Upr pathway

Upr pathway DEFAULT

The Unfolded Protein Response (UPR) is a regulatory system that protects the Endoplasmic Reticulum (ER) from overload. The UPR is provoked by the accumulation of improperly folded protein in the ER during times of unusually high secretion activity. Analysis of mutants with altered UPR, however, shows that the UPR is also required for normal development and function of secretory cells.
One level at which the URP operates is transcriptional and translational regulation: mobilization of ATF6, ATF6B, CREB3 factors and IRE1 leads to increased transcription of genes encoding chaperones, while mobilization of PERK (pancreatic eIF2alpha kinase, EIF2AK3) leads to phosphorylation of the translation initiation factor eIF2alpha and global down-regulation of protein synthesis.
ATF6, ATF6B, and CREB3 factors (CREB3 (LUMAN), CREB3L1 (OASIS), CREB3L2 (BBF2H7, Tisp40), CREB3L3 (CREB-H), and CREB3L4 (CREB4)) are membrane-bound transcription activators that respond to ER stress by transiting from the ER membrane to the Golgi membrane where their transmembrane domains are cleaved, releasing their cytosolic domains to transit to the nucleus and activate transcription of target genes. IRE1, also a resident of the ER membrane, dimerizes and autophosphorylates in response to ER stress. The activated IRE1 then catalyzes unconventional splicing of XBP1 mRNA to yield an XBP1 isoform that is targeted to the nucleus and activates chaperone genes.

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Unfolded protein response (UPR) integrated signaling networks determine cell fate during hypoxia

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Cellular & Molecular Biology Lettersvolume 25, Article number: 18 () Cite this article

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Abstract

During hypoxic conditions, cells undergo critical adaptive responses that include the up-regulation of hypoxia-inducible proteins (HIFs) and the induction of the unfolded protein response (UPR). While their induced signaling pathways have many distinct targets, there are some important connections as well. Despite the extensive studies on both of these signaling pathways, the exact mechanisms involved that determine survival versus apoptosis remain largely unexplained and therefore beyond therapeutic control. Here we discuss the complex relationship between the HIF and UPR signaling pathways and the importance of understanding how these pathways differ between normal and cancer cell models.

This article was specially invited by the editors and represents work by leading researchers.

Introduction

Aerobic organisms employ critical control strategies to ensure proper oxygen supply through various physiological and metabolic cellular signaling networks. The inability to meet cellular oxygen demands, termed hypoxia, results in the activation of specific cellular stress responses [1, 2]. Hypoxic stress induces global gene expression changes in order to help cells adapt and survive by altering the cell’s metabolic and angiogenic pathways and restoring oxygen homeostasis [3,4,5,6,7,8,9,10]. If these repair and adaptive mechanisms fail, cells modify their gene expression profiles to induce programmed cell death [11,12,13,14,15,16]. Although active hypoxia signaling networks are necessary during embryogenesis and development [17,18,19], hypoxic conditions either diminish normally, or they contribute to pathological events in mature organisms [20,21,22,23].

Efficient activation of hypoxia signaling and angiogenesis is critical, for example, after stroke [24], myocardial infarction [25], and other ischemic events [26,27,28,29]. Alternatively, metabolic adaptation to low oxygen levels and the related tissue revascularization allows for the survival and progression of the majority of human tumors [30,31,32], and contributes to macular degeneration [33,34,35,36], glaucoma progression [37], and diabetic retinopathy [38,39,40,41]. Thus, the discovery and development of therapeutic strategies exploiting hypoxia-related cellular networks are of great interest to modern medicine, as evidenced by the awarding of the Nobel Prize in Physiology or Medicine to Drs. Semenza, Ratcliffe, Kaelin on their research into how cells detect oxygen and react to hypoxia [42,43,44,45,46].

The main goal of the cellular response to hypoxia is to promote cell survival and restore oxygen homeostasis. This goal, however, is accompanied by deregulation of cellular organelle changes in mitochondria and endoplasmic reticulum (ER) function that are reflected in perturbations in protein folding and trafficking [4, 47,48,49,50,51,52,53]. Erratic protein folding activates another specific stress response pathway, the unfolded protein response (UPR). The UPR promotes cellular survival by restoring endoplasmic and mitochondrial homeostasis through its distinct signaling networks [54,55,56], but if unsuccessful, the UPR induces cell death [57,58,59].

Although activation of the UPR supports surviving hypoxia, it can also impair cellular survival [60]. The ER, for example, is responsible for folding and maturation of transmembrane and secretory proteins [61,62,63,64,65,66,67,68,69] that include proangiogenic receptors and ligands such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO) that are critical for hypoxia-induced angiogenesis and erythropoiesis, respectively [70,71,72]. Thus, although underappreciated, understanding mutual crosstalk between these stress response pathways is important for understanding and developing therapeutic interventions in cardiovascular diseases and cancer. Nevertheless, despite the extensive studies on both of these stress responses, the resulting consequences of their collective activation remain largely unexplained and are mainly limited to in vitro cell culture-based models. In this review, we summarize these two cell survival pathways and the implications of UPR involvement in the hypoxia cellular response pathway.

Hypoxia-inducible factor responses to hypoxia

The unmet cellular oxygen demand is reflected by the accumulation of functional heterodimeric α/β-subunit complexes of specific transcription factors called hypoxia-inducible factors (HIFs) [42,43,44,45,46]. HIFs mediate both the adaptive and apoptotic responses to hypoxia through transcriptional modulation of genes containing their specific target sequences that are termed hypoxia-response elements (HREs) [7, 73,74,75,76,77]. If cells are sufficiently supplied with the oxygen, the formation of active HIF complexes is inhibited by the limited availability of the alpha (α) subunits. Under normal oxygen pressure (normoxia), HIF-α subunits undergo oxygen-dependent post-translational modifications by proline-hydroxylases (PHDs) that mark these subunits for subsequent proteasomal degradation [42,43,44,45,46]. Furthermore, during normoxia there is another oxygen-dependent post-translational modification of α-subunits that is mediated by the factor inhibiting HIF (FIH) which impairs HIF transcriptional activity (Fig. 1) [78]. In contrast, the cellular levels of HIF-β subunits are oxygen independent [42,43,44,45,46]. During hypoxia, the post-translational modifications of HIF-α subunits are inhibited and lead to accumulation of the alpha subunit and the transcriptionally active HIF-αβ hetero-complexes. Despite the fact that HIF-1α is considered a major mediator of HIF signaling in higher metazoans, other tissue specific isoforms of α-subunits, HIF-2α and HIF-3α, are also known to participate in the cellular responses to hypoxia [7, 79,80,81,82,83,84].

Oxygen availability regulates HIF signaling. In normoxia, proline (P) residues on HIFα subunits are hydroxylated by PHDs that marks them for proteasomal degradation. Additionally, FIH-1 mediates hydroxylation of asparagine residues (N) on HIFα to prevent HIF transcriptional activity. Hypoxia impairs the ability of PHDs and FIH-1 to hydroxylate the HIFα subunits, and thus results in the accumulation of this subunit and its heterodimerization with the stable HIFβ subunits. In the nucleus, the HIFα/β complex binds to HRE elements in the HIF target genes and governs their expression in order to adapt the cells to hypoxic conditions

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The pro-survival pathway

During hypoxia, HIFs execute pro-survival transcriptomic strategies that allow cells to sustain energy levels via utilization of less efficient non-oxidative energy production. To sustain energy levels, HIFs upregulate glucose transporter genes and glycolytic enzymes, and inhibit oxidative phosphorylation (1) by preventing the conversion of pyruvate to acetyl-Co-A, (2) by reducing glucose oxidation and (3) by inhibiting β-oxidation of fatty acids [85,86,87,88]. Importantly, hypoxia-related utilization of this alternate metabolic pathway is accompanied by a HIF-mediated activation of the mechanisms that allow for a more efficient utilization of the anaerobic glycolytic pathway and that minimize its negative impact on the cell. The goal is to increase the electron transfer efficiency and to reduce reactive oxygen species (ROS) production. HIF-1 also regulates cytochrome c oxidase (COX) subunit composition to optimize the efficiency of respiration during hypoxia and to reduce ROS by promoting ROS scavenging pathways [89, 90]. Furthermore, since anaerobic glycolysis results in increased proton release, HIF-1 induces the expression of carbonic anhydrase 9 (CA-IX) and monocarboxylate transporter 4 (MCT4) to counteract acidosis by regulating intracellular pH [91, 92].

Since non-oxidative energy production of the cellular levels of ATP is less efficient than oxidative phosphorylation, HIFs activate pathways to decrease the cell’s energy needs. To accomplish this, HIFs selectively suppress translation and therefore decrease total protein production [93,94,95] and induce induction of autophagy [96, 97] and mitophagy [95, 98]. Notably, the mTOR pathway also reduces protein synthesis and cell growth and induces autophagy via a HIF-independent mechanism [48, 99].

In order to restore oxygen homeostasis and maintain the well-being of the endothelium, HIFs stimulate the expression of a number of angiogenic genes that include the vascular endothelial growth factor (VEGF) [9, ], heme oxygenase-1 (HMOX1) [], matrix metalloproteinases (MMP) 2 and 13 [], the stem cell factor OCT-3/4 [, ], angiopoietin 2 (ANGPT2) [], stromal derived factor 1 (SDF1) [], platelet-derived growth factor B (PDGFB) [], placental growth factor (PGF) [], and stem cell factor (SCF )[] and endothelial nitric oxide synthase (NOS3) [, ]. While HIF-induced angiogenesis ensures increased blood flow to hypoxic tissues, the oxygen caring capacity of the blood is enhanced via HIF-dependent upregulation of erythropoietin [,,]. Importantly, to secure proper cellular iron levels that are required for the efficient erythropoiesis, HIFs adjust the expression of transferrin as well as of other genes mediating iron homeostasis [, ]. Furthermore, EPO supports anti-apoptotic proteins and inhibits caspase activity [,,].

The UPR pathway responses to hypoxia

The fundamental function of the cellular response to hypoxia is surviving precarious conditions and restoring oxygen homeostasis. Hence, despite the HIF-related mechanisms to reduce the negative effects of anaerobic glycolysis and the reduced energy availability, this metabolic switch eventually disturbs cellular homeostasis. This energy deficiency limits the activity of ATP-dependent processes such as maintenance of ion homeostasis and the related redox potential, and limits protein and lipid synthesis, and post-translational protein folding capabilities due to the impaired disulfide-bond formation and ROS activity [4, ,,,,]. All of these factors can disturb endoplasmic reticulum homeostasis (termed as ER stress), and lead to the accumulation of unfolded or misfolded proteins in the ER []. The accumulation of misfolded proteins activate another specialized stress response signaling pathway called unfolded protein response (UPR) []. During hypoxia, there are critical changes in mitochondrial function that lead to elevated ROS levels. Furthermore, the proper folding of mitochondria-encoded as well as the import and corresponding refolding of mitochondrial nucleus-encoded proteins are crucial for mitochondrial function [, ]. Hence, prolonged hypoxia will eventually result in perturbations in mitochondrial protein folding and activation of a related specific stress response mechanism termed the mitochondrial unfolded protein response (UPRmt) [, ,,].

The three UPR signaling pathways

Controlling ER homeostasis relies on the interplay between three signaling pathways of UPR that are initiated by three distinctive transmembrane sensors []. Buildup of unfolded/misfolded proteins in the ER induces a higher demand for chaperone proteins that include glucose-regulated protein 78 (GRP78 also known as BiP (binding immunoglobin protein) [57]. BiP initiates the UPR by dissociating from luminal domains of three proteins, protein kinase RNA-like endoplasmic reticulum kinase (PERK), the inositol-requiring enzyme 1α (IRE1α), and with activating transcription factor 6 (ATF6) [57]. Upon BiP release, PERK and IRE1α are activated via multimerization and trans-autophosphorylation, whereas ATF6 is translocated to the Golgi apparatus where it is proteolytically processed to a cytoplasmically soluble and active ATF6f (p50) transcription factor (Fig. 2) [,,]. This activation cascade results in three distinctive UPR signaling pathways/axes that are mediated by the PERK, IRE1α and ATF6 sensors.

UPR and UPRmt signaling. Upon buildup of misfolded/unfolded proteins in ER, BIP is released from ER membrane to induce PERK dimerization and its subsequent autophosphorylation. Activated PERK phosphorylates the eIF2α, leading to global translation attenuation. Some transcripts, however, including ATF4 remain preferably translated. ATF4 provides the transcriptional signal to restore ER homeostasis, however, it can also induce proapoptotic CHOP. Similarly, accumulation of unfolded proteins in mitochondria leads to PERK activation and the induction of ATF4 signaling (UPRmt). Upon its dissociation from BIP, IRE1α undergoes oligomerization and autophosphorylation and thus gains endoribonuclease activity. To decrease the ER load, activated IRE1α degrades mRNAs and miRNAs (RIDD). IRE1α also performs splicing of XBP1 mRNA to release transcriptionally active XBP1s. XBP1s activates a transcriptional program to restore ER homeostasis. Alternatively, IRE1α can activate a proapoptotic kinase JNK1. Finally, BIP dissociation allows ATF6 translocation to Golgi, where cleavage of this protein results in release of transcriptionally active ATF6f. ATF6f activates a transcriptional program to restore ER homeostasis and support ERAD

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Active PERK phosphorylates the alpha subunit of the eukaryotic initiation factor eIF2, and this initiates the selective translation of certain proteins and repressing the translation others during stress conditions. Some of the selected proteins include activating transcription factor 4 (ATF4), growth arrest and DNA damage inducible protein (GADD34), and CCAAT/enhancer binding homologous protein (CHOP) [57, , ]. ATF4 modulates the expression of genes involved in amino acid biosynthesis, anti-oxidative responses, protein folding and in maintaining redox homeostasis []. Importantly, GADD34 mediates the dephosphorylation of eIF2, thus allowing the restoration of the protein synthesis upon stress recovery []. If the stress is persistent, ATF4 can also facilitate autophagy and stimulate transcription of the proapoptotic CHOP to induce cell death (Fig. 2) [, ].

Active IRE1α reduces protein synthesis through the degradation of selected mRNAs in a process referred to as regulated IRE1-dependent decay (RIDD) []. Notably, IRE1α endoribonuclease activity generates the active spliced isoform of the X-box binding-protein transcription factor (XBP1s) []. XBP1s modulates gene expression by increasing the ER’s folding capacity. XBP1s also promotes the expression of proteins that are involved in ER membrane biosynthesis, disulfide bond formation, as well as increasing the expression of chaperones and proteins involved in ER-associated degradation (EDEM) and vesicular trafficking [,,]. Furthermore, IRE1α kinase activity activates Janus N-terminal kinase (JNK) in order to activate the inflammatory response and to promote autophagy and apoptosis [, ] (Fig. 2).

ATF6f, on the other hand, initiates a transcriptional program to restore ER homeostasis and that includes the induction of BIP expression, promoting protein chaperone and lipid synthesis, stimulating ER-degradation, and enhancing N-glycosylation [, ]. ATF6f also induces CHOP expression and thus contributes to UPR-related cell death [, ] (Fig. 2). Notably however, a recent report has shown that IRE1α activation can deactivate the ATF6f pathway [].

Despite the fact that the UPR usually mediates cell death by activating the intrinsic apoptotic pathway, recent reports indicate that during unresolved ER stress, there is strong activation of the UPR that can lead to activation of programmed-necrosis pathways such as necroptosis [,,,,,]. Activation of these cell death pathways usually involves PERK signaling and is associated with a rapid depletion of intracellular ATP and a rapid release of ER-stored calcium [,,,,,]. Notably, the necroptosis pathway has been involved in modulation of both HIF-signaling and key glycolytic enzymes that include pyruvate dehydrogenase. This results in the enhancement of aerobic respiration and ROS generation, and thus can lead to impaired cellular adaptation to hypoxia [,,,]. That being said, the origins and role of necroptosis in both the UPR and the hypoxia response will require further studies.

Mitochondrial stress responses

Since mitochondria are separated from the cytosol and ER by their outer and inner membranes, they have to rely on their own stress response mechanisms for translating and folding proteins encoded in their genomes as well as refolding the imported nuclear-encoded proteins [, ]. In order to maintain their protein homeostasis, these organelles have a specific set of chaperones that includes heat shock protein 60 (HSP60) and LON peptidase 1 [,,]. Notably, it has been reported that events that lead to accumulation of unfolded/misfolded proteins in the mitochondria, or in impairment of energy dependent mitochondrial protein import, or in disturbances in mitochondrial protein synthesis and folding lead to the activation of a mitochondrial UPR (UPRmt) [, ,,].

To recover and preserve mitochondrial function, UPRmt modulates the expression of both mitochondria and nuclear encoded genes [, ,,]. However, if the stress is persistent, the UPRmt can contribute to the activation of intrinsic apoptosis pathways [, ,,]. In C. elegans, properly functional mitochondria import and subsequently degrade the stress sensor protein called activating transcription factor associated with stress (ATFS-1) []. Upon stress, however, ATFS-1 import to mitochondria is impaired, and this transcription factor accumulates in nucleus and activates a transcriptional program to restore mitochondrial homeostasis through upregulation of mitochondrial chaperons and proteases as well as components of both the protein import machinery and ROS scavenger pathway [].

Although the regulation of the mammalian UPRmt is poorly understood, it has been suggested that the import efficiency of activating transcription factors 5 and 4 (ATF5 and ATF4) can be sensors of mitochondrial protein disturbances [, ]. Upon stress, these transcription factors were shown to induce expression of mitochondrial chaperones and proteases. Furthermore, it has been shown that disturbances of mitochondrial protein homeostasis lead to activation of the PERK axis of the UPR, and this reduces global protein synthesis and selectively promotes expression of ATF4, ATF5 and the proapoptotic protein CHOP (Fig. 2) [, ,,, ]. However, the molecular mechanisms underpinning the integrated feedback between the UPR and the UPRmt will require further study.

The crosstalk between hypoxia and UPR in cancer versus normal cell models

Despite the fact that normal endothelial cells are the main effectors of the adaptive cellular response to hypoxia, the vast majority of current research regarding this signaling pathway is from cancer cells [31, 48, , ]. The mainstream reports of the interplay between hypoxia and UPR are limited to cancer models as well [71, 72, ,,,,]. Importantly, cancer progression and cancer cell survival often result from the deregulation of the cell fate decision mechanisms during both hypoxia and the UPR. Although hypoxia was shown to induce all three UPR signaling axes, and given their activation could also result from cancer cell-specific adaptations, it is important that the prosurvival consequences of the UPR need to be directly compared to normal cell types.

Hypoxia-related induction of BIP expression has been reported in both cancer and endothelial cells models [50, , ,,,,]. This suggests that hypoxia-induced perturbations in ER may increase BIP demand in both cell types and promote UPR induction. Indeed, activation of PERK signaling is also observed in both cancer and normal cells including endothelial cells, regardless of the hypoxia model applied [, ,,,,,]. PERK-mediated eIF2 phosphorylation was observed in cells within minutes after exposure to acute hypoxia (below % O2), whereas this reaction rate continuously declined with increasing oxygen concentrations []. Furthermore, activation of the PERK axis was also reported in transient (cyclic hypoxia) models that better resemble the fluctuating oxygen availability conditions that occur in solid tumors [,,,,]. Hence, it can be concluded that the hypoxia-required reduction of energy demand is partially achieved via UPR-mediated translational attenuation. Notably, this pathway was shown to be deactivated during prolonged hypoxia (16 h) as shown by dephosphorylation of eIF2 that is probably due to a negative feedback loop with GADD34 [, , ]. During prolonged hypoxia, HIF-1 signaling is only partially sustained by the HIF-2 activity during the transition from HIF-1 to HIF-2 expression [7, 76, 77]. This would suggest that the activation of PERK axis can only be modulated by the HIF-1, whereas during prolonged hypoxia, HIF-2 mediates the translational repression via an alternate mechanism []. However, this hypothesis will require further study. Interestingly, the PERK pathway was also shown to inhibit HIF-1α translation and thus prevent HIF-1 signaling in cancer cells [].

Besides attenuation of protein synthesis, the PERK pathway mediated by ATF4 activates genes supporting ER and mitochondrial homeostasis [, ,,, ]. Notably, however, the PERK pathway can induce cell death through CHOP accumulation []. Although CHOP accumulation and the potential induction of apoptotic response were observed in some hypoxia experiments (including lung endothelial cells) [,,], this protein and mRNA levels were much lower than those observed during ER stress []. Inhibition of the entire PERK axis during hypoxia, however, has more drastic effects on cell survival []. Furthermore, hypoxic PERK activation was shown to regulate carbonic anhydrase 9 (CA9) levels and thus is important for maintaining cellular pH [, ]. Importantly, CHOP also directly reduces the expression of the proangiogenic endothelial nitric synthase (NOS3, eNOS) []. The reduction of eNOS activity during hypoxia, however, may be required to prevent the uncoupling of this enzyme and the related ROS accumulation [, ]. Therefore, the evaluation of CHOP’s role in hypoxic cell survival requires careful consideration and further study [].

The activation of IRE1 axis and the role of XBP1s during hypoxia remain less clear. Despite some functionally relevant accumulation of XBP1s that supported cancer cell survival and tumor growth studies in cancers cell lines exposed to hypoxia, this effect was observed in acute and moderate hypoxia only [, ,,,,,,,,]. In contrast, impairment of XBP1 splicing under acute hypoxia was also reported []. Furthermore, although some potentially IRE1-related activity was observed in human pulmonary artery smooth muscle cells (PAMSCs), this did not result in direct hypoxia-induced XBP1s protein accumulation []. Hence, IRE1α involvement in cellular response may be very oxygen pressure- and cell type-specific and will require further studies in a wide range of primary endothelial cells [,,,,]. Finally, although numerous known ATF6 transcriptional targets were shown to be elevated in some experimental models by prolonged hypoxia and ischemia [, , ], the general direct hypoxic activation of the ATF6 UPR axis has been convincingly presented []. Hopefully, the novel ATF6 pathway inhibitors, Ceapins [], will be helpful in clarifying the role of this UPR branch during hypoxia.

The main function of adaptive HIF activity is with the induction of angiogenesis and erythropoiesis. The successful implementation of these cell rescue programs requires increased synthesis of proangiogenic factors (ligands and receptors) as well as increased erythropoietin production. All of these proteins fall into either the transmembrane or secretory proteins category, and as such they have to mature in the ER [52, , ,,,]. Hence, recovery from hypoxia absolutely depends on proper ER function []. Importantly, the PERK/ATF4 axis has been reported as a limiting factor for EPO production, and thus hypoxic UPR activation may limit adaptation to hypoxia [70].

In , Karali and coworkers described the mechanism potentially linking HIF transcriptional activity with the activation of PERK, ATF6 and IRE1 pathways in human endothelial cells []. They reported prosurvival UPR activation in VEGF (a HIF transcriptional target) treated human umbilical vein endothelial cells (HUVECs) []. In these studies, the authors proposed a mechanism in which VEGF-dependent phosphorylation of vascular endothelial growth factor receptors (VGFRs) leads to phospholipase C (PLC) activation and release of ER calcium, which activates all three axes of the UPR []. The active UPR promotes transcriptional expression of numerous proangiogenic genes that include VEGF that can be induced directly by ATF6f, XBP1s and ATF4 [, ,,,,,,,,,,

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Ubiquitination of Proteins and Protein Degradation

Structure and Molecular Mechanism of ER Stress Signaling by the Unfolded Protein Response Signal Activator IRE1

Introduction

The endoplasmic reticulum is a major site for protein folding and maturation within the eukaryotic cell. Proteins that reside in the ER, along with proteins destined for the Golgi, plasma membrane, and extracellular space are synthesized in ribosomes that are attached to the ER membrane. The newly translated polypeptide contains an N-terminal signal sequence that is recognized by signal recognition particle (SRP), which enables its insertion into the ER via the transolocon complex. Once inside the ER, the signal sequence is cleaved by signal peptidase and the translocated polypeptide undergoes post-translational modification and chaperone-assisted folding to help it to form its correct three-dimensional shape. There are various ER resident enzymes and chaperones that increase the efficiency of protein folding of the nascent polypeptide. One of the most abundant proteins within the ER is the Hsptype chaperone, BiP (binding-immunoglobulin protein aka GRP). BiP binds to nascent polypeptide chains to prevent their aggregation initially; and subsequently, facilitates their folding in order for the polypeptide to achieve its native conformation. Post-translational modifications are also critical for correct protein folding and one such modification is disulphide bond formation. This bond is important for maintaining tertiary and quaternary protein structure and is catalyzed by protein disulphide isomerase (PDI). Another essential modification is the attachment of N-linked oligosaccharides to the nascent chain, which occurs upon entry into the ER. Once in the ER, there are a number of glycosylating enzymes that either trim or add to the core N-linked oligosaccharide depending on the progress of protein folding. These alterations to the glycan chain help to monitor the folding status of the nascent polypeptide and act as an important quality control measure (Wang and Kaufman, ; Hetz and Papa, ).

A failure in the polypeptide chain to adopt its native conformation may lead to activation of degradation pathways, including ER-associated degradation (ERAD) (Hampton, ). In this process, misfolded protein is retro-translocated across the ER membrane into the cytosol, where it is ubiquitylated and targeted for degradation via the 26S proteasome. However, if the polypeptide adopts its correct shape, it can then transit to the Golgi and advance further through the secretory pathway.

The environment within the ER is more oxidizing than that of the cytosol. This is conducive to the formation of disulphide bonds, which occurs predominantly within the ER. Furthermore, a high concentration of calcium helps to buffer protein folding especially since many ER chaperones require calcium as a co-factor to operate effectively.

Protein folding requirements within the ER vary depending on cell type. For specialized secretory cells, such as plasma cells, insulin-producing ² cells, or highly proliferating malignant cells, which have increased protein synthesis rates, the demand for productive protein folding can be much higher than that for a typical cell. The inward flux of nascent polypeptides into the ER can overwhelm the protein-folding machinery, leading to an imbalance and the accumulation of misfolded protein, which is toxic for the cell. This imbalance is known as ER stress. Alongside an increase in protein synthesis, there are a number of factors that give rise to ER stress. These factors include: nutrient deprivation&#x;especially as protein folding is an energy-expending process; deficiencies in post-translational modifications; aberrations in calcium levels and redox homeostasis; inefficiencies in degradation pathways such as ERAD and autophagy; lipid bilayer stress; and low oxygen levels that result in hypoxia.

In order to restore protein folding capacity with protein synthesis requirements, a coordinated transcriptional and translational network termed the unfolded protein response (UPR) is initiated. The UPR monitors protein folding levels within the ER and readjusts folding capacity to match synthesis load, thus ensuring a successful balance for protein homeostasis (Wang and Kaufman, ).

A critical step in UPR signaling is the initial detection of ER stress, the process by which unfolded and misfolded proteins are recognized by UPR, leading to activation and downstream signaling. In this review, we discuss the molecular mechanisms that underlie this recognition process, and how this signal is then propagated to the cytosol.

UPR Signaling

In metazoans there are three key UPR signal activator proteins: inositol requiring enzyme 1±/² (IRE1) (Cox et al., ; Mori et al., ), PKR-like ER kinase (PERK) (Harding et al., ), and activating transcription factor 6±/² (ATF6) (Haze et al., ) (Figure 1). They consist of three domains: an ER luminal domain (LD), a single pass membrane spanning domain, and a cytosolic domain. The domain organization enables the proteins to traverse the ER membrane into the cytosol, with the LD either directly or indirectly involved in sensing misfolded proteins (Walter and Ron, ). PERK and IRE1 LD share sequence and structural similarity. Crystal structures of yeast (Credle et al., ) and human IRE1 LD (Zhou et al., ), along with crystal structures of PERK LD from both mouse and human species (Carrara et al., a) display similar architecture (Figure 2), thus suggesting a similar mechanism of action for both IRE1 and PERK that is conserved from yeast to humans. The cytosolic portion of IRE1 and PERK both possess kinase domains that autophosphorylate in trans (Shamu and Walter, ; Tirasophon et al., ; Harding et al., ; Prischi et al., ). For IRE1, this leads to the stimulation of endoribonuclease activity and the splicing of X-box-binding protein 1 (XBP1) mRNA to form a potent transcriptional activator, XBP1s (s refers to the spliced form) (Cox and Walter, ; Sidrauski and Walter, ; Calfon et al., ). This results in the upregulation of UPR-targeted genes that not only increase the cells&#x; capacity for protein folding, but also protein degradation and transport pathways, which help to alleviate the burden of misfolded protein within the ER. IRE1 activation can lead to promiscuous endoribonuclease activity, which causes mRNA decay at the ER membrane, thus helping to further reduce the protein load in a process called regulated IRE1 dependent decay (RIDD) (Hollien and Weissman, ).

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Figure 1. Overview of UPR signaling pathway. The UPR instigates a transcriptional and translational response to ER stress. The three UPR activator proteins, IRE1, PERK, and ATF6 give rise to three separate branches of the response, all of which aim to alleviate the burden of misfolded protein and to ensure successful ER protein homeostasis.

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Figure 2. Crystal structures of LD. (A) The dimer arrangement of IRE1 LD from both yeast (PDB 2BE1) and human (PDB 2HZ6) proteins, with dimer interface marked by dashed line. (B) PERK LD dimer structure shares similar architecture to IRE1 LD. PERK LD has also been visualized in a tetramer arrangement comprising two sets of dimers (PDB 4YZS and 4YZY), and PERK LD bound to peptide (PDB 5V1D).

PERK regulates the translation response of the UPR. PERK kinase activation leads to phosphorylation of eukaryotic translation initiation factor-2± (eIF2±), a component of the EIF2 complex, which results in ribosome inhibition and brief attenuation of global cell translation (Harding et al., ). Again, this helps in reducing the demands placed on the protein folding machinery. Although PERK activation results in the temporary attenuation of general protein synthesis, paradoxically, certain genes are upregulated, such as activation transcription factor 4 (ATF4) (Vattem and Wek, ). The expression of this gene directs an antioxidant response and contributes to a greater ER protein folding capacity.

The third member of UPR signal activators, ATF6, mediates a transcriptional response that promotes protein folding and ER-associated degradation pathways with a similar outcome to IRE1-XBP1 transcriptional activation (Yoshida et al., ). However, ATF6 contrasts significantly from both IRE1 and PERK in primary amino acid sequence, domain architecture, and mode of operation. Upon accumulation of misfolded proteins, ATF6 transits to the Golgi apparatus where it is cleaved by site-specific proteases S1P and S2P (Haze et al., ; Shen et al., ). This releases its cytosolic portion&#x;a bZIP transcription factor&#x;which migrates to the nucleus and mediates activation of UPR targeted genes, such as chaperones.

Chronic ER Stress and Apoptosis

The primary goal for the UPR is to restore ER protein homeostasis toward ensuring cell survival. However, persistent activation, caused by unmitigated severe ER Stress, leads to a signaling switch that favors apoptosis and a cell death output. Sustained activation of PERK leads to the upregulation of C/EBP-homologous protein (CHOP), a transcription factor implicated in the regulation of apoptosis. This, in turn, leads to the expression of the DNA damage-inducible protein 34 (GADD34), a factor that reverses eIF2± phosphorylation, thereby relieving translational inhibition and enabling the expression of genes, including those involved in ER stressed-induced apoptosis (Novoa et al., ).

The IRE1 arm of UPR is geared toward contributing to cell survival, but persistent activation can lead to it interacting with the tumor necrosis factor receptor-associated factor 2 (TRAF2), and inducing an apoptotic output. The interaction with TRAF2 results in the activation of apoptosis signal-regulating kinase (ASK-1) and downstream target c-jun NH2 terminal kinase (JNK) and p38 MAPK. JNK phosphorylation results in the stimulation of pro-apoptotic factors BID and BiM, whilst inhibiting anti-apoptotic factors BCL-2, BCL-XL and MCL-1 (Almanza et al., ).

ER Hsp70 Chaperone: BiP&#x;A Proximal Component of UPR Signaling?

BiP is the sole ER Hsp70 chaperone and one of the most abundant proteins within the ER, making it a major driving force for protein folding. Active BiP levels within the ER are carefully regulated by oligomer formation, post-translational modification such as AMPylation, and UPR induction (Preissler and Ron, ). Interestingly, BiP has also been directly implicated in UPR signaling (see below section ER stress sensing by IRE1). It comprises a classical Hsp70 architecture with a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) that is connected via a linker. BiP operates a typical Hsp70 chaperone substrate mechanism that involves cycling between an open ATP and closed ADP bound state, facilitated by co-chaperones (Kampinga and Craig, ; Hartl et al., ). Misfolded proteins are recruited to BiP SBD by a certain J-domain containing ERdj co-chaperones, when BiP is present in the open ATP bound state (high Kon, Koff). ERdj association stimulates BiP ATPase activity and leads to BiP converting to a closed ADP bound state that traps the misfolded protein substrate (low Kon and Koff). Nucleotide exchange factors (NEF) promote the exchange of ADP to ATP, with BiP reverting to open ATP form that enables the release of bound substrate (Behnke et al., ). Thus, BiP is dependent on co-chaperones for its protein folding ability.

ER Stress Sensing by IRE1

The principal function of the LD is to recognize misfolded proteins within the ER and translate that signal across the membrane to the cytosol. Whether the recognition of misfolded proteins occurs directly by IRE1 LD, or indirectly via the chaperone BiP, is contentious and unclear.

There are two established models, the direct association and BiP competition, that seek to explain how misfolded proteins induce UPR. More recently, an alternative BiP allosteric model has been proposed.

Direct Association Model

The direct association model postulates that misfolded proteins bind directly to the LD of IRE1 to activate UPR signaling (Credle et al., ; Kimata et al., ; Gardner and Walter, ; Promlek et al., ; Karagöz et al., ) (Figure 3A). The association of misfolded protein mediates conformational changes that result in the oligomerization of IRE1 LD and subsequent activation of UPR signaling (Credle et al., ; Karagöz et al., ; Karagoz et al., ). In this model, BiP is not involved in detecting ER stress, but plays a peripheral role by binding and sequestering inactive monomeric IRE1 (Pincus et al., ).

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Figure 3. A schematic representation of ER stress-sensing mechanisms. (A) Direct association model posits that misfolded proteins bind directly to IRE1 LD, resulting in oligomerization of IRE1 and activation of UPR. (B) In the competition model, IRE1 LD binds to BiP SBD in a chaperone-substrate type interaction. This is the same site that misfolded proteins bind to BiP, leading to a competition for this binding site. BiP interaction to IRE1 is mediated by ERdj4, which ultimately inhibits UPR signaling by facilitating the formation of IRE1 LD monomer. Thus, BiP acts as a repressor of UPR signaling, but is not a direct sensor of ER stress. (C) In the allosteric model, the binding of misfolded proteins and IRE1 LD to BiP occur on different domains; thus, obviating the requirement for competition. Misfolded protein binding induces a conformational change that releases BiP from IRE1, implicating BiP as a direct sensor of ER stress.

The direct association model is based on crystal structures of LD that suggest the formation of a peptide binding groove upon dimerization that resembles a major histocompatibility complex (MHC)-like fold (Credle et al., ). Mutation of residues within this groove impaired IRE1 signaling in yeast (Credle et al., ; Gardner and Walter, ). A peptide tiling array analysis identified peptides that interacted with yeast IRE1 LD in vitro that displayed a distinct amino acid composition similar to exposed polypeptide stretches found within the hydrophobic core of a protein (Gardner and Walter, ; Karagoz et al., ). The interaction with peptides also caused an increase in the LD oligomer species (Gardner and Walter, ). More recently, a structural and biochemical analysis indicated that human IRE1 LD was able to bind to both peptides and unfolded proteins in vitro, thus displaying similarities with yeast IRE1 LD (Karagöz et al., ). Also, nuclear magnetic resonance (NMR) experiments suggest conformational changes upon the binding of peptide to the MHC-like groove, with these conformational changes thought to facilitate IRE1 oligomerization and UPR activation (Karagöz et al., ). A recent crystal structure of PERK LD bound to misfolded peptide suggests that PERK can also bind to misfolded proteins (Figure 2B) (Wang et al., ).

BiP role in the direct association model is to fine-tune the activity of IRE1 sensor. A study that utilized a series of IRE1 deletion mutations suggested the binding site for BiP was proximal to the membrane (Kimata, ). Mutation of this site did not cause unrestrained UPR activation but displayed reaction kinetics consistent with BiP acting as a buffer of IRE1 activity in yeast (Kimata, ; Pincus et al., ; Karagoz et al., ).

However, although peptides bind to IRE1 LD, it is not clear whether the association was occurring at a single site or at multiple sites with less specificity (Preissler and Ron, ). Interestingly, the only crystal structure of LD bound to a peptide indicated that the peptide did not associate to the MHC-like groove but to a hydrophobic pocket that is required for PERK tetramer formation. This pocket is unlikely to have a stress-sensing role and it is surprising that the peptide did not bind to the MHC-like groove in this structure (Figure 2B) (Wang et al., ). Moreover, from an evolutionary perspective, it is difficult to rationalize why IRE1 would not evolve to utilize BiP as an ER stress detector (and not only as a buffer of IRE1 activity), especially since it interacts with BiP, whose primary function is to bind to misfolded proteins.

Competition Model

In this model, BiP binds IRE1 LD as a chaperone-substrate type interaction via its SBD to form a repressive complex (Bertolotti et al., ; Okamura et al., ; Ma et al., ; Kimata et al., ; Oikawa et al., ; Amin-Wetzel et al., ; Preissler and Ron, ) (Figure 3B). This interaction is mediated by ERdj4 and occurs on the same site that misfolded proteins bind to BiP. The formation of this complex stimulates BiP ATPase activity, resulting in ERdj4 dissociation and causing IRE1 LD to form monomers, leading to inhibition of UPR signaling (Amin-Wetzel et al., ). NEF facilitate the exchange of ADP to ATP, causing BiP to dissociate from IRE1 LD. Misfolded proteins now compete with IRE1 LD for binding to both free BiP and ERdj4. In high ER stress, BiP and ERdj4 become occupied with engaging misfolded proteins, thus impeding BiP association with IRE1 LD. This enables IRE1 to form dimers, which in turn activates UPR signaling (Amin-Wetzel et al., ). In this model, BiP acts as a repressor of UPR signaling by preventing dimerization of IRE1, but not as a direct ER stress sensor.

The central tenet of the competition model is that the binding between IRE1 and BiP is a chaperone-substrate type interaction that occurs via BiP SBD, the same site that misfolded proteins bind to BiP, resulting in a competition for this site (Preissler and Ron, ). This is analogous to competitive repression of the transcription factor, heat shock factor 1 (Hsf1), activity by Hsp70 in the cytosol (Abravaya et al., ). In the competition model, the actions of BiP are firmly based on the principles of nucleotide-dependent regulation of Hsp70 when it interacts with chaperone-substrate (Hartl et al., ; Mayer, ). An important facet of the mechanism is that ATP causes BiP dissociation from IRE1, and not misfolded proteins. This is in keeping with HspATP being the substrate loading and unloading state of the chaperone. Additionally, NEF facilitate the exchange of ADP to ATP, similar to how nucleotide exchange is achieved with the Hsp70 chaperone system. Moreover, ERdj4 functions as a recruitment factor that mediates interaction between BiP and IRE1, akin to how certain ERdj proteins recruit misfolded proteins to BiP (Behnke et al., ). Thus, in this model the interaction between IRE1 and BiP is governed by the principles of how Hsp70 interacts with a chaperone-substrate.

However, if the binding between IRE1 and BiP were not a chaperone-substrate type interaction and were to occur at another site on BiP i.e., the NBD, then the competition model would not hold true. This is due to the fact that it obviates the requirement for competitive binding between IRE1 and misfolded protein for BiP SBD and a nucleotide-dependent mechanism that underlie how Hsp70 interacts with chaperone-substrate (see below&#x;Allosteric model). Also, contrary to this model, a number of studies have observed binding between BiP and IRE1 independent of ERdj4 in vitro (Carrara et al., b; Kopp et al., ; Sepulveda et al., ). Furthermore, the current model suggests that there will be no difference in binding between folded IRE1 LD (LD has a high degree of secondary structure) and misfolded IRE1 LD to BiP.

Allosteric Model

The allosteric model indicates an interaction between BiP NBD and IRE1 LD (Todd-Corlett et al., ; Carrara et al., b; Kopp et al., ) (Figure 3C). This interaction is independent of nucleotides (Carrara et al., b; Kopp et al., ) and is distinct from the chaperone-substrate type interaction that occurs via BiP SBD (Amin-Wetzel et al., ). Misfolded proteins bind exclusively to the canonical BiP SBD, which leads to dissociation of BiP NBD from IRE1 LD via a conformational change to trigger UPR signaling (Carrara et al., b; Kopp et al., ). As misfolded proteins and IRE1 LD bind to different domains of BiP, there is no requirement for this process to be competitive. In this model, BiP acts as a direct sensor of ER stress (Carrara et al., b; Kopp et al., ).

Criticism of this model is centered on the observation that ATP does not cause the dissociation of BiP from IRE1, leading to the suggestion that the model does not obey the principles of nucleotide-dependent Hsp70 regulation (Preissler and Ron, ). This would be true if the interaction between IRE1 and BiP were a chaperone-substrate type interaction; however, in this model binding occurs via the BiP NBD. Hsp70 chaperones are primarily concerned with the protein folding processes, but they can specialize in certain roles within this remit, for example, in protein translocation (Craig, ). An analogy can be made with mitochondrial Hsp70 (mtHsp70) action during the translocation of polypeptide into the inner mitochondrial matrix. mtHsp70 associates with Tim44, a component of the translocon machinery, whilst awaiting the import of nascent polypeptide (Craig, ). The interaction with Tim44 primarily occurs via mtHsp70 NBD with contributions from the mtHsp70 SBD (but not as a chaperone-substrate interaction as it is still able to bind to misfolded peptide whilst bound to Tim44) (Krimmer et al., ; D&#x;Silva et al., ). More significantly, the interaction is independent of nucleotides (Liu et al., ). This is based on the observation that the addition of ATP and ADP failed to cause dissociation of the mtHspTim44 complex in vitro. It is only the addition of peptides binding to mtHsp70 SBD that caused mtHsp70 to release from Tim44 (Liu et al., ). Similarly, the interaction of IRE1 LD to BiP NBD is independent of nucleotides in vitro (Carrara et al., b; Kopp et al., ). The binding affinity between IRE1 LD and BiP in the absence or presence of nucleotides (ATP, ADP and AMP-PNP) were closely comparable (Kd 1&#x;2 ¼M). It is only with the addition of misfolded protein (CH1), binding to BiP SBD, that caused BiP to dissociate from IRE1 via BiP NBD (Carrara et al., b; Kopp et al., ). The observation that both mtHsp70 and ER Hsp70 interact with membrane-associated proteins via their NBD, and that the interaction is not influenced by nucleotides, with only misfolded protein/peptide binding to SBD causing dissociation, suggests mechanistic similarities between these two chaperones. Thus, the role of BiP in the allosteric model fits with known principles of Hsp70 mechanistic action, particularly when operating in specialized roles that interact with partner proteins in a non-chaperone-substrate type fashion.

However, there are a number of points yet to be addressed, such as whether nucleotides influence other aspects of the allosteric model, in particular, the ability of BiP to engage misfolded substrate whilst bound to IRE1 LD. Again, clues can be gleaned from the mtHsp70 system. The interaction between mtHsp70 and Tim44 is unaffected by nucleotides in vitro (Liu et al., ), but coimmunoprecipitation of mtHspTim44 complex from mitochondrial lysates were greatly sensitive to ATP and dissociated the complex (Krimmer et al., ; Liu et al., ). This is because there were polypeptides with exposed hydrophobic amino acids within the lysate that were able to associate with mtHspTim44 to cause dissociation of complex, and ATP enhanced the engagement of misfolded polypeptide substrates via SBD, without directly impacting the mtHsp70(NBD)-Tim44 interaction itself. A similar scenario likely occurs with BiP-IRE1 LD. This interaction is independent of nucleotides in vitro (Carrara et al., b; Kopp et al., ), but seems to be sensitive to ATP in cell lysate (Bertolotti et al., ). So, does ATP sensitize the BiP-IRE1 LD complex to engage misfolded proteins via BiP SBD leading to enhanced dissociation, without directly impacting BiP(NBD)-IRE1 LD interaction? Another interesting point to investigate is: how does BiP operate as a molecular chaperone and as an ER stress sensor? The allosteric model suggests that there will be differences in the way BiP interacts with folded IRE1 LD (via BiP NBD) and misfolded IRE1 LD (via SBD).

Oligomerization

After the detection of misfolded proteins, the signal is propagated across the ER membrane via a change in the oligomeric state of IRE1 and PERK, engendering cytosolic domain activation. There are numerous reports regarding the oligomeric state of LD and its transition upon activation induced by ER stress, including monomer to dimer transitions (Shamu and Walter, ; Welihinda and Kaufman, ; Bertolotti et al., ; Liu et al., ; Okamura et al., ; Oikawa et al., ; Zhou et al., ; Lee et al., ; Carrara et al., b; Amin-Wetzel et al., ), tetramer formation (Carrara et al., a), and higher oligomers (Credle et al., ; Kimata et al., ; Aragón et al., ; Korennykh et al., ; Gardner and Walter, ; Sundaram et al., ). Thus, it is likely that the LD is the primary determinant of IRE1 oligomeric status in response to ER stress, with contributions from both the transmembrane and cytosolic regions.

Overall, the mechanism by which IRE1 LD senses ER stress and induces UPR signaling is still not clearly understood. There are three models that provide contrasting mechanisms to explain how ER stress is detected. However, some aspects of these models are not mutually exclusive and could possibly operate synergistically (Kimata et al., ). Further studies are required to differentiate or reconcile between the contrasting models and to understand how this affects IRE1 oligomerization leading to cytosolic signal propagation.

Modulators of IRE1 Stress Signaling in the ER Lumen

Emerging evidence suggests that there may be factors that can bind to the IRE1 LD and influence its ability to detect or respond to ER stress. An ER resident PDI (PDIA6) has been suggested to attenuate UPR signaling by binding to IRE1 LD and reducing a disulphide bond. The oxidized form of the disulphide bond was associated with oligomer formation and UPR activation. The reduction of the bond facilitates the transition from oligomeric to monomeric IRE1, thereby preventing downstream IRE1 kinase phosphorylation and UPR signaling. In a similar fashion, PDIA6 also binds to PERK LD and attenuates its signaling, but does not interact with ATF6 and thus has no direct effect on this branch of UPR signaling (Eletto et al., ).

More recently, a study has suggested an interaction between IRE1 LD and the ER chaperone, Hsp47 (Sepulveda et al., ). Hsp47 belongs to the serine-protease (serpin) inhibitor family. It functions by binding to collagen and trafficking it from the ER to the Golgi in a pH-dependent fashion. Surprisingly, Hsp47 binds to IRE1 LD with high affinity and displaces BiP. This releases UPR repression by allowing the formation of IRE1 dimers to activate signaling.

The lipid composition of the ER membrane may also modulate UPR signaling. IRE1 contains an amphipathic transmembrane helix that is suggested to respond to different membrane lipid compositions by eliciting oligomerization in certain conditions. ATF6 has also been shown to be activated by lipids. In both cases, lipid-based activation occurs independently of proteotoxic stress mechanisms (Volmer et al., ; Halbleib et al., ; Tam et al., ).

Proteins that bind directly to IRE1 LD, along with membrane lipids, may modulate UPR signaling by ER stress-independent mechanisms. This may provide an extra level of regulation in which the UPR signal could be attenuated or strengthened. However, their exact integration with current ER stress-sensing mechanisms that regulate UPR signaling and ER homeostasis is yet to be determined.

IRE1 Cytosolic Stress Signaling

Upon ER stress, the UPR signal is propagated to the cytosolic portion via a change in its oligomeric status, stimulating IRE1 kinase and subsequently RNase activity. Both IRE1 and PERK cytosolic portions contain kinase domains that autophosphorylate in trans, suggesting that dimerization/oligomerization is required for activation and signaling.

IRE1 Autophosphorylation Crystal Structure

The crystal structure of the human cytosolic portion of IRE1 displays a dimer arrangement with each monomer orientated such that their kinase active sites face toward each other (Ali et al., ) (Figure 4A). In this face-to-face orientation, the kinase activation loop&#x;the loop that is phosphorylated&#x;points toward the active site of the opposing monomer in a manner that would allow autophosphorylation in trans to occur. This is reminiscent of similar kinases that have been structurally characterized to undergo dimerization dependent activation, including Chk2 and Lck (Oliver et al., ; Pike et al., ). The face-to-face orientation provides a rationale to how reciprocal autophosphorylation upon the activation loop may occur. In this particular crystal structure, IRE1 was de-phosphorylated, but a similar face-to-face arrangement has been described for phosphorylated murine IRE1 crystal structure (Sanches et al., ).

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Figure 4. Crystal structures of IRE1 cytosolic domain. (A) Schematic depicting the IRE1 cytosolic portion in a face-to-face dimer (PDB 3P23) that enables trans autophosphorylation, and in a back-to-back arrangement (PDB 2RIO), which is suggested to be the RNase active state. The red arrow represents the transition between these two states. (B) A comparison of crystal structures of IRE1 RNase domain when bound to a kinase inhibitor that prevents both kinase and RNase activation (gold, PDB 4YZ9) and when bound to a kinase inhibitor that activates RNase domain (cyan, PDB 4YZC). The small movements within the domain are suggested to enhance splicing activity.

IRE1 RNase Active Crystal Structures

The cytosolic portion of IRE1 has also been crystallized in an alternative dimeric arrangement (Lee et al., ; Concha et al., ; Joshi et al., ) (Figure 4A), in which their kinase active sites face away from each other, enabling a more substantial contact between the RNase domains of the monomers (Lee et al., ). This back-to-back arrangement may represent the RNase activated form. Within these structures, there were differences in the way the RNase domain aligned with each other depending on whether the kinase domain was in an active or inactive/inhibited conformation (Concha et al., ; Joshi et al., ). This resulted in small differences in the hydrogen bonding network between RNase monomers, with more substantial interactions favoring higher splicing activity, thus suggesting that RNase dimer interface movements could provide contrasting splicing outputs (Concha et al., ; Joshi et al., ) (Figure 4B). Aside from the dimer state of the cytosolic domain, there is a crystal structure of yeast IRE1 cytosolic domain forming a large helical arrangement utilizing the dimer as a building block (Korennykh et al., ), which may represent oligomerization events upon ER stress.

So far, crystal structures provide the basis for mechanistic interpretation, but there is still the need for greater clarity. IRE1 cytosolic portion predominantly forms dimers without the influence of the LD. The dimer has been visualized in two separate orientations that seem to be distinct from each other (Lee et al., ; Ali et al., ). It is plausible these arrangements are interconvertible, with the initial state being the autophosphoryl competent face-to-face orientation. This is in keeping with the requirement for phosphorylation to occur at the activation loop to stimulate RNase activity (Prischi et al., ), which then transits to the back-to-back arrangement, and consequently larger oligomeric structures (Joshi et al., ). Although how this would work remains to be resolved, particularly since the cytosolic domain rearrangements would depend upon the LD, and thus far the LD dimer structures seem to suggest that it is present in only one stable form. Additionally, why the RNase domains are required to interact, considering that the core catalytic residues are present in each monomer, remains to be elucidated.

Inhibitors of IRE1 Enzymatic Activity

IRE1 ability to influence cellular fate has made it a target for pharmacological intervention in disease. Chemical compounds that modulate IRE1 activity specifically target its kinase or endoribonuclease enzymatic function in order to influence the levels of spliced XBP1. So far, small molecules that target the RNase activity are aldehyde derivatives that covalently modify the active site leading to inhibition (Papandreou et al., ; Volkmann et al., ; Cross et al., ; Mimura et al., ; Sanches et al., ). These compounds directly interact with the lysine , a key RNase catalytic residue (Tirasophon et al., ), forming a stable imine that prevents XBP1 splicing.

Small molecule inhibitors that target IRE1 kinase activity work by competitively binding to the kinase active site and displacing ATP, thereby preventing the kinase trans autophosphorylation reaction. However, these ATP competitive inhibitors have differing effects on the RNase activity, with some compounds increasing RNase splicing (Papa et al., ; Korennykh et al., ; Concha et al., ; Feldman et al., ), and others inhibiting RNase activity (Wang et al., ; Concha et al., ). A subset of compounds that inhibit activity were based on imidazopyrazine scaffold and were termed kinase inhibiting RNase attenuators (KIRA) (Wang et al., ). Another study reported inhibition by a compound with a spirodecane core (Concha et al., ).

Mechanistically, ATP competitive inhibitors impact kinase activity by displacing key secondary structural elements (±C helix and DFG motif) within the active sites that are required for productive phosphorylation to occur. This results in the kinase domain adopting an inactive conformation, with corresponding realignment of the RNase domain and inhibition of RNase splicing (Concha et al., ). The engagement of KIRA compounds to the kinase active site prevents the formation of dimers, keeping IRE1 in a monomeric state (Wang et al., ). Surprisingly, KIRA compounds seem to be able to dictate the IRE1 oligomerization status. This suggests that IRE1 C-terminal stimulus may regulate LD multimer formation, in contrast to ER stress-induced LD oligomerization, and may represent a novel and unusual method for communicating from cytosol to ER.

IRE1 Cytosolic Domain Interacting Proteins

There are a growing number of studies that have identified proteins that interact with IRE1 cytosolic portion to influence UPR signaling output. There are three categories that the IRE1 interacting proteins fall into: proteins that inhibit IRE1 signaling; proteins that activate IRE1 signaling; and proteins that bind to IRE1 as a scaffold and recruit other proteins.

Inhibitory Interactions

Interacting proteins that exert an inhibitory effect include the apoptosis- and tumor-linked factor: Fortilin. Binding between Fortilin and IRE1 occurs only when IRE1 is phosphorylated, and its interaction inhibits both kinase and RNase activity, possibly by blocking IRE1 dimer formation and autophosphorylation (Pinkaew et al., ). This action attempts to prevent IRE1-induced apoptosis signaling, by reducing phosphorylated IRE1 levels. In a similar manner, the apoptosis regulator Bax inhibitor 1 (BI-1) has been suggested to interact with IRE1 and again inhibits apoptosis signaling. In contrast to Fortilin, this attenuation is achieved by obstructing TRAF2 binding (Castillo et al., ). The underlying inhibitory mechanism here is to prevent dimer formation or to displace other interacting proteins.

Stimulatory Interactions

Proteins that have been suggested to stimulate IRE1 activity include Abelson tyrosine protein kinase 1 (ABL1 or c-abl), a tyrosine kinase implicated in a diverse range of cell signaling processes. Under stress conditions, it engages the cytosolic portion of IRE1 to induce oligomerization, leading to hyperactivation of the RNase function (Morita et al., ). Similarly, Non-muscle myosin-IIb (NMIIB) and Filamin A, are two proteins that are involved in actin cytoskeleton remodeling. NMIIB interaction is dependent on ER stress and leads to IRE1 oligomerization (He et al., ). Filamin A interaction is independent of ER stress and IRE1 stress signaling; its binding to monomeric IRE1, at a distal C-terminal region, possibly leads to dimer formation and recruitment of PKC, which enables Filamin A phosphorylation (Urra et al., ). This acts to increase actin cytoskeletal remodeling. Thus, IRE1 stimulatory proteins are suggested to shift the monomer state to a higher oligomeric assembly.

Scaffold Interactions

Scaffold proteins that engage IRE1 include TRAF2 (Urano et al., ). Its binding facilitates the recruitment of JNK to IRE1 and influences an apoptotic outcome by signaling via the ASK1 pathway and caspase cascade (Urano et al., ; Castillo et al., ). TRAF2 recognizes and specifically engages the phosphorylated IRE1, although it is not known how this occurs. Moreover, what influences scaffold proteins have upon the oligomeric status of IRE1 is not known. Other IRE1 scaffold proteins include; Nck, a cell signaling adaptor protein, which recruits Nuclear Factor ºB (NF-ºB) (Nguyen et al., ); and CHIP, an E3 ligase that ubiquitylates IRE1. This modification may enhance TRAF2/JNK signaling, because in CHIP knockdown cells, IRE1 phosphorylation and IRE1-TRAF2 interactions were nearly abolished (Zhu et al., ). Interestingly, a recent study has suggested an interaction between IRE1 and Sec61 translocon. The formation of the complex provides a platform for the recruitment of XBP1 mRNA, enabling more efficient splicing by IRE1 at the ER membrane (Plumb et al., ). The molecular details of this interaction are yet to be determined and its elucidation could possibly provide further molecular clues into IRE1 splicing activity.

Proteins that interact with the IRE1 cytosolic region may provide an auxiliary way of modulating signaling output and cell fate. Also, scaffolding proteins provide a way to link IRE1 and UPR signaling to other signaling networks and processes. However, the details of such interactions are yet to be determined and highlight the need for a better understanding of the molecular mechanism.

Conclusions

Great progress has been made toward understanding the mechanism of IRE1 stress signaling since its role was first described as mediating a rectifying signal that restores ER homeostasis. Crystal structures have formed the basis of our molecular understanding. The general approach has been to dissect IRE1 into its component parts&#x;the LD and the cytosolic domain&#x;based on the cellular compartment to which they originate from, toward understanding the roles that IRE1 plays in ER stress recognition and UPR signal propagation. The mechanism by which IRE1 detects ER stress is still not clearly understood with three alternative models put forth, highlighting the need for further studies that either provide support or offer reconciliation between models. The cytosolic domain structures have helped to inform and guide drug development programs that aim to target IRE1 kinase and RNase activity, and have provided substantial insights into the mechanism. However, additional experiments are required to provide more molecular detail into IRE1 enzymatic activity. Future structural studies would benefit from understanding how the two domains, residing in two separate cellular compartments, communicate with each other in the absence and presence of ER stress. This communication may be coupled to transitions in oligomeric status of IRE1, emphasizing the importance of understanding this mechanistic step. Also, it would be very interesting to learn the molecular basis of how modulators that bind both the IRE1 LD and cytosolic domain influence output. Overall, although significant progress has been made toward the understanding of IRE1 stress signaling, there still remain many unresolved questions that require further experimentation. Such research may yet provide significant and novel mechanistic insights and discoveries into the IRE1 function.

Author Contributions

CA, MK, NL, PN, and MA read and reviewed final version of the work, and participated in bibliographical research and design. CA assisted in writing and figure preparation. MA wrote paper and did the figures.

Funding

We acknowledge senior cancer research UK fellowship funding to MA (C/A) and (C/A).

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Keywords: unfolded protein response (UPR), IRE1 inositol-requiring enzyme 1, ER stress, crystal structures, Hsp70, BiP

Citation: Adams CJ, Kopp MC, Larburu N, Nowak PR and Ali MMU () Structure and Molecular Mechanism of ER Stress Signaling by the Unfolded Protein Response Signal Activator IRE1. Front. Mol. Biosci. doi: /fmolb

Received: 27 November ; Accepted: 15 February ;
Published: 12 March

Copyright © Adams, Kopp, Larburu, Nowak and Ali. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Maruf M. U. Ali, [email protected]

Sours: https://www.frontiersin.org/articles//fmolb/full

Pathway upr

The Role of the PERK/eIF2α/ATF4/CHOP Signaling Pathway in Tumor Progression During Endoplasmic Reticulum Stress

Hypoxia is a major hallmark of the tumor microenvironment that is strictly associated with rapid cancer progression and induction of metastasis. Hypoxia inhibits disulfide bond formation and impairs protein folding in the Endoplasmic Reticulum (ER). The stress in the ER induces the activation of Unfolded Protein Response (UPR) pathways via the induction of protein kinase RNA-like endoplasmic reticulum kinase (PERK). As a result, the level of phosphorylated Eukaryotic Initiation Factor 2 alpha (eIF2α) is markedly elevated, resulting in the promotion of a pro-adaptive signaling pathway by the inhibition of global protein synthesis and selective translation of Activating Transcription Factor 4 (ATF4). On the contrary, during conditions of prolonged ER stress, pro-adaptive responses fail and apoptotic cell death ensues. Interestingly, similar to the activity of the mitochondria, the ER may also directly activate the apoptotic pathway through ER stress-mediated leakage of calcium into the cytoplasm that leads to the activation of death effectors. Apoptotic cell death also ensues by ATF4-CHOP- mediated induction of several pro-apoptotic genes and suppression of the synthesis of anti-apoptotic Bcl-2 proteins. Advancing molecular insight into the transition of tumor cells from adaptation to apoptosis under hypoxia-induced ER stress may provide answers on how to overcome the limitations of current anti-tumor therapies. Targeting components of the UPR pathways may provide more effective elimination of tumor cells and as a result, contribute to the development of more promising anti-tumor therapeutic agents.

Sours: https://pubmed.ncbi.nlm.nih.gov//
What is the Unfolded Protein Response?

Unfolded protein response

The unfolded protein response (UPR) is a cellular stress response related to the endoplasmic reticulum (ER) stress.[1] It has been found to be conserved between all mammalian species,[2] as well as yeast[1][3] and worm organisms.

The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum. In this scenario, the UPR has three aims: initially to restore normal function of the cell by halting protein translation, degrading misfolded proteins, and activating the signalling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time span or the disruption is prolonged, the UPR aims towards apoptosis.

Sustained overactivation of the UPR has been implicated in prion diseases as well as several other neurodegenerative diseases, and inhibiting the UPR could become a treatment for those diseases.[4] Diseases amenable to UPR inhibition include Creutzfeldt–Jakob disease, Alzheimer's disease, Parkinson's disease, and Huntington's disease.[5][better&#;source&#;needed]

Protein folding in the endoplasmic reticulum[edit]

Protein synthesis[edit]

The term protein folding incorporates all the processes involved in the production of a protein after the nascent polypeptides have become synthesized by the ribosomes. The proteins destined to be secreted or sorted to other cell organelles carry an N-terminal signal sequence that will interact with a signal recognition particle (SRP). The SRP will lead the whole complex (Ribosome, RNA, polypeptide) to the ER membrane. Once the sequence has “docked”, the protein continues translation, with the resultant strand being fed through the polypeptide translocator directly into the ER. Protein folding commences as soon as the polypeptide enters to the luminal environment, even as translation of the remaining polypeptide continues.

Protein folding and quality control[edit]

Protein folding steps involve a range of enzymes and molecular chaperones to coordinate and regulate reactions, in addition to a range of substrates required in order for the reactions to take place. The most important of these to note are N-linked glycosylation and disulfide bond formation. N-linked glycosylation occurs as soon as the protein sequence passes into the ER through the translocon, where it is glycosylated with a sugar molecule that forms the key ligand for the lectin molecules calreticulin (CRT; soluble in ER lumen) and calnexin (CNX; membrane bound).[6] Favoured by the highly oxidizing environment of the ER, protein disulfide isomerases facilitate formation of disulfide bonds, which confer structural stability to the protein in order for it to withstand adverse conditions such as extremes of pH and degradative enzymes.

The ER is capable of recognizing misfolding proteins without causing disruption to the functioning of the ER. The aforementioned sugar molecule remains the means by which the cell monitors protein folding, as the misfolding protein becomes characteristically devoid of glucose residues, targeting it for identification and re-glycosylation by the enzyme UGGT (UDP-glucose:glycoprotein glucosyltransferase).[6] If this fails to restore the normal folding process, exposed hydrophobic residues of the misfolded protein are bound by the protein glucose regulate protein 78 (Grp78), a heat shock protein 70kDa family member[7] that prevents the protein from further transit and secretion.[8]

Where circumstances continue to cause a particular protein to misfold, the protein is recognized as posing a threat to the proper functioning of the ER, as they can aggregate to one another and accumulate. In such circumstances the protein is guided through endoplasmic reticulum-associated degradation (ERAD). The chaperone EDEM guides the retrotranslocation of the misfolded protein back into the cytosol in transient complexes with PDI and Grp[9] Here it enters the ubiquitin-proteasome pathway, as it is tagged by multiple ubiquitin molecules, targeting it for degradation by cytosolic proteasomes.

A simplified diagram of the processes involved in protein folding. The polypeptide is translated from its ribosome directly into the ER, where it is glycosylated and guided through modification steps to reach its desired conformation. It is then transported from the ER to the Golgi apparatus for final modifications. Where misfolding proteins continually breach quality control, chaperones including Grp78 facilitate its removal from the ER through retrotranslocation, where it is broken down by the ubiquitin-proteasome pathway as part of the ERAD system.

Successful protein folding requires a tightly controlled environment of substrates that include glucose to meet the metabolic energy requirements of the functioning molecular chaperones; calcium that is stored bound to resident molecular chaperones and; redox buffers that maintain the oxidizing environment required for disulfide bond formation.[10]

Unsuccessful protein folding can be caused by HLA-B27, disturbing balance of important (IL and TNF) signaling proteins. At least some disturbances are reliant on correct HLA-B27 folding.[11]

However, where circumstances cause a more global disruption to protein folding that overwhelms the ER's coping mechanisms, the UPR is activated.

Molecular mechanism[edit]

Initiation[edit]

The molecular chaperone BiP/Grp78 has a range of functions within the ER. It maintains specific transmembrane receptor proteins involved in initiation of the downstream signalling of the UPR in an inactive state by binding to their luminal domains. An overwhelming load of misfolded proteins or simply the over-expression of proteins (e.g. IgG)[12] requires more of the available BiP/Grp78 to bind to the exposed hydrophobic regions of these proteins, and consequently BiP/Grp78 dissociates from these receptor sites to meet this requirement. Dissociation from the intracellular receptor domains allows them to become active. PERK dimerizes with BiP in resting cells and oligomerizes in ER-stressed cells.

Although this is traditionally the accepted model, doubts have been raised over its validity. It has been argued that the genetic and structural evidence supporting the model simply shows BiP dissociation to be merely correlated with Ire1 activation, rather than specifically causing it.[13] An alternative model has been proposed, whereby unfolded proteins interact directly with the ER-lumenal domain of Ire1, causing oligomerization and transautophosphorylation.[13]

Functions[edit]

The initial phases of UPR activation have two key roles:

Translation Attenuation and Cell Cycle Arrest by the PERK Receptor This occurs within minutes to hours of UPR activation to prevent further translational loading of the ER. PERK (protein kinase RNA-like endoplasmic reticulum kinase) activates itself by oligomerization and autophosphorylation of the free luminal domain. The activated cytosolic domain causes translational attenuation by directly phosphorylating the α subunit of the regulating initiator of the mRNA translation machinery, eIF2.[14] This also produces translational attenuation of the protein machinery involved in running the cell cycle, producing cell cycle arrest in the G1 phase.[15] PERK deficiency may have a significant impact on physiological states associated with ER stress.

A simplified diagram of the initiation of the UPR by prolonged and overwhelming protein misfolding. Grp78 recruitment to chaperone the misfolded proteins results in Grp78 dissociation from its conformational binding state of the transmembrane receptor proteins PERK, IRE1 and ATF6. Dissociation results in receptor homodimerisation and oligomerisation to an active state. The activated cytosolic domain of PERK phosphorylates the eIF2alpha, inhibiting translation and resulting in cell cycle arrest. The activated cytosolic domain of IRE1 cleaves the 26bp intron from its substrate XBP1, facilitating its translation to form the transcription factor XBP1. Activated ATF6 translocates to the Golgi, cleaved by proteases to form an active 50kDa fragment (ATF6 p50). ATF6 p50 and XBP1bind ERSE promoters in the nucleus to produce upregulation of the proteins involved in the unfolded protein response.

Increased Production of Proteins Involved in the Functions of the UPR UPR activation also results in upregulation of proteins involved in chaperoning malfolding proteins, protein folding and ERAD, including further production of Grp Ultimately this increases the cell's molecular mechanisms by which it can deal with the misfolded protein load. These receptor proteins have been identified as:

  • Inositol-requiring kinase 1,[16] whose free luminal domain activates itself by homodimerisation and transautophosphorylation.[17] The activated domain is able to activate the transcription factor XBP1(Xbox binding protein) mRNA (the mammalian equivalent of the yeast Hac1 mRNA) by cleavage and removal of a 26bp intron. The activated transcription factor upregulates UPR 'stress genes' by directly binding to stress element promoters in the nucleus.[18]
  • ATF6 (activating transcription factor 6) is a basic leucine zipper transcription factor.[19] Upon Grp78 dissociation, the entire 90kDa protein translocates to the Golgi, where it is cleaved by proteases to form an active 50kDa transcription factor[20] that translocates to the nucleus. It binds to stress element promoters upstream of genes that are upregulated in the UPR.[21]

The aim of these responses is to remove the accumulated protein load whilst preventing any further addition to the stress, so that normal function of the ER can be restored as soon as possible.

If the UPR pathway is activated in an abnormal fashion, such as when obesity triggers chronic ER stress and the pathway is constitutively active, this can lead to insensitivity to insulin signaling and thus insulin resistance. Individuals suffering from obesity have an elevated demand placed on the secretory and synthesis systems of their cells. This activates cellular stress signaling and inflammatory pathways because of the abnormal conditions disrupting ER homeostasis.

A downstream effect of the ER stress is a significant decrease in insulin-stimulated phosphorylation of tyrosine residues of insulin receptor substrate (IRS-1), which is the substrate for insulin tyrosine kinase (the insulin receptor). C-Jun N-terminal kinase (JNK) is also activated at high levels by IRE-1α, which itself is phosphorylated to become activated in the presence of ER stress. Subsequently, JNK phosphorylates serine residues of IRS-1, and thus inhibits insulin receptor signaling. IRE-1α also recruits tumor necrosis factor receptor-associated factor 2 (TRAF2). This kinase cascade that is dependent on IRE-1α and JNK mediates ER stress–induced inhibition of insulin action.[22]

Obesity provides chronic cellular stimuli for the UPR pathway as a result of the stresses and strains placed upon the ER, and without allowing restoration to normal cellular responsiveness to insulin hormone signaling, an individual becomes very likely to develop type 2 diabetes.

Skeletal muscles are sensitive to physiological stress, as exercise can impair ER homeostasis. This causes the expression of ER chaperones to be induced by the UPR in response to the exercise-induced ER stress. Muscular contraction during exercise causes calcium to be released from the sarcoplasmic reticulum (SR), a specialized ER network in skeletal muscles. This calcium then interacts with calcineurin and calcium/calmodulin-dependent kinases that in turn activate transcription factors. These transcription factors then proceed to alter the expression of exercise-regulated muscle genes. PGC-1alpha, a transcriptional coactivator, is a key transcription factor involved in mediating the UPR in a tissue-specific manner in skeletal muscles by coactivating ATF6alpha. Therefore, PGC-1alpha gets expressed in muscles after acute and long-term exercise training. The function of this transcription factor is to increase the number and function of mitochondria, as well as to induce a switch of skeletal fibers to slow oxidative muscle fibers, as these are fatigue-resistant. Therefore, this UPR pathway mediates changes in muscles that have undergone endurance training by making them more resistant to fatigue and protecting them from future stress.[23]

Initiating apoptosis[edit]

In conditions of prolonged stress, the goal of the UPR changes from being one that promotes cellular survival to one that commits the cell to a pathway of apoptosis. Proteins downstream of all 3 UPR receptor pathways have been identified as having pro-apoptotic roles. However, the point at which the 'apoptotic switch' is activated has not yet been determined, but it is a logical consideration that this should be beyond a certain time period in which resolution of the stress has not been achieved. The two principal UPR receptors involved are Ire1 and PERK.

By binding with the protein TRAF2, Ire1 activates a JNK signaling pathway,[24] at which point human procaspase 4 is believed to cause apoptosis by activating downstream caspases.

Although PERK is recognised to produce a translational block, certain genes can bypass this block. An important example is that the proapoptotic protein CHOP (CCAAT/-enhancer-binding protein homologous protein), is upregulated downstream of the bZIP transcription factor ATF4 (activating transcription factor 4) and uniquely responsive to ER stress.[25] CHOP causes downregulation of the anti-apoptotic mitochondrial protein Bcl-2,[26] favouring a pro-apoptotic drive at the mitochondria by proteins that cause mitochondrial damage, cytochrome c release and caspase 3 activation.

Diseases

Diseases amenable to UPR inhibition include Creutzfeldt–Jakob disease, Alzheimer's disease, Parkinson's disease, and Huntington's disease.[5]

Endoplasmic reticulum stress was reported to play a major role in non‐alcoholic fatty liver disease (NAFLD) induction and progression. High fat diet fed rats showed increased ER stress markers CHOP, XBP1, and GRP ER stress is known to activate hepatic de novo lipogenesis, inhibit VLDL secretion, promote insulin ressistance and inflammatory process, and promote cell apoptosis. Thus it increase the level of fat accumulation and worsens the NAFLD to a more serious hepatic state.[27]Zingiber officinale (ginger) extract and omega‐3 fatty acids were reported to ameliorate endoplasmic reticulum stress in a nonalcoholic fatty liver rat model.[27]

Chemical inducers[edit]

Biological inducers[edit]

  • Dengue virus induces PERK dependent ER stress as part of virus induced response in infected cells to favor replication.[29]
  • Influenza virus requires endoplasmic reticulum protein kD (ERp57) for replication and apoptosis induction in infected cells.[30]

See also[edit]

References[edit]

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Sours: https://en.wikipedia.org/wiki/Unfolded_protein_response

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