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At least 19 recordsLinked to original sources

PubMed1995

PDI-, PPI- and chaperone-catalyzed refolding of recombinant human IL-2 and GM-CSF.

The studies on PDI-, PPI- and chaperone-catalyzed refolding of recombinant human IL-2 and GM-CSF show that PDI can prevent the mismatch of disulfide bonds and formation of aggregates by interchains linkage; furthermore, PDI can correct the mismatching of disulfide bonds in IL-2 isomers. PPI can increase the rate of folding reaction while chaperone can prevent the aggregation during the folding process. In addition, there is a synergistic effect between them.

Amino Acid Isomerases↗

PubMed1994

Hormonal regulation of protein disulfide isomerase and chaperone synthesis in the rat exocrine pancreas.

Folding and assembly of polypeptides translocated into the rough endoplasmic reticulum (RER) is facilitated by a set of resident proteins in the lumen of the RER. We studied the regulation of synthesis of the RER luminal proteins immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), and of the cytosolic stress 70 protein (hsc70) after hormonal stimulation of the pancreatic exocrine secretory pathway. Their rate of synthesis was assessed at both mRNA and protein levels and under two experimental conditions that are associated with large increases in exocrine production. After in vivo stimulation of the pancreas by either endogenous release of cholecystokinin (CCK) following proteinase inhibitor feeding (FOY-305) or by in vivo infusion of the pancreatic secretagogue cerulein, the relative rates of synthesis detected for BiP and PDI were enhanced 2.5 to 4-fold compared to control. Interestingly, the kinetics and the degree of hsc70 mRNA induction were almost identical to those of BiP and PDI, suggesting coordinated hormonal regulation of BiP, PDI as hormonal stimulation was even twice that following heat shock treatment. The mRNA levels of calreticulin (CaBP3) increased up to 2.3-fold with a kinetic comparable to that of BiP, PDI and hsc 70, while CaBP1 and the RER membrane proteins, ribophorin I and the signal recognition particle receptor did not show any changes in their relative mRNA amounts after hormonal stimulation. The increase in the rates of PDI and chaperone biosynthesis exceeds the associated increase in total protein biosynthesis. In vitro experiments, using transformed rat acinar cells (AR4-2J) in which pancreatic enzyme synthesis can be induced by glycocorticoid hormones, demonstrated that induction of PDI and chaperone mRNA synthesis preceded extensive mRNA expression of secretory proteins.

Amino Acid Sequence↗

PubMed1993

Protein disulfide isomerase is both an enzyme and a chaperone.

Protein disulfide isomerase (PDI) catalyzes the formation of native disulfides of peptide chains from either the reduced form or randomly joined disulfides. So that thiols situated at distant parts of the polypeptide chain can be joined together to form the native disulfides, the polypeptide chain has to be folded, at least to some extent, into the native conformation. It is suggested that PDI promotes folding of the chains as well as formation of the disulfides and plays a role similar to the chaperones in the folding process. PDI is known to be a multifunctional protein and capable of nonspecific peptide binding. These properties are closely connected to its possible function as a chaperone. Thioredoxin, which has an active site sequence similar to that of PDI but lacks the property of peptide binding, is much less efficient as a disulfide isomerase.

Biological Evolution↗

PubMed1994

The role of the thiol/disulfide centers and peptide binding site in the chaperone and anti-chaperone activities of protein disulfide isomerase.

The complexity of protein folding is often aggravated by the low solubility of the denatured state. The inefficiency of the oxidative refolding of reduced, denatured lysozyme results from a kinetic partitioning of the unfolded protein between pathways leading to aggregation and pathways leading to the native structure. Protein disulfide isomerase (PDI), a resident foldase of the endoplasmic reticulum, catalyzes the in vitro oxidative refolding of reduced, disulfide-containing proteins, including denatured lysozyme. Depending on the concentrations of foldase and denatured substrate and the order in which they are added to initiate folding, PDI can exhibit either a chaperone activity or an anti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Chem 269, 7764-7771). PDI's chaperone activity leads to quantitative recovery of native lysozyme. Its anti-chaperone activity diverts substrate away from productive folding and facilitates disulfide cross-linking of lysozyme into large, inactive aggregates that specifically incorporate PDI. A mutant PDI (NmCm-PDI), in which both the N- and C-terminal active site cysteines have been changed to serines, loses all chaperone activity and behaves as an anti-chaperone at all substrate and PDI concentrations tested. The dithiol/disulfide sites of PDI are essential for the chaperone activity observed at high PDI concentrations, but they are not required for the anti-chaperone activity found at low PDI concentrations. Inactivation of PDI's peptide/protein binding site by a specific photoaffinity label (Noiva, R., Freedman, R. B., and Lennarz, W. J. (1993) J. Biol. Chem. 268, 19210-19217) inhibits the disulfide isomerase and chaperone activity, but the protein still retains its anti-chaperone activity. In a glutathione redox buffer, lysozyme-PDI aggregates are disulfide cross-linked; however, disulfide cross-linking is not required for aggregate formation or for the incorporation of PDI into the aggregates. Although both the peptide binding site and the catalytic active sites of PDI are required for chaperone and disulfide isomerase activity, neither of these sites are involved in PDI's anti-chaperone activity. PDI's anti-chaperone activity could serve as a quality control device by providing an efficient mechanism to retain misfolded proteins in the endoplasmic reticulum (Marquardt, T., and Helenius, A. (1992) J. Cell. Biol. 117, 505-513).

Animals↗

PubMed1994

Protein disulfide isomerase exhibits chaperone and anti-chaperone activity in the oxidative refolding of lysozyme.

Reduced, denatured lysozyme tends to aggregate at neutral pH, and competition between productive folding and aggregation substantially reduces the efficiency of refolding (Goldberg, M.E., Rudolph, R., and Jaenicke, R. (1991) Biochemistry 30, 2790-2797). Protein disulfide isomerase (PDI), a catalyst of oxidative protein folding, has a variety of effects on the yield of native lysozyme during the oxidative refolding of the reduced, denatured protein. Depending on the concentration of lysozyme, the concentration of PDI, and the order in which lysozyme and PDI are added to initiate folding, PDI can produce a substantial increase or a substantial decrease in the recovery of native lysozyme, when compared with the uncatalyzed reaction. In the presence of a glutathione redox buffer, denatured lysozyme (1-10 microM) partitions almost equally between productive folding leading to native lysozyme (50-63%) and non-productive fates including the formation of disulfide cross-linked aggregates. At the higher lysozyme concentrations examined (5-10 microM), substoichiometric concentrations of PDI (0.5-1 microM) exhibit "anti-chaperone" activity; PDI actively diverts most of the denatured lysozyme away from productive folding so that only 17 +/- 9% of the lysozyme is recovered as native enzyme. PDI's anti-chaperone activity results in extensive intermolecular disulfide crosslinking of lysozyme into large, inactive aggregates. On the other hand, if PDI is initially present at a large molar excess (5-10-fold) when denatured lysozyme is diluted to initiate folding, PDI demonstrates a chaperone-like activity that prevents aggregate formation and promotes correct folding. When PDI's chaperone activity is dominant, virtually all of the denatured lysozyme is correctly folded. The schizophrenic chaperone/anti-chaperone nature of PDI activity accounts for a number of observations on in vivo protein folding, including the necessity for maintaining a high concentration of PDI in the endoplasmic reticulum and the formation of disulfide cross-linked aggregates in the endoplasmic reticulum during the expression of disulfide-containing proteins (deSilva, A., Braakman, I., and Helenius, A. (1993) J. Cell. Biol. 120, 647-655).

Animals↗

PubMed1995

Chaperone-like activity of protein disulfide-isomerase in the refolding of rhodanese.

Protein disulfide-isomerase (PDI) in near stoichiometric concentrations promotes reactivation and prevents aggregation of guanidine-hydrochloride-denatured rhodanese during refolding upon dilution. PDI also suppresses aggregation of rhodanese during thermal inactivation. The above-mentioned properties displayed by PDI completely satisfy the definition of chaperone and provide additional evidence to confirm the hypothesis proposed previously [Wang, C. C. & Tsou, C. L. (1993) FASEB J. 7, 1515-1517] that PDI is both an enzyme and a chaperone. Since rhodanese contains no disulfide bonds, the chaperone-like activity of PDI acting on rhodanese is independent of its disulfide-isomerase activity.

Animals↗

PubMed1995

Independence of the chaperone activity of protein disulfide isomerase from its thioredoxin-like active site.

Protein disulfide isomerase (PDI) alkylated at thiols of the thioredoxin-like -CHC- active sites is devoid of isomerase activity, but its chaperone-like activity to increase the reactivation yield and prevent the aggregation of guanidine hydrochloride-denatured D-glyceraldehyde-3-phosphate dehydrogenase upon dilution is unimpaired. A peptide of 28 amino acids markedly inhibits both the enzyme and the chaperone activities of PDI. The above results indicate that the -CGHC- active site is necessary for the isomerase activity but not required for the chaperone activity of PDI, whereas the peptide binding site is essential for both activities.

Alkylation↗

PubMedDate not supplied

Alteration of hybridoma viability and antibody secretion in transfectomas with inducible overexpression of protein disulfide isomerase.

Monoclonal antibody (mAb)-secreting transfectomas with dexamethasone inducible expression of the mammalian endoplasmic reticulum foldase and chaperone protein disulfide isomerase (PDI, ERp59) were generated from the murine 9.2.27 hybridoma in order to obtain in vivo evidence of whether alteration of the level of PDI, believed to be involved in immunoglobulin (Ig) assembly, results in alteration of mAb secretion kinetics. Using an RNase refolding assay, the specific activity of endogenous PDI in the 9.2.27 hybridoma was found to be constant during batch growth. An expression vector for glucocorticoid-inducible overexpression of PDI, pMMTVPDI, was constructed from pMAMneo using a rat PDI cDNA. Cell lysates of stable transfectomas contained 2-4-fold higher levels of PDI mRNA and increased levels of PDI protein, detected by immunoblotting, following induction with 0.1 microM dexamethasone. Monoclonal antibody secretion kinetics were evaluated in 12.5 mL shake flasks, a 100 mL spinner, and a 1 L aerated batch reactor. A transfectoma was found with altered mAb secretion kinetics during cell growth following dexamethasone induction of PDI overexpression. Specific mAb secretion rate was not significantly increased following dexamethasone induction; however, hybridoma viability was sustained longer during the stationary phase of cell growth and hence total antibody yield was increased in comparison to the parent 9.2.27 hybridoma.

Animals↗

PubMed1994

Influence of protein disulfide isomerase (PDI) on antibody folding in vitro.

The role of eucaryotic protein disulfide isomerase (PDI) in the folding and reoxidation of proteins in vitro was investigated using an antibody Fab fragment as a model substrate, since PDI is known to participate in the disulfide bond formation of immunoglobulins in vivo. PDI has no effect on the folding of the Fab fragment with intact disulfide bonds, suggesting that, at least in this system, PDI is not able to influence the folding process in a chaperone-like manner. Instead, the role of PDI is limited to disulfide bond formation as demonstrated for the folding of the denatured and reduced Fab fragment. Here, PDI influences the yield of reactivation enormously with a maximum effect at about stoichiometric amounts of PDI and Fab. Furthermore, PDI changes the redox dependence of the reaction. In the presence of PDI, formation of the correct disulfide bonds is possible at higher oxidizing conditions compared to the spontaneous reaction. The requirements both for stoichiometric amounts of PDI and for the presence of PDI during the first seconds of refolding suggest that there is a kinetic competition between rapid structure formation of the antibody domains and interaction of PDI with cysteine residues in the folding protein.

Animals↗

PubMed1992

Renaturation of a single-chain immunotoxin facilitated by chaperones and protein disulfide isomerase.

B3(Fv)-PE38KDEL, a recombinant immunotoxin, forms inclusion bodies when produced in Escherichia coli. In renaturation experiments, nonspecific aggregation of non-native polypeptide chains, and the formation of incorrect disulfide linkages lead to inactive molecules. To prevent these side reactions, we added molecular chaperones and protein disulfide isomerase (PDI) to the refolding buffer. Both DnaK and GroEL/S influenced the reactivation process. GroEL alone inhibited reactivation, but in the presence of ATP, GroEL and GroES significantly increased the yield of active protein. DnaK also increased the yield of properly folded protein and the stimulating effect of DnaK was also observed using immobilized DnaK, which can be used repeatedly without significant loss of activity. PDI, which catalyzes disulfide bridging of proteins, also stimulated reactivation of the immunotoxin. Under optimum conditions, reactivation yields in the presence of PDI were about twice that obtained with nonenzymatic disulfide bond formation. Furthermore, DnaK and PDI were additive when renaturation was performed in the presence of both proteins.

Antibodies, Monoclonal↗

PubMed2024

The combined effect of the gene copy number and chaperone overexpression on the recombinant bovine chymosin production in Pichia pastoris, with mutant ADH2 promoter.

Chymosin is an enzyme used to coagulate milk, in the cheese industry. This study aimed to increase recombinant production of the chymosin in Pichia pastoris by determining the optimum copy number and overproduction of a Protein Disulfide Isomerase (PpPDI) chaperon protein. Bos taurus chymosin was expressed under the control of a mutant ADH2 promoter. The clones containing 1-4 gene copy numbers of the chymosin were constructed using the in vitro cloning method, and the effect of chaperone protein on chymosin secretion was investigated. The enzyme production levels are 4, 6.3, 4.5, and 3 IMCU/mL for 1, 2, 3, and 4-copy clones. The secreted chymosin levels increased up to two copies, and increasing the number of copies decreased the secretion level. Therefore, PpPDI was over-expressed in the clones regulated with the ADH2 promoter. The over-expression of PDI gene increased chymosin secretion in clones compared to the counterpart host. However, the highest chymosin level was obtained with C2 (2-copy chymosin containing clone; 6.3 IMCU/mL) and C2P2 (2-copy chymosin/2-copy PDI containing clone; 8.2 IMCU/mL). The maximum production was 39 IMCU/mL with the clone C2P2 in the fermenter scale production. The enzyme activity increased approximately 2-fold by adding two copies of the chaperone protein. The combined effect of gene copy number and chaperone overexpression on chymosin production was investigated. Two copies of the chymosin and PpPDI genes were the optimum among the tested clones.

Animals↗

PubMed1994

Anti-chaperone behavior of BiP during the protein disulfide isomerase-catalyzed refolding of reduced denatured lysozyme.

Folding catalysts of the endoplasmic reticulum (ER), such as protein disulfide isomerase (PDI), accelerate the slow chemical steps, such as disulfide bond formation, that accompany protein folding. Molecular chaperones of the ER, notably the heavy chain-binding protein, BiP (grp78), bind and release unfolded proteins in an ATP-dependent fashion. In vitro, the fate of reduced, denatured lysozyme is dependent on whether the substrate interacts first with BiP or PDI. Depending on the ratio of PDI to substrate and order in which the components of the reaction are mixed, PDI can exhibit a foldase/chaperone activity, which increases the rate and extent of lysozyme refolding, or it can function as an anti-chaperone that promotes the formation of inactive, disulfide-linked lysozyme aggregates (Puig, A., and Gilbert, H.F. (1994) J. Biol. Chem. 269, 7764-7771). Reduced, denatured lysozyme, but not the native protein, interacts with BiP and efficiently stimulates its peptide-dependent ATPase activity. When present at substoichiometric amounts, BiP, like PDI, facilitates the formation of large, inactive lysozyme aggregates that are non-covalently associated with BiP. BiP and PDI compete for a limited number of sites in these insoluble aggregates. If BiP is present at a high molar excess, the chaperone binds unfolded lysozyme and inhibits its aggregation by maintaining it in a soluble, yet inactive, conformation, both in the presence or absence of ATP. Increasing concentrations of BiP decrease the extent, but not the initial rate, of refolding, suggesting that BiP and PDI compete for unfolded lysozyme and that the BiP-lysozyme complex is not a very good substrate for PDI either in the presence or absence of ATP. Depending on the BiP and PDI concentrations, unfolded lysozyme may either be efficiently refolded into the native conformation in a PDI-catalyzed reaction, or it may form both soluble and insoluble BiP-lysozyme complexes. In vitro, PDI- and BiP-facilitated aggregation, as well as the competition of the two proteins for substrate, reproduces many of the features of the quality control system of the ER.

Adenosine Triphosphate↗

PubMed1995

Purification, characterization, and intracellular localization of glycosylated protein disulfide isomerase from wheat grains.

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.

Amino Acid Sequence↗

PubMed1994

A major phosphoprotein of the endoplasmic reticulum is protein disulfide isomerase.

One of the effects of ATP in the endoplasmic reticulum is to induce the phosphorylation of several proteins among which a 57-kDa protein (pp57) prevails in our labeling conditions. We provide evidence that pp57 is protein disulfide isomerase (PDI), an abundant ubiquitous protein of the endoplasmic reticulum involved in various important cellular functions. This phosphorylation does not result from the activity of a microsomal protein kinase but from an autophosphorylation as described for other microsomal proteins such as chaperones. Phosphoamino acid analysis and cyanogen bromide cleavage indicate that the modification site lies on a threonine residue within the central region of the protein outside the thioredoxin-like domains. For the pure PDI, only the dimer is able to phosphorylate, while some experiments suggest that within the endoplasmic reticulum the phosphorylated form of PDI is mainly mobilized in larger size oligomers. Thus a possible role for this phosphorylation may be to modulate the association of PDI with its different partners.

Amino Acid Sequence↗

PubMed1995

Protein disulfide isomerase mutant lacking its isomerase activity accelerates protein folding in the cell.

We investigated the effect of protein disulfide isomerase (PDI) on in vivo protein folding of human lysozyme (h-LZM) in a specially constructed yeast coexpression system. Coexpression with PDI increased the amounts of intracellular h-LZM with the native conformation, leading to an increase in h-LZM secretion. The results indicated that PDI is a real catalyst of protein folding in the cell. The secretion of h-LZM increased even when both active sites of PDI were disrupted, suggesting that the effect of PDI resulted from a function other than the formation of disulfide bonds. This is the first finding that PDI without isomerase activity accelerates protein folding in vivo.

Base Sequence↗

PubMed1994

A single purification procedure for the major resident proteins of the ER lumen: endoplasmin, BiP, calreticulin and protein disulfide isomerase.

We have developed a single purification procedure for the four major resident endoplasmic reticulum (ER) proteins: protein disulfide isomerase (PDI), BiP, endoplasmin, and calreticulin. Three of these proteins are thought to play a role in protein folding in vivo, whereas calreticulin is thought to be the major calcium binding protein in the ER. The proteins were purified from fresh bovine liver by taking advantage of individual characteristics of the proteins. Liver microsomes were prepared and then premeabilized to release the lumenal contents. After ammonium sulfate precipitation, the proteins were purified by chromatography; BiP was purified by affinity chromatography on ATP-agarose, and both endoplasmin and calreticulin were purified by affinity chromatography on Con A-Sepharose. PDI was purified by anionic ion exchange chromatography.

Amino Acid Sequence↗

PubMed1993

Changes in levels of pancreatic endoplasmic reticulum proteins that function in translocation and maturation of secretory proteins in response to cholecystokinin.

Two pathways operate to target newly-synthesised proteins to the endoplasmic reticulum. In one, the signal recognition particle attaches to the signal sequences of nascent chains on ribosomes and slows or stops translation until contact is made with the docking protein at the membrane. The second operates via molecular chaperons. The pathways converge at the level of a 43 kDa signal binding protein integrated into the membrane, where translocation through a proteinaceous pore is initiated. In the lumen, proteins fold and disulphide formation is catalysed by the enzyme protein disulphide isomerase. The heavy chain binding protein may attach to unassembled or unfolded proteins and prevent their exit from the ER to the Golgi. Cholecystokinin (CCK) treatment increases the biosynthesis and secretion of pancreatic proteins, increases the levels of PDI and the 43 kDa binding protein, and reduces levels of BiP. These proteins may be possible targets for genetic manipulation to improve processing of heterologous proteins from cultured mammalian cells.

Acids↗

PubMed1992

Heavy chain binding protein (BiP/GRP78) and endoplasmin are exported from the endoplasmic reticulum in rat exocrine pancreatic cells, similar to protein disulfide-isomerase.

Previously we found that in rat exocrine pancreatic cells, protein disulfide-isomerase (PDI), one of the major resident proteins in the lumen of the endoplasmic reticulum (ER) of many cells, is localized not only in the ER but also in the Golgi apparatus, secretory granules, plasma membranes, and even in the glandular lumens, despite possessing the ER retention signal KDEL (Lys-Asp-Glu-Leu) at the carboxyl terminus. In this report, we examined whether other ER luminal proteins bearing the KDEL signal at their C-termini, such as BiP/GRP78 and endoplasmin/GRP94 are also exported from the ER. We prepared two kinds of affinity-purified polyclonal antibodies; one against a synthetic peptide with 12 amino acids which is identical to the carboxyl terminus of BiP and another against purified endoplasmin. Immunoblot analysis using these two antibodies showed that BiP and endoplasmin exist in both the plasma membrane and the microsomal fractions, similar to the intracellular distribution of PDI in rat exocrine pancreas. The ratios of the amount of the three proteins in the two fractions, however, were variable, suggesting that the KDEL-bearing proteins such as PDI, BiP, and endoplasmin are exported from the ER with different efficiencies. Postembedding protein A-immunogold electron microscopy revealed that endoplasmin was exported from the ER and secreted to the extracellular space. The secretion of PDI in rat pancreatic lobules was inhibited by Brefeldin A (BFA) and by guanidino acid esters (FOY-305), which are known to be the inhibitors of the intracellular transport. Taken together with the previous immunogold electron microscopic analyses by Akagi et al. (1988), it is strongly suggested that in rat exocrine pancreatic cells PDI and the other KDEL-bearing proteins found in the extracellular space were not artificially released by cell damage during incubation but were secreted via the normal secretory pathway.

Amino Acid Sequence↗