Depletion of Cyclophilins B and C Leads to Dysregulation of Endoplasmic Reticulum Redox Homeostasis

Protein folding within the endoplasmic reticulum is assisted by molecular chaperones and folding catalysts that include members of the protein-disulfide isomerase and peptidyl-prolyl isomerase families. In this report, we examined the contributions of the cyclophilin subset of peptidyl-prolyl isomerases to protein folding and identified cyclophilin C as an endoplasmic reticulum (ER) cyclophilin in addition to cyclophilin B. Using albumin and transferrin as models of cis-proline-containing proteins in human hepatoma cells, we found that combined knockdown of cyclophilins B and C delayed transferrin secretion but surprisingly resulted in more efficient oxidative folding and secretion of albumin. Examination of the oxidation status of ER protein-disulfide isomerase family members revealed a shift to a more oxidized state. This was accompanied by a >5-fold elevation in the ratio of oxidized to total glutathione. This “hyperoxidation” phenotype could be duplicated by incubating cells with the cyclophilin inhibitor cyclosporine A, a treatment that triggered efficient ER depletion of cyclophilins B and C by inducing their secretion to the medium. To identify the pathway responsible for ER hyperoxidation, we individually depleted several enzymes that are known or suspected to deliver oxidizing equivalents to the ER: Ero1αβ, VKOR, PRDX4, or QSOX1. Remarkably, none of these enzymes contributed to the elevated oxidized to total glutathione ratio induced by cyclosporine A treatment. These findings establish cyclophilin C as an ER cyclophilin, demonstrate the novel involvement of cyclophilins B and C in ER redox homeostasis, and suggest the existence of an additional ER oxidative pathway that is modulated by ER cyclophilins.     

The biosynthesis of rat transferrin. Evidence for rapid glycosylation, disulfide bond formation, and tertiary folding

The transit time of newly synthesized transferrin in the liver is markedly longer than that of albumin. We sought to learn the basis of this difference by the use of labeled leucine and mannose in vivo and by isolation of newly formed transferrin from rough microsomes of rat liver. Albumin and alpha 1-antitrypsin, a second glycoprotein, were also studied for comparison. Minimal hepatic transit times were 17, 23, and 31 min for albumin, alpha 1-antitrypsin, and transferrin, respectively. The delay in the case of transferrin was found to occur chiefly in the rough endoplasmic reticulum and to be paralleled by an increase in the amount of transferrin relative to albumin in that organelle. Initial glycosylation of transferrin was as rapid as that of alpha 1-antitrypsin, and essentially all of the transferrin in the rough endoplasmic reticulum contained glycans which bound to concanavalin A and were removed by endoglycosidase H. Only 3% of the transferrin isolated from the rough microsomes came from the plasma by endocytosis or adsorption. Rapidity of disulfide bond formation in rough microsomes was evident from the presence of only 1.3 cysteine thiols/molecule of rough microsomal transferrin (total of 19 cystines) and the absence of mixed disulfides. Peptide patterns upon mild proteolysis were consistent with a native configuration of disulfide bond pairing. The ability of rough microsomal transferrin to bind and deliver iron through interaction with transferrin receptors on reticulocytes suggests that considerable tertiary structure is present. Thus, initial glycosylation, disulfide bridging, and tertiary folding are all rapid processes. The cause for the slow release of transferrin from the rough endoplasmic reticulum may lie with a rate-limiting transfer mechanism.     

Protein folding and translocation across the endoplasmic reticulum membrane (Review)

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

Calcium is required for folding of newly made subunits of the asialoglycoprotein receptor within the endoplasmic reticulum.

By resolving immunoprecipitates on nonreducing sodium dodecyl sulfate gels, we have detected several disulfide-bonded intermediates in folding within the endoplasmic reticulum of newly made H1 subunits of the asialoglycoprotein receptor. H1 in the endoplasmic reticulum (ER) can be partially unfolded by treatment of cells with dithiothreitol, but H1 in Golgi or post-Golgi organelles is resistant to such unfolding. This defines a late step in H1 folding that occurs just prior to exit from the ER. Depletion of calcium from the endoplasmic reticulum, either by treatment with A23187 or thapsigargin, has no effect on folding or secretion of newly made albumin, but totally blocks H1 maturation from the ER. No ER intermediates in H1 folding are formed in cells treated with A23187 or thapsigargin, indicating that at least an early step in H1 folding requires a high Ca2+ concentration in the ER lumen. As judged by cross-linking experiments, formation of H1 dimers and trimers occurs immediately after biosynthesis of the peptide chain, before monomer folding, and occurs normally in cells in which ER Ca2+ is reduced and where the monomer never folds properly. Calcium is essential for the asialoglycoprotein receptor to bind galactose, and our results suggest that Ca2+ is also essential for the receptor polypeptides to fold in the ER.     

Protein folding and translocation across the endoplasmic reticulum membrane

Proteins destined for secretion are translocated across or inserted into the endoplasmic reticulum membrane whereupon they fold and assemble to their native state before their subsequent transport to the Golgi apparatus. Proteins that fail to fold correctly are translocated back across the endoplasmic reticulum membrane to the cytosol where they become substrates for the cytosolic degradative machinery. Central to translocation is a protein pore in the membrane called the translocon that allows passage of proteins in and out of the endoplasmic reticulum. It is clear that the conformation of the polypeptide chain influences the translocation process and that there is a temporal relationship between modification of the chain, translocation and folding. This review will consider when and how the polypeptide chain folds, and how this might influence translocation into and out of the ER; and discuss how protein folding might affect post-translational modification of the polypeptide chain following translocation into the ER lumen.     

Elevated expression temperature in a mesophilic host results in increased secretion of a hyperthermophilic enzyme and decreased cell stress

Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins.     

Expression of calreticulin P-domain results in impairment of secretory pathway in Leishmania donovani and reduced parasite survival in macrophages

The secretory proteins of Leishmania are thought to be involved in the parasite survival inside the insect vector or mammalian host. It is clear from studies in higher eukaryotes that proper folding in the endoplasmic reticulum and targeting out of the endoplasmic reticulum is critical for the function of secretory proteins. The endoplasmic reticulum chaperones such as calreticulin play an important role in the quality control of secretory proteins. However, very little is known about the secretory pathway of trypanosomatid parasites such as Leishmania. In the present study, we show that overexpression of the P-domain of Leishmania donovani calreticulin in transfected L. donovani resulted in a significant reduction in the secretion of the parasite secretory acid phosphatases. This effect is associated with an intracellular accumulation of active enzyme in these transfected parasites. In addition, parasites expressing the P-domain calreticulin showed a significant decrease in survival inside human macrophages. This study suggests that altering the function of an endoplasmic reticulum chaperone such as calreticulin in Leishmania may affect the targeting of proteins that are associated with the virulence of the parasite during their trafficking through the parasite secretory pathway.     

Elevated synthesis of human alpha 1-antitrypsin hinders the secretion of murine alpha 1-antitrypsin from hepatocytes of transgenic mice

alpha 1-Antitrypsin (AAT) is a major hepatic secretory protein. The elevated synthesis of human AAT within hepatocytes of transgenic mice results in its accumulation within a subset of distended cisternae of the rough endoplasmic reticulum. The protein does not accumulate in large insoluble aggregates as is the case for the human PiZ AAT variant. Furthermore, the accumulated protein is not associated with immunoglobulin heavy chain binding protein. Transgenic animals exhibiting an elevated synthesis and subsequent intrahepatic accumulation of human AAT exhibit reduced serum levels of murine AAT as a result of its hindered secretion and accumulation within the rough endoplasmic reticulum. Interestingly, the secretion of murine transferrin and albumin which represent glycosylated and non-glycosylated hepatic secretory proteins, respectively, is unaffected. Overall, these results demonstrate that the elevated synthesis of human AAT can hinder the export of murine AAT from the hepatic rough endoplasmic reticulum in an apparently specific manner.     

Protein folding includes oligomerization - examples from the endoplasmic reticulum and cytosol

A correct three-dimensional structure is a prerequisite for protein functionality, and therefore for life. Thus, it is not surprising that our cells are packed with proteins that assist protein folding, the process in which the native three-dimensional structure is formed. In general, plasma membrane and secreted proteins, as well as those residing in compartments along the endocytic and exocytic pathways, fold and oligomerize in the endoplasmic reticulum. The proteins residing in the endoplasmic reticulum are specialized in the folding of this subset of proteins, which renders this compartment a protein-folding factory. This review focuses on protein folding in the endoplasmic reticulum, and discusses the challenge of oligomer formation in the endoplasmic reticulum as well as the cytosol.     

Rescue and Stabilization of Acetylcholinesterase in Skeletal Muscle by N-terminal Peptides Derived from the Noncatalytic Subunits

The vast majority of newly synthesized acetylcholinesterase (AChE) molecules do not assemble into catalytically active oligomeric forms and are rapidly degraded intracellularly by the endoplasmic reticulum-associated protein degradation pathway. We have previously shown that AChE in skeletal muscle is regulated in part post-translationally by the availability of the noncatalytic subunit collagen Q, and others have shown that expression of a 17-amino acid N-terminal proline-rich attachment domain of collagen Q is sufficient to promote AChE tetramerization in cells producing AChE. In this study we show that muscle cells, or cell lines expressing AChE catalytic subunits, incubated with synthetic proline-rich attachment domain peptides containing the endoplasmic reticulum retrieval sequence KDEL take up and retrogradely transport them to the endoplasmic reticulum network where they induce assembly of AChE tetramers. The peptides act to enhance AChE folding thereby rescuing them from reticulum degradation. This enhanced folding efficiency occurs in the presence of inhibitors of protein synthesis and in turn increases total cell-associated AChE activity and active tetramer secretion. Pulse-chase studies of isotopically labeled AChE molecules show that the enzyme is rescued from intracellular degradation. These studies provide a mechanistic explanation for the large scale intracellular degradation of AChE previously observed and indicate that simple peptides alone can increase the production and secretion of this critical synaptic enzyme in muscle tissue.     

Depletion of cellular calcium accelerates protein degradation in the endoplasmic reticulum.

In this study the effects of A23187 and thapsigargin on the degradation of T-cell antigen receptor-beta (TCR-beta) and CD3-delta in the endoplasmic reticulum have been studied. Preliminary experiments showed that these drugs had different effects on the secretory pathway. Depletion of cellular calcium pools by incubation of cells with A23187 in calcium-free medium blocked transport between the endoplasmic reticulum and the Golgi apparatus whereas thapsigargin caused a modest increase in transport. When added to cells transfected with TCR-beta or CD3-delta the drugs caused an immediate stimulation of proteolysis of presynthesized protein and at maximum effective concentrations caused a 3-fold increase in the rate of degradation. They did not affect the lag period of 1 h which precedes degradation of newly synthesized proteins. Chelation of cytosolic calcium also accelerated degradation, suggesting that depletion of calcium from the endoplasmic reticulum was the main stimulus of proteolysis and that increased degradation was not caused by a transient increase in cytosolic calcium levels. The selectivity of degradation in the endoplasmic reticulum was maintained. A23187 had no effect on the stability of CD3-gamma nor co-transfected epsilon-beta dimers. Calcium depletion increased the overall rate of degradation in the endoplasmic reticulum and increased the rate of proteolysis of an "anchor minus" beta chain. The results suggested that proteolysis within the endoplasmic reticulum may be regulated by the high concentrations of Ca2+ which are stored in the organelle. Ca2+ may be required for protein folding. Calcium depletion may have caused the beta and delta chains to adopt a conformation that was more susceptible to proteolysis. Alternatively, calcium depletion may have disrupted the lumenal content of the endoplasmic reticulum and increased the access of proteases to potential substrates.     

A study on the intracellular transport of prothrombin, albumin and transferrin in rat

The intracellular transport of prothrombin in rat has been studied and compared with the transport of albumin and transferrin. The proteins were immunoisolated from plasma samples after pulse labelling with [3H]leucine and the secretion kinetics were determined. The half-times for secretion (t1/2) were approx. 30, 53 and 75 min for albumin, prothrombin and transferrin, respectively, whereas the minimal transit time for prothrombin was approx. 30 min, and those for albumin and transferrin 15-20 min. After injection of vitamin K-1 into warfarin-treated rats, the accumulated prothrombin precursor was gamma-carboxylated and secreted with a t1/2 of 37 min. This indicates that the gamma-carboxylation of prothrombin in rough endoplasmic reticulum cannot account for the delay in the transport of prothrombin as compared to albumin. Comparison of the incorporation of [3H]leucine and [3H]glucosamine into plasma prothrombin and transferrin suggested that transferrin is secreted randomly from an intracellular pool, whereas prothrombin is transported in a more orderly sequence. Moreover, treatment of rough microsomes with 0.05% sodium deoxycholate indicated that prothrombin is more tightly associated with the membranes of rough endoplasmic reticulum than albumin and transferrin.     

Promotion of transferrin folding by cyclic interactions with calnexin and calreticulin.

Calnexin, an abundant membrane protein, and its lumenal homolog calreticulin interact with nascent proteins in the endoplasmic reticulum. Because they have an affinity for monoglucosylated N-linked oligosaccharides which can be regenerated from the aglucosylated sugar, it has been speculated that this repeated oligosaccharide binding may play a role in nascent chain folding. To investigate the process, we have developed a novel assay system using microsomes freshly prepared from pulse labeled HepG2 cells. Unlike the previously described oxidative folding systems which required rabbit reticulocyte lysates, the oxidative folding of transferrin in isolated microsomes could be carried out in a defined solution. In this system, addition of a glucose donor, UDP-glucose, to the microsomes triggered glucosylation of transferrin and resulted in its cyclic interaction with calnexin and calreticulin. When the folding of transferrin in microsomes was analyzed, UDP-glucose enhanced the amount of folded transferrin and reduced the disulfide-linked aggregates. Analysis of transferrin folding in briefly heat-treated microsomes revealed that UDP-glucose was also effective in elimination of heat-induced misfolding. Incubation of the microsomes with an alpha-glucosidase inhibitor, castanospermine, prolonged the association of transferrin with the chaperones and prevented completion of folding and, importantly, aggregate formation, particularly in the calnexin complex. Accordingly, we demonstrate that repeated binding of the chaperones to the glucose of the transferrin sugar moiety prevents and corrects misfolding of the protein.     

Procollagen folding and assembly: the role of endoplasmic reticulum enzymes and molecular chaperones

Procollagen assembly occurs within the endoplasmic reticulum, where the C-propeptide domains of three polypeptide alpha-chains fold individually, and then interact and trimerise to initiate folding of the triple helical region. This highly complex folding and assembly pathway requires the co-ordinated action of a large number of endoplasmic reticulum-resident enzymes and molecular chaperones. Disease-causing mutations in the procollagens disturb folding and assembly and lead to prolonged interactions with molecular chaperones, retention in the endoplasmic reticulum, and intracellular degradation. This review focuses predominantly on prolyl 1-hydroxylase, an essential collagen modifying enzyme, and HSP47, a collagen-specific binding protein, and their proposed roles as molecular chaperones involved in fibrillar procollagen folding and assembly, quality control, and secretion.     

Variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and Golgi in human hepatoma cells

We have previously shown that newly synthesized liver secretory proteins are exported at three distinct characteristic rates, with intracellular retention half-times of 110-120 min (e.g. transferrin), 75-80 min (e.g. ceruloplasmin), and 30-40 min (e.g. alpha 1-protease inhibitor) (J. B. Parent, H. Bauer, and K. Olden (1985) Biochim. Biophys. Acta, in press). In the present study we have determined the average time required for specific glycoproteins to move through the various compartments of the intracellular transport pathway, consisting of endoplasmic reticulum and Golgi complex. Localization in particular compartments was monitored by the use of the following complementary approaches: (i) Percoll density gradient fractionation of the subcellular organelles, (ii) sensitivity of the glycan moiety of N-linked glycosylation to endo-beta-N-acetylglucosaminidase H, and (iii) by the lectin-binding characteristics. The cell fractionation studies revealed that alpha 1-protease inhibitor, ceruloplasmin, and transferrin were transported from the rough endoplasmic reticulum with a retention half-time of 10, 30, or 45 min, respectively. Measurements of the rate at which newly synthesized glycoprotein became endo H-resistant (an event localized near the medial region of Golgi) demonstrated that it took 60-70, 30, and 18 min for 50% of transferrin, ceruloplasmin, and alpha 1-protease inhibitor, respectively, to reach the medial Golgi. Consistent with this finding, maximal binding of transferrin to wheat germ agglutinin (also a medial Golgi event) and Ricinus communis agglutinin I (a trans Golgi event) required 75 and 90 min, respectively, and maximal binding of ceruloplasmin to both lectins occurred in approximately 30 min. Maximal binding of alpha 1-protease inhibitor to wheat germ agglutinin and Ricinus communis agglutinin I required 15 and 30 min, respectively. The results presented here clearly indicate that (i) the time required for protein secretion cannot be entirely accounted for by lag in transport from the rough endoplasmic reticulum to the Golgi since the glycoproteins examined are retained in the former organelle for no more than two-fifths of the total intracellular retention half-time, and (ii) the variability in rates of protein secretion is not due solely to differences in rates of transport from the rough endoplasmic reticulum to the Golgi as variability in retention within the Golgi is also demonstrated. The results are discussed in terms of their compatibility with receptor-mediated transport of glycoproteins in both the endoplasmic reticulum and Golgi.     

PHO5-LACZ hybrid proteins block translocation of native acid phosphatase in Saccharomyces cerevisiae

A set of protein hybrids composed of variable portions of the amino-terminal residues of the yeast phosphate-repressible acid phosphatase (product of PHO5) and an active fragment of bacterial beta-galactosidase has been constructed. When these PHO5-LACZ hybrids are expressed in a yeast strain carrying an intact chromosomal PHO5 gene, they show a size-dependent interference with the secretion of native acid phosphatase. Hybrid proteins containing approximately 50 residues of acid phosphatase do not affect secretion of native acid phosphatase. Hybrids containing greater than 200 residues of acid phosphatase reduce the amount of secreted acid phosphatase more than by 50%. The interference with secretion is specific for acid phosphatase. The hybrids do not affect secretion of invertase, and do not confer a growth-deficient phenotype on yeast. Both the hybrid proteins and acid phosphatase accumulate in non-glycosylated, membrane-bound forms which are sensitive to proteolysis from the cytoplasmic side of the membrane. The hybrids and accumulated acid phosphatase co-migrate on Percoll density gradients with markers of the endoplasmic reticulum, but not with markers of the Golgi or secretory vesicles. These results suggest that PHO5-LACZ hybrid proteins specifically block secretion of native acid phosphatase by interfering with enzyme after targeting but before translocation across the endoplasmic reticulum.     

The effect of dexamethasone on transferrin secretion by cultured fetal rat hepatocytes

Cultured fetal rat hepatocytes derived from 12, 15 and 19-day gestation rats are capable of secreting transferrin. When dexamethasone is added to the medium an increased secretion rate is observed. The changes in secretion rates in control as well as dexamethasone-treated cells during culture have been shown to correlate with the level of mRNA coding for transferrin. Immunocytochemical experiments show that initially all hepatocytes contain transferrin which is localized in the lumina of the perinuclear space, rough endoplasmic reticulum and in the saccules and vesicles of the Golgi apparatus. During culture, particularly in control cells, the intensity of labelling varies from cell to cell. In addition, adjacent cells are observed to label more intensely in different intracellular organelles.     

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.     

HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells

Hsp40 co-chaperones, characterized by the presence of a highly conserved J domain, are involved in nearly all aspects of protein synthesis, folding, and secretion. Within the lumen of the endoplasmic reticulum, these chaperones are also involved in reverse translocation and degradation of misfolded proteins. We describe here the cloning and characterization of a novel Hsp40 chaperone, which we named HEDJ. Epitope-tagged HEDJ was demonstrated by confocal microscopy to be localized to the endoplasmic reticulum. Protease susceptibility, glycosidase treatment, and detergent solubility assays demonstrated that the molecule was luminally oriented and membrane-associated. In vitro experiments demonstrated that the J domain interacted with the endoplasmic reticulum-associated Hsp70, Bip, in an ATP-dependent manner and was capable of stimulating its ATPase activity. HEDJ mRNA expression was detected in all human tissues examined. Highly homologous sequences were found in mouse, Drosophila, and Caenorhabditis elegans data bases. These results suggest potential roles for HEDJ in protein import, folding, or translocation within the endoplasmic reticulum.