Alter Endoplasmic Reticulum Homeostasis Research Articles

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant

The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have previously reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.

Newcastle disease virus NP and P proteins induce autophagy via the endoplasmic reticulum stress-related unfolded protein response.

Newcastle disease virus (NDV) can replicate and trigger autophagy in human tumor cells. Our previous study confirmed the critical role of autophagy in NDV infection. Here we studied the role of NDV structural proteins in the induction of autophagy through endoplasmic reticulum (ER) stress-related unfolded protein response (UPR) pathways. Ectopic expression of the NDV nucleocapsid protein (NP) or phosphoprotein (P) was sufficient to induce autophagy. NP or P expression also altered ER homeostasis. The PERK and ATF6 pathways, but not the XBP1 pathway, all of which are components of the UPR, were activated in both NDV-infected and NP or P-transfected cells. Knockdown of PERK or ATF6 inhibited NDV-induced autophagy and reduced the extent of NDV replication. Collectively, these data suggest not only roles for the NDV NP and P proteins in autophagy, but also offer new insights into the mechanisms of NDV-induced autophagy through activation of the ER stress-related UPR pathway.

Altered ER Homeostasis and Mitochondria ER Network in PINK1 Deficient Parkinson's Disease Models

Parkinson's disease (PD) is one of the most common neurodegenerative diseases, affecting 1% of the population over 60 years old. The pathological hallmarks of PD include loss of dopaminergic neruons in the substantia nigra pars compacta and formation of alpha-synuclein positive intracellular inclusions named Lewy bodies. Although majority of PD cases are sporadic, mutations in numerous genes (e.g. alpha-synuclein, LRRK2, PINK1, Parkin) have been linked to the development of familial PD. Growing evidence from genetic and toxicological models as well as human PD brain studies suggests that endoplasmic reticulum (ER) stress is one of the features which contributes to neurodegeneration in PD. Increased ER stress has been descried in post-mortem brain tissues from PD patients as well as cellular models of PD with overexpression of mutant alpha-synuclein. However, it is still unclear whether altered ER homeostasis play a role in receive forms of PD. Here we investigated ER function and mitochondria-ER network in PINK1-deficient cellular models for Parkinson's disease. We observed and increased ER calcium release upon stimulation with SERCA inhibitor thapsigargin which is accompanied by increase mitochondria calcium uptake in PINK1 deficient SH-SY5Y neuroblastoma cells, as measured by genetically encode ER calcium probe (ERD1) and X-Rhod 1 respectively. These results indicated an increased contact between mitochondria and ER. We further confirmed this using both immunohistochemistry staining with GRP75 as mitochondria maker and calnexin as ER marker and live cell imaging using TMRM as mitochondria marker and ER-GFP as maker for ER. Furthermore, altered ER stress markers were also detected in PINK1 deficient cells. Further examination of the impact of altered ER-mitochondria contact on neuronal survival will be presented at the conference. Altogether, our results suggests an altered ER homeostasis in PINK1-deficient cellular models of PD.

Abstract 1255: Mifepristone causes endoplasmic reticulum stress, triggers the unfolded protein response, and increases autophagic flux in ovarian cancer cells

Abstract Mifepristone (MF) is a synthetic steroid that blocks the growth of ovarian cancer cells causing cytostasis or lethality depending on its concentration. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes up-regulated by MF and that are involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) 78, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP); both proteins are known for balancing ER stress in a ‘yin’ (CHOP; pro-cell death factor) ‘yang’ (GRP78; pro-survival factor) manner. GRP78 and CHOP were up-regulated in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that MF caused expansion of the ER and triggered the UPR. The three UPR associated signaling pathways, PERK, IRE1α, and ATF6, were activated by MF. While low-dose MF caused cytostasis with compensatory UPR activation, high-dose MF induced lethality with concurrent UPR activation, up-regulation of pro-apoptotic factor Bim, down-regulation of anti-apoptotic factor Bcl-2, and cleavage of PARP, a downstream target for executer caspases. MF-induced lethality but not cytostasis was abrogated by the ER stress inhibitor salubrinal, indicating that ER stress is required when MF causes cell death but not when its action is limited to cell cycle arrest. Because ER stress involves the accumulation of misfolded/unfolded proteins that alter ER homeostasis, the UPR engages compensatory mechanisms including short-term attenuation of the rate of protein translation, and degradation of surplus proteins by the ubiquitin proteasome system (UPS) and/or autophagy. Contrary to our expectation, MF-induced UPR associated with short-term increase rather than decrease, in protein translation rate. Moreover, the accumulation of poly-ubiquitinated proteins-a surrogate marker of the activity of the UPS-was slightly decreased by MF, suggesting that ER-associated protein degradation (ERAD) may be operating at a faster pace. Finally, MF increased the accumulation of LC3-II, which results from the fusion of LC3-I and phosphatidylethanolamine along the process of autophagosome formation. MF-induced LC3-II accumulation also augmented in the presence of the lysosomal degradation inhibitor bafilomycin A1, indicating that MF increases autophagic flux. In MF-treated cells, the induction of autophagy may counterbalance and alleviate ER expansion induced by ER stress. The stimulation by MF of ER stress, UPR, and autophagy offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to aggravators of the ER and/or inhibitors of autophagy. Citation Format: Lei Zhang, Alicia Goyeneche, Rekha Srinivasan, Carlos Telleria. Mifepristone causes endoplasmic reticulum stress, triggers the unfolded protein response, and increases autophagic flux in ovarian cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1255. doi:10.1158/1538-7445.AM2015-1255

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

How does protein misfolding in the endoplasmic reticulum affect lipid metabolism in the liver?

The endoplasmic reticulum (ER) maintains cellular metabolic homeostasis by coordinating protein synthesis, secretion activities, lipid biosynthesis and calcium (Ca²⁺) storage. In this review, we will discuss how altered ER homeostasis contributes to dysregulation of hepatic lipid metabolism and contributes to liver-associated metabolic diseases. Perturbed ER functions or accumulation of unfolded protein in the ER leads to the activation of the unfolded protein response (UPR) to protect the cell from ER stress. Recent findings pinpoint the key regulatory role of the UPR in hepatic lipid metabolism and demonstrate the potential causal mechanism of ER stress in metabolic dysregulation including diabetes and obesity. A wide range of factors can alter the protein-folding environment in the ER of hepatocytes and contribute to dysregulation of hepatic lipid metabolism and liver disease. The UPR constitutes a series of adaptive programs that preserve ER protein-folding environment and maintain hepatic lipid homeostasis. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human liver-associated metabolic diseases.

The Adaptive Endoplasmic Reticulum Stress Response to Lipotoxicity in Progressive Human Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7%-14% of the U.S. population through a second "hit" in the form of increased oxidative stress and inflammation. Endoplasmic reticulum (ER) stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress-associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty), and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0 ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis, and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress-associated lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress-associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor X-box binding protein 1 spliced (XBP-1s) were significantly elevated among NASH samples, whereas other downstream ER stress proteins including CHOP, ATF4, and phosphorylated JNK and eIF2α were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD.

The endoplasmic reticulum stress response in the pancreatic β‐cell

Eukaryotic cells respond to stress in the endoplasmic reticulum (ER) resulting from insufficient protein folding capacity or altered ER homeostasis by activating the unfolded protein response (UPR). In mammalian cells the UPR is mediated by at least three ER-localized sensors/transducers, and the cellular response and susceptibility to ER stress is likely to be cell-type specific to some degree. Here, we review the response of pancreatic β-cells or islets to ER stress induced by pharmacological agents, misfolded insulin expression, excessive nutrient exposure and in animal models of type 2 diabetes. This review highlights the particular importance of PERK-mediated translational control and the transcriptional response in pancreatic β-cells and how these relate to the highly specialized function of β-cells, namely glucose-regulated insulin secretion and production. We examine how chronic ER stress may prematurely 'age' the β-cell or cause its genetic reprogramming to either reduce its ability to mount a cell survival response to ER stress, or impair normal function. Both could contribute to β-cell failure in diabetes. We also explore the therapeutic potential of targeting the UPR to preserve β-cell function.

La réponse UPR

The endoplasmic reticulum (ER) is the first compartment in the secretory pathway. In the ER, proteins fold into their native configuration and are modified by post-translational modifications. Perturbations that alter ER homeostasis therefore disrupt folding and lead to the accumulation of unfolded proteins. These perturbations include modifications of Ca2+ homeostasis, increased demand for protein folding due to elevated synthesis of proteins in specialized cells or expression of a mutant misfolded protein. To limit accumulation of unfolded proteins, the cells have developed a specialized pathway : the unfolded protein response (UPR). UPR involves the activation of three transmembrane proteins of the ER : the PKR-like ER protein kinase (PERK), the activating transcription factor 6 (ATF6) and the inositol requiring enzyme 1 (IRE-1). The activation of all three components of the UPR depends on the dissociation of the luminal chaperone BiP/GRP78 from the luminal part of these proteins. Once activated, these pathways down-regulate protein synthesis through the phosphorylation of eiF2 (eucaryotic translation initiation factor 2) and up-regulate the transcription of genes which encode ER chaperones, protein folding enzymes and components of the ER-associated degradation system (ERAD). Growing evidences indicate that UPR signaling plays critical roles in nutrient sensing, differentiation of secretory cells such as pancreatic b cell and antibody producing plasma cells, glucose homeostasis and in the development of pathologies linked to accumulation of aggregated proteins.

Endoplasmic Reticulum Stress Induction of Insulin-like Growth Factor-binding Protein-1 Involves ATF4

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

The unfolded protein response.

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 (UPs), which is a threat to all living cells. To cope with the stress, cells