.08 after pecPDI neutralization ( P < 0.05), together with an enhanced coefficient of variation for distribution of force modules, suggesting increased noise. Moreover, in a single cell model, pecPDIA1 neutralization impaired migration persistence without affecting total distance or velocity, whereas siRNA-mediated total PDIA1 silencing disabled all such variables of VSMC migration. Neither expression nor total activity of the master mechanotransmitter/regulator RhoA was affected by pecPDIA1 neutralization. However, cyclic stretch-induced focal distribution of membrane-bound RhoA was disrupted by pecPDI inhibition, which promoted a nonpolarized pattern of RhoA/caveolin-3 cluster colocalization. Accordingly, FRET biosensors showed that pecPDIA1 supports localized RhoA activity at cell protrusions versus perinuclear regions. Thus, pecPDI acts as a thiol redox-dependent organizer and noise reducer mechanism of cytoskeletal repositioning, oxidant generation, and localized RhoA activation during a variety of VSMC mechanoresponses. NEW & NOTEWORTHY Effects of a peri/epicellular pool of protein disulfide isomerase-A1 (pecPDIA1) during mechanoregulation in vascular smooth muscle cells (VSMCs) were highlighted using approaches such as equibiaxial and uniaxial stretch, random single cell migration, and traction force microscopy. pecPDIA1 regulates organization of the cytoskeleton and minimizes the noise of cell alignment, migration directionality, and persistence. pecPDIA1 mechanisms involve redox control of ß1-integrin and localized RhoA activation. pecPDIA1 acts as a novel organizer of mechanoadaptation responses in VSMCs.

Human Milk Oligosaccharides Increase Mucin Expression in Experimental Necrotizing Enterocolitis.

SCOPE: Necrotizing enterocolitis (NEC) is a leading cause of morbidity and death in preterm infants, occurring more often in formula-fed than breastfed infants. Studies in both rats and humans show that human milk oligosaccharides (HMOs) lower the incidence of NEC, but the mechanism underlying such protection is currently unclear. METHODS AND RESULTS: By extracting HMOs from pooled human breastmilk, the impact of HMOs on the intestinal mucin levels in a murine model of NEC are investigated. To confirm the results, the findings are validated by exposing human intestinal epithelial cells and intestinal organoids to HMOs and evaluated for mucin expression. HMO-gavage to pups increases Muc2 levels and decreases intestinal permeability to macromolecular dextran. HMO-treated cells have increased Muc2 expression, decreased bacterial attachment and dextran permeability during challenge by enteric pathogens. To identify the mediators involved in HMO induction of mucins, it is demonstrated that HMOs directly induce the expression of chaperone proteins including protein disulfide isomerase (PDI). Suppression of PDI activity removes the protective effects of HMOs on barrier function in vitro as well as NEC protection in vivo. CONCLUSIONS: Taken together, the results provide insights to the possible mechanisms by which HMOs protect the neonatal intestine through upregulation of mucins.

Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis.

BACKGROUND: Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. RESULTS: In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. CONCLUSIONS: Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.

Endoplasmic Reticulum Protein TXNDC5 Augments Myocardial Fibrosis by Facilitating Extracellular Matrix Protein Folding and Redox-Sensitive Cardiac Fibroblast Activation.

RATIONALE: Cardiac fibrosis plays a critical role in the pathogenesis of heart failure. Excessive accumulation of extracellular matrix (ECM) resulting from cardiac fibrosis impairs cardiac contractile function and increases arrhythmogenicity. Current treatment options for cardiac fibrosis, however, are limited, and there is a clear need to identify novel mediators of cardiac fibrosis to facilitate the development of better therapeutics. Exploiting coexpression gene network analysis on RNA sequencing data from failing human heart, we identified TXNDC5 (thioredoxin domain containing 5), a cardiac fibroblast (CF)-enriched endoplasmic reticulum protein, as a potential novel mediator of cardiac fibrosis, and we completed experiments to test this hypothesis directly. OBJECTIVE: The objective of this study was to determine the functional role of TXNDC5 in the pathogenesis of cardiac fibrosis. METHODS AND RESULTS: RNA sequencing and Western blot analyses revealed that TXNDC5 mRNA and protein were highly upregulated in failing human left ventricles and in hypertrophied/failing mouse left ventricle. In addition, cardiac TXNDC5 mRNA expression levels were positively correlated with those of transcripts encoding transforming growth factor ß1 and ECM proteins in vivo. TXNDC5 mRNA and protein were increased in human CF (hCF) under transforming growth factor ß1 stimulation in vitro. Knockdown of TXNDC5 attenuated transforming growth factor ß1-induced hCF activation and ECM protein upregulation independent of SMAD3 (SMAD family member 3), whereas increasing expression of TXNDC5 triggered hCF activation and proliferation and increased ECM protein production. Further experiments showed that TXNDC5, a protein disulfide isomerase, facilitated ECM protein folding and that depletion of TXNDC5 led to ECM protein misfolding and degradation in CF. In addition, TXNDC5 promotes hCF activation and proliferation by enhancing c-Jun N-terminal kinase activity via increased reactive oxygen species, derived from NAD(P)H oxidase 4. Transforming growth factor ß1-induced TXNDC5 upregulation in hCF was dependent on endoplasmic reticulum stress and activating transcription factor 6-mediated transcriptional control. Targeted disruption of Txndc5 in mice (Txndc5-/-) revealed protective effects against isoproterenol-induced cardiac hypertrophy, reduced fibrosis (by ≈70%), and markedly improved left ventricle function; post-isoproterenol left ventricular ejection fraction was 59.1±1.5 versus 40.1±2.5 (P<0.001) in Txndc5-/- versus wild-type mice, respectively. CONCLUSIONS: The endoplasmic reticulum protein TXNDC5 promotes cardiac fibrosis by facilitating ECM protein folding and CF activation via redox-sensitive c-Jun N-terminal kinase signaling. Loss of TXNDC5 protects against ß agonist-induced cardiac fibrosis and contractile dysfunction. Targeting TXNDC5, therefore, could be a powerful new therapeutic approach to mitigate excessive cardiac fibrosis, thereby improving cardiac function and outcomes in patients with heart failure.

Pseudogene PDIA3P1 promotes cell proliferation, migration and invasion, and suppresses apoptosis in hepatocellular carcinoma by regulating the p53 pathway.

Pseudogenes are a subclass of long non-coding (lnc) RNAs that arose from protein-coding genes, but have lost the ability to produce proteins. Pseudogenes play an important role in the pathogenesis of various tumors; however, the role of pseudogenes in hepatocellular carcinoma (HCC) is poorly understood. In this study, we investigated a novel pseudogene, PDIA3P1, which was upregulated in HCC tissues compared with paired normal adjacent tissues. The expression of PDIA3P1 was significantly correlated with tumor size, metastasis, TNM stage, and overall stage. Knockdown of PDIA3P1decreased proliferation, migration, and invasion of HCC cells. PDIA3P1 knockdown also promoted cell apoptosis and inhibited tumor growth in vivo. We performed a GeneChip assay to investigate the underlying mechanism of PDIA3P1 action on biological function, and our results suggested that PDIA3P1 may promote proliferation and inhibit apoptosis of liver cancer cells by inhibiting the p53 pathway. Thus, our data suggest that PDIA3P1 acts as an oncogene in HCC and could be a potential prognostic marker and therapeutic target for HCC.

SNOWY COTYLEDON 2 Promotes Chloroplast Development and Has a Role in Leaf Variegation in Both Lotus japonicus and Arabidopsis thaliana.

Plants contain various factors that transiently interact with subunits or intermediates of the thylakoid multiprotein complexes, promoting their stable association and integration. Hence, assembly factors are essential for chloroplast development and the transition from heterotrophic to phototrophic growth. Snowy cotyledon 2 (SCO2) is a DNAJ-like protein involved in thylakoid membrane biogenesis and interacts with the light-harvesting chlorophyll-binding protein LHCB1. In Arabidopsis thaliana, SCO2 function was previously reported to be restricted to cotyledons. Here we show that disruption of SCO2 in Lotus japonicus results not only in paler cotyledons but also in variegated true leaves. Furthermore, smaller and pale-green true leaves can also be observed in A. thaliana sco2 (atsco2) mutants under short-day conditions. In both species, SCO2 is required for proper accumulation of PSII-LHCII complexes. In contrast to other variegated mutants, inhibition of chloroplastic translation strongly affects L. japonicus sco2 mutant development and fails to suppress their variegated phenotype. Moreover, inactivation of the suppressor of variegation AtClpR1 in the atsco2 background results in an additive double-mutant phenotype with variegated true leaves. Taken together, our results indicate that SCO2 plays a distinct role in PSII assembly or repair and constitutes a novel factor involved in leaf variegation.

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

Study question: Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? Summary answer: ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. What is known already: A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. Study design, size, duration: The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Participants/materials, setting, methods: Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Main results and the role of chance: Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. Large scale data: N/A. Limitations, reasons for caution: The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Wider implications of the findings: Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. Study funding/competing interest(s): This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests.

A prominent role of PDIA6 in processing of misfolded proinsulin.

Despite its critical role in maintaining glucose homeostasis, surprisingly little is known about proinsulin folding in the endoplasmic reticulum. In this study we aimed to understand the chaperones involved in the maturation and degradation of proinsulin. We generated pancreatic beta cell lines expressing FLAG-tagged proinsulin. Several chaperones (including BiP, PDIA6, calnexin, calreticulin, GRP170, Erdj3 and ribophorin II) co-immunoprecipitated with proinsulin suggesting a role for these proteins in folding. To investigate the chaperones responsible for targeting misfolded proinsulin for degradation, we also created a beta cell line expressing FLAG-tagged proinsulin carrying the Akita mutation (Cys96Tyr). All chaperones found to be associated with wild type proinsulin also co-immunoprecipitated with Akita proinsulin. However, one additional protein, namely P58(IPK), specifically precipitated with Akita proinsulin and approximately ten fold more PDIA6, but not other PDI family members, was bound to Akita proinsulin. The latter suggests that PDIA6 may act as a key reductase and target misfolded proinsulin to the ER-degradation pathway. The preferential association of PDIA6 to Akita proinsulin was also confirmed in another beta cell line (ßTC-6). Furthermore, for the first time, a physiologically relevant substrate for PDIA6 has been evidenced. Thus, this study has identified several chaperones/foldases that associated with wild type proinsulin and has also provided a comprehensive interactome for Akita misfolded proinsulin.

A sucrose transporter-interacting protein disulphide isomerase affects redox homeostasis and links sucrose partitioning with abiotic stress tolerance.

Sucrose accumulation in leaves in response to various abiotic stresses suggests a specific role of this disaccharide for stress tolerance and adaptation. The high-affinity transporter StSUT1 undergoes substrate-induced endocytosis presenting the question as to whether altered sucrose accumulation in leaves in response to stresses is also related to enhanced endocytosis or altered activity of the sucrose transporter. StSUT1 is known to interact with several stress-inducible proteins; here we investigated whether one of the interacting candidates, StPDI1, affects its subcellular localization in response to stress: StPDI1 expression is induced by ER-stress and salt. Both proteins, StSUT1 and StPDI1, were found in the detergent resistant membrane (DRM) fraction, and this might affect internalization. Knockdown of StPDI1 expression severely affects abiotic stress tolerance of transgenic potato plants. Analysis of these plants does not reveal modified subcellular localization or endocytosis of StSUT1, but rather a disturbed redox homeostasis, reduced detoxification of reactive oxygen species and effects on primary metabolism. Parallel observations with other StSUT1-interacting proteins are discussed. The redox status in leaves seems to be linked to the sugar status in response to various stress stimuli and to play a role in stress tolerance.

Antrodia cinnamomea Inhibits Migration in Human Hepatocellular Carcinoma Cells: The Role of ERp57 and PGK-1.

Evidences suggest that ERp57 and PGK-1 signaling lead to cancer cell proliferation and migration. We hypothesized that ERp57 and PGK-1 down-regulation may inactivate matrix metalloproteinase (MMP)-2, -9 expressions and inhibit hepatocellular carcinoma (HCC) migration. Antrodia cinnamomea is widely prescribed as an adjuvant to treat HCC in Taiwan. We aimed to investigate if ethanol extract of fruiting bodies of Antrodia cinnamomea (EEAC) and its active ingredients (i.e., zhankuic acid A, cordycepin, and adenosine) can modulate HCC cancer cells migration through ERp57 and PGK-1 and other molecular pathways such as PI3K/Akt and MAPK. ERp57 and PGK-1 siRNA were transfected into HCC to determine effects on MMP-2/-9 expressions and cell migration. We then examined the inhibitory effects of EEAC and its active ingredients on HCC migration and its related mechanisms including ERp57, PGK-1, PI3K/Akt, and MAPK signaling pathways. Down-regulation of ERp57 and PGK-1 by siRNA decreased MMP-2, -9 expressions and Transwell cell migration in HCC. Nontoxic EEAC markedly inhibited migration of HCC, and significantly inhibited activities and protein expressions of MMP-2 and -9, while the expression of the endogenous inhibitors (TIMP-1 and TIMP-2) of these proteins increased. Nontoxic EEAC and its active ingredients decreased ERp57, GLUD-1, GST-pi, and PGK-1 protein expressions. Finally, nontoxic EEAC inhibited the phosphorylated FAK, PI3K/Akt, and MAPK signaling. Our findings first indicate that EEAC and its ingredients effectively suppress HCC migration. Additionally, the molecular mechanisms appear to be mediated, in part, through the down-regulation of ERp57, PGK-1, MAPK, and PI3K/Akt.

The P5 disulfide switch: taming the aging unfolded protein response.

Aging cells are characterized by a loss of proteostasis and a decreased ability to survive under environmental stress. Regulation of the UPR in aging cells has been under much scrutiny, and studies have shown that the UPR in these cells differs considerably from younger cells with regard to the induction of apoptosis and chaperone activity. The role of IRE-1 and PERK in UPR-associated apoptosis makes the regulation of these signaling cascades an important target of study. The seemingly contradictory findings regarding the role of P5 in activating and deactivating these responses warrant further investigation and may hold the key to unlocking the role of this protein in various pathological conditions. Another important target for study with regard to P5 is the effects of the localization of this protein in the mitochondria and the consequences, if any, of these effects on the activation of the UPR.

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.

Identification of miR-494 direct targets involved in senescence of human diploid fibroblasts.

Cellular senescence is a permanent cell cycle arrest triggered by different stimuli. We recently identified up-regulation of microRNA (miR)-494 as a component of the genetic program leading to senescence of human diploid IMR90 fibroblasts. Here, we used 2-dimensional differential gel electrophoresis (2D-DIGE) coupled to mass spectrometry to profile protein expression changes induced by adoptive overexpression of miR-494 in IMR90 cells. miR-494 induced robust perturbation of the IMR90 proteome by significantly (P≤0.05) down-regulating a number of proteins. Combination of mass spectrometry-based identification of down-regulated proteins and bioinformatic prediction of the miR-494 binding sites on the relevant mRNAs identified 26 potential targets of miR-494. Among them, computational analysis identified 7 potential evolution-conserved miR-494 targets. Functional miR-494 binding sites were confirmed in 3'-untranslated regions (UTRs) of 4 of them [heterogeneous nuclear ribonucleoprotein A3 (hnRNPA3), protein disulfide isomerase A3 (PDIA3), UV excision repair protein RAD23 homolog B (RAD23B), and synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP)/heterogeneous nuclear ribonucleoprotein Q (hnRNPQ)]. Their reduced expression correlated with miR-494 up-regulation in senescent cells. RNA interference-mediated knockdown of hnRNPA3 and, to a lesser extent, RAD23B mirrored the senescent phenotype induced by miR-494 overexpression, blunting cell proliferation and causing up-regulation of SA-ß-galactosidase and DNA damage. Ectopic expression of hnRNPA3 or RAD23B slowed the appearance of the senescent phenotype induced by miR-494. Overall, these findings identify novel miR-494 direct targets that are involved in cellular senescence.

Inactivation of mammalian Ero1α is catalysed by specific protein disulfide-isomerases.

Disulfide formation within the endoplasmic reticulum is a complex process requiring a disulfide exchange protein such as PDI (protein disulfide-isomerase) and a mechanism to form disulfides de novo. In mammalian cells, the major pathway for de novo disulfide formation involves the enzyme Ero1α (endoplasmic reticulum oxidase 1α) which couples oxidation of thiols to the reduction of molecular oxygen to form hydrogen peroxide (H2O2). Ero1α activity is tightly regulated by a mechanism that requires the formation of regulatory disulfides. These regulatory disulfides are reduced to activate and reform to inactivate the enzyme. To investigate the mechanism of inactivation we analysed regulatory disulfide formation in the presence of various oxidants under controlled oxygen concentration. Neither molecular oxygen nor H2O2 was able to oxidize Ero1α efficiently to form the correct regulatory disulfides. However, specific members of the PDI family, such as PDI or ERp46 (endoplasmic reticulum-resident protein 46), were able to catalyse this process. Further studies showed that both active sites of PDI contribute to the formation of regulatory disulfides in Ero1α and that the PDI substrate-binding domain is crucial to allow electron transfer between the two enzymes. The results of the present study demonstrate a simple feedback mechanism of re-gulation of mammalian Ero1α involving its primary substrate.