Stress tolerance of misfolded carboxypeptidase Y requires maintenance of protein trafficking and degradative pathways

The accumulation of aberrantly folded proteins can lead to cell dysfunction and death. Currently, the mechanisms of toxicity and cellular defenses against their effects remain incompletely understood. In the endoplasmic reticulum (ER), stress caused by misfolded proteins activates the unfolded protein response (UPR). The UPR is an ER-to-nucleus signal transduction pathway that regulates a wide variety of target genes to maintain cellular homeostasis. We studied the effects of ER stress in budding yeast through expression of the well-characterized misfolded protein, CPY*. By challenging cells within their physiological limits to resist stress, we show that the UPR is required to maintain essential functions including protein translocation, glycosylation, degradation, and transport. Under stress, the ER-associated degradation (ERAD) pathway for misfolded proteins is saturable. To maintain homeostasis, an "overflow" pathway dependent on the UPR transports excess substrate to the vacuole for turnover. The importance of this pathway was revealed through mutant strains compromised in the vesicular trafficking of excess CPY*. Expression of CPY* at levels tolerated by wild-type cells was toxic to these strains despite retaining the ability to activate the UPR.     

Cellular response to unfolded proteins in the endoplasmic reticulum of plants

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero-oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal-transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.     

ER stress protects from retinal degeneration

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 H₂O₂, 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.     

ER stress and its functional link to mitochondria: role in cell survival and death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.     

Activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.     

Drosophila as a model for unfolded protein response research

Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms. [BMB Reports 2015; 48(8): 445-453]     

Endoplasmic Reticulum Enrollment in Alzheimer’s Disease

Alzheimer??s disease (AD) poses a huge challenge for society and health care worldwide as molecular pathogenesis of the disease is poorly understood and curative treatment does not exist. The mechanisms leading to accelerated neuronal cell death in AD are still largely unknown, but accumulation of misfolded disease-specific proteins has been identified as potentially involved. In the present review, we describe the essential role of endoplasmic reticulum (ER) in AD. Despite the function that mitochondria may play as the central major player in the apoptotic process, accumulating evidence highlights ER as a critical organelle in AD. Stress that impairs ER physiology leads to accumulation of unfolded or misfolded proteins, such as amyloid ?? (A??) peptide, the major component of amyloid plaques. In an attempt to ameliorate the accumulation of unfolded proteins, ER stress triggers a protective cellular mechanism, which includes the unfolded protein response (UPR). However, when activation of the UPR is severe or prolonged enough, the final cellular outcome is pathologic apoptotic cell death. Distinct pathways can be activated in this process, involving stress sensors such as the JNK pathway or ER chaperones such as Bip/GRP94, stress modulators such as Bcl-2 family proteins, or even stress effectors such as caspase-12. Here, we detail the involvement of the ER and associated stress pathways in AD and discuss potential therapeutic strategies targeting ER stress.     

Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death

A variety of debilitating diseases including diabetes, Alzheimer's, Huntington's, Parkinson's, and prion-based diseases are linked to stress within the endoplasmic reticulum (ER). Using S. cerevisiae, we sought to determine the relationship between protein misfolding, ER stress, and cell death. In the absence of ERV29, a stress-induced gene required for ER associated degradation (ERAD), misfolded proteins accumulate in the ER leading to persistent ER stress and subsequent cell death. Cells alleviate ER stress through the unfolded protein response (UPR); however, if stress is sustained the UPR contributes to cell death by causing the accumulation of reactive oxygen species (ROS). ROS are generated from two sources: the UPR-regulated oxidative folding machinery in the ER and mitochondria. Our results demonstrate a direct mechanism(s) by which misfolded proteins lead to cellular damage and death.     

Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.     

Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR.     

The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis

Recently, there has been an increasing amount of literature published on the effects of 4-phenylbutyric acid (4-PBA) in various biological systems. 4-PBA is currently used clinically to treat urea cycle disorders under the trade name Buphenyl. Recent studies however have explored 4-PBA in the context of a low weight molecular weight chemical chaperone. Its properties as a chemical chaperone prevent misfolded protein aggregation and alleviate endoplasmic reticulum (ER) stress. As the ER is responsible for folding proteins targeted for use in membranes or secreted out of the cell, failure of maintaining adequate ER homeostasis may lead to protein misfolding and subsequent cell and organ pathology. Accumulation of misfolded proteins within the ER activates the unfolded protein response (UPR), a molecular repair response. The activation of the UPR aims to restore ER and cellular proteostasis by regulating the rate of synthesis of newly formed proteins as well as initiating molecular programs aimed to help fold or degrade misfolded proteins. If proteostasis is not restored, the UPR may initiate pro-apoptotic pathways. It is suggested that 4-PBA may help fold proteins in the ER, attenuating the activation of the UPR, and thus potentially alleviating various pathologies. This review discusses the biomedical research exploring the potential therapeutic effects of 4-PBA in various in vitro and in vivo model systems and clinical trials, while also commenting on the possible mechanisms of action.     

Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

Unfolded protein response

In eukaryotic cells, the endoplasmic reticulum (ER) is a membrane-enclosed interconnected organelle responsible for the synthesis, folding, modification, and quality control of numerous secretory and membrane proteins. The processes of protein folding and maturation are highly assisted and scrutinized but are also sensitive to changes in ER homeostasis, such as Ca(2+) depletion, oxidative stress, hypoxia, energy deprivation, metabolic stimulation, altered glycosylation, activation of inflammation, as well as increases in protein synthesis or the expression of misfolded proteins or unassembled protein subunits. Only properly folded proteins can traffic to the Golgi apparatus, whereas those that misfold are directed to ER-associated degradation (ERAD) or to autophagy. The accumulation of unfolded/misfolded proteins in the ER activates signaling events to orchestrate adaptive cellular responses. This unfolded protein response (UPR) increases the ER protein-folding capacity, reduces global protein synthesis, and enhances ERAD of misfolded proteins.     

Regulation of the unfolded protein response by microRNAs

The unfolded protein response (UPR) is an adaptive response to the stress that is caused by an accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). It is an important component of cellular homeostasis. During ER stress, the UPR increases the protein-folding capacity of the endoplasmic reticulum to relieve the stress. Failure to recover leads to apoptosis. Specific cellular mechanisms are required for the cellular recovery phase after UPR activation. Using bioinformatics tools, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR. In this review, we discuss the potential role of microRNAs as key regulators of this pathway and describe how microRNAs may play an essential role in turning off the UPR after the stress has subsided.     

The unfolded protein response as a target for cancer therapy

Various physiological and pathological conditions generate an accumulation of misfolded proteins in the endoplasmic reticulum (ER). This results in ER stress followed by a cellular response to cope with this stress and restore homeostasis: the unfolded protein response (UPR). Overall, the UPR leads to general translational arrest and the induction of specific factors to ensure cell survival or to mediate cell death if the stress is too severe. In multiple cancers, components of the UPR are overexpressed, indicating increased dependence on the UPR. In addition, the UPR can confer resistance to anti-cancer treatment. Therefore, modification of the UPR should be explored for its anti-cancer properties. This review discusses factors associated with the UPR that represent potential therapeutic targets.     

The potential contribution of stromal cell-derived factor 2 (SDF2) in endoplasmic reticulum stress response in severe preeclampsia and labor-onset

Endoplasmic reticulum (ER) stress occurs when the protein folding machinery in the cell is unable to cope with newly synthesized proteins, which results in an accumulation of misfolded proteins in the ER lumen. In response, the cell activates a cellular signaling pathway known as the Unfolded Protein Response (UPR), aiming to restore cellular homeostasis. Activation and exacerbation of the UPR have been described in several human pathologies, including cancer and neurological disorders, and in some gestational diseases such as preeclampsia and gestational diabetes. This review explores the participation of stromal cell-derived factor 2 (SDF2) in UPR pathways, shows new information and discusses its exacerbation regarding protein expression in severe preeclampsia and labor, both of which are associated with ER stress.