Interplays Between Covalent Modifications in the Endoplasmic Reticulum Increase Conformational Diversity in Nascent Prion Protein

Prion protein (PrP), the causative agent of transmissible spongiform encephalopathies, is synthesized in the endoplasmic reticulum (ER) where it undergoes numerous covalent modifications. Here we investigate the interdependence and regulation of PrP oxidative folding, N-glycosylation and GPI addition in diverse ER conditions. Our results show that formation of the single disulphide bond is a pivotal event, essential for PrP transport, and can occur post-translationally. Retarding its formation enhances N-glycosylation and GPI-anchoring. In contrast, lowering ER Ca²⁺ concentration inhibits N-glycosylation and GPI-anchoring. These data reveal tight interplays between the different ER covalent modifications, which collectively increase of PrP conformational diversity and may be important for its propagation.Key Words: Ca²⁺ homeostasis, ER-golgi transport, GPI-anchoring, N-glycosylation, oxidative folding, redox regulation     

Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

Conserved Roles of the Prion Protein Domains on Subcellular Localization and Cell-Cell Adhesion

Analyses of cultured cells and transgenic mice expressing prion protein (PrP) deletion mutants have revealed that some properties of PrP -such as its ability to misfold, aggregate and trigger neurotoxicity- are controlled by discrete molecular determinants within its protein domains. Although the contributions of these determinants to PrP biosynthesis and turnover are relatively well characterized, it is still unclear how they modulate cellular functions of PrP. To address this question, we used two defined activities of PrP as functional readouts: 1) the recruitment of PrP to cell-cell contacts in Drosophila S2 and human MCF-7 epithelial cells, and 2) the induction of PrP embryonic loss- and gain-of-function phenotypes in zebrafish. Our results show that homologous mutations in mouse and zebrafish PrPs similarly affect their subcellular localization patterns as well as their in vitro and in vivo activities. Among PrP’s essential features, the N-terminal leader peptide was sufficient to drive targeting of our constructs to cell contact sites, whereas lack of GPI-anchoring and N-glycosylation rendered them inactive by blocking their cell surface expression. Importantly, our data suggest that the ability of PrP to homophilically trans-interact and elicit intracellular signaling is primarily encoded in its globular domain, and modulated by its repetitive domain. Thus, while the latter induces the local accumulation of PrPs at discrete punctae along cell contacts, the former counteracts this effect by promoting the continuous distribution of PrP. In early zebrafish embryos, deletion of either domain significantly impaired PrP’s ability to modulate E-cadherin cell adhesion. Altogether, these experiments relate structural features of PrP to its subcellular distribution and in vivo activity. Furthermore, they show that despite their large evolutionary history, the roles of PrP domains and posttranslational modifications are conserved between mouse and zebrafish.     

Conditions of endoplasmic reticulum stress favor the accumulation of cytosolic prion protein

After signal sequence-dependent targeting to the endoplasmic reticulum (ER), prion protein (PrP) undergoes several post-translational modifications, including glycosylation, disulfide bond formation, and the addition of a glycosylphosphatidylinositol anchor. As a result, multiple isoforms are generated. Because of the intrinsic weakness of the PrP signal sequence, a fraction of newly synthesized molecules fails to translocate and localizes to the cytosol. The physiopathologic role of this cytosolic isoform is still being debated. Here we have shown that, in both cultured cell lines and primary neurons, ER stress conditions weaken PrP co-translational translocation, favoring accumulation of aggregation-prone cytosolic species, which retain the signal sequence but lack N-glycans and disulfides. Inhibition of proteasomes further increases the levels of cytosolic PrP. Overexpression of spliced XBP1 facilitates ER translocation, suggesting that downstream elements of the Ire1-XBP1 pathway are involved in PrP targeting. These studies reveal a link between ER stress and the formation of cytosolic PrP isoforms potentially endowed with novel signaling or cytotoxic functions.     

Cysteineless non-glycosylated monomeric blue fluorescent protein,secBFP2, for studies in the eukaryotic secretory pathway

Fluorescent protein (FP) technologies suitable for use within the eukaryotic secretory pathway are essential for live cell and protein dynamic studies. Localization of FPs within the endoplasmic reticulum (ER) lumen has potentially significant consequences for FP function. All FPs are resident cytoplasmic proteins and have rarely been evolved for the chemically distinct environment of the ER lumen. In contrast to the cytoplasm, the ER lumen is oxidizing and the site where secretory proteins are post-translationally modified by disulfide bond formation and N-glycosylation on select asparagine residues. Cysteine residues and N-linked glycosylation consensus sequences were identified within many commonly utilized FPs. Here, we report mTagBFP is post-translationally modified when localized to the ER lumen. Our findings suggest these modifications can grossly affect the sensitivity and reliability of FP tools within the secretory pathway. To optimize tools for studying events in this important intracellular environment, we modified mTagBFP by mutating its cysteines and consensus N-glycosylation sites. We report successful creation of a secretory pathway-optimized blue FP, secBFP2.     

Post-translational import of the prion protein into the endoplasmic reticulum interferes with cell viability: a critical role for the putative transmembrane domain

Aberrant folding of the mammalian prion protein (PrP) is linked to prion diseases in humans and animals. We show that during post-translational targeting of PrP to the endoplasmic reticulum (ER) the putative transmembrane domain induces misfolding of PrP in the cytosol and interferes with its import into the ER. Unglycosylated and misfolded PrP with an uncleaved N-terminal signal sequence associates with ER membranes, and, moreover, decreases cell viability. PrP expressed in the cytosol, lacking the N-terminal ER targeting sequence, also adopts a misfolded conformation; however, this has no adverse effect on cell growth. PrP processing, productive ER import, and cellular viability can be restored either by deleting the putative transmembrane domain or by using a N-terminal signal sequence specific for co-translational ER import. Our study reveals that the putative transmembrane domain features in the formation of misfolded PrP conformers and indicates that post-translational targeting of PrP to the ER can decrease cell viability.     

N-glycans and glycosylphosphatidylinositol-anchor act on polarized sorting of mouse PrP(C) in Madin-Darby canine kidney cells

The cellular prion protein (PrP(C)) plays a fundamental role in prion disease. PrP(C) is a glycosylphosphatidylinositol (GPI)-anchored protein with two variably occupied N-glycosylation sites. In general, GPI-anchor and N-glycosylation direct proteins to apical membranes in polarized cells whereas the majority of mouse PrP(C) is found in basolateral membranes in polarized Madin-Darby canine kidney (MDCK) cells. In this study we have mutated the first, the second, and both N-glycosylation sites of PrP(C) and also replaced the GPI-anchor of PrP(C) by the Thy-1 GPI-anchor in order to investigate the role of these signals in sorting of PrP(C) in MDCK cells. Cell surface biotinylation experiments and confocal microscopy showed that lack of one N-linked oligosaccharide leads to loss of polarized sorting of PrP(C). Exchange of the PrP(C) GPI-anchor for the one of Thy-1 redirects PrP(C) to the apical membrane. In conclusion, both N-glycosylation and GPI-anchor act on polarized sorting of PrP(C), with the GPI-anchor being dominant over N-glycans.     

Drosophila segment polarity gene product porcupine stimulates the posttranslational N-glycosylation of wingless in the endoplasmic reticulum

Wnt is a family of cysteine-rich secreted glycoproteins, which controls the fate and behavior of the cells in multicellular organisms. In the absence of Drosophila segment polarity gene porcupine (porc), which encodes an endoplasmic reticulum (ER) multispanning transmembrane protein, the N-glycosylation of Wingless (Wg), one of Drosophila Wnt family, is impaired. In contrast, the ectopic expression of porc stimulates the N-glycosylation of both endogenously and exogenously expressed Wg. The N-glycosylation of Wg in the ER occurs posttranslationally, while in the presence of dithiothreitol, it efficiently occurs cotranslationally. Thus, the cotranslational disulfide bond formation of Wg competes with the N-glycosylation by an oligosaccharyl transferase complex. Porc binds the N-terminal 24-amino acid domain (residues 83-106) of Wg, which is highly conserved in the Wnt family and stimulates the N-glycosylation at surrounding sites. Porc is also necessary for the processing of Drosophila Wnt-3/5 in both embryos and cultured cells. Thus, Porc binds the N-terminal specific domain of the Wnt family and stimulates its posttranslational N-glycosylation by anchoring them at the ER membrane possibly through acylation.     

Recombinant anti-hCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues

Plant-based expression systems are attractive for the large-scale production of pharmaceutical proteins. However, glycoproteins require particular attention as inherent differences in the N-glycosylation pathways of plants and mammals result in the production of glycoproteins bearing core-xylose and core-alpha(1,3)-fucose glyco-epitopes. For treatments requiring large quantities of repeatedly administered glycoproteins, the immunological properties of these non-mammalian glycans are a concern. Recombinant glycoproteins could be retained within the endoplasmic reticulum (ER) to prevent such glycan modifications occurring in the late Golgi compartment. Therefore, we analysed cPIPP, a mouse/human chimeric IgG1 antibody binding to the beta-subunit of human chorionic gonadotropin (hCG), fused to a C-terminal KDEL sequence, to investigate the efficiency of ER retrieval and the consequences in terms of N-glycosylation. The KDEL-tagged cPIPP antibody was expressed in transgenic tobacco plants or Agrobacterium-infiltrated tobacco and winter cherry leaves. N-Glycan analysis showed that the resulting plantibodies contained only high-mannose (Man)-type Man-6 to Man-9 oligosaccharides. In contrast, the cPIPP antibody lacking the KDEL sequence was found to carry complex N-glycans containing core-xylose and core-alpha(1,3)-fucose, thereby demonstrating the secretion competence of the antibody. Furthermore, fusion of KDEL to the diabody derivative of PIPP, which contains an N-glycosylation site within the heavy chain variable domain, also resulted in a molecule lacking complex glycans. The complete absence of xylose and fucose residues clearly shows that the KDEL-mediated ER retrieval of cPIPP or its diabody derivative is efficient in preventing the formation of non-mammalian complex oligosaccharides.     

Glycoprotein folding and quality-control mechanisms in protein-folding diseases

Biosynthesis of proteins – from translation to folding to export – encompasses a complex set of events that are exquisitely regulated and scrutinized to ensure the functional quality of the end products. Cells have evolved to capitalize on multiple post-translational modifications in addition to primary structure to indicate the folding status of nascent polypeptides to the chaperones and other proteins that assist in their folding and export. These modifications can also, in the case of irreversibly misfolded candidates, signal the need for dislocation and degradation. The current Review focuses on the glycoprotein quality-control (GQC) system that utilizes protein N-glycosylation and N-glycan trimming to direct nascent glycopolypeptides through the folding, export and dislocation pathways in the endoplasmic reticulum (ER). A diverse set of pathological conditions rooted in defective as well as over-vigilant ER quality-control systems have been identified, underlining its importance in human health and disease. We describe the GQC pathways and highlight disease and animal models that have been instrumental in clarifying our current understanding of these processes.KEY WORDS: N-glycosylation, Glycoprotein folding, ER quality control, ER-associated degradation, ER export     

Membrane topology influences N-glycosylation of the prion protein

The glycosylation state of the glycosyl-phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N-glycosylation of PrP by expressing a C-terminal transmembrane anchored form, PrP-CTM, an N-terminal transmembrane anchored form, PrP-NTM, a double-anchored form, PrP-DA, and a truncated form, PrPDeltaGPI, in human neuroblastoma SH-SY5Y cells. Wild-type PrP, PrP- CTM and PrP-DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP-NTM, although membrane anchored and localized at the cell surface, was not N-glycosylated. PrPDeltaGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4-fold slower rate at which PrPDeltaGPI was trafficked through the cell compared with wild-type PrP was due to the absence of the GPI anchor not the lack of N-glycans. Retention of PrPDeltaGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C-terminal membrane anchorage is required for N-glycosylation of PrP.     

Glycosylation disorders of membrane trafficking

During evolution from prokaryotic to eukaryotic cells, compartmentalization of cellular functions has been achieved with a high degree of complexity. Notably, all secreted and transmembrane proteins travel through endoplasmic reticulum (ER) and Golgi apparatus, where they are synthesized, folded and subjected to covalent modifications, most particularly glycosylation. N-glycosylation begins in the ER with synthesis and transfer of glycan onto nascent protein and proceeds in Golgi apparatus where maturation occurs. This process not only requires the precise localization of glycosyltransferases, glycosidases and substrates but also an efficient, finely regulated and bidirectional vesicular trafficking among membrane-enclosed organelles. Basically, it is no surprise that alterations in membrane transport or related pathways can lead to glycosylation abnormalities. During the last few years, this has particularly been highlighted in genetic diseases called CDG (Congenital Disorders of Glycosylation). Alterations in mechanisms of vesicle formation due to COPII coat component SEC23B deficiency, or in vesicles tethering, caused by defects of the COG complex, but also impaired Golgi pH homeostasis due to ATP6V0A2 defects have been discovered in CDG patients. This mini review will summarize these fascinating discoveries.     

Spontaneous deamidation and isomerization of Asn108 in prion peptide 106-126 and in full-length prion protein.

In prion-related encephalopathies, the cellular prion protein (PrP(C)) undergoes a change in conformation to become the scrapie prion protein (PrP(Sc)) which forms infectious deposits in the brain. Conceivably, the conformational transition of PrP(C) to PrP(Sc) might be linked with posttranslational alterations in the covalent structure of a fraction of the PrP molecules. We tested a synthetic peptide corresponding to residues 106-126 of human PrP for the occurrence of spontaneous chemical modifications. The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days. The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected by the protein-l-isoaspartyl methyltransferase assay (t(1/2) approximately 30 days). Mass spectrometry and Edman degradation of Lys-C fragments identified Asn108 in the amino-terminal flexible part of the protein to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226' which is only present in rodents.     

Trapping prion protein in the endoplasmic reticulum impairs PrPC maturation and prevents PrPSc accumulation

The conversion of the normal cellular prion protein (PrP(C)) into the abnormal scrapie isoform (PrP(Sc)) is a key feature of prion diseases. The pathogenic mechanisms and the subcellular sites of the conversion are complex and not completely understood. In particular, little is known on the role of the early compartment of the secretory pathway in the processing of PrP(C) and in the pathogenesis of prion diseases. In order to interfere with the intracellular traffic of endogenous PrP(C) we have generated two anti-prion single chain antibody fragments (scFv) directed against different epitopes, each fragment tagged either with a secretory leader or with the ER retention signal KDEL. The stable expression of these constructs in PC12 cells allowed us to study their specific effects on the synthesis, maturation, and processing of endogenous PrP(C) and on PrP(Sc) formation. We found that ER-targeted anti-prion scFvs retain PrP(C) in the ER and inhibit its translocation to the cell surface. Retention in the ER strongly affects the maturation and glycosylation state of PrP(C), with the appearance of a new aberrant endo-H sensitive glycosylated species. Interestingly, ER-trapped PrP(C) acquires detergent insolubility and proteinase K resistance. Furthermore, we show that ER-targeted anti-prion antibodies prevent PrP(Sc) accumulation in nerve growth factor-differentiated PC12 cells, providing a new tool to study the molecular pathology of prion diseases.     

Control of N-linked carbohydrate unit synthesis in thyroid endoplasmic reticulum by membrane organization and dolichyl phosphate availability

Thyroid rough endoplasmic reticulum (ER) has been shown to contain a highly organized multienzyme system capable of carrying out the N-glycosylation of newly synthesized proteins. These reactions were studied in isolated ER vesicles and found to be controlled to a large extent by the availability of a key substrate, dolichyl phosphate (Dol-P), as well as by the amount of endogenous polypeptide acceptor present. Although in intact vesicles UDP-Glc was utilized in an efficient manner to form Dol-P-Glc and glucosylated oligosaccharide-lipid, after disruption of vesicle integrity, even with low concentrations of Triton X-100, the coupling of Dol-P-Glc formation to lipid-linked oligosaccharide assembly and subsequent N-glycosylation was substantially impaired. Increased incubation temperatures also resulted in a decreased effectiveness of glucose transfer from Dol-P-Glc to lipid-oligosaccharide, presumably because of a decline in the extent of structural organization of the ER membranes. The limited availability of endogenous Dol-P was demonstrated by the pronounced stimulation in Dol-P-Glc formation resulting from the addition of this lipid acceptor to Triton-disrupted ER membranes as well as by its generation in intact vesicles. The latter was accomplished by stimulating recycling of endogenous Dol-P through the addition of a peptide (Tyr-Asn-Leu-Thr-Ser-Val) which is an N-glycosylation substrate. The inhibition of Dol-P-Glc synthesis from UDP-Glc observed in the presence of elevated levels of GDP-Man which could be relieved in Triton-disrupted or intact ER vesicles by the addition or generation, respectively, of Dol-P, is considered to be the result of a competing requirement for Dol-P by the mannosyltransferase. Moreover GTP, by selectively inhibiting the mannosyltransferase, prevented the decrease of Dol-P-Glc formation caused by GDP-Man. Since addition of the acceptor peptide to intact vesicles stimulated Dol-P-P-GlcNAc as well as Dol-P-Glc and Dol-P-Man synthesis it would appear that a pool of Dol-P available in common to all three enzymes responsible for dolichol-linked monosaccharide synthesis exists in the ER membranes.     

Prion protein contains a second endoplasmic reticulum targeting signal sequence located at its C terminus

Prion protein (PrP) is synthesized at the membrane of the endoplasmic reticulum (ER) in three different topological forms as follows: a fully translocated one ((sec)PrP) and two with opposite orientations in the membrane ((Ntm)PrP and (Ctm)PrP). We asked whether other signal sequences exist in the PrP, other than the N-terminal signal sequence, that contribute to its topological diversity. In vitro translocation assays showed that PrP lacking its N-terminal signal sequence could still translocate into ER microsomes, although at reduced efficiency. Deletion of each of the two hydrophobic regions in PrP revealed that the C-terminally located hydrophobic region (TM2) can function as second signal sequence in PrP. Translocation mediated by the TM2 alone can occur post-translationally and yields mainly (Ctm)PrP, which is implicated in some forms of neurodegeneration in prion diseases. We conclude that, in vitro, PrP can insert into ER membranes co- and post-translationally and can use two different signal sequences. We propose that the unusually complex topology of PrP results from the differential utilization of two signal sequences in PrP.     

Thioredoxin converts the Syrian hamster (29-231) recombinant prion protein to an insoluble form

The prion protein (PrP) is an essential, and probably the only, component of the infectious agent responsible for the transmissible spongiform encephalopathies. In its cellular (PrP(C)) form, it is a soluble, alpha-helix-rich protein of yet unknown function attached to the outer membrane of neurons through a glycosylphosphatidyl inositol anchor. In its pathogenic, "scrapie" form (PrP(Sc)), it appears as an aggregate showing no detectable covalent modifications but displaying a profoundly altered conformation enriched in beta-sheet structure. Reduction of the single disulfide bridge in the prion protein with millimolar concentrations of dithiothreitol results in transformation of the alpha-helix-rich to the beta-sheet-rich conformation, with concomitant decrease in solubility. We report here that thioredoxin coupled with thioredoxin reductase and NADPH efficiently reduces recombinant Syrian hamster (29-231) prion protein under physiologically relevant conditions. The reduced prion protein immediately becomes insoluble and precipitates, although it does not gain significant resistance to proteinase K. The thioredoxin/thioredoxin reductase system is approximately 7000 times more efficient than dithiothreitol.     

Proteasomal dysfunction and endoplasmic reticulum stress enhance trafficking of prion protein aggregates through the secretory pathway and increase accumulation of pathologic prion protein

A conformational change of the cellular prion protein (PrP(c)) underlies formation of PrP(Sc), which is closely associated with pathogenesis and transmission of prion diseases. The precise conformational prerequisites and the cellular environment necessary for this post-translational process remain to be completely elucidated. At steady state, glycosylated PrP(c) is found primarily at the cell surface, whereas a minor fraction of the population is disposed of by the ER-associated degradation-proteasome pathway. However, chronic ER stress conditions and proteasomal dysfunctions lead to accumulation of aggregation-prone PrP molecules in the cytosol and to neurodegeneration. In this study, we challenged different cell lines by inducing ER stress or inhibiting proteasomal activity and analyzed the subsequent repercussion on PrP metabolism, focusing on PrP in the secretory pathway. Both events led to enhanced detection of PrP aggregates and a significant increase of PrP(Sc) in persistently prion-infected cells, which could be reversed by overexpression of proteins of the cellular quality control. Remarkably, upon proteasomal impairment, an increased fraction of misfolded, fully glycosylated PrP molecules traveled through the secretory pathway and reached the plasma membrane. These findings suggest a novel pathway that possibly provides additional substrate and template necessary for prion formation when protein clearance by the proteasome is impaired.     

Local environmental effects on the structure of the prion protein

Prion diseases are neurodegenerative diseases causally linked to the partial unfolding and subsequent misfolding and aggregation of the prion protein (PrP). While most proteins fold into a single low energy state, PrP can fold into two distinct isoforms. In its innocuous state, denoted as PrPC, the protein has predominantly alpha-helical secondary structure, however, PrPC can misfold into an isoform rich in extended structure capable of forming toxic and infectious aggregates. While prion disease is believed to be a protein-only disease, one not requiring any non-protein elements for propagation, the different environments the protein finds itself in vivo likely influence its ability to misfold and aggregate. In this review we will examine various molecules, covalent modifications and environments PrP faces in vivo and the effect they have on PrP's local environment and, potentially, conformation. Included in this discussion are: (1) pH, (2) carbohydrates, (3) lipid membranes, (4) metal ions, and (5) small molecules.     

Familial CJD associated PrP mutants within transmembrane region induced Ctm-PrP retention in ER and triggered apoptosis by ER stress in SH-SY5Y cells

Background

Genetic prion diseases are linked to point and inserted mutations in the prion protein (PrP) gene that are presumed to favor conversion of the cellular isoform of PrP (PrP^C) to the pathogenic one (PrP^Sc). The pathogenic mechanisms and the subcellular sites of the conversion are not completely understood. Here we introduce several PRNP gene mutations (such as, PrP-KDEL, PrP-3AV, PrP-A117V, PrP-G114V, PrP-P102L and PrP-E200K) into the cultured cells in order to explore the pathogenic mechanism of familial prion disease.

Methodology/Principal Findings

To address the roles of aberrant retention of PrP in endoplasmic reticulum (ER), the recombinant plasmids expressing full-length human PrP tailed with an ER signal peptide at the COOH-terminal (PrP-KDEL) and PrP with three amino acids exchange in transmembrane region (PrP-3AV) were constructed. In the preparations of transient transfections, 18-kD COOH-terminal proteolytic resistant fragments (Ctm-PrP) were detected in the cells expressing PrP-KDEL and PrP-3AV. Analyses of the cell viabilities in the presences of tunicamycin and brefeldin A revealed that expressions of PrP-KDEL and PrP-3AV sensitized the transfected cells to ER stress stimuli. Western blots and RT-PCR identified the clear alternations of ER stress associated events in the cells expressing PrP-KDEL and PrP-3AV that induced ER mediated apoptosis by CHOP and capase-12 apoptosis pathway. Moreover, several familial CJD related PrP mutants were transiently introduced into the cultured cells. Only the mutants within the transmembrane region (G114V and A117V) induced the formation of Ctm-PrP and caused the ER stress, while the mutants outside the transmembrane region (P102L and E200K) failed.

Conclusions/Significance

The data indicate that the retention of PrP in ER through formation of Ctm-PrP results in ER stress and cell apoptosis. The cytopathic activities caused by different familial CJD associated PrP mutants may vary, among them the mutants within the transmembrane region undergo an ER-stress mediated cell apoptosis.