Maturation Of Secretory Proteins Research Articles

Insights into the interaction between UGGT, the gatekeeper of folding in the ER, and its partner, the selenoprotein SEP15

The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.

Heat shock protein Hspa13 regulates endoplasmic reticulum and cytosolic proteostasis through modulation of protein translocation

Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.

Understanding ER homeostasis and the UPR to enhance treatment efficacy of acute myeloid leukemia.

Protein biogenesis, maturation and degradation are tightly regulated processes that are governed by a complex network of signaling pathways. The endoplasmic reticulum (ER) is responsible for biosynthesis and maturation of secretory proteins. Circumstances that alter cellular protein homeostasis, determine accumulation of misfolded and unfolded proteins in the ER, a condition defined as ER stress. In case of stress, the ER activates an adaptive response called unfolded protein response (UPR), a series of pathways of major relevance for cancer biology. The UPR plays a preeminent role in adaptation of tumor cells to the harsh conditions that they experience, due to high rates of proliferation, metabolic abnormalities and hostile environment scarce in oxygen and nutrients. Furthermore, the UPR is among the main adaptive cell stress responses contributing to the development of resistance to drugs and chemotherapy. Clinical management of Acute Myeloid Leukemia (AML) has improved significantly in the last decade, thanks to development of molecular targeted therapies. However, the emergence of treatment-resistant clones renders the rate of AML cure dismal. Moreover, different cell populations that constitute the bone marrow niche recently emerged as a main determinant leading to drug resistance. Herein we summarize the most relevant literature regarding the role played by the UPR in expansion of AML and ability to develop drug resistance and we discuss different possible modalities to overturn this adaptive response against leukemia. To this aim, we also describe the interconnection of the UPR with other cellular stress responses regulating protein homeostasis. Finally, we review the newest findings about the crosstalk between AML cells and cells of the bone marrow niche, under physiological conditions and in response to therapies, discussing in particular the importance of the niche in supporting survival of AML cells by favoring protein homeostasis.

The Toxoplasma Vacuolar H+-ATPase Regulates Intracellular pH and Impacts the Maturation of Essential Secretory Proteins

SUMMARYVacuolar-proton ATPases (V-ATPases) are conserved complexes that couple the hydrolysis of ATP to the pumping of protons across membranes. V-ATPases are known to play diverse roles in cellular physiology. We studied the Toxoplasma gondii V-ATPase complex and discovered a dual role of the pump in protecting parasites against ionic stress and in the maturation of secretory proteins in endosomal-like compartments. Toxoplasma V-ATPase subunits localize to the plasma membrane and to acidic vesicles, and characterization of conditional mutants of the a1 subunit highlighted the functionality of the complex at both locations. Microneme and rhoptry proteins are required for invasion and modulation of host cells, and they traffic via endosome-like compartments in which proteolytic maturation occurs. We show that the V-ATPase supports the maturation of rhoptry and microneme proteins, and their maturases, during their traffic to their corresponding organelles. This work underscores a role for V-ATPases in regulating virulence pathways.

Characterization of Genes Encoding Protein Disulfide Isomerase in Wheat Including the Specific Role of PDI in the Formation of Gluten.

The results of phylogenetic analyses revealed that the family of plant PDI may comprise of at least eight different subfamilies with varying numbers and positions of active centers while retention signals in the endoplasmic reticulum may be present or absent. At least one gene has been cloned for each phylogenetic group. Other phylogenetic analyses have indicated that the family of PDIlike proteins consists of ten classes, the first five of which include proteins equipped with two thioredoxin domains. These results indicate complexity and diversity of the family of protein disulfide isomerase in plants. The study of molecular characteristics of PDI in some cereal species have shown that this enzyme participates in the maturation of secretory proteins and also in the formation of albuminous substances in endosperm, in the mechanism of formation of disulfide bonds and polymerization of gluten polypeptides in wheat. The mechanism of formation of disulfide linkage was tested through in vitro experiments. However, it is not entirely certain whether it reflects their formation in vivo. The results of researches suggest that protein folding and disulfide bond formation occurs in the endoplasmic reticulum. PDI plays a prominent role among enzymes involved in posttranslational modification of proteins. The main goal of this work is to present research data on protein disulfide isomerase, which may be a leading research objective in the area of wheat gluten and the impact of PDI on the baking quality of wheat flour.

Abstract A01: ER stress disrupts circadian rhythms via miR-211

Abstract The Endoplasmic Reticulum, ER, senses both intracellular and extracellular stimuli that perturb the folding and maturation of secretory proteins that transit the ER. In the context of neoplastic growth, oncogenes such as c-Myc, H-Ras and BRAF trigger ER stress and activate the Unfolded Protein Response (UPR), a cell adaptive signaling pathway composed of three primary signal transducers, PERK, IRE-1 and ATF6. Bmal1 and Clock form a core heterodimeric transcription complex regulates both central and peripheral circadian rhythms. There are multiple lines of evidence suggesting that deregulation of circadian rhythms contribute to neoplastic cell growth, altered cellular metabolism and tumorigenesis. Analogously, UPR signaling contributes to tumor cell survival and tumor growth under conditions of micro-environmental and metabolic stress. However, whether UPR interactively regulates stress with the circadian clock is largely unknown. Herein, we describe the molecular mechanisms whereby UPR signaling integrates with the circadian clock. We provide evidence that ER stress-dependent activation of PERK alters circadian oscillations via micro-RNA 211-dependent suppression of BMAL1 and Clock. We provide evidence that the mechanism of regulation of BMAL1 versus clock is distinct, reflecting novel ability of miR-211 to function in both the nucleus and cytoplasm. We provide additional support for this model through using a mouse model to interrogate the role of PERK signaling in a liver specific response to ER stress. With regard to the impact of PERK signaling and circadian clock disruption in cancer, we assessed PERK-miR-211 signaling in c-Myc and MLL-AF9 driven malignancy. Here, expression of either c-Myc or MLL-AF9 expression was directly associated with activation of PERK-miR-211 and loss of both BMAL1 and Clock expression. We also found that Myc driven human Burkitt's lymphoma has PERK hyperactivation, higher miR-211 expression, and loss of circadian oscillations. As the Bmal1 promoter is hypermethylated in multiple lymphoma cancer cell lines, we hypothesize that this is likely to reflect the action of nuclear miR-211. Consistently, anti-miR211 partially released Bmal1 repression and Inhibition PERK or suppression miR-211 rescued circadian rhythms in lymphoma cells thereby affecting their survival and proliferation. Our data suggested a model where ER stress disrupts peripheral circadian rhythms via PERK-miR211-Bmal1/Clock pathway. Targeting activated ER stress or abnormal miR-211 expression to rescue circadian rhythms could be a new potential therapy for Myc- (perhaps also other oncogenes) driven hematological malignancy. Citation Format: Yiwen Bu, John Alan Diehl. ER stress disrupts circadian rhythms via miR-211. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A01.

Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.

Molecular Intricacies and the Role of ER Stress in Diabetes

Molecular Intricacies and the Role of ER Stress in Diabetes

A New Branch of Endoplasmic Reticulum Stress Signaling and the Osmotic Signal Converge on Plant-specific Asparagine-rich Proteins to Promote Cell Death

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene glycol treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activation of the UPR by ER stress inducers, but did not affect activation of NRPs. We also found that this integrated pathway transduces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senescence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmentation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde production, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytokinin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

A High-Throughput Turbidometric Assay for Screening Inhibitors of Protein Disulfide Isomerase Activity

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.

Characterization of a foldase, protein disulfide isomerase A, in the protein secretory pathway of Aspergillus niger.

Protein disulfide isomerase (PDI) is important in assisting the folding and maturation of secretory proteins in eukaryotes. A gene, pdiA, encoding PDIA was previously isolated from Aspergillus niger, and we report its functional characterization here. Functional analysis of PDIA showed that it catalyzes the refolding of denatured and reduced RNase A. pdiA also complemented PDI function in a Saccharomyces cerevisiae Deltapdi1 mutant in a yeast-based killer toxin assay. Levels of pdiA mRNA and PDIA protein were raised by the accumulation of unfolded proteins in the endoplasmic reticulum. This response of pdiA mRNA levels was slower and lower in magnitude than that of A. niger bipA, suggesting that the induction of pdiA is not part of the primary stress response. An increased level of pdiA transcripts was also observed in two A. niger strains overproducing a heterologous protein, hen egg white lysozyme (HEWL). Although overexpression of PDI has been successful in increasing yields of some heterologous proteins in S. cerevisiae, overexpression of PDIA did not increase secreted yields of HEWL in A. niger, suggesting that PDIA itself is not limiting for secretion of this protein. Downregulation of pdiA by antisense mRNA reduced the levels of microsomal PDIA activity by up to 50%, lowered the level of PDIA as judged by Western blots, and lowered the secreted levels of glucoamylase by 60 to 70%.

The protein disulphide-isomerase family: unravelling a string of folds

The mammalian protein disulphide-isomerase (PDI) family encompasses several highly divergent proteins that are involved in the processing and maturation of secretory proteins in the endoplasmic reticulum. These proteins are characterized by the presence of one or more domains of roughly 95-110 amino acids related to the cytoplasmic protein thioredoxin. All but the PDI-D subfamily are composed entirely of repeats of such domains, with at least one domain containing and one domain lacking a redox-active -Cys-Xaa-Xaa-Cys- tetrapeptide. In addition to their known roles as redox catalysts and isomerases, the last few years have revealed additional functions of the PDI proteins, including peptide binding, cell adhesion and perhaps chaperone activities. Attention is now turning to the non-redox-active domains of the PDIs, which may play an important role in all of the known activities of these proteins. Thus the presence of both redox-active and -inactive domains within these proteins portends a complexity of functions differentially accommodated by the various family members.

Role of endoproteolytic dibasic proprotein processing in maturation of secretory proteins in Trichoderma reesei.

Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.

Calumenin, a Ca2+-binding protein retained in the endoplasmic reticulum with a novel carboxyl-terminal sequence, HDEF.

We have identified and characterized a cDNA encoding a novel Ca2+-binding protein named calumenin from mouse heart by the signal sequence trap method. The deduced amino acid sequence (315 residues) of calumenin contains an amino-terminal signal sequence and six Ca2+-binding (EF-hand) motifs and shows homology with reticulocalbin, Erc-55, and Cab45. These proteins seem to form a new subset of the EF-hand protein family expressed in the lumen of the endoplasmic reticulum (ER) and Golgi apparatus. Purified calumenin had Ca2+-binding ability. The carboxyl-terminal tetrapeptide His-Asp-Glu-Phe was shown to be responsible for retention of calumenin in ER by the retention assay, immunostaining with a confocal laser microscope, and the deglycosylation assay. This is the first report indicating that the Phe residue is included in the ER retention signal. Calumenin is expressed most strongly in heart of adult and 18.5-day embryos. The calumenin gene (Calu) was mapped at the proximal portion of mouse chromosome 7.

Effect of serum-free and serum-containing medium on cellular levels of ER-based proteins in various mouse hybridoma cell lines

BiP, GRP94 and PDI, three endoplasmic reticulum (ER) based proteins are involved in the maturation of secretory proteins and might represent a bottleneck in the secretory pathway of monoclonal antibodies (MAB). With the three hybridoma cell lines tested, MAB production kinetics were significantly increased for the batch cultures done in serum-free medium (SFM) with respect to those done in serum-containing medium (SCM). It could be established that there was a correlation between the cellular levels of PDI and GRP94 and the specific MAB production rate. With respect to BiP, no correlation with the MAB production rate was observed. The non-producing myeloma cell line X63, used as a reference, showed increased cellular PDI levels when cultivated in SFM. However, in this cell, the cellular GRP94 levels were not significantly influenced by the medium composition.It was concluded that SFM induced an increase of cellular PDI levels and this elevation seemed to be responsible for the increase in the specific MAB production rates. On the other hand, only MAB producing cells showed an increase in the cellular GRP94 levels which might be a result of increased MAB sythesis. Indeed, I.13.17 cultivated in SFM supplemented with serum showed a significantly reduced (about 50%) specific MAB production rate in comparison to I.13.17 cultivated in non-serum supplemented SFM. The cellular PDI and BiP levels did not vary between these conditions of culture, whereas the cellular GRP94 level was about two-fold lower in I.13.17 cultivated in SFM when supplemented with serum than in I.13.17 cultivated in SFM without futher supplementation. These results are discussed with respect to the medium composition as well as in the context of apparent and potential bottlenecks within the secretory pathway of MAB.

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

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.

Reconstitution of a tandem Co- and post-translational processing pathway with rat liver subcellular fractions.

Previously we showed that smooth microsomes from a variety of tissues effectively cleaved, sequestered, and "core" glycosylated nascent chains of secretory proteins. To further characterize the role of smooth membranes in the biosynthesis of secretory polypeptides, rat liver smooth microsomes were separated into smooth endoplasmic reticulum and Golgi fractions. Membranes of the smooth endoplasmic reticulum cleaved the signal peptide of pre-placental lactogen, attached the high mannose core to the alpha subunit of chorionic gonadotropin, and sequestered the processed proteins. None of these processing steps were performed by Golgi membranes. However, processing of asparagine-linked oligosaccharides and the coincident addition of terminal sugars was performed by Golgi but not by smooth endoplasmic reticulum membranes. The properties of this post-translational reaction are very similar to those described for the reactions in vivo. These observations demonstrate that the enzymes for co-translational (pre-protein processing) and posttranslational (oligosaccharide maturation) processing events are localized in the endoplasmic reticulum and Golgi apparatus, respectively. This functional differentiation of Golgi and endoplasmic reticulum membranes is an important feature of the secretory process in eukaryotic cells. Restriction of the recognition and transport of nascent secretory proteins to the endoplasmic reticulum establishes the polarity necessary for the ordered sequence of post-translational steps involved in the synthesis and maturation of secretory proteins.