Pharmacological Research 155 (2020) 104702 Contents lists available at ScienceDirect Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs 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