Inhibitor Of Protein Glycosylation Research Articles

Endoplasmic Reticulum Stress and Obesity.

In recent years, the world has seen an alarming increase in obesity and is closely associated with insulin resistance, which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) plays in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably many causes for obesity-related insulin resistance and inflammation. One of the faulty mechanisms is protein homeostasis, protein quality control system included protein folding, chaperone activity, and ER-associated degradation leading to endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens, or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.

Use of metabolic glycoengineering and pharmacological inhibitors to assess lipid and protein sialylation on cells.

Metabolic glycoengineering (MGE) has been developed to visualize carbohydrates on live cells. The method allows the fluorescent labeling of sialic acid (Sia) sugar residues on neuronal plasma membranes. For instance, the efficiency of glycosylation along neurite membranes has been characterized as cell health measure in neurotoxicology. Using human dopaminergic neurons as model system, we asked here, whether it was possible to separately label diverse classes of biomolecules and to visualize them selectively on cells. Several approaches suggest that a large proportion of Sia rather incorporated in non-protein components of cell membranes than into glycoproteins. We made use here of deoxymannojirimycin (dMM), a non-toxic inhibitor of protein glycosylation, and of N-butyl-deoxynojirimycin (NBdNM) a well-tolerated inhibitor of lipid glycosylation, to develop a method of differential labeling of sialylated membrane lipids (lipid-Sia) or sialylated N-glycosylated proteins (protein-Sia) on live neurons. The time resolution at which Sia modification of lipids/proteins was observable was in the range of few hours. The approach was then extended to several other cell types. Using this technique of target-specific MGE, we found that in dopaminergic or sensory neurons >60% of Sia is lipid bound, and thus polysialic acid-neural cell adhesion molecule (PSA-NCAM) cannot be considered the major sialylated membrane component. Different from neurons, most Sia was bound to protein in HepG2 hepatoma cells or in neural crest cells. Thus, our method allows visualization of cell-specific sialylation processes for separate classes of membrane constituents.

Inhibitors of Protein Glycosylation Are Active against the Coronavirus Severe Acute Respiratory Syndrome Coronavirus SARS-CoV-2

Repurposing clinically available drugs to treat the new coronavirus disease 2019 (COVID-19) is an urgent need in the course of the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV-2) pandemic, as very few treatment options are available. The iminosugar Miglustat is a well-characterized drug for the treatment of rare genetic lysosome storage diseases, such as Gaucher and Niemann-Pick type C, and has also been described to be active against a variety of enveloped viruses. The activity of Miglustat is here demonstrated in the micromolar range for SARS-CoV-2 in vitro. The drug acts at the post-entry level and leads to a marked decrease of viral proteins and release of infectious viruses. The mechanism resides in the inhibitory activity toward α-glucosidases that are involved in the early stages of glycoprotein N-linked oligosaccharide processing in the endoplasmic reticulum, leading to a marked decrease of the viral Spike protein. Indeed, the antiviral potential of protein glycosylation inhibitors against SARS-CoV-2 is further highlighted by the low-micromolar activity of the investigational drug Celgosivir. These data point to a relevant role of this approach for the treatment of COVID-19.

Inhibition of protein glycosylation is a novel pro-angiogenic strategy that acts via activation of stress pathways

Endothelial cell (EC) metabolism is thought to be one of the driving forces for angiogenesis. Here we report the identification of the hexosamine D-mannosamine (ManN) as an EC mitogen and survival factor for bovine and human microvascular EC, with an additivity with VEGF. ManN inhibits glycosylation in ECs and induces significant changes in N-glycan and O-glycan profiles. We further demonstrate that ManN and two N-glycosylation inhibitors stimulate EC proliferation via both JNK activation and the unfolded protein response caused by ER stress. ManN results in enhanced angiogenesis in a mouse skin injury model. ManN also promotes angiogenesis in a mouse hindlimb ischemia model, with accelerated limb blood flow recovery compared to controls. In addition, intraocular injection of ManN induces retinal neovascularization. Therefore, activation of stress pathways following inhibition of protein glycosylation can promote EC proliferation and angiogenesis and may represent a therapeutic strategy for treatment of ischemic disorders.

Proteomic analysis of the cardiac myocyte secretome reveals extracellular protective functions for the ER stress response

The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.

Implication of protein glycosylation impairment in cellular cholesterol accumulation caused by Presenilin deficiency

BackgroundMajor genetic causes of familial Alzheimer disease (FAD) are mutations in genes encoding presenilin (PS) 1 and 2, respectively. PS1 and PS2 are transmembrane proteins known to constitute the catalytic subunits of the γ‐secretase complex, a membrane protease involved in proteolytic processing of amyloid precursor protein (APP) that gives rise to amyloid β‐protein (Aβ) secretion. In addiction, PSs are known to be involved in cellular cholesterol metabolism, which is fundamental for physiological cellular functions. Alterations in cholesterol metabolism could be implicated in AD development, indicated by the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PSs play a role in cellular cholesterol metabolism is still not fully clarified. In this study, we characterized cholesterol metabolism in the cells deficient in either PS1 or PS2.MethodsWe have studied PS1‐ or PS2‐knock out (KO) MEFs by mass spectrometry (MS) for sterols including cholesterol and by microscopy with a cholesterol binding compound, filipin. Expression level of proteins involved in cellular cholesterol metabolism was analyzed by Western blotting (WB).ResultsCellular level of cholesterol as well as its precursors, desmosterol and lathosterol, were elevated in PS1‐ and PS2‐KO as compared to WT MEFs. Fluorescence microscopy also revealed intracellular cholesterol accumulation in PS1 and PS2 KO cells. Interestingly, the subcellular distribution of cholesterol was different between PS1‐ and PS2‐KO MEFs. We further analyzed expression of proteins involved in cellular cholesterol metabolism, including NPC1, an intracellular cholesterol transporter whose deficiency leads to lysosomal cholesterol storage and neurodegeneration in Niemann‐Pick disease type C. The deficiency of PS1‐ and PS2‐KO MEFs was associated with altered glycosylation of NPC1. The glycosylation and expression of other membrane proteins, such as N‐cadherin, LAMP2, and LRP1 was also dependent on the PS genotype. To investigate the role of protein glycosylation in cellular cholesterol metabolism, we chronically treated wild‐type MEFs with several inhibitors of protein glycosylation targeting different steps of glycosylation processes. The inhibitors differentially affected protein glycosylation pattern and subcellular cholesterol distribution. Importantly, some of the inhibitors partially mimicked the cellular cholesterol accumulation as well as the protein glycosylation pattern observed in PS1‐ and PS2‐KO MEFs.Discussion/ConclusionsThese results suggest that deficiency of PS1 or PS2 differentially affect cellular cholesterol metabolism via impairment of protein glycolsylation. The exact mechanisms of altered expression of NPC1 as well as γ‐secretase activity in the impairment of cholesterol metabolism and protein glycosylation remain to be elucidated. It will be interesting to further dissect the role of protein glycosylation and cholesterol mismetabolism in the pathogenesis of AD.Support or Funding InformationGerman Research Foundation (DFG) (Project number: 348207315, Naoto Oikawa)

Sphingolipid/Pkh1/2-TORC1/Sch9 Signaling Regulates Ribosome Biogenesis in Tunicamycin-Induced Stress Response in Yeast.

Reduced ribosome biogenesis in response to environmental conditions is a key feature of cell adaptation to stress. For example, ribosomal genes are transcriptionally repressed when cells are exposed to tunicamycin, a protein glycosylation inhibitor that induces endoplasmic reticulum stress and blocks vesicular trafficking in the secretory pathway. Here, we describe a novel regulatory model, in which tunicamycin-mediated stress induces the accumulation of long-chain sphingoid bases and subsequent activation of Pkh1/2 signaling, which leads to decreased expression of ribosomal protein genes via the downstream effectors Pkc1 and Sch9. Target of rapamycin complex 1 (TORC1), an upstream activator of Sch9, is also required. This pathway links ribosome biogenesis to alterations in membrane lipid composition under tunicamycin-induced stress conditions. Our results suggest that sphingolipid/Pkh1/2-TORC1/Sch9 signaling is an important determinant for adaptation to tunicamycin-induced stress.

Mineralocorticoid receptor blockade improves arginine transport and nitric oxide generation through modulation of cationic amino acid transporter-1 in endothelial cells

Blockade of the mineralocorticoid receptor (MCR) has been shown to improve endothelial function far beyond blood pressure control. In the current studies we have looked at the effect of MCR antagonists on cationic amino acid transporter-1 (CAT-1), a major modulator of endothelial nitric oxide (NO) generation. Using radio-labeled arginine, {[3H] l-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with either spironolactone or eplerenone with or without silencing of MCR. Western blotting for CAT-1, PKCα and their phosphorylated forms were performed. NO generation was measured by using Griess reaction assay. Both Spironolactone and eplerenone significantly increased endothelial arginine transport, an effect which was further augmented by co-incubation with aldosterone, and blunted by either silencing of MCR or co-administration of amiloride. Following MCR blockade, we identified two bands for CAT-1. The addition of tunicamycin (an inhibitor of protein glycosylation) or MCR silencing resulted in disappearance of the extra band and prevented the increase in arginine transport. Only spironolactone decreased CAT-1 phosphorylation through inhibition of PKCα (CAT-1 inhibitor). Subsequently, incubation with either MCR antagonists significantly augmented NO2/NO3 levels (stable NO metabolites) and this was attenuated by silencing of MCR or tunicamycin. GO 6076 (PKCα inhibitor) intensified the increase of NO metabolites only in eplerenone treated cells. In conclusion spironolactone and eplerenone augment arginine transport and NO generation through modulation of CAT-1 in endothelial cells. Both MCR antagonists activate CAT-1 by inducing its glycosylation while only spironolactone inhibits PKCα.

TERT enhances the survival rate of human fibroblasts under endoplasmic reticulum, Golgi apparatus, and lysosomal stresses.

The exposure of organelles, such as the endoplasmic reticulum (ER), Golgi apparatus (GA), and lysosomes, to stress activates death mechanisms. Recently, telomerase reverse transcriptase (TERT) has been shown to be involved in cell survival. However, the relationship between TERT and the stress responses is still unclear. Here, we aimed to clarify the possible mechanisms of action through which TERT promotes cell survival by studying its effect on the stresses faced by multiple organelles in human fibroblasts. We found that TERT enhanced the survival rate of cells under ER stress, regardless of ER stress inducers such as tunicamycin (protein glycosylation inhibitor), thapsigargin (Ca2+-ATPase inhibitor), brefeldin A (protein transport inhibitor), or dithiothreitol (disulfide bond formation inhibitor). We also found that TERT enhanced the survival rate of cells under GA and lysosomal stresses. Collectively, these results suggest that TERT suppresses cell stress and promotes cell survival via different mechanisms. These findings may offer new insights into the implications of TERT in the treatment of stress-induced conditions such as aging, obesity, and neurodegenerative diseases.

Endoplasmic Reticulum Stress and Obesity.

In recent years, the world has seen an alarming increase in obesity and closely associated with insulin resistance which is a state of low-grade inflammation, the latter characterized by elevated levels of proinflammatory cytokines in blood and tissues. A shift in energy balance alters systemic metabolic regulation and the important role that chronic inflammation, endoplasmic reticulum (ER) dysfunction, and activation of the unfolded protein response (UPR) play in this process.Why obesity is so closely associated with insulin resistance and inflammation is not understood well. This suggests that there are probably other causes for obesity-related insulin resistance and inflammation. One of these appears to be endoplasmic reticulum (ER) stress.The ER is a vast membranous network responsible for the trafficking of a wide range of proteins and plays a central role in integrating multiple metabolic signals critical in cellular homeostasis. Conditions that may trigger unfolded protein response activation include increased protein synthesis, the presence of mutant or misfolded proteins, inhibition of protein glycosylation, imbalance of ER calcium levels, glucose and energy deprivation, hypoxia, pathogens or pathogen-associated components and toxins. Thus, characterizing the mechanisms contributing to obesity and identifying potential targets for its prevention and treatment will have a great impact on the control of associated conditions, particularly T2D.

Nuclear extrusion precedes discharge of genomic DNA fibers during tunicamycin-induced neutrophil extracellular trap-osis (NETosis)-like cell death in cultured human leukemia cells.

We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR.

Expression of Arabidopsis Bax Inhibitor-1 in transgenic sugarcane confers drought tolerance.

SummaryThe sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor‐1 from Arabidopsis thaliana (AtBI‐1), can confer increased tolerance of sugarcane plants to long‐term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C4 grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long‐term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.

Purvalanol induces endoplasmic reticulum stress-mediated apoptosis and autophagy in a time-dependent manner in HCT116 colon cancer cells.

Purvalanol, a novel cyclin-dependent kinase inhibitor, is referred to as a strong apoptotic inducer which causes cell cycle arrest in various cancer cells such as prostate, breast and colon cancer cell lines. Various physiological and pathological conditions such as glucose starvation, inhibition of protein glycosylation and oxidative stress may cause an accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and autophagy. Lacking proteosomal function on aggregates of unfolded proteins, ER stress may induce autophagic machinery. Autophagy, an evolutionarily conserved process, is characterized by massive degradation of cytosolic contents. In the present study, our aim was to determine the time-dependent, ER-mediated apoptotic and autophagy induction of purvalanol in HCT 116 colon cancer cells. Fifteen micromoles of purvalanol induced a reduction in cell viability by 20 and 35% within 24 and 48 h, respectively. HCT 116 colon cancer cells were exposed to purvalanol, which activated ER stress via upregulation of PERK, IRE1α gene expression, eIF-2α phosphorylation and ATF-6 cleavage at early time-points in the HCT 116 colon cancer cells. Moreover, we determined that during purvalanol-mediated ER stress, autophagic machinery was also activated prior to apoptotic cell death finalization. Beclin-1 and Atg-5 expression levels were upregulated and LC3 was cleaved after a 6 h purvalanol treatment. Purvalanol induced mitochondrial membrane potential loss, caspase-7 and caspase-3 activation and PARP cleavage following a 48 h treatment. Thus, we conclude that the anticancer effect of purvalanol in HCT 116 cells was due to ER stress-mediated apoptosis; however, purvalanol triggered autophagy, which functions as a cell survival mechanism at early time-points.

Seipin mutation at glycosylation sites activates autophagy in transfected cells via abnormal large lipid droplets generation.

Seipin is a protein that resides in endoplasmic reticulum, and involved in both lipid metabolic disorders and motor neuropathy. The aim of this study was to investigate the effects of mutant seipin on autophagy system and the morphology of lipid droplets in vitro. HEK-293, H1299 and MES23.5 cells were transfected with the plasmids of mutated seipin at glycosylation sites (N88S or S90L) and GFP-LC3 plasmids. The cells were subjected to immunofluorescence and flow cytometry assays, and the cell lysates were subjected to immunoblot analysis. Nile Red was used to stain the lipid droplets in the cells. Overexpression of the mutated seipin proteins N88S or S90L activated autophagy in the 3 cell lines, and substantially altered the sub-cellular distribution of the autophagosome marker GFP-LC3, leading to a number of large vacuoles appearing in the cytoplasm. The sub-cellular location of GFP-LC3 and mutated seipin proteins highly overlapped. Moreover, and the mutated seipin proteins caused diffuse small lipid droplets to fuse into larger lipid droplets. Treatment of mutated seipin-transfected cells with the autophagy inhibitor 3-MA (5 mmol/L) facilitated the fusion of mutated seipin-induced large vacuoles. The protein glycosylation inhibitor tunicamycin could mimic the mutated seipin-induced effects, and treatment of the wild-type seipin-transfected cells with tunicamycin (2.5 μg/mL) produced similar morphological and biochemical properties as in the mutated seipin-transfected cells. The mutation of seipin at glycosylation sites disrupt its function in regulating lipid droplet metabolism, and the autophagy acts as an adaptive response to break down abnormal lipid droplets. The interruption of autophagy would accelerate the fusion of abnormal lipid droplets.

Improvement of chemotherapeutic drug efficacy by endoplasmic reticulum stress.

Tunicamycin (TUN), an inhibitor of protein glycosylation and therefore a potent stimulator of endoplasmic reticulum (ER) stress, has been used to improve anticancer drug efficacy, but the underlying mechanism remains obscure. In this study, we show that acute administration of TUN in mice induces the unfolded protein response and suppresses the levels of P21, a cell cycle regulator with anti-apoptotic activity. The inhibition of P21 after ER stress appears to be C/EBP homologous protein (CHOP)-dependent because in CHOP-deficient mice, TUN not only failed to suppress, but rather induced the expression of P21. Results of promoter-activity reporter assays using human cancer cells and mouse fibroblasts indicated that the regulation of P21 by CHOP operates at the level of transcription and involves direct binding of CHOP transcription factor to the P21 promoter. The results of cell viability and clonogenic assays indicate that ER-stress-related suppression of P21 expression potentiates caspase activation and sensitizes cells to doxorubicin treatment, while administration of TUN to mice increases the therapeutic efficacy of anticancer therapy for HepG2 liver and A549 lung cancers.

Anti‐angiogenic and anti‐cancer property of a protein N‐glycosylation inhibitor (607.4)

Tumor growth is angiogenesis dependent. Current therapeutics targets either tumor cells or microvasculature but not both. The patients therefore enjoy disease free survival only for a limited period of time, irrespective of a good initial prognosis and pathological response rates. Aberrant glycosylation has been observed in tumors and we have hypothesized that an asparagine‐linked (N‐linked) protein glycosylation inhibitor could target both endothelial cells and tumor cells and eliminate tumor progression. Our objective is to test the efficacy of Tunicamycin. The results support Tunicamycin treatment causes cell cycle arrest, develops endoplasmic reticulum (ER) stress and induces unfolded protein response (upr)‐mediated apoptosis. Analysis of C=O stretching vibrations of Amide I of proteome by Raman spectroscopy concluded protein denaturation. Tunicamycin treatment (i) prevented the colony formation, (ii) failed to reverse the inhibition of angiogenesis by VEGF165; and (iii) indiscriminately inhibited the progression of double‐ and a triple‐negative breast cancers by ~55‐65% in three weeks. Proliferation of MDA‐MB‐231, MDA‐MB‐468, BT‐20, BT‐474 and ZR‐731 human breast cancer cells was also inhibited. Induction of apoptosis in MDA‐MB‐23 confirmed translational attenuation of upr. Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD007583 (KB).Grant Funding Source: Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD0

Canonical and non‐canonical pathway of unfolded protein response signaling (607.5)

The endoplasmic reticulum (ER) is a principal site for folding and maturation of transmembrane, secretory and ER‐resident proteins. Perturbations that alter ER homeostasis can lead to accumulation of unfolded proteins, and a threat to all living cells. To cope with such stress, cells activate an intracellular signaling pathway ‐ the unfolded protein response (upr). It is an integrated intracellular signaling pathway that transmits information about the protein folding status in the ER lumen to the cytoplasm and the nucleus. If the protein‐folding defect is not corrected, cells undergo apoptosis. We have used Tunicamycin, a chemical mediator, a potent asparagine‐linked protein glycosylation inhibitor to induce upr and study the capillary endothelial cell fate, i.e., angiogenesis, a hallmark for tumor progression. Our results indicate ER stress developed due to protein denaturation, causes upr‐mediated cell cycle arrest in G1 and induction of apoptosis. Most importantly, Tunicamycin prevented double‐ and triple‐negative breast cancer progression ~55‐65%. To enhance the efficacy, we have examined nano‐formulated Tunicamycin. The results indicated gold anoparticles (20nm‐50nm) of Tunicamycin (1μg/ml) were three times more potent in inhibiting angiogenesis. There was upr‐mediated cell cycle arrest but no activation of apoptosis. This raised the possibility of a canonical and non‐canonical pathway.Grant Funding Source: Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 2G12MD0

Inhibition of PKR protects against tunicamycin-induced apoptosis in neuroblastoma cells

Endoplasmic reticulum (ER) dysfunction is thought to play a significant role in several neurological disorders, including Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, amyotrophic lateral sclerosis, cerebral ischemia, and the prion diseases. ER dysfunction can be mimicked by cellular stress signals such as disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds, which results in accumulation of misfolded proteins in the ER and leads to cell death by apoptosis. Tunicamycin, which is an inhibitor of protein glycosylation, induces ER stress and apoptosis. In this study, we examined the involvement of double stranded (ds) RNA-activated protein kinase PKR in tunicamycin-induced apoptosis. We used overexpression of the trans-dominant negative, catalytically inactive mutant K296R to inhibit PKR activity in neuroblastoma cells. We demonstrate that inhibition of PKR activation in response to tunicamycin protects neuronal cells from undergoing apoptosis. Furthermore, K296R overexpressing cells show defective PKR activation, delayed eIF2α phosphorylation, dramatically delayed ATF4 expression. In addition, both caspase-3 activation and C/EBP homologous protein (CHOP, also known as GADD153) induction, which are markers of apoptotic cells, are absent from K296R overexpression cells in response to tunicamycin. These results establish that PKR activation plays a major regulatory role in induction of apoptosis in response to ER stress and indicates the potential of PKR as possible target for neuroprotective therapeutics.

HDLs protect the MIN6 insulinoma cell line against tunicamycin-induced apoptosis without inhibiting ER stress and without restoring ER functionality

HDLs protect pancreatic beta cells against apoptosis induced by several endoplasmic reticulum (ER) stressors, including thapsigargin, cyclopiazonic acid, palmitate and insulin over-expression. This protection is mediated by the capacity of HDLs to maintain proper ER morphology and ER functions such as protein folding and trafficking. Here, we identified a distinct mode of protection exerted by HDLs in beta cells challenged with tunicamycin (TM), a protein glycosylation inhibitor inducing ER stress. HDLs were found to inhibit apoptosis induced by TM in the MIN6 insulinoma cell line and this correlated with the maintenance of a normal ER morphology. Surprisingly however, this protective response was neither associated with a significant ER stress reduction, nor with restoration of protein folding and trafficking in the ER. These data indicate that HDLs can use at least two mechanisms to protect beta cells against ER stressors. One that relies on the maintenance of ER function and one that operates independently of ER function modulation. The capacity of HDLs to activate several anti-apoptotic pathways in beta cells may explain their ability to efficiently protect these cells against a variety of insults.

Phenyl acyl acids attenuate the unfolded protein response in tunicamycin-treated neuroblastoma cells.

Understanding how neural cells handle proteostasis stress in the endoplasmic reticulum (ER) is important to decipher the mechanisms that underlie the cell death associated with neurodegenerative diseases and to design appropriate therapeutic tools. Here we have compared the sensitivity of a human neuroblastoma cell line (SH-SY5H) to the ER stress caused by an inhibitor of protein glycosylation with that observed in human embryonic kidney (HEK-293T) cells. In response to stress, SH-SY5H cells increase the expression of mRNA encoding downstream effectors of ER stress sensors and transcription factors related to the unfolded protein response (the spliced X-box binding protein 1, CCAAT-enhancer-binding protein homologous protein, endoplasmic reticulum-localized DnaJ homologue 4 and asparagine synthetase). Tunicamycin-induced death of SH-SY5H cells was prevented by terminal aromatic substituted butyric or valeric acids, in association with a decrease in the mRNA expression of stress-related factors, and in the accumulation of the ATF4 protein. Interestingly, this decrease in ATF4 protein occurs without modifying the phosphorylation of the translation initiation factor eIF2α. Together, these results show that when short chain phenyl acyl acids alleviate ER stress in SH-SY5H cells their survival is enhanced.