Identification of mitogen-activated protein kinase signaling pathways that confer resistance to endoplasmic reticulum stress in Saccharomyces cerevisiae

Hypoxia activates all components of the unfolded protein response (UPR), a stress response initiated by the accumulation of unfolded proteins within the endoplasmic reticulum (ER). Our group and others have shown previously that the UPR, a hypoxia-inducible factor-independent signaling pathway, mediates cell survival during hypoxia and is required for tumor growth. Identifying new genes and pathways that are important for survival during ER stress may lead to the discovery of new targets in cancer therapy. Using the set of 4,728 homozygous diploid deletion mutants in budding yeast, Saccharomyces cerevisiae, we did a functional screen for genes that conferred resistance to ER stress-inducing agents. Deletion mutants in 56 genes showed increased sensitivity under ER stress conditions. Besides the classic UPR pathway and genes related to calcium homeostasis, we report that two additional pathways, including the SLT2 mitogen-activated protein kinase (MAPK) pathway and the osmosensing MAPK pathway, were also required for survival during ER stress. We further show that the SLT2 MAPK pathway was activated during ER stress, was responsible for increased resistance to ER stress, and functioned independently of the classic IRE1/HAC1 pathway. We propose that the SLT2 MAPK pathway is an important cell survival signaling pathway during ER stress. This study shows the feasibility of using the yeast deletion pool to identify relevant mammalian orthologues of the UPR.     

ER stress and the unfolded protein response

Conformational diseases are caused by mutations altering the folding pathway or final conformation of a protein. Many conformational diseases are caused by mutations in secretory proteins and reach from metabolic diseases, e.g. diabetes, to developmental and neurological diseases, e.g. Alzheimer's disease. Expression of mutant proteins disrupts protein folding in the endoplasmic reticulum (ER), causes ER stress, and activates a signaling network called the unfolded protein response (UPR). The UPR increases the biosynthetic capacity of the secretory pathway through upregulation of ER chaperone and foldase expression. In addition, the UPR decreases the biosynthetic burden of the secretory pathway by downregulating expression of genes encoding secreted proteins. Here we review our current understanding of how an unfolded protein signal is generated, sensed, transmitted across the ER membrane, and how downstream events in this stress response are regulated. We propose a model in which the activity of UPR signaling pathways reflects the biosynthetic activity of the ER. We summarize data that shows that this information is integrated into control of cellular events, which were previously not considered to be under control of ER signaling pathways, e.g. execution of differentiation and starvation programs.     

Endoplasmic reticulum stress response in yeast and humans

Stress pathways monitor intracellular systems and deploy a range of regulatory mechanisms in response to stress. One of the best-characterized pathways, the UPR (unfolded protein response), is an intracellular signal transduction pathway that monitors ER (endoplasmic reticulum) homoeostasis. Its activation is required to alleviate the effects of ER stress and is highly conserved from yeast to human. Although metazoans have three UPR outputs, yeast cells rely exclusively on the Ire1 (inositol-requiring enzyme-1) pathway, which is conserved in all Eukaryotes. In general, the UPR program activates hundreds of genes to alleviate ER stress but it can lead to apoptosis if the system fails to restore homoeostasis. In this review, we summarize the major advances in understanding the response to ER stress in Sc (Saccharomyces cerevisiae), Sp (Schizosaccharomyces pombe) and humans. The contribution of solved protein structures to a better understanding of the UPR pathway is discussed. Finally, we cover the interplay of ER stress in the development of diseases.     

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.     

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.     

A novel role in cytokinesis reveals a housekeeping function for the unfolded protein response

The unfolded protein response (UPR) pathway helps cells cope with endoplasmic reticulum (ER) stress by activating genes that increase the ER's functional capabilities. We have identified a novel role for the UPR pathway in facilitating budding yeast cytokinesis. Although other cell cycle events are unaffected by conditions that disrupt ER function, cytokinesis is sensitive to these conditions. Moreover, efficient cytokinesis requires the UPR pathway even during unstressed growth conditions. UPR-deficient cells are defective in cytokinesis, and cytokinesis mutants activate the UPR. The UPR likely achieves its role in cytokinesis by sensing small changes in ER load and making according changes in ER capacity. We propose that cytokinesis is one of many cellular events that require a subtle increase in ER function and that the UPR pathway has a previously uncharacterized housekeeping role in maintaining ER plasticity during normal cell growth.     

CHOP is a multifunctional transcription factor in the ER stress response

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) induces ER stress. To restore ER homeostasis, cells possess a highly specific ER quality-control system called the unfold protein response (UPR). In the case of prolonged ER stress or UPR malfunction, apoptosis signalling is activated. This ER stress-induced apoptosis has been implicated in the pathogenesis of several conformational diseases. CCAAT-enhancer-binding protein homologous protein (CHOP) is induced by ER stress and mediates apoptosis. Recent studies by the Gotoh group have shown that the CHOP pathway is also involved in ER stress-induced cytokine production in macrophages. The multifunctional roles of CHOP in the ER stress response are discussed below.     

NADPH Oxidase-Dependent Production of Reactive Oxygen Species Induces Endoplasmatic Reticulum Stress in Neutrophil-Like HL60 Cells

Reactive oxygen species (ROS) primarily produced via NADPH oxidase play an important role for killing microorganisms in neutrophils. In this study we examined if ROS production in Human promyelocytic leukemia cells (HL60) differentiated into neutrophil-like cells (dHL60) induces ER stress and activates the unfolded protein response (UPR). To cause ROS production cells were treated with PMA or by chronic hyperglycemia. Chronic hyperglycemia failed to induce ROS production and did not cause activation of the UPR in dHL60 cells. PMA, a pharmacologic NADPH oxidase activator, induced ER stress in dHL60 cells as monitored by IRE-1 and PERK pathway activation, and this was independent of calcium signaling. The NADPH oxidase inhibitor, DPI, abolished both ROS production and UPR activation. These results show that ROS produced by NADPH oxidase induces ER stress and suggests a close association between the redox state of the cell and the activation of the UPR in neutrophil-like HL60 cells.     

Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ire1p.

Conditions that stress the endoplasmic reticulum (ER) in Saccharomyces cerevisiae can elicit a combination of an unfolded protein response (UPR) and an inositol response (IR). This results in increased synthesis of ER protein-folding factors and of enzymes participating in phospholipid biosynthesis. It was suggested that in cells grown on glucose or galactose medium, the UPR and the IR are linked and controlled by the ER stress sensor Ire1p. However, our studies suggest that during growth on oleate the IR is controlled both by an Ire1p-dependent pathway and by an Ire1p-independent pathway.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Signal integration in the endoplasmic reticulum unfolded protein response

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways - cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate and the metabolism of proteins, amino acids and lipids. The arms of the UPR are integrated to provide a response that remodels the secretory apparatus and aligns cellular physiology to the demands imposed by ER stress.     

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.