Pharmacological Research 155 (2020) 104702

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Pharmacological Research

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Review Endoplasmic reticulum stress signaling in cancer and neurodegenerative disorders: Tools and strategies to understand its complexity T

Daniela Correia da Silva, Patrícia Valentão, Paula B. Andrade, David M. Pereira*

REQUIMTE/LAQV, Laboratório de Farmacognosia, Departamento de Química, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050- 213, Porto, Portugal

ARTICLE INFO ABSTRACT

Chemical compounds studied in this article: The endoplasmic reticulum (ER) comprises a network of tubules and vesicles that constitutes the largest orga- Thapsigargin (PubChem CID: 126969181) nelle of the eukaryotic cell. Being the location where most proteins are synthesized and folded, it is crucial for Tunicamycin (PubChem CID: 56927848) the upkeep of cellular homeostasis. Disturbed ER homeostasis triggers the activation of a conserved molecular Palmitic acid (PubChem CID: 985) machinery, termed the unfolded protein response (UPR), that comprises three major signaling branches, in- Brefeldin (PubChem CID: 5287620) itiated by the protein kinase RNA-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 (IRE1) DTT (PubChem CID: 446094) and the activating transcription factor 6 (ATF6). Given the impact of this intricate signaling network upon an Salubrinal (PubChem CID: 5717801) 4-Phenylbutyric acid (PubChem CID: 4775) extensive list of cellular processes, including protein turnover and autophagy, ER stress is involved in the onset and progression of multiple diseases, including cancer and neurodegenerative disorders. There is, for this reason, Keywords: an increasing number of publications focused on characterizing and/or modulating ER stress, which have re- Unfolded protein response sulted in a wide array of techniques employed to study ER-related molecular events. This review aims to sum up Endoplasmic reticulum stress the essentials on the current knowledge of the molecular biology of endoplasmic reticulum stress, while high- PERK lighting the available tools used in studies of this nature. IRE-1 ATF6 Autophagy

1. Endoplasmic reticulum (ER) function, structure and dynamics the absence of microtubules, peripheral ER tubules may also move along actin filaments. Furthermore, microtubule polymerization may, The endoplasmic reticulum (ER) is the largest organelle of eu- by itself, push the movement of the ER tubules [4]. Altogether, mi- karyotic cells, comprising an intricate and highly dynamic network of crotubule and actin filament dynamics influence the tubule-to-sheet tubules and branches that emerge from the nucleus and are distributed ratio of the ER, as has been shown by cell imaging/ 3D-electron mi- throughout the cytoplasm. croscopy techniques [5]. From a structural point of view, the ER comprises the rough ER, The ER plays a role of the utmost importance in the homeostasis of a constituted by sheets, and the smooth ER, constituted by tubules, each wide array of cellular processes, even though it is classically associated structure being related to the type of processes that takes place at the to its main function: the de novo synthesis and folding of proteins site. The rough ER is easily distinguished from the smooth ER due to the (mainly in the rough ER). It is in the ER that the synthesis of most density of ribosomes it presents on its cytosolic surface, while the proteins takes place, mainly secreted and transmembrane proteins, but smooth ER lodges few ribosomes and presents smoother and more also some cytosolic ones. In the presence of a signal recognition particle curved surfaces [1–3]. (SRP), ribosomes containing mRNAs to be translated are recruited to The reticular architecture is highly dynamic, with new tubules being bind the surface of the ER and proceed with their translation [1]. The formed at constant rates, as well as their cytoskeletal transport along next step is protein folding, which encompasses the formation of dis- microtubules and homotypic fusion. New tubules branch from the older ulfide bonds between cysteine residues of peptides at the ER lumen. ones and slide along the others, being that the microtubule motors After this, post-translational modifications, such as N-linked glycosy- govern the expansion of the ER towards the cell membrane, while the lation, also take place [6]. As discussed later (Section 7), the inability to inwards movements are microtubule-independent [4]. Nonetheless, in properly conduct this folding step is the basis of a number of

⁎ Corresponding author. E-mail address: dpereira@ff.up.pt (D.M. Pereira). https://doi.org/10.1016/j.phrs.2020.104702 Received 16 December 2019; Received in revised form 10 February 2020; Accepted 13 February 2020 Available online 14 February 2020 1043-6618/ © 2020 Elsevier Ltd. All rights reserved. D.C. da Silva, et al. Pharmacological Research 155 (2020) 104702 proteinopathies, notably several neurodegenerative diseases. Recently, a promising alternative to conventional antibodies has The ER is also involved in the transport of newly-synthesized pro- emerged, namely the use of nanobodies or single-domain antibodies. tein and their delivery to target site through the secretory pathway, These are fragments of antibodies that can be used in fluorescence which involves the rough ER, ER exit sites, the ER-to-Golgi intermediate microscopy and in the investigation of protein functions and interac- compartment, the Golgi apparatus itself and post-Golgi carriers that tions [27]. Such approach has already been used to study the plasma transport the proteins to their final target site [7]. Although on a membrane junctions with the ER on mammalian neurons [28] and to smaller scale, the synthesis and transportation of phospholipids and analyze retrograde transport to the Golgi apparatus [29]. There is also a steroids also takes place in this organelle, mostly in the smooth ER [1]. report concerning the development of a toolkit of functionalized na- Furthermore, the ER constitutes the most important reservoir of ionic nobodies to study calcium dynamics [30]. Even though currently this is calcium in the cell, as discussed in depth in Section 5. not (yet) a common approach to study ER morphology, it is likely that will be the case in the years to come. This tool offers new possibilities to 1.1. Studying ER morphology accurately study and visualize protein function in real time on live cells, posing a set of major advantages when compared to traditional anti- Changes to the reticular morphology are commonly evaluated in bodies. Briefly, these single domain antibodies are more stable in the studies regarding ER stress. Concerning ER labeling, even though the cellular environment, more soluble, more resistant to varying thermal number of selective probes towards the ER is scarce, there are a few of chemical conditions and possess higher affinity towards the antigen commercial options for direct ER imaging. Assessing ER morphology [31]. Nanobodies are highly specific due to their size, which allows resorting to this sort of probe can be useful to detect ER stress by ob- them to bind cavities on the surface of the antigen, such as ligand- serving its characteristic traits, such as dilation, expansion, granulation binding sites of receptors or catalytic sites on enzymes, and thus re- or vacuolization of the organelle [8,9]. For instance, the ER is reported ducing nonspecific background binding. Furthermore, the production to expand in response to thapsigargin [10] or cyclosporine [11]. On the yield of this type of antibody considerably higher [32,33]. fungus Pisolithus tinctorius, ER expansion and modified localization were observed upon treatment with brefeldin [12]. 1.2. Assessing misfolded proteins ER trackers include ER-Tracker™ Blue-White, DPX ER-Tracker™ Green (glibenclamide BODIPY® FL) and ER-Tracker™ Red (glib- Chaperones aid newly.synthesized proteins acquire their correct enclamide BODIPY® TR). The green and red options consist of a dye of tridimensional conformation [34]. In order to fulfill its role in protein the selected spectrum bound to glibenclamide, which binds sulfony- folding, the ER relies on three chaperone families: i) the heat shock lurea receptors of ATP-sensitive K+ channels in the ER, potentially family, such as BiP, a member of the heat shock protein 70 kDa family bearing the disadvantage of impairing the normal function of the or- (HSP70), also termed glucose regulated protein 78 (GRP78), and the ganelle. On the other hand, the blue-white option is more en- glucose-related protein-94 (GRP94), which belongs to the heat shock vironmentally sensitive and decreases its quantum yield on the pre- protein 90 kDa family (HSP90); ii) lectins, such as calreticulin, calnexin sence of highly polar solvents, which can pose a great disadvantage and the ER degradation-enhancing α-mannosidase-like protein (EDEM) when working with live cells [13–15]. Choosing the color of the probe and iii) the protein disulfide isomerase family (PDI) [35]. relies solely on the preference of the researcher, being that it should be BiP regulates one of the two major chaperone systems by re- of a distinct color when the user intends to co-stain with dyes for other cognizing exposed hydrophobic regions rich in tryptophan, phenylala- organelles. nine or leucine, typical of misfolded proteins. This is a monomeric Another option is CellLight® products, that transfect green or red protein that possesses two binding domains, one peptide-binding and fluorescent protein-based constructs fused to an ER retention signal one ATP-binding. The second system is led by the two lectin chaperones that, in both cases, is calreticulin and KDEL, into cells. These options calnexin (integral membrane protein) and calreticulin (luminal protein) are to be used on live cells only, unlike ER trackers, which allow ER that present glycoproteins to ERp57, a member of the PDI family, for visualization on both live or fixed cells [16,17]. Furthermore, in 2016, disulfide bond formation and isomerization [35,36]. Whenever the McDonald et al. reported the first two fluorescent flavonoids that ac- calnexin-calreticulin deglycosylation-glycosylation cycle fails to pro- cumulate in the ER lumen. This accumulation is reported to be selec- duce correctly assembled peptides, the aberrant proteins are forwarded tive, as it was comparable with the localization of ER-Tracker™ Red. to endoplasmic reticulum-associated degradation (ERAD), that involves However, the authors did not evaluate if these molecules accumulated ubiquitination of targeted proteins and their subsequent degradation by in other cellular compartments, such as mitochondria or the Golgi ap- the proteasome [37]. paratus, reason for which the there is still room for improving the The BiP system differs from calnexin in that it only detects the un- knowledge regarding the selectivity of these compounds [18]. Meinig folded regions mentioned above, while calnexin/calreticulin system et al. synthesized analogues of the flurophore rhodol that display the also detects N-linked glycans [36,38]. Hence, BiP is strongly associated same properties [19]. The number of selective probes towards the ER is to the detection of unfolded non-glycosylated peptides. Furthermore, rather scarce. Furthermore, they are associated to a few limitations, this chaperone is important for maintaining the permeability of the ER since they may not be the most specific labeling tool and the manu- translocon. Once recognized as misfolded, proteins are directed to the facturer may not clearly indicate their target, resulting in constraints cytosol via a protein channel formed by Sec61p, where the 26S pro- when interpreting results. For these reasons, immunocytochemistry is teasome can be found. Substrates recognized by calnexin/calreticulin often used to image the ER. The use of antibodies anti-reticular proteins are transferred to EDEM, while peptides associated to BiP are freed combined with secondary antibodies with fluorogenic properties are from the folding enzyme ERdj3/6 and bound to ERdh4/5 for de- the most frequent instances of this procedure. There are published ex- gradation [36,38]. Even though the mechanism by which the ER can amples for several ER proteins, such as PDI [20], calpain-1 [21], cal- distinguish and target misfolded proteins from nascent polypeptides is nexin [22,23], calreticulin [24] or the binding immunoglobulin protein yet to be clarified, chaperone GRP94 is considered to have a role on the (BiP) [25]. The selectivity inherent to this sort of method is higher than process, since its knockdown inhibits the degradation of known ERAD that of the previously mentioned ER probes in that it allows visualiza- substrates. It is therefore proposed that this chaperone interacts with tion of a specific antigen, providing information regarding its expres- ERAD sensor proteins [39]. Like other members of the HSP90 family, sion at the protein level while simultaneously allowing to capture its GRP94 displays an N-terminal domain, and acidic linker domain, a subcellular localization [26]. Differently, some commercially available middle domain and a C-terminal domain and functions as a dimer. probes do not always inform on the biological basis of the ER-affinity, Being more selective, it possesses a shorter client list than other cha- which may lead to problems regarding selectivity. perones [39].

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The accumulation of protein aggregates in the ER lumen is a hall- aggregation of misfolded and/or unfolded proteins in the ER lumen. At mark of ER stress, reason for which its detection and quantification is a this point, the unfolded protein response (UPR) is triggered, a chain of powerful tool to detect compromised reticular homeostasis, particularly molecular events that has evolved to restore homeostatic conditions. in fields in which proteotoxicity is pivotal, such as neurodegeneration Briefly, the UPR i) decreases the rate of protein synthesis to alleviate [40]. Thioflavin T (ThT, Ex. 458 nm excitation, Em. 480–520 nm) is a protein overload, while promoting the correct processing of synthesized small molecule that issues a fluorescent response when bound to pro- proteins and preventing aggregation in the ER lumen, ii) promotes tein aggregates. Techniques based on this molecule can be employed for ERAD and iii) boosts the expansion of the ER network. Nevertheless, if both imaging and quantitation of misfolded protein accumulation. the stress upon the ER is of such intensity or duration that it cannot be Nonetheless, this approach does not provide insight into the molecular repaired, the UPR signaling switches from survival to pro-death me- machinery activated during that disturbance. ThT is reported to have a chanisms [56]. The major molecular events resulting from UPR acti- particular affinity towards β-sheets [41]. In a study designed to assess vation will be discussed hereafter. the feasibility of its use to detect ER stress, it was successfully employed BiP senses the accumulation of misfolded polypeptides in the ER by in a cell culture system with mouse embryonic fibroblasts (MEFs), binding their exposed hydrophobic residues to its C-terminal domain, human hepatocarcinoma cells (HepG2), and human aortic endothelial thus acting as a chaperone. When misfolded proteins are sensed on its cells (HAEC), and also on ApoE-/-mouse liver sections. In thapsigargin C-terminal domain, ATP is hydrolyzed at the N-terminal domain, in- (a potent ER stress inducer)-challenged cells (1 μM, 12 h), 5 μM ThT creasing the affi nity of the C-terminal domain towards its incorrectly resulted in maximum differences of 5.5 fold against control cells. It also processed substrate, thus keeping it in the ER lumen for a more ex- responded to treatments with other known ER stress inducers, namely tended period of time, in order to allow for other mechanism to inter- dithiothreitol (DTT), glucosamine and palmitate. Increasing thapsi- vene and correct the mistake. Under homeostatic conditions, BiP loca- gargin concentration (from 0 to 1 μM) revealed a linear response, re- lizes on the ER lumen, binding the luminal domain of all three major latable to the degree of ER stress. This study also develops a staining transmembrane proteins that sense ER stress. The classic UPR model method employing ThT, which allows visualization of the fluorescent indicates that, upon ER stress and increased levels of misfolded pro- compound in the ER lumen, where misfolded proteins accumulate [42]. teins, BiP detaches from these sensors, subsequently leading to their Anti-KDEL staining has been used to visualize increased chaperone activation [57,58]. These sensor proteins are the protein kinase RNA- protein levels under ER stress, using monoclonal antibodies that bind like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme 1 these ER stress biomarkers. KDEL represents the aminoacid sequence (IRE1) and the activating transcription factor 6 (ATF6). Their me- Lys-Asp-Glu-Leu, characteristic of ER-synthesized proteins for the se- chanisms of action will be detailed below and are schematized in Fig. 1. cretory pathway [42,43]. The use of this technique is vastly described By minimizing or even silencing gene expression, interfering RNA in the literature, using several ER stress biomarkers [44–49]. However, (RNAi) is a powerful tool of gene knockdown. An example of one of even though KDEL is a retention signal to the ER, it also works as a these instances was developed to study the role of BiP on palate de- retrieval signal from the Golgi apparatus, and hence anti-KDEL staining velopment. Towards this goal, a small RNAi of BiP was inserted on a may suffer some interference from peptides that are in the latter or- fluorescent vector and injected on palate explants. This injection re- ganelle to be retrotransported to the ER [50]. sulted, as expected, on reduced UPR gene expression [59]. PDIs play a pivotal role in protein folding. For this reason, cell-free enzymatic assays have been developed to analyze the ability of candi- 2.1. PERK branch date molecules to modulate the activity of these enzymes, which can be a demanding task considering PDIs can mediate up to four distinct re- The first of the three major branches of the UPR discussed here is actions. Isomerase activity is measured when substrates presents initiated by PERK. When BiP dissociates from this sensor, PERK is ac- scrambled disulfide bonds. PDI converts it to its native state, restoring tivated by homodimerization and trans-autophosphorylation. Then, it its activity. Oxidase activity occurs when the substrate is in a reduced phosphorylates the α subunit of the eukaryotic initiation factor 2 and PDI performs its oxidative refolding. Reductase activity, on the (eIF2), which, in turn, attenuates protein translation in order to de- other hand, occurs when the substrate is oxidized. Finally, chaperone crease the load of proteins in the ER lumen that await processing. This activity can be measured using substrates that do not contain disulfide pro-survival signaling may allow the cell to cope with the stress con- bonds. This last can be determined through recovery of substrate ac- ditions it is under, thus helping restore homeostasis. However, if these tivity, thus its subsequent determination is required (such as the re- stress conditions are too severe or last for too long, this branch can ductase or isomerase) and also through changes in substrate aggrega- switch to pro-apoptotic signaling by activating the activating tran- tion [51,52]. A summary of the substrates that are commonly used for scription factor 4 (ATF4). ATF4 is a basic leucine zipper transcription the determination of each type of PDI activity can be found in Table 1. factor responsible for enhancing the expression of genes related to amino acid metabolism, nutrient uptake, anti-oxidation, protein folding 2. The unfolded protein response and apoptosis, working along with other transcription factors in the UPR [60,61]. This transcription factor induces the expression of genes In cases where homeostasis of any of the aforementioned ER-based like the one encoding for the CCAAT-enhancer-binding protein homo- processes is disturbed, the resulting stress conditions may compromise logous protein (CHOP), which triggers apoptosis [62]. Although the this organelle’s ability to correctly assemble and process peptides. mechanisms through which CHOP triggers regulated cell death are still Eventually, the amount of newly-synthesized proteins surpasses the not completely understood, it is accepted that there is a correlation amount awaiting folding in the ER, leading to the accumulation and between CHOP-induced cell death and downregulation of the pro-

Table 1 Common substrates for the determination of each type of PDI activity.

Activity Substrates References

Reductase Insulin along with DTT or glutathione; di-(o-aminobenzoyl)-GSSG (diabz-GSSG); dieosinGSSG (Di-E-GSSG) [53] Oxidase RNAse; bovine pancreatic trypsin inhibitor (BPTI) lysozyme [51,52] Isomerase Scrambled RNAse; riboflavin-binding protein [51,52] Chaperone D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); [54,55] lactate dehydrogenase; rhodanase or citrate synthase; alcohol dehydrogenase

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technique is robust and widespread throughout the literature regarding ER stress. In order to infer about the role of a given pathway in in the UPR, some selective pharmacological inhibitors are available. Most of these molecules have been discovered or developed in the attempt to create drugs acting specifically in the ER, yet their toxicity deemed their use for research only. GSK2606414 is a selective inhibitor of PERK. In in vivo models of prion-disease, GSK2606414 restored protein synthesis, resulting in neuroprotection and prevention of disease onset. However, the com- pound presented marked toxicity, inducing weight loss and hypergly- cemia [69]. Salubrinal is a pharmacological inhibitor of eIF2α depho- sphorylation that acts by blocking the activity of the protein complex growth arrest and GADD34/protein phosphatase 1 (PP1) [70]. There is a considerable number of studies indicating that salubrinal is able to protect cells against a set of toxic compounds that act by triggering ER stress. Multiple studies rely in the co-incubation of cells with a toxic compound in the presence of salubrinal and subsequently measure cell Fig. 1. Major molecular machinery involved in the activation of the unfolded viability or death pathways. Concentrations of salubrinal used are – μ protein response (UPR). usually in the 5 100 M range and, in some cases, this molecule is pre- incubated one to two hours before the target toxic compound [70–75]. Salubrinal has also been tested in vivo, being injected via intracerebral survival protein B-cell lymphoma-2 (Bcl-2), concurrently with upregu- ventricle to study the impact of ER stress in autophagy induced by brain lation of caspase-3, Bcl-X (Bcl-2 like protein 1), Bax (Bcl-2 associated X- ischemic preconditioning. The concentrations of salubrinal tested in protein), GADD34 (growth arrest and DNA damage-inducible protein), this study are considerably lower (75–150 pM) than usually reported as well as the EOR1α (ER oxidoreductin 1α) and TRB3 genes [63,64]. [76]. In different reports, however, salubrinal was administrated GADD34 restores protein synthesis rates by stimulating eIF2α depho- through intraperitoneal injection at 1 mg/kg [77,78], in the intra-ar- sphorylation, which, in turn, results in proteotoxicity by increased re- ticular space at 1.5 mg/kg [73] or intravenously at 2.0 mg/kg [75]. active oxygen species (ROS) production and ATP depletion [64]. The This significant variation in protocols calls for a standard in the field relationship between oxidative stress and ER stress will be more de- which can contribute to improve the benchmarking between distinct tailed on Section 4. TRB3 is another CHOP-induced gene known to be studies. There are also in vivo studies that reported the high toxicity of involved in cell death and to downregulate its own expression by re- the compound, reason for which its use is currently reserved for re- pressing CHOP activity [65]. Finally, ERO1 sets off the CHOP-EOR1α- search purposes [69,79]. IP3R-calcium-calcium/calmodulin-dependent protein kinase II (CaMKII) pathway, which culminates in mitochondria permeabilization and release of Bax/Bak [63]. 2.2. IRE1 branch Phosphorylation of eIF2α favors the translation of a few genes, designated by integrated stress response (ISR) genes [66]. These genes The IRE1 branch of the UPR is initiated upon activation of the ki- include ATF4 and CHOP. As is it associated to triggering stress-induced nase through autophosphorylation and homodimerization or oligo- apoptosis, basal levels of CHOP are usually low. However, they quickly merization. Of all the UPR transducers, IRE1 is the most conserved rise upon ATF4 activation, even though it can also be induced in a [80]. PERK-, IRE1- or ATF6-dependent manner. This transcription factor is Not unlike PERK, this branch can also promote survival or trigger crucial for ensuing ER stress-induced apoptotic signaling [67]. Mole- cell death, as summarized in Fig. 3. In addition to its kinase function, cular events ensuing the activation of the PERK branch of the UPR are IRE1 also has RNAse activity and it splices the mRNA encoding for X- schematized in Fig. 2. box binding protein 1 (XBP1), removing its introns and thus leading to Badr et al. employed Western Blotting to analyze UPR activation by the formation of spliced XBP1 (XBP1s) and its subsequent translation assessing ATF4 protein levels and distinguish between phosphorylated into a transcription factor. and unphosphorylated eIF2α, using a specific antibody for the phos- This requires the cooperation of IRE1 units and is involved in its – phorylated form and a pan-eIF2α antibody [68]. In brief, this sort of pro-survival action [80 84]. Its pro-death action, however, can be carried out by a single unit of the enzyme, and consists of a process

Fig. 3. Molecular events ensuing the activation of the IRE1 and ATF6 branches Fig. 2. Molecular events ensuing the activation of the PERK branch of the UPR. of the UPR.

4 D.C. da Silva, et al. Pharmacological Research 155 (2020) 104702 termed regulated IRE1-dependent decay (RIDD), ultimately resulting in 4μ8C is one of the pharmacological inhibitors available that acts cell death. RIDD involves the preferential cleavage of ER-associated upon the IRE1 branch of the UPR. It inhibits both the kinase and RNAse mRNAs encoding for growth-promoting proteins, the resulting frag- activity of IRE1 by binding its kinase domain at K599 and its RNAse ments being rapidly degraded by the action of exoribonucleases domain at K907 [98,99]. In vitro, the concentration range in which 4μ8C [80–82]. It is worth mentioning that IRE1 occurs in two different iso- is used is quite wide [72,100–102], including a report of 150 nM as its forms (IRE1α and –β) and, even though both are activated upon ER IE50 (50 % inhibition efficacy) towards XBP1 splicing inhibition in stress conditions, IRE1α is the most relevant, since it occurs ubiqui- macrophages [103], to concentrations as high as 100 μM described for tously in the organism, unlike IRE1β, which is restricted to gastro- the same cell type [92]. In vivo, it has been used at 10 mg/kg, ad- intestinal and respiratory epithelial cells [80,85,86]. ministrated by intraperitoneal injection [104]. XBP1s is a potent transcription factor that binds the endoplasmic STF083010, like 4μ8C, acts as an IRE1 inhibitor. However, it in- reticulum stress response element (ERSE) and unfolded protein re- hibits only its RNAse domain, thus allowing it to retain its kinase ac- sponse element (UPRE) and, consequently, enhances the expression of tivity while preventing XBP1 mRNA splicing and RIDD. In vitro,itis genes comprised in the ER machinery, such as BiP. Although its clea- commonly used at 50 or 60 μM[92,104–106]. In vivo, it has been used vage is associated to the IRE1 pathway, its expression can also be in- at 30 mg/kg via intraperitoneal injection [104,107]. duced by ATF6 [87,88]. A luminescence-based high-throughput IRE1 activity assay has been designed to identify its potential modulators. The authors identified 2.3. ATF6 branch peptides derived from the kinase domain of IRE1 that can modulate its activity and performed in vitro (in human hepatocellular carcinoma The transcription factor ATF6 is embedded in the ER membrane, cells) and in vivo (in Caenorhabditis elegans) experiments that conclude being released upon accumulation of misfolded proteins in the ER that these peptides can enhance IRE1 oligomerization and promote cell lumen. Once activated, it translocates to the Golgi apparatus, where it is survival upon ER stress conditions by enhancing XBP1 splicing but si- cleaved by site-1 and site-2 proteases, originating p50ATF6. It then multaneously limiting JNK activation and RIDD. This poses the possi- translocates to the nucleus, where it is responsible for inducing the bility of selectively modulating of IRE1 activity, being that the estab- transcription of ER chaperones by binding ERSE in an attempt to restore lishment of modulators of the pro-survival activity of this protein may homeostasis [108–111]. Under optimal conditions, ATF6 is maintained build a new strategy against the nefarious effects of ER stress [89,90]. in the ER binding BiP, which inhibits Golgi localization signals [112]. In the case of XBP1, to simply determine total mRNA amounts by The events resulting from the activation of this transcription factor are qPCR is not illustrative of its activity, since its activation as a tran- depicted on Fig. 3. scription factor involves mRNA splicing. As so, experimental frame- Not unlike other UPR transducers, simply assessing gene expression works should compare the amounts of spliced and unspliced XBP1 levels of ATF6 may not provide enough information, being that it is mRNA to be able to draw any conclusions [87]. There is a report on a important to determine its activation by analyzing its cleavage and spliced-XBP1 specific RT-PCR technique, which allows to separate localization. Another possible approach is to determine gene expression spliced and unspliced mRNA in a human monocytic cell line (THP-1) levels of its downstream genes, such as HYOU1 and HERPUD [113]. [87]. There are also several reports of green fluorescent protein constructs Gene expression levels of IRE1 may also be misleading, since it is with ATF6 that can be useful in assessing its subcellular localization, more assertive to infer about its activation by determining its phos- and therefore infer about its activation status [114]. phorylation status by Western Blotting. There are numerous IRE1-spe- There are not many examples in the literature of the use of phar- cific antibodies available, not only to determine the whole IRE1 protein macological inhibitors towards the ATF6 branch of the UPR. However, but also to specifically analyze the phosphorylated portion of the latter in 2016, Gallagher et al. discovered that a class of pyrazole amides [91,92]. For this reason, researchers have resorted to the determination named ceapins can inhibit ATF6 signaling, without affecting other of mRNA levels of genes like ERDJ4, which is an ERAD-related gene proteins involved in the UPR, and thus allowing for the analysis of the downstream of XBP1 [93]. impact of all the three major UPR branches separately. The IC50 of Reporter genes assays have also been employed to study the IRE1 ceapins are 4.9 μM for ceapin A1 and 2.6 μM and 0.59 μM for ceapin A6 branch of the UPR. One such instance are Venus-based constructs, a and ceapin A7, respectively [115]. There have not been noteworthy variant of green fluorescent protein. By fusing it to XBP1, it is possible advances since this, and thus there is still a significant need for new to analyze its splicing by IRE1 under ER stress conditions, by detecting ATF6 modulators. There are, however, reports of a pharmacological the fluorescence of the translated protein [94]. Furthermore, XBP1 has inducer of this transcription factor, discovered among a library of over been implied in the antimyeloma activity of bortezomib via RNAi 600,000 molecules [116], and subsequent studies on knockout mice knockdown in cell lines [95]. This type of technique has also been used models. The discovery of an ATF6 inducer led to the hypothesis that on XBP1 and IRE1 to study the mechanism of RIDD [96]. There are also stimulation of this signaling branch may greatly benefit proteostasis reports of dominant-negative IRE1 transgenes expressed in cell lines, as under pathological conditions in which it is compromised [117]. well as IRE1 knockout mice models [97]. Mice models have been an important tool to the achievement of the current knowledge of ER stress and the UPR. There are available in vivo

Table 2 Description of the activation and mechanisms of action of the major proteins involved in the UPR.

Protein Class Mechanism of action References

ATF4 Transcription factor Upregulates pro-apoptotic genes such as CHOP or activating transcription factor 3 (ATF3) / upregulates pro-survival genes [68] like BiP ATF6 Transcription factor Upregulation of UPR machinery and CHOP [119,120] BiP Chaperone Binding of unfolded/misfolded proteins, [57] CHOP Transcription factor Triggers apoptosis via Bcl-2 downregulation [67] eIF2α Translation initiation factor Impairment of translation initiation, selectively upregulation of a few UPR proteins [121] IRE1 Kinase and RNAse Splicing of the XBP1mRNA [81] PERK Kinase Phosphorylation of eIF2α [122,123] XBP1s Transcription factor Binding of ERSE and UPRE, upregulating UPR machinery [88]

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Table 3 Name, NCBI reference sequence, chromosomal location and function of the most widely-used UPR genes, according to the GenBank database.

Gene/Protein NCBI Reference Sequence Chromosomal Location Expression/Function

ATF4/ATF4 Var1: NM_001675.4 22q13.1 Ubiquitous expression. Broadly expressed in bone marrow. Var2: NM_182810.2 ATF6/ATF6 NM_007348.4 1q23.3 Ubiquitous expression, particularly high on thyroid, placenta, brain, kidneys and appendix. DDIT3/CHOP Var1: NM_001195053.1 12q13.3 Expressed in virtually every type of tissue, notably on thyroid and bone marrow. Var2: NM_001195054.1 Var3: NM_001195055.1 Var4: NM_001195056.1 Var5: NM_004083.5 Var6: NM_001195057.1 EIF2AK3/PERK Var1: NM_004836.6 2p11.2 Higher expression in thyroid, stomach, colon and bone marrow. Var2: NM_001313915.1 EIF2S1/eIF2α NM_004094.5 14q23.3 Expressed in all tissues, highlighting testis and esophagus. ERN1/IRE1 NM_001433.5 17q23.3 Ubiquitously expressed. Broadly expressed in adrenal glands and pancreas HSPA5/BiP NM_005347.5 9q33.3 Broadly expressed in every tissue, particularly in thyroid, bone marrow and placenta. XBP1/XBP1 Var1: NM_005080.3 22q12.1; 22q12 Ubiquitous expression, albeit especially high in salivary glands, liver and urinary bladder. Var2: NM_001079539.1 knockout models to study multiple UPR transducers, like Atf6, Ire1, lumen into the mitochondria, since chaperone GRP75 directly links

Xbp1, Perk, eIF2α, Atf4 and Chop, as reviewed elsewhere [118]. IP3R on the ER membrane and the voltage-dependent anion channel A brief summary of the functions of the major proteins involved in (VDAC) on the OMM. Under ER stress, this transfer may be over- the UPR can be found in Table 2. whelming for mitochondria, leading to the depolarization of the inner Assessment of changes in the mRNA levels of UPR target genes is mitochondrial membrane (IMM) and potentially triggering regulated one of the strategies most commonly found in the literature cell death mechanisms, as depicted on Fig. 4 [147]. Currently, it is [44,45,48,68,124–127]. Table 3 presents the most relevant UPR genes, possible to isolate MAMs resorting to subcellular fractionation techni- which expression is often evaluated in ER stress-related studies. ques [148]. In the case of several key mediators of the UPR, mRNA expression Depolarization of IMM disrupts its otherwise low permeability and by itself does not provide sufficient information. For example, in the results in the release of cytochrome c and other pro-apoptotic factors. case of IRE1, PERK or eIF2α, the phosphorylation status should also be Such proteins include the BH3-only proteins of the Bcl-2 family; Bid, determined, in order to correctly infer about their activity. Bad, Bim and Puma, which bind other proteins of the Bcl-2 and block Analogously, the gene expression of ATF6 or XBP1 provides little in- their pro-survival action [149,150]. Apoptotic cell death is classically formation about its activity as a transcription factor, since it depends on divided in two pathways: the intrinsic, or mitochondrial, and the ex- proteolytic or mRNA cleavage, respectively. Thus, it is necessary to trinsic, or death receptor pathway. While ER stress is known to trigger distinguish between the native and cleaved forms, rather than just as- the intrinsic pathway, ER stress-mediated pathway has been associated sessing total mRNA amount. to distinct apoptotic models, namely the cell death receptor-mediated In order to understand and modulate ER stress, several molecules of apoptosis [151]. ER stress-induced regulated cell death involves the different origins were established as modulators of one or more of its activation of the inflammatory caspase-4 in humans (caspase-12 in signaling pathways. The most commonly used molecules on research rodents [152]), which resides in the ER in homeostatic conditions are listed on Table 4, along with a brief description of their mechanism [127,153]. Shortly, the involvement of the UPR in the process is known of action, in order to provide insights about their use on assays aiming to involve changes on PERK and IRE1α signaling, as well as calcium to analyze ER stress. For a deeper insight on the subject of ER stress release from the ER lumen, elements of the Bcl-2 family and MAPK- modulators, the reader is referred to a few available reviews [128–132]. kinases, namely the c-Jun N-terminal kinase (JNK) [64,154]. In order to study the communication between the ER and the mi- tochondria, the most extensively used techniques have been electron 3. The ER-mitochondria axis microscopy, assessment of calcium dynamics and evaluation of ROS production [155]. The ER and mitochondria mutually communicate through zones collectively termed mitochondria-associated ER membrane (MAM). Both organelles are dynamic structures that can relocate within the 4. Crosstalk between ER stress and ROS cytoplasm by moving through the cytoskeleton according to the needs of the cell. For this reason, under ER stress MAM surface increases, Recent research has established that oxidative stress can trigger ER particularly on the perinuclear region. MAM encloses a significant stress, the opposite event also being possible [156]. In fact, it is cur- portion of the outer mitochondrial membrane (OMM), up to 20 %. At rently known that protein folding and generation of ROS are insepar- these sites, there are direct channels for calcium transfer from the ER able events. As mentioned before, protein folding implicates the

Table 4 ER stress modulators classified according to their respective mechanism of action.

Molecule Effect Mechanism of action References

4-Phenylbutyric acid Protector Chemical chaperone. [125,133,134,135] Salubrinal Protector Inhibition of eIF2α dephosphorylation [70,71,76,136,137] Brefeldin Inducer Inhibition of ER-Golgi transport. [138,139,140] DTT Inducer Impairment of protein folding [141] Palmitate Inducer Activation of caspase-4 and JNK, induction of ATF4 and CHOP. [142,143] Thapsigargin Inducer Irreversible SERCA pump inhibition. [144,145] Tunicamycin Inducer N-linked glycosylation inhibition. [146]

6 D.C. da Silva, et al. Pharmacological Research 155 (2020) 104702

reduction of the ER redox potential and UPR activation [164]. This construct contained KDEL, the mammalian ER retrieval signal, on the C- terminus of the reporter, and the sequence of BiP on its N-terminus. It was observed that, in vivo, the probe is nearly completely oxidized under homeostatic conditions, and that impaired ERO1 or peroxir- edoxin 4 activities compromise the oxidizing potential of the ER [165]. Subsequent reports mention green fluorescent protein-based constructs (roGFP1-iL) detect oxidizing and reducing changes between -190 and −130 mV (as determined in 3T3-L1 fibroblasts) by glutathione-medi- ated formation of disulfide bonds and dimerization, signi ficantly in- creasing the molecule’s reduction potential [157]. A similar green- fluorescent protein probe for measuring the ER redox potential has been developed by Hoseki et al. by substituting specific amino acids in order to increase folding efficacy of the protein. This new probe was named ER-targeted roGFP-S4 and was tested in HeLa cells. Like the previously mentioned reporter, this probe presents two excitations maxima de- pending on the surrounding redox status. This study reports that an enhanced ERO1 redox cycle increases ER oxidation. Consequently,

protein folding is enhanced, since this enzyme generates H2O2 via transference of electrons to H2O molecules. As expected and like in the Fig. 4. ER-induced mitochondria-dependent regulated cell death. Dynamin- previously mentioned studies, the ER redox state was found to reduce fi related protein 1 (Drp1) mediates mitochondrial membrane ssion. after treatment with reference ER stress inducers (thapsigargin and Phosphofurin acidic cluster sorting protein 2 (PACS-2) is necessary for the tunicamycin) [166]. connection between the ER and mitochondria. Apoptosis signal-regulating ki- nase 1 (ASK1) is a member of the mitogen-activated protein kinase MAPK fa- 5. Role of the ER in calcium homeostasis mily, as well as dual specificity mitogen-activated protein kinase kinases -4 and -7 (MKK4/7). Maintenance of proteostasis is not the only role that the ER plays in the cellular upkeep. This organelle is the main calcium reservoir of the fi formation of disul de bonds between cysteine pairs and allows the cell and is involved in the tight regulation of its levels, which in turn are protein to acquire its correct tertiary structure. The highly oxidizing involved in processes such as cell proliferation, differentiation, meta- redox potential of the ER is pivotal for oxidative protein folding to take bolism, apoptosis and gene expression [167]. The normal concentration place, namely disulfide bond formation, and thus the accumulation of of calcium outside the cell can be as high as 2 mM, while in the cytosol unfolded proteins on the ER lumen is accompanied by changes on its it is estimated to be around 100 nM [167,168]. redox status, beyond UPR activation. The redox status of this organelle Fundamental mechanisms of calcium concentration upkeep in the is actively regulated by a number of thiol oxidoreductases and low- ER involve three types of proteins: i) pumps to import Ca2+ ions from molecular weight redox-active electron carriers, which regulate the the cytosol, ii) luminal proteins to bind calcium and iii) channels to activities of the ER-resident oxidases peroxiredoxin 4 and ERO1 [157]. release these ions according to the cellular context, controlled by an ROS are physiologically present in the ER as a product of the trans- electrochemical gradient. Several calcium-binding ER proteins are in- ference of electrons between PDI and ERO1. When the ER is overloaded volved on calcium buffering, like BiP and other chaperones, such as with misfolded proteins, ROS are overproduced and interact with ER GRP94 and calreticulin, as well as proteins from the PDI family. One of enzymes, chaperones and calcium channels, leading to the leakage of the best-known pumps involved in this process is the sarco/en- ionic calcium into the cytosol, leading the mitochondria to generate 2+ doplasmic reticulum Ca -ATPase (SERCA). This ATPase, regulated by more ROS and disrupting the mitochondrial membrane potential and calnexin and calreticulin, is in charge of calcium uptake by the ER, thus inducing programmed cell death events or triggering autophagy importing two calcium ions from the cytosol for each ATP molecule – [158 160]. In a thapsigargin-induced model of cell death, over- hydrolyzed [169]. On the other hand, the release of calcium ions into expression of antioxidant enzymes like superoxide dismutase has been the cytosol requires the inositol 1,4,5-triphosphate receptor (IP3R) and observed to protect neuronal cells, thus evidencing the role of ROS in the ryanodine receptor (RyR) [170]. The relationship between ionic ER-mediated cell death [161]. In vitro and in vivo studies with Chop-/- calcium levels and proteostasis is further established, since insufficient mice have elucidated that this gene is required for the response to calcium on the ER will impair the function of these proteins. oxidative stress and subsequent progression of apoptosis, since its Binding of calcium to chaperones results in two major con- knockout protects against the induction of UPR related genes, such as sequences: i) buffering ER calcium levels and ii) regulation of the cal- EDEM1, while simultaneously reducing the expression of ERO1 cium-dependent function of these proteins. GRP94 represents 5–10% of [159,162]. Furthermore, CHOP contributes to oxidative stress by in- the pool of luminal proteins in the ER and possesses 19 calcium binding hibiting the expression of Bcl-2 and enhancing JNK signaling [163]. sites. BiP has a lower capacity to bind calcium (1–2 mol of calcium per fl Redox-sensitive green uorescent proteins (GFP) have been suc- mol). However, due to its high expression, it contributes to about 25 % cessfully employed to evaluate redox changes on the ER both in vitro of the calcium storage capacity of the ER. This monomeric protein and in vivo. These reporters, termed eroGFP (ER-targeted redox-sensi- possesses two binding domains, one peptide-binding and one ATP- tive GFP), possess a cysteine pair that becomes disulfide-linked when in binding. PDI and PDI-like proteins also contribute to calcium buffering fl and oxidizing environment, changing its uorescence excitability in the reticular environment. Examples of such PDI-like proteins are maximum from 490 to 400 nm. These reporters are therefore ad- calcistorin, calsequestrin, ERp72 and ERp44 [37]. vantageous in that they allow the determination of the ratio between Evidently, intracellular calcium levels are tightly regulated within fl the uorescence excitation of both wavelengths, hence allowing to the cell, being that the occurrence of ER stress may lead to its dis- minimize interferences such as photobleaching or operator errors. placement from the ER lumen into the cytosol. It also works on the fl Combining this reporter with a red uorescent protein-based construct opposite direction, i.e., temporal and/or spatial imbalances on calcium designed to evaluate UPR activation, the authors conclude that un- upkeep may lead to ER stress. For this reason, disturbances in calcium folded protein accumulation induced by ER stressors results in homeostasis are frequently analyzed in ER stress studies. Fura-2-

7 D.C. da Silva, et al. Pharmacological Research 155 (2020) 104702 acetoxymethyl ester or Fura-2/AM, a membrane-permeable derivative the lysosome and iii) selective autophagy, that eliminates mitochon- of the original calcium dye Fura-2, is the most commonly employed dria, endoplasmic reticulum, lipid droplets and invading pathogens calcium probe in the ER-related literature nowadays. It is a ratiometric [181]. Autophagy is triggered by mammalian target of rapamycin dye that is used both for intracellular calcium imaging and quantita- (mTOR) inactivation, resulting in activation of the autophagy-related tion; the fact that it is ratiometric confers it a high reliability, due to protein 1 (ATG1) and that phosphorylation of ATG13, which in turn decreased errors brought by uneven pipetting, photobleaching, or dif- recruits other ATG proteins, ultimately leading to the formation of the ferences in cell lines. Once in the cell, cellular esterases cleave the autophagosome. Detailed insights on the molecular and cell biology of acetoxymethyl group; then, the calcium-bound molecule will display its autophagy surpasses the scope of this article and thus the reader is excitation wavelength at approximately 340 nm, while the free form is referred to comprehensive reviews on the subject [182–184]. centered around 380 nm, being that the emission is maximum at 510 Early in the current century, the crosstalk between this catabolic nm in both cases. Hence, the amount of intracellular calcium is pre- process and the UPR was first reported, when it was noticed that ER sented as the F340/510/F380/510 ratio [171–173].This fluorescent dye has stress inducers could trigger the formation of autophagosomes been used extensively in a wide set of cell lines in the 3–5 μM range [185,186]. Activation of any of the three major signaling branches of [72,174–176]. the UPR can impact the autophagic process. Activated IRE1 induces There are other fluorescent calcium probes commercially available. autophagy by phosphorylating the MAPK8, that in turn promotes the One example is Fura-PE3/AM, a variant of Fura-2/AM that is designed activity of the downstream autophagy mediator Beclin-1. In addition, to avoid possible compartmentalization in the cell. Suppliers report that UPR-mediated JNK activation (either by the IRE1 or PERK branch) also hydrolysis of this molecule by esterases will trap it inside the cytosol results in MAPK8 phosphorylation by this kinase. JNK itself has a direct and make it cell membrane impermeable [177,178]. Other methodol- effect on autophagy by stimulating the expression of ATG5. Further- ogies available include quantitation of radiolabeled 45Ca2+ in ER mi- more, this kinase activates c-Jun, which plays a role in increasing the crosomes and whole cells [168]. expression of the autophagosome initiator Beclin-1 by interacting with Assays to determine SERCA pump activity are not found in the lit- its promoter. Finally, JNK also prevents the association of Beclin-1 to erature quite as often as other less direct approaches to infer about Bcl-2 by phosphorylating the latter. This will result in complexation of changes on calcium homeostasis. A reason for this may be the technical Beclin-1 with the phosphoinositide-3-kinase-kinase (PI3K) and leads to difficulties posed by direct measurements of its activity. There are, autophagosome formation. IRE1/JNK/-cJun signaling pathway is, for however, a few reports concerning the indirect determination of SERCA this reason, a major mechanism by which the UPR influences autop- activity. Towards this goal, McMullen and co-workers developed a 96- hagy. A role for XBP1 in autophagy, by indirectly regulating Bcl-2 and well plate colorimetric assay to quantify the amount of inorganic directly inducing Beclin-1 expressions, is also in place [187–191]. phosphate that is released by the pump following ATP hydrolysis, The involvement of the PERK branch of the UPR on the autophagic which then complexes with ammonium molybdate and malachite process is mediated its transcription factors ATF4 and CHOP, which green. The authors proceeded to isolate microsomes from brain tissue increase the expression of autophagic proteins such as ATG5, ATG12 and prepared an inorganic phosphate quantification reagent that binds and Beclin-1. Furthermore, as a result of overall impaired protein with inorganic phosphate, whereas the resulting complex issues a green synthesis induced by the activation of the PERK branch, Bcl-2 levels are color that can be measured at 660 nm in a spectrophotometer [168]. reduced and, consequently, Beclin-1 is released. In addition, ATF4 in- Another approach for the analysis of SERCA pump activity in vitro is duces the expression of microtubule-associated protein 1 light chain 3β based on the chemiluminescent calcium-binding protein aequorin tar- (LC3β), a structural protein in autophagosomes [188–190,192–194]. geted to the ER. This allows for the analysis of the rate of calcium Since it leads to the expansion of the ER machinery, including CHOP import into the ER lumen, which is related to SERCA activity. The same and XBP1, ATF6 activation can also result in the occurrence of autop- authors propose a protocol for determining SERCA activity in vivo, hagy [188–190,195,196]. through isolation of ER vesicles and their analysis of their calcium The exit of ionic calcium from the ER lumen into the cytosol leads uptake resorting to the fluorescent calcium dye Fluo-3 [46]. the activation of autophagic enzymes, such as the on calcium/calmo- dulin-dependent protein kinase kinase 2 (CAMKK2), that leads to the 6. A role for ER stress and the UPR in autophagy inhibition of mTORC1, consequently resulting in autophagy initiation. In fact, this is the mechanism behind the calcium-induced autophagy Autophagy, a highly conserved catabolic mechanism, is critical for elicited by thapsigargin [197,198]. In recent years many paths con- organism development and cellular homeostasis by assuring the protein necting ER stress, the UPR and autophagy have been described [184]. turnover of the cell, refurnishing it with the amino acids that result Learning how to modulate ER stress may constitute a strategy to fine- from the breakdown of intracellular components like soluble peptides, tune autophagy, and consequently pave the way for new therapeutic protein aggregates or even organelles. The process begins with the strategies, particularly given the increasingly recognized potential of formation of the phagophore, often arising from the ER membrane, and autophagy modulation in pathologies such as immune diseases, cancer comes to an end with the elimination of the autophagosome [179]. and neurodegenerative diseases [180,184,189]. This interplay between Autophagy is involved on the pathogenesis of several diseases. This the UPR and autophagy is summarized in Fig. 5. process progressively subsides with age, leading to the reduced for- The analysis of autophagy incorporated the identification of au- mation of autophagic vesicles and their impaired fusion with lysosomes tophagic structures and measuring of the autophagic flux, as reviewed [180]. In fact, the accumulation of toxic protein aggregates that are elsewhere [199]. characteristic of some neurodegenerative diseases is connected to un- The selective lysosomal degradation of ER fragments and/or protein successful autophagy following the cell’s inability to properly fold these aggregates is termed reticulophagy [200]. Together with the UPR and proteins. However, on the case of neoplastic disease, a dual role is at- ERAD, reticulophagy is another major quality control pathway that tributed to the autophagic pathway: on one hand, it suppresses initial bridges ER turnover and homeostasis. FAM134B (family with sequence tumor development, on the other hand, it may protect it from the im- similarity 134, member B)/RETREG1 is a reticulophagy receptor, and mune response [181]. thus its inhibition enhances proteotoxicity. In malignant cells, it is re- Autophagy can be sorted in three different types, i. e., i) macro- ported that this receptor inhibits tumoral growth. Concurrently, it is autophagy, that implies the enclosure of cytoplasmic content in double- known that aggressive cancers often carry mutations on the corre- walled vesicles destined to fuse with lysosomes and is generally referred sponding gene. It may also play a role on several other types of chronic to simply as autophagy, ii) microautophagy, that occurs when the cy- disease and on viral infection. For these reasons, it is a potential bio- toplasmic content, labeled with a signal peptide, is directly gorged by marker of disease [201]. Its expression has been evaluated at gene and

8 D.C. da Silva, et al. Pharmacological Research 155 (2020) 104702

ER stress in cancer cells can be an effective strategy for eliciting cancer cell death, both with stand-alone ER stress inducers [72, 220–222] and in combination with well-established anticancer drugs [223,224]. A significant number of neurodegenerative diseases are, from a pathophysiological point of view, proteinopathies which symptoms arise from upstream protein aggregation or misfolding. Each disease has its typically-involved proteins, such as huntingtin in Huntington’s dis- ease, amyloid-β in Alzheimer’s (AD) or α-synuclein in Parkinson’s dis- ease (PD). AD and PD are the two neurodegenerative disorders of highest incidence worldwide. For many neurodegenerative diseases ER stress and the activation of the UPR have been established as hallmarks of the aforementioned disorders, being also biomarkers of these conditions [56]. Increased levels of phosphorylated PERK and IRE1α have been found in the hippocampus of patients with AD, where they colocalize with phos- phorylated tau, one of the hallmarks of the disease [225,226]. In cul- β β Fig. 5. Molecular crosstalk between the UPR and autophagy. tured human neurons, exposure to amyloid- 42 (A 42), the major component of amyloid plaques, induces CHOP expression, while treatment with CHOP antisense RNA results in increased survival, thus protein level through through qPCR and Western Blotting, respectively suggesting a role for the ER in Aβ42-mediated cell death and hence a [202], while knockout mouse embryonic fibroblasts are currently potential pharmaceutical target [226,227]. The involvement of ER available [203,204]. Recently, an increasing number of such receptors stress in neuronal death in AD is further strengthened if we consider have been identified, including the cargo receptor Atlastin GTPase3 that administration of Aβ42 results in activation of the ER-resident (ATL3), SEC63, the long splice variant of RTN3 (RTN3L and CCPG1) caspases-4/12, which are involved in ER stress-induced apoptosis [205]. [228]. Promisingly, co-incubation with the ER protector salubrinal re- sults in decreased toxicity of Aβ42 in primary neurons [229]. 7. ER stress in health and disease Disturbances in the secretory pathway are known to be significant to the onset and progression of PD [230,231]. A few in vivo reports ER stress has been observed throughout the pathogenesis of a establish a relationship between UPR activation and the progression of number of severe diseases of considerable prevalence worldwide, such PD. A number of works show that α-synuclein accumulation at the ER as cancer, neurodegenerative diseases, cardiovascular diseases, obesity of dopaminergic neuros is accompanied by UPR activation and that ER and diabetes mellitus. Furthermore, the link between ER stress and protectors, such as salubrinal, improve motor performance, alleviate α- inflammation has already been established. synuclein accumulation at the ER and ultimately improve the lifespan The latest numbers available show that in 2016 there were some of PD models [232]. staggering 17.2 million cancer cases worldwide and 8.9 million deaths Even though the existence of a relationship between ER stress and [206]. The enhanced metabolic rates of cancer, resulting from aberrant neurodegenerative disorders is fairly documented in the literature, a lot proliferation under a low vascularized state, results in an adverse mi- remains to be described in what seems to be a vicious cycle of cause- croenvironment that includes acidic pH, low oxygen and also and low effect in which ER stress triggers and is triggered by the progression of glucose, as well as other nutrient deficiencies [126]. These conditions these diseases. Regardless, the fact that pharmacological intervention result in the accumulation of misfolded proteins, reason for which with molecules capable of alleviating ER stress show clinical gains in cancer cells depend heavily on adequate proteostasis. For this reason, animal models [225,226,229] may represent an exciting and promising acute states of the UPR may benefit tumor progression by enhancing the strategy. protein folding capacity of malignant cells, hence maintaining pro- teostasis. However, if the UPR fails to restore ER proteostasis, or if it is 8. Conclusions and future perspectives augmented beyond a tolerability threshold, tumor cell apoptosis ensues [207]. The performance and UPR signature of the ER are crucial to the Building on this information, the pivotal role of the UPR in tumor physiology of the cell and to its built-in stress response mechanisms. Its survival can be exploited from the standpoint of diagnosis and treat- importance towards stress recognition and response relies on complex ment. For example, GRP78 is frequently found to be highly expressed in signaling processes that have evolved in eukaryotic organisms. breast, lung cancer and prostate, as well as other cancers [208–210], Regardless of the intense research that has focused in the signaling being clinically associated to poor prognosis [211,212]. Interestingly, pathways of the ER and its involvement in disease onset, it is clear that this overexpression of GRP78 in the surface of cancer cells is not found no universal biomarker of UPR activation is currently known. This is to in non-cancer cells [213], which may pave the way for a UPR-guided be expected, considering the distinct branches of the UPR and the sorting of cancer cells. Other branches of the UPR are equally known to myriad of pathways that connect the events taking place during the be involved. For example, increased XBP1 expression and splicing have UPR to many other cellular events. Specific UPR signatures have al- been described in breast cancer and hepatocellular carcinoma ready been identified in cancer cells and have been used as a prognostic [214–216], which can in turn be a consequence of its regulatory marker and as a tool to predict response to chemotherapy. When con- function over GRP78. We refer the reader to previous works that have sidering the tight interconnection of cancer to upregulated UPR sig- reviewed the evidence of ER stress and UPR activation in various nature markers, a provocative line of research could be the clarification human cancers [216,217]. There are promising results that suggest this of potentially oncogenic nature of misfolded proteins. role of UPR branches in tumor progression can be exploited as a ther- Likewise, brain tissue of AD and PD patients have shown to over- apeutic strategy. For example, the XBP1 splicing inhibitor MKC-3946 express a number of UPR proteins. Are these proteins a consequence of has reduced the growth of MM and potentiate the pro-apoptotic effect the diseases, or can they perhaps be involved in their very onset? This is of the anticancer drug bortezomib [218], a result also found for the still unclear nowadays, however the next few years are expected to shed IRE1 endonuclease inhibitor STF-083010 [219]. a light on this apparent cause-effect cycle. It could be the case that both There is sufficient evidence in literature suggesting that triggering hypotheses are correct and that the explanation lies in the kinetics of

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