Stress-sensing and regulatory mechanism of the endoplasmic-stress sensors Ire1 and PERK

Ageing is driven by the inexorable and stochastic accumulation of damage in biomolecules vital for proper cellular function. Although this process is fundamentally haphazard and uncontrollable, senescent decline and ageing is broadly influenced by genetic and extrinsic factors. Numerous gene mutations and treatments have been shown to extend the lifespan of diverse organisms ranging from the unicellular Saccharomyces cerevisiae to primates. It is becoming increasingly apparent that most such interventions ultimately interface with cellular stress response mechanisms, suggesting that longevity is intimately related to the ability of the organism to effectively cope with both intrinsic and extrinsic stress. Here, we survey the molecular mechanisms that link ageing to main stress response pathways, and mediate age-related changes in the effectiveness of the response to stress. We also discuss how each pathway contributes to modulate the ageing process. A better understanding of the dynamics and reciprocal interplay between stress responses and ageing is critical for the development of novel therapeutic strategies that exploit endogenous stress combat pathways against age-associated pathologies.

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

The unfolded protein response (UPR) is a specific cellular process that allows the cell to cope with the overload of unfolded/misfolded proteins in the endoplasmic reticulum (ER). ER stress is commonly associated with degenerative pathologies, but its role in disease progression is still a matter for debate. Here, we found that mutations in the ER-resident chaperone, neither inactivation nor afterpotential A (NinaA), lead to mild ER stress, protecting photoreceptor neurons from various death stimuli in adult Drosophila. In addition, Drosophila S2 cultured cells, when pre-exposed to mild ER stress, are protected from H(2)O(2), cycloheximide- or ultraviolet-induced cell death. We show that a specific ER-mediated signal promotes antioxidant defences and inhibits caspase-dependent cell death. We propose that an immediate consequence of the UPR not only limits the accumulation of misfolded proteins but also protects tissues from harmful exogenous stresses.

The endoplasmic reticulum (ER) is susceptible to various stresses that provoke the accumulation of unfolded proteins in the ER. Excessive or long-termed stresses in the ER result in apoptotic cell death involving activation of caspase-12 and -3 and the Ask-1-JNK pathway. Eukaryotic cells can adapt for survival to deal with an accumulation of unfolded proteins in the ER by increasing transcription of genes encoding ER-resident chaperones such as GRP78/BiP to facilitate protein folding. The induction system is termed the unfolded protein response (UPR). It has been reported that IRE1 and PERK, transmembrane kinases, and ATF6, a transmembrane transcription factor, are mediators of the UPR through sensing accumulation of unfolded proteins. Cell fates after ER stress are regulated by the balance of both apoptosis and the UPR signaling. In the nervous systems, astrocytes are well known to be resistant to ER stresses induced by ischemia and hypoxia. These findings raise the possibility that astrocytes possess a novel UPR signaling different from that of neuronal cells. Recently, we identified a novel ER stress sensor, OASIS, which is specifically expressed in astrocytes. This protein is a transmembrane protein containing the bZIP domain. The functional analyses of OASIS showed that 1) it was cleaved within the ER membrane in response to the ER stress, 2) overexpression of OASIS induced the transcription of GRP78/BiP mRNA through the activation of cyclic AMP responsive element (CRE) and ER stress responsive element (ERSE), and 3) its stable cell lines were resistant to ER stress compared with the control cells. These results indicate that the ER-resident transcription factor OASIS may be a candidate for leading astrocytes to protect against ER stress.

Background and Significance: Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities such as gene transcription, proliferation and apoptosis. Endoplasmic Reticulum (ER) is the Ca2+ reservoir inside the cell that strictly orchestrate Ca2+ gradient across the Plasma Membrane(PM). Importantly, intracellular Ca2+ flux, is a key to orderly regulate cell proliferation, if altered can promote uncontrolled proliferation leading to cancer. Ca2+ entry through the [Ca2+]PM channels during [Ca2+]ER depletion is called the Store Operated Ca2+ Entry (SOCE). Impairment of endogenous SOCE leads to sustained [Ca2+]ER depletion and results in accumulation of unfolded proteins in the ER (known as ER stress). Understanding the basic molecular mechanism regulating PM to ER Ca2+ flow and developing novel strategies to target ER Ca2+ signaling could be beneficial from cancer therapeutic perspective. We have identified Ecdysoneless (ECD), an evolutionary conserved novel cell cycle regulator protein. ECD is overexpressed in several cancers, including breast and pancreatic cancers and its overexpression in breast cancer correlates with poor prognosis and short survival of patients. Recently our lab demonstrates that ECD is a negative regulator of ER stress; however, there is no mechanistic insights of how ECD senses and acts to maintain ER homeostasis. Experimental Design: We have utilized mouse embryonic fibroblasts (MEFs) from Tet(O)-FhECD-IRES-eGFP-rtTA and Ecdflox/flox mice transgenic mice to regulate ECD levels in cells to assess the role of ECD in Ca2+ signaling in a highly controlled system. Structured illumination microscopy(SIM) and biochemical fractionation techniques were performed to identify localization of ECD. Live cell imaging of cells stained with Ca2+ binding dyes like Fluo4-AM and Fluo5N-AM were used to access [Ca2+]Cyto and [Ca2+]ER levels respectively. Fluorescence resonance energy transfer (FRET) and immunoprecipitation methods were conducted to analyze SOCE pathway. Western blotting was used to assess the levels of various proteins involved in Ca2+ signaling. Results: We demonstrate several novel findings: (i) ECD is a smooth ER associated protein; (ii) ECD regulates assembly of ER-PM protein complex to drive Ca2+ entry through SOCE into the ER, maintaining [Ca2+]ER homeostasis; iii) ECD is overexpressed upon Ca2+ depletion and provides survival benefit to cells; v) ECD regulates Ca2+ mediated cell proliferation via CaMKII phosphorylation. Conclusion: Our results demonstrate ECD is a sensor of ER stress, induced by Ca2+deprivation, and overexpressed ECD promotes cell proliferation and survival under ER stress. These findings suggest ECD could be a potential target of Cancer therapy. Citation Format: Aniruddha Sarkar, Irfana Saleem, Sameer Mirza, Keshore Bidasee, Rajini Rao, Hamid Band, Vimla Band. The novel role of ecdysoneless (ECD) in maintain ER Ca2+ homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 448.

Stress management: How the unfolded protein response impacts fatty liver disease

Bacterial infection often leads to cellular damage, primarily marked by loss of cellular integrity and cell death. However, in recent years, it is being increasingly recognized that, in individual cells, there are graded responses collectively termed cell-autonomous defense mechanisms that induce cellular processes designed to limit cell damage, enable repair, and eliminate bacteria. Many of these responses are triggered not by detection of a particular bacterial effector or ligand but rather by their effects on key cellular processes and changes in homeostasis induced by microbial effectors when recognized. These in turn lead to a decrease in essential cellular functions such as protein translation or mitochondrial respiration and the induction of innate immune responses that may be specific to the cellular deficit induced. These processes are often associated with specific cell compartments, e.g., the endoplasmic reticulum (ER). Under non-infection conditions, these systems are generally involved in sensing cellular stress and in inducing and orchestrating the subsequent cellular response. Thus, perturbations of ER homeostasis result in accumulation of unfolded proteins which are detected by ER stress sensors in order to restore the normal condition. The ER is also important during bacterial infection, and bacterial effectors that activate the ER stress sensors have been discovered. Increasing evidence now indicate that bacteria have evolved strategies to differentially activate different arms of ER stress sensors resulting in specific host cell response. In this review, we will describe the mechanisms used by bacteria to activate the ER stress sensors and discuss their role during infection.

Members of the yeast p24 family, including Emp24p and Erv25p, exist as heteromeric complexes that have been proposed to cycle between the endoplasmic reticulum (ER) and Golgi compartments. The specific functions and sites of action of p24 proteins are still unknown. Here we identified a human homolog of the yeast p24 family of proteins, named ERS25 (endoplasmic reticulum stress-response protein 25), and investigated its role in stress response. ERS25 is predicted to have an ER localization signal peptide, a GOLD (Golgi dynamics) domain, which is found in several eukaryotic Golgi and lipid-trafficking proteins, a coiled-coil region, and a transmembrane domain. We demonstrate that ERS25 is localized to the ER and is induced by ER-specific stress, heat shock, and oxidative stress. The selective induction of ERS25 by brefeldin A, but not tunicamycin, implicates the involvement of ERS25 in protein trafficking between the ER and the Golgi. Small interfering RNA-mediated inhibition of ERS25 results in a significant decrease in apoptosis as well as a reduction of reactive oxygen species induced by oxidative stress. Moreover, ERS25 depletion results in a significant increase in the levels of the ER chaperone HSP70 in response to heat-shock stress through increased levels of HSF-1. We also found that inhibition of ERS25 induction in response to heat shock enhanced the binding of HSP70 to Apaf-1, which is likely to interfere in stress-mediated apoptosis. Together, the data presented here demonstrate that ERS25 may play a critical role in regulation of heat-shock response and apoptosis.

Endoplasmic reticulum (ER) stress, defined as prolonged disturbances in protein folding and accumulation of unfolded proteins in the ER. Perturbation of the ER, such as distribution of oxidative stress, iron imbalance, Ca2+ leakage, protein overload, and hypoxia, can cause ER stress. The cell reacts to ER stress by activating protective pathways, called the unfolded protein response (UPR), which is comprised of cellular mechanisms aimed for maintaining cellular homeostasis or, in case of excessively severe stress, at the initiation of cellular apoptosis. The three UPR signaling pathways from the ER stress sensors are initiated by activating transcription factor 6, inositol requiring enzyme 1, and protein kinase RNA-activated-like ER kinase. A number of physiological and pathological conditions, environmental toxicants and variety of pharmacological agents showed disruption of proper ER functions and thereby cause ER stress in male reproductive organ in rat model. The present review summarizes the existing data concerning the molecular and biological mechanism of ER stress in male reproduction and male infertility. ER stress initiated cell death pathway has been related to several diseases, including hypoxia, heath disease, diabetes, and Parkinson's disease. Although there is not enough evidence to prove the relationship between ER stress and male infertility in human, most studies in this review found that ER stress was correlated with male reproduction and infertility in animal models. The ER stress could be novel signaling pathway of regulating male reproductive cellular apoptosis. Infertility might be a result of disturbing the ER stress response during the process of male reproduction.

Nature is unlikely to maintain systems that are detrimental to survival. Any pathway that causes illness must also have a meaningful role. Endoplasmic reticulum (ER) stress, which mediates obesity-induced insulin resistance, is no exception as it helps maintain nutrient homeostasis. About one-third of all proteins are synthesized and folded into their native conformation in ER (1). Unfortunately, as much as 30% of all newly synthesized proteins, especially those abundant in cells, are misfolded. These are generally repaired or cleared during the maturation processes (2). ER is also responsible for protein quality control (3). Unfolded proteins in the ER lumen are identified by several ER stress sensors, including activating transcription factor (ATF) 6, protein kinase RNA-like ER kinase (PERK), and inositol-requiring enzyme 1 (IRE-1). These ER stress sensors trigger cellular adaptation to unfolded protein accumulation that restores normal cell function. This is called the unfolded protein response (UPR). The UPR initially 1 ) activates a signaling pathway that induces a large number of ER chaperones that assist protein folding, 2 ) halts protein translation and transcription, and 3 ) ubiquitinates and degrades unfolded proteins by 26S proteasome and through autophagy. The latter process is called ER-associated degradation. If these adaptations fail to maintain cellular homeostasis, the UPR leads the cell toward apoptosis or dedifferentiation (3). Chronic UPRs are causally linked to the pathogenesis of human metabolic disease including obesity and type 2 diabetes. Accumulating evidence suggests that obesity promotes ER stress, which is detected as enhanced UPR signaling. In turn, this activates c-Jun NH2-terminal kinase (JNK) and impairs insulin signaling at the level of insulin receptor substrates (IRSs) in the liver and adipose tissue (4). However, the signals that activate UPR under this condition are still unknown. Defective autophagy (5) and proteasome dysfunction (6) have recently been …

Stresses increasing the load of unfolded proteins that enter the endoplasmic reticulum (ER) trigger a protective response termed the unfolded protein response (UPR). Stromal cell-derived factor2 (SDF2)-type proteins are highly conserved throughout the plant and animal kingdoms. In this study we have characterized AtSDF2 as crucial component of the UPR in Arabidopsis thaliana. Using a combination of biochemical and cell biological methods, we demonstrate that SDF2 is induced in response to ER stress conditions causing the accumulation of unfolded proteins. Transgenic reporter plants confirmed induction of SDF2 during ER stress. Under normal growth conditions SDF2 is highly expressed in fast growing, differentiating cells and meristematic tissues. The increased production of SDF2 due to ER stress and in tissues that require enhanced protein biosynthesis and secretion, and its association with the ER membrane qualifies SDF2 as a downstream target of the UPR. Determination of the SDF2 three-dimensional crystal structure at 1.95 A resolution revealed the typical beta-trefoil fold with potential carbohydrate binding sites. Hence, SDF2 might be involved in the quality control of glycoproteins. Arabidopsis sdf2 mutants display strong defects and morphological phenotypes during seedling development specifically under ER stress conditions, thus establishing that SDF2-type proteins play a key role in the UPR.

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 stress sensors ATF6, IRE1, and PERK monitor deviations from homeostatic conditions in the endoplasmic reticulum (ER), a protein biogenesis compartment of eukaryotic cells. Their activation elicits unfolded protein responses (UPR) to re-establish proteostasis. UPR have been extensively investigated in cells exposed to chemicals that activate ER stress sensors by perturbing calcium, N-glycans, or redox homeostasis. Cell responses to variations in luminal load with unfolded proteins are, in contrast, poorly characterized. Here, we compared gene and protein expression profiles in HEK293 cells challenged with ER stress-inducing drugs or expressing model polypeptides. Drug titration to limit up-regulation of the endogenous ER stress reporters heat shock protein family A (Hsp70) member 5 (BiP/HSPA5) and homocysteine-inducible ER protein with ubiquitin-like domain 1 (HERP/HERPUD1) to levels comparable with luminal accumulation of unfolded proteins substantially reduced the amplitude of both transcriptional and translational responses. However, these drug-induced changes remained pleiotropic and failed to recapitulate responses to ER load with unfolded proteins. These required unfolded protein association with BiP and induced a much smaller subset of genes participating in a chaperone complex that binds unfolded peptide chains. In conclusion, UPR resulting from ER load with unfolded proteins proceed via a well-defined and fine-tuned pathway, whereas even mild chemical stresses caused by compounds often used to stimulate UPR induce cellular responses largely unrelated to the UPR or ER-mediated protein secretion.

The inositol requiring enzyme (IRE1) is an endoplasmic reticulum (ER) stress sensor. When activated, it splices the bZIP60 mRNA, producing a truncated transcription factor that upregulates genes involved in the unfolded protein response. Bax inhibitor 1 (BI-1) is another ER stress sensor that regulates cell death in response to environmental assaults. The potyvirus 6K2 and potexvirus TGB3 proteins are known to reside in the ER, serving, respectively, as anchors for the viral replicase and movement protein complex. This study used green fluorescent protein (GFP)-tagged Turnip mosaic virus (TuMV), Plantago asiatica mosaic virus (PlAMV), Potato virus Y (PVY), and Potato virus X (PVX) to determine that the IRE1/bZIP60 pathway and BI-1 machinery are induced early in virus infection in Arabidopsis thaliana, Nicotiana benthamiana, and Solanum tuberosum. Agrodelivery of only the potyvirus 6K2 or TGB3 genes into plant cells activated bZIP60 and BI-1 expression in Arabidopsis thaliana, N. benthamiana, and S. tuberosum. Homozygous ire1a-2, ire1b-4, and ire1a-2/ire1b-4 mutant Arabidopsis plants were inoculated with TuMV-GFP or PlAMV-GFP. PlAMV accumulates to a higher level in ire1a-2 or ire1a-2/ire1b-4 mutant plants than in ire1b-4 or wild-type plants. TuMV-GFP accumulates to a higher level in ire1a-2, ire1b-4, or ire1a-2/ire1b-4 compared with wild-type plants, suggesting that both isoforms contribute to TuMV-GFP infection. Gene silencing was used to knock down bZIP60 and BI-1 expression in N. benthamiana. PVX-GFP and PVY-GFP accumulation was significantly elevated in these silenced plants compared with control plants. This study demonstrates that two ER stress pathways, namely IRE1/bZIP60 and the BI-1 pathway, limit systemic accumulation of potyvirus and potexvirus infection. Silencing BI-1 expression also resulted in systemic necrosis. These data suggest that ER stress-activated pathways, led by IRE1 and BI-1, respond to invading potyvirus and potexviruses to restrict virus infection and enable physiological changes enabling plants to tolerate virus assault.

ER stress: Can the liver cope?