Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Robust post-translocational N-glycosylation at the extreme C-terminus of membrane and secreted proteins in Xenopus laevis oocytes and HEK293 cells

N-Glycosylation is normally a co-translational process that occurs as soon as a nascent and unfolded polypeptide chain has emerged ~12 residues into the lumen of the endoplasmic reticulum (ER). Here, we describe the efficient utilization of an N-glycosylation site engineered within the luminal extreme C-terminal residues of distinct integral membrane glycoproteins, a native ER resident protein and an engineered secreted protein. This N-glycan addition required that the acceptor asparagine within an Asn-Trp-Ser sequon be located at least four residues away from the C-terminus of the polypeptide and, in the case of membrane proteins, at least 13 residues away from the lumenal side of the transmembrane segment. Pulse-chase assays revealed that the natural N-glycans of the proteins studied were attached co-translationally, whereas C-terminal N-glycosylation occurred post-translocationally within a time frame of hours in Xenopus laevis oocytes and minutes in human embryonic kidney 293 (HEK293) cells. In oocyte and HEK cell expression systems, affinity tag-driven C-terminal N-glycosylation may facilitate the determination of orientation of the C-terminal tail of membrane proteins relative to the membrane.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.     

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.     

N-glycan structure dictates extension of protein folding or onset of disposal

The endoplasmic reticulum (ER) is the site of folding for proteins that are resident in the ER or that are destined for the Golgi, endosomes, lysosomes, the plasma membrane, or secretion. Cotranslational addition of preassembled glucose(3)-mannose(9)-N-acetylglucosamine(2) core oligosaccharides (N-glycosylation) is a common event for polypeptides synthesized in this compartment. Protein-bound oligosaccharides are exposed to several ER glycanases that sequentially remove terminal glucose or mannose residues. Their activity must be tightly regulated because the N-glycan composition determines whether the associated protein is subjected to folding attempts in the ER lumen or whether it is retrotranslocated into the cytosol and degraded.     

Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ～20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.     

Topology, subcellular localization, and sequence diversity of the Mlo family in plants

Barley Mlo defines the founder of a novel class of plant integral membrane proteins. Lack of the wild type protein leads to broad spectrum disease resistance against the pathogenic powdery mildew fungus and deregulated leaf cell death. Scanning N-glycosylation mutagenesis and Mlo-Lep fusion proteins demonstrated that Mlo is membrane-anchored by 7 transmembrane (TM) helices such that the N terminus is located extracellularly and the C terminus intracellularly. Fractionation of leaf cells and immunoblotting localized the protein to the plant plasma membrane. A genome-wide search for Mlo sequence-related genes in Arabidopsis thaliana revealed approximately 35 family members, the only abundant gene family encoding 7 TM proteins in higher plants. The sequence variability of Mlo family members within a single species, their topology and subcellular localization are reminiscent of the most abundant class of metazoan 7 TM receptors, the G-protein-coupled receptors.     

Autocatalytic cleavage of human gamma-glutamyl transpeptidase is highly dependent on N-glycosylation at asparagine 95

γ-Glutamyl transpeptidase (GGT) is a heterodimeric membrane enzyme that catalyzes the cleavage of extracellular glutathione and other γ-glutamyl-containing compounds. GGT is synthesized as a single polypeptide (propeptide) that undergoes autocatalytic cleavage, which results in the formation of the large and small subunits that compose the mature enzyme. GGT is extensively N-glycosylated, yet the functional consequences of this modification are unclear. We investigated the effect of N-glycosylation on the kinetic behavior, stability, and functional maturation of GGT. Using site-directed mutagenesis, we confirmed that all seven N-glycosylation sites on human GGT are modified by N-glycans. Comparative enzyme kinetic analyses revealed that single substitutions are functionally tolerated, although the N95Q mutation resulted in a marked decrease in the cleavage efficiency of the propeptide. However, each of the single site mutants exhibited decreased thermal stability relative to wild-type GGT. Combined mutagenesis of all N-glycosylation sites resulted in the accumulation of the inactive propeptide form of the enzyme. Use of N-glycosylation inhibitors demonstrated that binding of the core N-glycans, not their subsequent processing, is the critical glycosylation event governing the autocleavage of GGT. Although N-glycosylation is necessary for maturation of the propeptide, enzymatic deglycosylation of the mature wild-type GGT does not substantially impact either the kinetic behavior or thermal stability of the fully processed human enzyme. These findings are the first to establish that co-translational N-glycosylation of human GGT is required for the proper folding and subsequent cleavage of the nascent propeptide, although retention of these N-glycans is not necessary for maintaining either the function or structural stability of the mature enzyme.     

Signals and mechanisms for protein retention in the endoplasmic reticulum

After their co-translational insertion into the ER lumen or the ER membrane, most proteins are transported via the Golgi apparatus downstream on the secretory pathway while a few protein species are retained in the ER. Polypeptide retention in the ER is either signal-independent or depends on specific retention signals encoded by the primary sequence of the polypeptide. A first category, i.e. the newly synthesized polypeptides that are unable to reach their final conformation, are retained in the ER where this quality control generally results in their degradation. A second category, namely the ER-resident proteins escape the bulk flow of secretion due to the presence of a specific N- or C-terminal signal that interacts with integral membrane or soluble receptors. ER retention of soluble proteins mediated by either KDEL, HDEL or related sequences and membrane receptors has been relatively well characterized in plants. Recent efforts has been relatively well characterized in plants. Recent efforts have aimed at a characterization of the retention signal(s) of type I membrane proteins in the plant ER.     

Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation

In eukaryotic cells, surface expression of most type I transmembrane proteins requires translation and simultaneous insertion of the precursor protein into the endoplasmic reticulum (ER) membrane for subsequent routing to the cell surface. This co-translational translocation pathway is initiated when a hydrophobic N-terminal signal peptide (SP) on the nascent protein emerges from the ribosome, binds the cytosolic signal recognition particle (SRP), and targets the ribosome-nascent chain complex to the Sec61 translocon, a universally conserved protein-conducting channel in the ER-membrane. Despite their common function in Sec61 targeting and ER translocation, SPs have diverse but unique primary sequences. Thus, drugs that recognise SPs could be exploited to inhibit translocation of specific proteins into the ER. Here, through flow cytometric analysis the small-molecule macrocycle cyclotriazadisulfonamide (CADA) is identified as a highly selective human CD4 (hCD4) down-modulator. We show that CADA inhibits CD4 biogenesis and that this is due to its ability to inhibit co-translational translocation of CD4 into the lumen of the ER, both in cells as in a cell-free in vitro translation/translocation system. The activity of CADA maps to the cleavable N-terminal SP of hCD4. Moreover, through surface plasmon resonance analysis we were able to show direct binding of CADA to the SP of hCD4 and identify this SP as the target of our drug. Furthermore, CADA locks the SP in the translocon during a post-targeting step, possibly in a folded state, and prevents the translocation of the associated protein into the ER lumen. Instead, the precursor protein is routed to the cytosol for degradation. These findings demonstrate that a synthetic, cell-permeable small-molecule can be developed as a SP-binding drug to selectively inhibit protein translocation and to reversibly regulate the expression of specific target proteins.     

Membrane topology of S2P, a protein required for intramembranous cleavage of sterol regulatory element-binding proteins.

In sterol-depleted mammalian cells, a two-step proteolytic process releases the NH(2)-terminal domains of sterol regulatory element-binding proteins (SREBPs) from membranes of the endoplasmic reticulum (ER). These domains translocate into the nucleus, where they activate genes of cholesterol and fatty acid biosynthesis. The SREBPs are oriented in the membrane in a hairpin fashion, with the NH(2)- and COOH-terminal domains facing the cytosol and a single hydrophilic loop projecting into the lumen. The first cleavage occurs at Site-1 within the ER lumen to generate an intermediate that is subsequently released from the membrane by cleavage at Site-2, which lies within the first transmembrane domain. A membrane protein, designated S2P, a putative zinc metalloprotease, is required for this cleavage. Here, we use protease protection and glycosylation site mapping to define the topology of S2P in ER membranes. Both the NH(2) and COOH termini of S2P face the cytosol. Most of S2P is hydrophobic and appears to be buried in the membrane. All three of the long hydrophilic sequences of S2P can be glycosylated, indicating that they all project into the lumen. The HEIGH sequence of S2P, which contains two potential zinc-coordinating residues, is contained within a long hydrophobic segment. Aspartic acid 467, located approximately 300 residues away from the HEIGH sequence, appears to provide the third coordinating residue for the active site zinc. This residue, too, is located in a hydrophobic sequence. The hydrophobicity of these sequences suggests that the active site of S2P is located within the membrane in an ideal position to cleave its target, a Leu-Cys bond in the first transmembrane helix of SREBPs.     

Biogenesis and transmembrane topology of the CHIP28 water channel at the endoplasmic reticulum

CHIP28 is a 28-kD hydrophobic integral membrane protein that functions as a water channel in erythrocytes and renal tubule epithelial cell membranes. We examined the transmembrane topology of CHIP28 in the ER by engineering a reporter of translocation (derived from bovine prolactin) into nine sequential sites in the CHIP28 coding region. The resulting chimeras were expressed in Xenopus oocytes, and the topology of the reporter with respect to the ER membrane was determined by protease sensitivity. We found that although hydropathy analysis predicted up to seven potential transmembrane regions, CHIP28 spanned the membrane only four times. Two putative transmembrane helices, residues 52-68 and 143-157, reside on the lumenal and cytosolic surfaces of the ER membrane, respectively. Topology derived from these chimeric proteins was supported by cell-free translation of five truncated CHIP28 cDNAs, by N-linked glycosylation at an engineered consensus site in native CHIP28 (residue His69), and by epitope tagging of the CHIP28 amino terminus. Defined protein chimeras were used to identify internal sequences that direct events of CHIP28 topogenesis. A signal sequence located within the first 52 residues initiated nascent chain translocation into the ER lumen. A stop transfer sequence located in the hydrophobic region from residues 90-120 terminated ongoing translocation. A second internal signal sequence, residues 155-186, reinitiated translocation of a COOH-terminal domain (residues 186-210) into the ER lumen. Integration of the nascent chain into the ER membrane occurred after synthesis of 107 residues and required the presence of two membrane-spanning regions. From this data, we propose a structural model for CHIP28 at the ER membrane in which four membrane- spanning alpha-helices form a central aqueous channel through the lipid bilayer and create a pathway for water transport.     

The signal peptide as a new target for drug design

Many current and potential drug targets are membrane-bound or secreted proteins that are expressed and transported via the Sec61 secretory pathway. They are targeted to translocon channels across the membrane of the endoplasmic reticulum (ER) by signal peptides (SPs), which are temporary structures on the N-termini of their nascent chains. During translation, such proteins enter the lumen and membrane of the ER by a process known as co-translational translocation. Small molecules have been found that interfere with this process, decreasing protein expression by recognizing the unique structures of the SPs of particular proteins. The SP may thus become a validated target for designing drugs for numerous disorders, including certain hereditary diseases.     

Molecular analysis of an auxin binding protein gene located on chromosome 4 of Arabidopsis.

We have isolated a cDNA clone from Arabidopsis, At-ERabp1, for the Arabidopsis auxin binding protein located in the lumen of the endoplasmic reticulum (ER). This cDNA clone codes for a protein related to the major auxin binding protein from maize, Zm-ERabp1. A single open reading frame, 594 bases in length, predicts a protein of 198 amino acid residues and a molecular mass of 22,044 D. The primary amino acid sequence contains an N-terminal hydrophobic signal sequence of 33 amino acids. We demonstrated by in vitro studies that the At-ERabp1 protein is translocated into ER-derived microsomes. The protein was processed, and the cleavage site for the N-terminal signal peptide was determined by radiosequencing. The mature protein is composed of 165 amino acid residues, with a molecular mass of 18,641 D. The At-ERabp1 protein contains potential N-glycosylation sites (Asn46-Ile-Ser and Asn130-Ser-Thr). In vitro transport studies demonstrated cotranslational glycosylation. Retention within the lumen of the ER correlates with an additional signal located at the C terminus and represented by the amino acids Lys196-Asp-Glu-Leu, well known to be essential for active retrieval of proteins into the lumen of the ER. DNA gel blot analysis of genomic DNA revealed single hybridizing bands, suggesting that only a single At-ERabp1 gene is present in the Arabidopsis genome. Restriction fragment length polymorphism mapping indeed revealed a single locus mapping to chromosome 4.     

Identification of the hydrophobic glycoproteins of Caenorhabditis elegans

Hydrophobic proteins such as integral membrane proteins are difficult to separate, and therefore to study, at a proteomics level. However, the Asn-linked (N-linked) carbohydrates (N-glycans) contained in membrane glycoproteins are important in differentiation, embryogenesis, inflammation, cancer and metastasis, and other vital cellular processes. Thus, the identification of these proteins and their sites of glycosylation in a well-characterized model organism is the first step toward understanding the mechanisms by which N-glycans and their associated proteins function in vivo. In this report, a proteomics method recently developed by our group was applied to identify 117 hydrophobic N-glycosylated proteins of Caenorhabditis elegans extracts by analysis of 195 glycopeptides containing 199 Asn-linked oligosaccharides. Most of the proteins identified are involved in cell adhesion, metabolism, or the transport of small molecules. In addition, there are 18 proteins for which no function is known or predictable by sequence homologies and two proteins which were previously predicted to exist only on the basis of genomic sequences in the C. elegans database. Because N-glycosylation is initiated in the lumen of the endoplasmic reticulum (ER), our data can be used to reassess the previously predicted subcellular localizations of these proteins. As well, the identification of N-glycosylation sites helps establish the membrane topology of the associated glycoproteins. Caenorhabditis elegans strains are presently available with mutations in 17 of the genes we have identified. The powerful genetic tools available for C. elegans can be used to make other strains with mutations in genes encoding N-glycosylated proteins and thereby determine N-glycan function.     

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.     

O-mannosylation protects mutant alpha-factor precursor from endoplasmic reticulum-associated degradation

Secretory proteins that fail to fold in the endoplasmic reticulum (ER) are transported back to the cytosol and degraded by proteasomes. It remains unclear how the cell distinguishes between folding intermediates and misfolded proteins. We asked whether misfolded secretory proteins are covalently modified in the ER before export. We found that a fraction of mutant alpha-factor precursor, but not the wild type, was progressively O-mannosylated in microsomes and in intact yeast cells by protein O-mannosyl transferase 2 (Pmt2p). O-Mannosylation increased significantly in vitro under ER export conditions, i.e., in the presence of ATP and cytosol, and this required export-proficient Sec61p in the ER membrane. Deletion of PMT2, however, did not abrogate mutant alpha-factor precursor degradation but, rather, enhanced its turnover in intact yeast cells. In vitro, O-mannosylated mutant alpha-factor precursor was stable and protease protected, and a fraction was associated with Sec61p in the ER lumen. Thus, prolonged ER residence allows modification of exposed O-mannosyl acceptor sites in misfolded proteins, which abrogates misfolded protein export from the ER at a posttargeting stage. We conclude that there is a limited window of time during which misfolded proteins can be removed from the ER before they acquire inappropriate modifications that can interfere with disposal through the Sec61 channel.     

The signal recognition particle receptor alpha subunit assembles co-translationally on the endoplasmic reticulum membrane during an mRNA-encoded translation pause in vitro.

Many proteins, including the alpha subunit of the signal recognition particle receptor (SR alpha), are targeted within the cell by poorly defined mechanisms. A 140 residue N-terminal domain of SR alpha targets and anchors the polypeptide to the endoplasmic reticulum membrane by a mechanism independent of the pathway involving the signal recognition particle. To investigate the mechanism of membrane anchoring, translation pause sites on the SR alpha mRNA were used to examine the targeting of translation intermediates. A strong pause site at nucleotide 507 of the mRNA open reading frame corresponded with the shortest nascent SR alpha polypeptide able to assemble on membranes. An mRNA sequence at this pause site that resembles a class of viral -1 frameshift sequences caused translation pausing when transferred into another mRNA context. Site-directed mutagenesis of the mRNA greatly reduced translation pausing without altering the polypeptide sequence, demonstrating unambiguously a role for this mRNA sequence in translation pausing. SR alpha polypeptides synthesized from the non-pausing mRNA were impaired in co-translational membrane anchoring. Furthermore, co-translational membrane assembly of SR alpha appears to anchor polysomes translating SR alpha to membranes.     

Identification of functional domains of Mid1, a stretch-activated channel component, necessary for localization to the plasma membrane and Ca2+ permeation

The Saccharomyces cerevisiae MID1 gene product (Mid1) is a stretch-activated Ca(2+)-permeable channel component required for Ca2+ influx and the maintenance of viability of cells exposed to the mating pheromone, alpha-factor. It is composed of 548-amino-acid (aa) residues with four hydrophobic segments, H1 (aa 2-22), H2 (aa 92-111), H3 (aa 337-356) and H4 (aa 366-388). It also has 16 putative N-glycosylation sites. In this study, sequentially truncated Mid1 proteins conjugated with GFP were expressed in S. cerevisiae cells. The truncated protein containing the region from H1 to H3 (Mid1(1-360)-GFP) localized normally in the plasma and endoplasmic reticulum (ER) membranes and complemented the low viability and Ca(2+)-uptake activity of the mid1 mutant, whereas Mid1(1-133)-GFP containing the region from H1 to H2 did not. Mid1(Delta3-22)-GFP lacking the H1 region failed to localize in the plasma membrane. Membrane fractionation showed that Mid1(1-22)-GFP containing only H1 localized in the plasma membrane in the presence of alpha-factor, suggesting that H1 is a signal sequence responsible for the alpha-factor-induced Mid1 delivery to the plasma membrane. The region from H1 to H3 is required for the localization of Mid1 in the plasma and ER membranes. Finally, trafficking of Mid1-GFP to the plasma membrane was dependent on the N-glycosylation of Mid1 and the transporter protein Sec12.     

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H₂O₂ in the endoplasmic reticulum (ER). Approaches to quantifying H₂O₂ directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H₂O₂ concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H₂O₂. With the expression of ER-Catalase N244, a highly effective H₂O₂ inactivation within the ER was achieved for the first time. Catalase has a high H₂O₂-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H₂O₂ on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H₂O₂.