scholarly journals Transient aggregation of nascent thyroglobulin in the endoplasmic reticulum: relationship to the molecular chaperone, BiP.

1992 ◽  
Vol 118 (3) ◽  
pp. 541-549 ◽  
Author(s):  
P S Kim ◽  
D Bole ◽  
P Arvan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
Subsequent Removal ◽  
Long Time ◽  
Early Interaction ◽  
Potential Substrate

Because of its unusual length, nascent thyroglobulin (Tg) requires a long time after translocation into the endoplasmic reticulum (ER) to assume its mature tertiary structure. Thus, Tg is an ideal molecule for the study of protein folding and export from the ER, and is an excellent potential substrate for molecular chaperones. During the first 15 min after biosynthesis, Tg is found in transient aggregates with and without interchain disulfide bonds, which precede the formation of free monomers (and ultimately dimers) within the ER. By immunoprecipitation, newly synthesized Tg was associated with the binding protein (BiP); association was maximal at the earliest chase times. Much of the Tg released from BiP by the addition of Mg-ATP was found in aggregates containing interchain disulfide bonds; other BiP-associated Tg represented non-covalent aggregates and unfolded free monomers. Importantly, the immediate precursor to Tg dimer was a compact monomer which did not associate with BiP. The average stoichiometry of BiP/Tg interaction involved nearly 10 BiP molecules per Tg molecule. Cycloheximide was used to reduced the ER concentration of Tg relative to chaperones, with subsequent removal of the drug in order to rapidly restore Tg synthesis. After this treatment, nascent Tg aggregates were no longer detectable. The data suggest a model of folding of exportable proteins in which nascent polypeptides immediately upon translocation into the ER interact with BiP. Early interaction with BiP may help in presenting nascent polypeptides to other helper molecules that catalyze folding, thereby preventing aggregation or driving aggregate dissolution in the ER.

Endoplasmic reticulum stress regulates glutathione metabolism and activities of glutathione related enzymes in Arabidopsis

2018 ◽  
Vol 45 (2) ◽  
pp. 284 ◽  
Author(s):  
Baris Uzilday ◽  
Rengin Ozgur ◽  
A. Hediye Sekmen ◽  
Ismail Turkan
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Er Stress ◽  
Disulfide Bonds ◽  
Degradation Pathway ◽  
Glutathione Metabolism ◽  
Unfolded Proteins ◽  
Glutathione S Transferase ◽  
Glutathione Biosynthesis ◽  
Protein Disulfide

Stress conditions generate an extra load on protein folding machinery in the endoplasmic reticulum (ER) and if the ER cannot overcome this load, unfolded proteins accumulate in the ER lumen, causing ER stress. ER lumen localised protein disulfide isomerase (PDI) catalyses the generation of disulfide bonds in conjugation with ER oxidoreductase1 (ERO1) during protein folding. Mismatched disulfide bonds are reduced by the conversion of GSH to GSSG. Under prolonged ER stress, GSH pool is oxidised and H2O2 is produced via increased activity of PDI-ERO1. However, it is not known how glutathione metabolism is regulated under ER stress in plants. So, in this study, ER stress was induced with tunicamycin (0.15, 0.3, 0.45 μg mL–1 Tm) in Arabidopsis thaliana (L.) Heynh. Glutathione content was increased by ER stress, which was accompanied by induction of glutathione biosynthesis genes (GSH1, GSH2). Also, the apoplastic glutathione degradation pathway (GGT1) was induced. Further, the activities of glutathione reductase (GR), dehydroascorbate reductase (DHAR), glutathione peroxidase (GPX) and glutathione S-transferase (GST) were increased under ER stress. Results also showed that chloroplastic GPX genes were specifically downregulated with ER stress. This is the first report on regulation of glutathione metabolism and glutathione related enzymes in response to ER stress in plants.

Calnexin: a molecular chaperone with a taste for carbohydrate

1995 ◽  
Vol 73 (3-4) ◽  
pp. 123-132 ◽  
Author(s):  
David B. Williams
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Secretory Pathway ◽  
Quaternary Structure ◽  
Integral Membrane Protein ◽  
Secretory Proteins ◽  
Chaperone Function ◽  
Folded Proteins

Calnexin is an integral membrane protein of the endoplasmic reticulum (ER) that binds transiently to a wide array of newly synthesized membrane and secretory proteins. It also exhibits prolonged binding to misfolded or incompletely folded proteins. Recent studies have demonstrated that calnexin functions as a molecular chaperone to facilitate the folding and assembly of proteins in the ER. It is also a component of the quality control system that prevents proteins from progressing along the secretory pathway until they have acquired proper tertiary or quaternary structure. Most proteins that are translocated into the ER are glycosylated at Asn residues, and calnexin's interactions are almost exclusively restricted to proteins that possess this posttranslational modification. The preference for glycoproteins resides in calnexin's ability to function as a lectin with specificity for the GlC1Man9GlcNAc2 oligosaccharide, an early intermediate in the processing of Asn-linked oligosaccharides. Calnexin also has the capacity to bind to polypeptide segments of unfolded glycoproteins. Available evidence suggests that calnexin utilizes its lectin property during initial capture of a newly synthesized glycoprotein and that subsequent association (and chaperone function) is mediated through polypeptide interactions. Unlike other molecular chaperones that are soluble proteins, calnexin is an intrinsic component of the ER membrane. Its unique ability to capture unfolded glycoproteins through their large oligosaccharide moieties may have evolved as a means to overcome accessibility problems imposed by being constrained within a lipid bilayer.Key words: protein folding, molecular chaperones, calnexin, quality control, endoplasmic reticulum.

scholarly journals Molecular Chaperones in the Yeast Endoplasmic Reticulum Maintain the Solubility of Proteins for Retrotranslocation and Degradation

2001 ◽  
Vol 153 (5) ◽  
pp. 1061-1070 ◽  
Author(s):  
Shuh-ichi Nishikawa ◽  
Sheara W. Fewell ◽  
Yoshihito Kato ◽  
Jeffrey L. Brodsky ◽  
Toshiya Endo
Keyword(s):  
Endoplasmic Reticulum ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Elevated Temperatures ◽  
Carboxypeptidase Y ◽  
Mutant Form ◽  
Genes Encoding

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the process by which aberrant proteins in the ER lumen are exported back to the cytosol and degraded by the proteasome. Although ER molecular chaperones are required for ERAD, their specific role(s) in this process have been ill defined. To understand how one group of interacting lumenal chaperones facilitates ERAD, the fates of pro–α-factor and a mutant form of carboxypeptidase Y were examined both in vivo and in vitro. We found that these ERAD substrates are stabilized and aggregate in the ER at elevated temperatures when BiP, the lumenal Hsp70 molecular chaperone, is mutated, or when the genes encoding the J domain–containing proteins Jem1p and Scj1p are deleted. In contrast, deletion of JEM1 and SCJ1 had little effect on the ERAD of a membrane protein. These results suggest that one role of the BiP, Jem1p, and Scj1p chaperones is to maintain lumenal ERAD substrates in a retrotranslocation-competent state.

Different rates of formation of secondary and tertiary structure during renaturation of urea-denatured human serum albumin

1989 ◽  
Vol 54 (9) ◽  
pp. 2542-2549 ◽  
Author(s):  
Josef Chmelík
Keyword(s):  
Protein Folding ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Disulfide Bonds ◽  
Tertiary Structure ◽  
Hydrophobic Interactions ◽  
Protein Structures ◽  
Three Dimensional ◽  
Comparison Of The Results

A comparison of the results of our polarimetric measurements with the polarographic experiments reported earlier shows that the restoration of the secondary structure during the renaturation of human serum albumin is a process which is faster than the formation of the tertiary structure. These results, which are in agreement with the data on the kinetic control of protein folding, are discussed from the viewpoint of the importance of the individual types of interactions which take place during the formation and stabilization of three-dimensional protein structures. We have been able to demonstrate the great importance of electrostatic and hydrophobic interactions which together with the disulfide bonds are essential for the reversibility of the denaturation phenomena. The discussion also shows the essential role which evolution processes play in the selection of the mode of protein folding.

Antimyeloma In Vitro Activity Of 15-Deoxy-D12,14-Prostaglandin J2 Is Associated With Endoplasmic Reticulum Stress In a Reactive Oxygen Species-Dependent Fashion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4913-4913
Author(s):  
Ana Paula Demasi ◽  
Marcelo Henrique Napimoga ◽  
Elizabeth Ferreira Martinez ◽  
Adriana Silva Santos Duarte ◽  
Fernando V Pericole ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Multiple Myeloma ◽  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Research Funding ◽  
Misfolded Proteins ◽  
Oxygen Species

Abstract Multiple myeloma (MM) is an incurable malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow, usually accompanied by continuous monoclonal immunoglobulin production. This production requires proper folding and assembly of immunoglobulin chains, conducted by disulfide bonds formation in the endoplasmic reticulum (ER). Disulfide bonds are formed via electron transfer from thiol groups in a protein relay system, from protein disulfide isomerase (PDI) to ER oxidoreductase1 (ERO1) and finally to molecular oxygen, generating one molecule of hydrogen peroxide (H2O2) as secondary product for each disulfide bond formed. The high ER protein-folding demand usually placed on MM cells predisposes them to accumulation of unfolded proteins, which results in ER stress and also oxidative stress, both capable to induce cell death. Cellular defense systems for these specific forms of stress, known as the unfolded protein response (UPR) and oxidative stress response, have been explored as therapeutic targets for MM. 15-Deoxy-D12,14-prostaglandin J2 (15d-PGJ2) is a member of J2 series of PGs, characterized by the presence of an electrophilic carbonyl group in the cyclopentenone ring, which confers them biological properties that are different from those of other components of the PG family. These include anti-inflammatory, anti-proliferative and pro-apoptotic activities, depending on the concentration and cell type. Starting from the observation that15d-PGJ2 is emerging as the most potent antineoplastic agent of the J2 series of PGs, in the present study we investigated its effect on MM cells. We found that 15d-PGJ2 displayed superior cytotoxicity than dexamethasone in MM cell lines. Using flow cytometry, we demonstrated intracellular reactive oxygen species (ROS) formation in these cells after 15d-PGJ2 treatment, which was associated with an augmented ratio of oxidized to reduced glutathione (GSH), indicating that the cells were under oxidative stress. Due to its electrophilic property, it has been suggested that 15d-PGJ2 may directly react with GSH and/or other thiol compounds. Such reactions would deplete essential cellular antioxidants thereby leading to oxidative stress in MM cells. Results from qPCR analysis showed that 15d-PGJ2 induces transcription of UPR genes, including GRP78, GRP94, EDEM1 and also PRDX4. GRP78/BiP and GRP94 are ER-resident chaperones, members of the heat shock protein (HSP) 70 family and HSP 90 family, respectively. These chaperones were described to bind to immunoglobulin chains assisting their assembly. The ER degradation-enhancer mannosidase-like protein 1 (Edem1) is involved in the degradation of misfolded proteins to prevent their aggregation and cytotoxicity. PRDX4 encodes the ER-localized peroxiredoxin (Prx) IV, an enzyme not yet recognized as an UPR component, but recently demonstrated to use the H2O2 produced by ERO1 during disulfide bond formation to generate another pair of disulfide, with the additional benefit of reducing H2O2 to water. Increased transcription of these genes indicates an attempt to preserve ER-processing function and MM cells survival in response to accumulated misfolded proteins. In this regard, 15d-PGJ2-induced oxidative stress, by provoking oxidative modification of proteins, could have intensified the formation of misfolded proteins, thereby activating UPR signaling. In agreement with 15d-PGJ2-mediated accumulation of misfolded proteins, which are prevented from progressing further along the secretory pathway, our results from ELISA have shown a massive decrease in the concentration of light chain immunoglobulin secreted by MM cells in the presence of 15d-PGJ2, but not dexamethasone. These data suggest that 15d-PGJ2 favors ROS accumulation and protein misfolding, especially threatening the survival of cells that have a high protein-folding load and/or are susceptible to oxidative stress. In this sense, multiple myeloma and related disorders may benefit from 15d-PGJ2 therapy. Disclosures: Demasi: FAPESP: Research Funding. Napimoga:FAPESP: Research Funding.

scholarly journals Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum.

1995 ◽  
Vol 128 (1) ◽  
pp. 29-38 ◽  
Author(s):  
P S Kim ◽  
P Arvan
Keyword(s):  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Disulfide Bonds ◽  
Secretory Proteins ◽  
Chaperone Function ◽  
Er Chaperone ◽  
Sds Page ◽  
Membrane Bound ◽  
Fraction Bound

Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER.

scholarly journals Prion propagation and molecular chaperones

1999 ◽  
Vol 32 (4) ◽  
pp. 309-370 ◽  
Author(s):  
Ralph Zahn
Keyword(s):  
Protein Folding ◽  
Prion Protein ◽  
Molecular Chaperone ◽  
Molecular Chaperones ◽  
Cellular Prion Protein ◽  
Theoretical Point ◽  
General Terms ◽  
Protein X ◽  
Prion Propagation ◽  
Central Paradigm

1. Introduction 3102. Protein-only hypothesis 3123. The scrapie prion protein PrPSc3133.1 Purification of PrP 27–30 3133.2 Proteinase K resistance 3143.3 Scrapie-associated fibrils 3143.4 Smallest infectious unit 3163.5 Conformational properties 3163.6 Dissociation and stability 3194. The cellular prion protein PrPC3214.1 Prnp expression 3214.2 Biosynthetic pathway 3224.3 NMR structures 3244.4 Copper binding 3265. Post-translational PrP conversion 3275.1 Conformational isoforms 3275.2 Location of propagation 3295.3 Minimal PrP sequence 3305.4 Prion species barrier 3315.5 Prion strains 3326. Effect of familial TSE mutations 3336.1 Thermodynamic stability of PrPC 3346.2 De novo synthesis of PrPSc 3356.3 Transmembrane PrP forms 3377. Physical properties of synthetic PrP 3377.1 Amyloidogenic peptides 3377.2 Folding intermediates 3398. Hypothetical protein X 3408.1 Two species-specific epitopes 3408.2 Mapping the protein X epitope 3419. Chaperone-mediated PrP conversion 3439.1 Hsp60 and Hsp10 chaperonins 3439.2 GroEL promoted PrP-res formation 3459.3 Membrane-associated chaperonins 3459.4 Preference of GroEL for positive charges 3479.5 Potential GroEL/Hsp60 epitopes on PrP 3479.6 Conformations of chaperonin-bound PrP 3499.7 Conserved Hsp60 substrate binding sites 3499.8 Requirement of ATP-hydrolysis 3519.9 Hsp60-mediated prion propagation 35410. Template-assisted annealing model 35511. Acknowledgments 35712. References 357Although the central paradigm of protein folding (Anfinsen, 1973), that the unique three-dimensional structure of a protein is encoded in its amino acid sequence, is well established, its generality has been questioned due to two recent developments in molecular biology, the ‘prion’ and ‘molecular chaperone’. Biochemical characterization of infectious scrapie material causing central nervous system (CNS) degeneration indicates that the necessary component for disease propagation is proteinaceous (Prusiner, 1982), as first outlined by Griffith (1967) in general terms, and involves a conversion from a cellular prion protein, denoted PrPC, into a toxic scrapie form, PrPSc, which is facilitated by PrPSc acting as a template for PrPC to form new PrPSc molecules (Prusiner, 1987). The ‘protein-only’ hypothesis implies that the same polypeptide sequence, in the absence of any post-translational modifications, can adopt two considerably different stable protein conformations (Fig. 1). Thus, in the case of prions it is possible, although not proven, that they violate the central paradigm of protein folding. There is some indirect evidence that another factor, provisionally named ‘protein X’, might be involved in the conformational conversion process (Prusiner et al. 1998), which includes a dramatic change from α-helical into β-sheet secondary structure (Fig. 1). This factor has not been identified yet, but it has been proposed that protein X may act as a molecular chaperone. The idea that molecular chaperones play a critical role in the generation of PrPSc is appealing also from a theoretical point of view, because PrPSc formation involves changes in protein folding and possibly intermolecular aggregation (Fig. 1), processes in which chaperones are known to participate (Musgrove & Ellis, 1986). The discovery and functional analysis of more than a dozen molecular chaperones made it clear that these proteins do not complement folding information that is not already contained in the genetic code (Ellis et al. 1989); rather they facilitate the folding and assembly of proteins by preventing misfolding and refolding misfolded proteins (Hartl, 1996). Whether a molecular chaperone or another type of macromolecule is identified as the conversion factor, therefore, the molecular chaperone concept is likely to contribute to the understanding of the molecular nature of PrPC to PrPSc conversion.In this review I consider the prion concept from the view of a structural biologist whose main interest focuses on spontaneous and chaperone-mediated conformational changes in proteins.

scholarly journals Ero1–PDI interactions, the response to redox flux and the implications for disulfide bond formation in the mammalian endoplasmic reticulum

2013 ◽  
Vol 368 (1617) ◽  
pp. 20110403 ◽  
Author(s):  
Adam M. Benham ◽  
Marcel van Lith ◽  
Roberto Sitia ◽  
Ineke Braakman
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Terminal Electron Acceptor ◽  
Post Translational Modifications

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

scholarly journals Enhanced Binding to the Molecular Chaperone BiP Slows Thyroglobulin Export from the Endoplasmic Reticulum

1998 ◽  
Vol 12 (3) ◽  
pp. 458-467 ◽  
Author(s):  
Zoia Muresan ◽  
Peter Arvan
Keyword(s):  
Endoplasmic Reticulum ◽  
B Cells ◽  
Unfolded Protein Response ◽  
Molecular Chaperone ◽  
Disulfide Bonds ◽  
Chinese Hamster ◽  
Hormone Synthesis ◽  
Unfolded Protein ◽  
Hsp70 Family ◽  
Protein Response

Abstract To examine how binding of BiP (a molecular chaperone of the hsp70 family that resides in the endoplasmic reticulum) influences the conformational maturation of thyroglobulin (Tg, the precursor for thyroid hormone synthesis), we have developed a system of recombinant Tg stably expressed in wild-type Chinese hamster ovary (CHO) cells and CHO-B cells genetically manipulated for selectively increased BiP expression. The elevation of immunoreactive BiP in CHO-B cells is comparable to that seen during the unfolded protein response in the thyrocytes of certain human patients and animals suffering from congenital hypothyroid goiter with defective Tg. However, in CHO-B cells, we expressed Tg containing no mutations that induce misfolding (i.e. no unfolded protein response), so that levels of all other endoplasmic reticulum chaperones were normal. Increased availability of BiP did not accelerate Tg secretion; rather, the export of newly synthesized Tg was delayed. Tg detained intracellularly was concentrated in the endoplasmic reticulum. By coimmunoprecipitation, BiP exhibited enhanced binding to Tg in CHO-B cells. Moreover, two-dimensional gel analysis showed that BiP associated especially well with intracellular Tg containing mispaired disulfide bonds, thought to represent early Tg folding intermediates. An endoplasmic reticulum chaperone of the hsp90 family, GRP94, was also associated in Tg-chaperone complexes. The results suggest that increased binding of BiP to Tg leads to its delayed conformational maturation in the endoplasmic reticulum.

The endoplasmic reticulum sulfhydryl oxidase Ero1β drives efficient oxidative protein folding with loose regulation

2011 ◽  
Vol 434 (1) ◽  
pp. 113-121 ◽  
Author(s):  
Lei Wang ◽  
Li Zhu ◽  
Chih-chen Wang
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Feedback Regulation ◽  
Unfolded Protein ◽  
Oxidative Protein Folding ◽  
Genes Encoding ◽  
The Unfolded Protein Response ◽  
Protein Response

In eukaryotes, disulfide bonds are formed in the endoplasmic reticulum, facilitated by the Ero1 (endoplasmic reticulum oxidoreductin 1) oxidase/PDI (protein disulfide-isomerase) system. Mammals have two ERO1 genes, encoding Ero1α and Ero1β proteins. Ero1β is constitutively expressed in professional secretory tissues and induced during the unfolded protein response. In the present work, we show that recombinant human Ero1β is twice as active as Ero1α in enzymatic assays. Ero1β oxidizes PDI more efficiently than other PDI family members and drives oxidative protein folding preferentially via the active site in the a′ domain of PDI. Our results reveal that Ero1β oxidase activity is regulated by long-range disulfide bonds and that Cys130 plays a critical role in feedback regulation. Compared with Ero1α, however, Ero1β is loosely regulated, consistent with its role as a more active oxidase when massive oxidative power is required.

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