Biotechnol Lett DOI 10.1007/s10529-014-1537-y
REVIEW
The endoplasmic reticulum and unfolded protein response in the control of mammalian recombinant protein production Hirra Hussain • Rodrigo Maldonado-Agurto Alan J. Dickson
•
Received: 20 February 2014 / Accepted: 10 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression. Keywords Autophagy Endoplasmic reticulum ER-associated degradation ER expansion Oxidative stress Recombinant protein production Unfolded protein response Introduction There is a growing demand for the production of recombinant proteins, either as biotherapeutics or
research tools and, as a result, the need for efficient protein expression has vastly increased (Walsh 2010). Mammalian cell lines, such as Chinese Hamster Ovary (CHO), remain major expression systems for production of biopharmaceuticals due to their ability to perform appropriate post-translational modifications (Walsh 2010, Kim et al. 2012). In mammalian cells, the pathway from the gene through to the production of fully processed and folded protein involves multiple steps (Fig. 1). Several studies suggest that folding and post-translational modification of proteins in the ER is the rate-limiting stage for production of recombinant proteins since the overexpression of folding regulators and machinery involved in protein processing increases the specific productivity of secreted proteins in different cell lines (Tigges and Fussenegger 2006; Campos-Da-Paz et al. 2008; Khan and Schroder 2008; Dreesen and Fussenegger 2011). The literature surrounding endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) has been reviewed previously (Cudna and Dickson 2003; Khan and Schroder 2008); this review discusses new developments, links to other cell processes and related approaches to improve recombinant protein production. The endoplasmic reticulum (ER) and ER stress
H. Hussain R. Maldonado-Agurto A. J. Dickson (&) Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK e-mail: [email protected]
The ER is the central site for synthesis and processing of proteins and lipids in the secretory pathway (Lynes and Simmen 2011). The ER is a complex membranous
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Fig. 1 Protein expression pathway. Recombinant protein production is controlled at multiple steps (arrow). Each step represents a possible bottleneck for recombinant protein production (blue boxes) and different strategies have been employed to overcome these. If the amount of messenger RNA and translation are not limiting factors, the nascent polypeptide chain is transported into the ER. Once in the ER, the
recombinant protein undergoes folding and processing steps to form mature protein and subsequently secreted. If the protein load exceeds the capacity of the ER the unfolded protein response is activated to increase the folding and processing capacity of the ER. In addition, degradation pathways are upregulated to decrease protein load
Fig. 2 The endoplasmic reticulum (ER) architecture and organelle biogenesis. The ER is firstly categorised as rough ER (purple) and smooth ER (blue) due to the respective presence and absence of ribosomes on the surface. The rough ER gives rise to the nuclear envelope and autophagosomes whilst the smooth ER generates lipid droplets and peroxisomes. Russell bodies (grey) can form both the smooth and rough ER.
The smooth ER can be sub-divided into domains based on their distinct functions and interactions with other organelles, such as the mitochondrial associated membrane (MAM), ER-quality control compartment (ERQC), plasma membrane-associated membrane (PAM), ER exit sites (ERES), ER-Golgi intermediate compartment (ERGIC)
structure categorised as rough ER and smooth ER. The rough ER and smooth ER are further sub-categorised into several distinct domains (Fig. 2). The domains include ER quality control compartment (ERQC), ER exit sites (ERES), mitochondrial-associated membrane (MAM) and plasma membrane-associated ER (PAM). Each domain is associated with a different function, as well as forming links with other organelles and structures (Lynes and Simmen 2011).
The ER also plays a part in the biogenesis of organelles, such as Russell bodies, peroxisomes and lipid droplets (Fig. 2). Russell bodies store insoluble immunoglobulin aggregates (Stoops et al. 2012). They can form from either the rough or smooth ER (Mattioli et al. 2006) and form in CHO and mouse myeloma (NS0) cells (Stoops et al. 2012). Lipids produced in the ER can also change the size of the ER. The ER has also been implicated in autophagosome formation and can
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change its structure and function in response to nutrient availability, energy state and demands of the cell to maintain flow of proteins and lipids through the secretory pathway (Lynes and Simmen 2011). In the secretory pathway, newly-synthesised polypeptide chains enter the ER and undergo different folding and processing steps. The proteins are then exported to the Golgi apparatus and eventually are targeted for secretion. The accumulation of unfolded or mis-folded protein in the ER lumen can induce ER stress. ER stress can be detrimental and affect multiple aspects of cellular function, including metabolism and apoptosis (Fribley et al. 2009; Wang et al. 2011). As well as sensing the presence of aberrant protein, ER stress responses can also be triggered by nutrient deprivation, infection, chemical insult, DNA mutations or altered metabolism (Moore and Hollien 2012). The ER homeostasis can potentially be affected by cell culture conditions where changes to the environment occur such as pH alteration and osmotic stress which can affect recombinant protein production (Schroder and Kaufman 2005; Chakrabarti et al. 2011). In response to ER stress, signalling pathways such as the unfolded protein response (UPR) can be activated to restore ER homeostasis. The unfolded protein response (UPR) The UPR is a co-ordinated response to restore ER homeostasis in the event of ER stress (Cao and Kaufman 2012). In the ER membrane there are three well-characterised transmembrane sensors that mediate the UPR: pancreatic ER kinase-PKR-like kinase (PERK), activating transcription factor 6 (ATF6) and Inositol-requiring enzyme 1 (IRE1; Fig. 3). Under non-stressed homeostatic conditions these three sensors are inactive and are bound to the molecular chaperone binding immunoglobulin protein (BiP). In the presence of increased mis-folded protein, BiP dissociates from the sensors and binds with greater affinity to the mis-folded protein. Upon dissociation of BiP, PERK and IRE1 homodimerise and activate their cytoplasmic kinase domains via auto-phosphorylation. Subsequently, the phosphorylated residues act as docking sites for signalling molecules leading to cell signalling pathways to be initiated in order to restore ER homeostasis (Bertolotti et al. 2000). IRE1 is one of the most studied UPR sensors due to the presence of a homologue in yeast (Cox et al. 1993; Mori et al. 1993).
It is responsible for splicing and activating X-box binding protein 1 (XBP1). The UPR acts to overcome ER stress by inhibiting protein synthesis and up-regulating the capacity of cells to fold proteins. UPR mechanisms can also activate degradation pathways to maintain homeostasis (Walter and Ron 2011). These UPR mechanisms act largely through the activation of specific transcription events (Walter and Ron 2011). If the amount of ER stress is excessive, UPR signalling pathways can induce cell death via apoptosis. PERK-mediated phosphorylation of eukaryotic translation initiation factor 2a (eIF2a), attenuates global protein translation whilst increasing translation of some mRNA species such as activating transcription factor 4 (ATF4) which in turn induces transcription of CCAAT/-enhancerbinding protein homologous protein (CHOP). Together, ATF4 and CHOP increase the expression of genes that are involved in protein synthesis such as aminoacyl-tRNA synthetases, and translation initiation factors (Han et al. 2013). If the UPR is able to adjust to the increased protein load, protein homeostasis is restored promoting cell survival. Conversely, if the protein load is too high for the cell to adjust via the initial UPR event, the increased protein synthesis causes a change to the energy balance of the cell, depletion of ATP and generation of reactive oxygen species (ROS) which can, in turn, promote cell death via apoptosis (Han et al. 2013). In recent years our understanding of the complexity of the UPR signaling pathway has increased and a wide range of ER-stress-induced microRNAs (miRNA) have been identified in mammalian cells (Fig. 4; Chitnis et al. 2012; Behrman et al. 2011). miRNAs are small non-coding RNAs (*22 nucleotides) (Klanert et al. 2013) that regulate cell physiology through posttranscriptional regulation of gene expression. The expression of these ER-stress-induced miRNAs is cell-type dependent and cellular functions regulated by these miRNAs have been covered in different reviews (Bartoszewska et al. 2013; Chitnis et al. 2013; Maurel and Chevet 2013). The application of miRNAs as a tool to improve the production of recombinant proteins in mammalian cells has been highlighted in different publications (Strotbek et al. 2013; Sanchez et al. 2013; Jadhav et al. 2012). A single miRNA can regulate the expression of multiple genes and, as a result, it offers a targeted way for regulation of gene networks. The use of miRNAs to
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Fig. 3 The unfolded protein response (UPR). ER stress is sensed by three ER-membrane bound sensors (PERK, ATF6 and IRE1). Under conditions of ER stress, mis-folded protein accumulates in the ER which causes the dissociation of Binding immunoglobulin protein (BiP) from the sensors. The dissociation of BiP leads to the initiation of a coordinated signalling
pathway, the UPR, to restore ER homeostasis. The UPR acts via different mechanisms to inhibit protein synthesis and increase the protein folding capacity of the cell to overcome ER stress. When the UPR is unable to restore ER homeostasis, UPR activation can lead to apoptosis
engineer recombinant cell lines presents an advantage over protein-coding mRNAs, as miRNAs do not represent an additional translational burden for the cell (Hackl et al. 2012). Therefore, miRNAs are an attractive tool to regulate the activity of signaling pathways important for protein secretion, like the UPR. The UPR can also regulate gene expression in a post-transcriptional manner through the endoribonuclease activity of IRE1. This process, called regulated IRE1-dependant decay (RIDD), degrades mRNAs encoding proteins that either localise to the ER or transit through the ER as part of the secretory pathway (Hollien et al. 2009; Hollien and Weissman 2006). RIDD can also regulate protein expression through the degradation of miRNAs (Fig. 3; Upton et al. 2012). Degradation of mRNA via RIDD is thought to limit the amount of protein entering the ER and decrease the ER unfolded protein load (Hollien et al. 2009; Ali et al. 2011).
ER-associated degradation (ERAD)
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Accumulation of mis-folded or improperly processed proteins in the ER also activates protein degradation through ER-associated degradation (ERAD) (Stolz and Wolf 2010). The mis-folded proteins are transported from the ER to the cytoplasm through translocons, after which they are ubiquitinated and then degraded via the proteasome (Fig. 4; Stolz and Wolf 2010). One of the mechanisms by which mis-folded proteins are targeted for degradation results from interactions of exposed hydrophobic faces which bind to chaperones such as BiP (Stolz and Wolf 2010). Under non-stressed conditions, ERAD acts to dispose any inappropriate (mis-folded) proteins and this prevents accumulation of undesirable proteins in the ER (and their potential release from the cell). To maintain basal ERAD activity under non-stressed conditions, ERAD is regulated by the removal of ERAD machinery from the ER and subsequent degradation via proteases in the
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Fig. 4 Physiologic consequences of unfolded protein response (UPR) activation. Different stimuli that perturb ER function can lead to the accumulation of mis-folded protein activating the UPR. The UPR inhibits general translation and regulates transcriptional responses to increase the protein folding capacity of the ER and restore homeostasis. Increased phospholipid synthesis and subsequent ER membrane expansion is also a
consequence of UPR activation. During protein folding, the formation of disulphide bonds produces reactive oxygen species (ROS) which activates the UPR. Other consequences of the UPR involve the up-regulation of degradation processes such as ERAD and autophagy, which act to decrease the amount of misfolded protein in the ER
lysosome (Cali et al. 2008; Bernasconi and Molinari 2011). Under ER stress, ERAD activity is increased by inhibition of degradation of ERAD machinery which, in turn, allows increased removal of unfolded or misfolded protein. UPR activation takes a more prolonged time period (e.g. several hours) than ERAD activation; therefore ERAD acts to remove aberrant proteins rapidly (Bernasconi and Molinari 2011). Regulation of ERAD in this way prevents substantial accumulation of protein in the ER. As a consequence of ER stress and the activation of the UPR, physiological changes occur within the cell in order to maintain homeostasis. These include the consequences of the UPR on the ER and the cell as a whole that then determine the efficiency of the cell to secrete heterologous proteins.
2013). Lipids can be used as a source of energy, in addition to involvement in processes such as membrane biogenesis, membrane trafficking, cell signalling and regulation of membrane protein activity (Shevchenko and Simons 2010). Membrane biogenesis involves the coordination of many metabolic and lipid synthesis pathways (Nohturfft and Zhang 2009). Phospholipids produced by the ER and Golgi are important components in all membranes in mammalian cells (Brewer and Jackowski 2012). The lipid composition of a membrane dictates its physical properties (Brewer and Jackowski 2012). Phosphatidylcholine (PC) is the most abundant phospholipid species in mammalian membranes. Newly synthesised lipids produced by the ER are inserted into the ER membrane and are rapidly transported to their target destinations within the cell (van Meer et al. 2008). The main method by which membrane lipids are transported to different organelles in the secretory pathway is via vesicular transport (van Meer et al. 2008).
Lipid synthesis and membrane expansion The ER and Golgi are the main intracellular sites for lipid synthesis and secretion (Lagace and Ridgway
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Membrane biogenesis is essential for cell growth and can be increased in response to many cellular processes such as increased protein synthesis and secretion (Nohturfft and Zhang 2009). Certain cell types with high secretory capacities respond to environmental stimulation by changing the size and organisation of organelles. This is exemplified by B lymphocytes that differentiate into antibody-secreting plasma cells where increased protein synthesis and secretion coincides with increased lipid synthesis and ER expansion (Fagone et al. 2007). Other studies have shown that the smooth ER in liver cells increased in amount in the presence of toxins. As a result there was an increase in the amount of detoxifying enzymes associated with the smooth ER to decrease ROS (Iseri et al. 1966; Lieber 2004). The UPR can influence lipid metabolism under conditions of ER stress (Fig. 4). The spliced form of XBP1 (XBP1S), a mediator within the UPR signalling pathway, increases lipid metabolism and ER membrane biogenesis. XBP1S is essential for the development of ‘‘professional’’ secretory cells (e.g. B lymphocytes, macrophages and pancreatic islets) (Lee et al. 2005, 2008). In antibody-secreting B lymphocytes, the amount of XBP1 transcript increased in response to UPR activation and the transcript underwent splicing to produce XBP1S mRNA (and, subsequently, protein). As well as activating expression of UPR target genes in the secretory pathway, XBP1S induced expression of enzymes involved in lipid synthesis. For example, XBP1S induced expression of PC via the CDP-choline pathway which is required for ER development (Sriburi et al. 2004, 2007; Shaffer et al. 2004). However, more research is needed to uncover the precise mechanisms by which XBP1S regulates lipid synthesis. In XBP1 deficient situations, the amount of membrane lipids is significantly decreased (Sriburi et al. 2004). ATF6a, another UPR mediator, also influences ER membrane biogenesis as overexpression of ATF6a increased phospholipid and fatty acid synthesis, a contributory factor in ER expansion (Bommiasamy et al. 2009). Early studies in CHO cells treated with sodium butyrate showed increased production of recombinant protein as well as an expanded ER (Dorner et al. 1989). Engineering CHO cells to express XBP1S constitutively resulted in an expanded ER and Golgi with increased recombinant protein production capacity (Tigges and Fussenegger 2006). In another
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publication, the expression of three monoclonal antibodies (mABs) in CHO DUXB11 and CHOK1 cell lines was compared (Hu et al. 2013). CHOK1 clones produced up to 10 times more mAB than CHO DUXB11 clones. Interestingly, the ER and mitochondrial content was higher in CHOK1 than CHO DUXB1 (Hu et al. 2013) suggesting that ER expansion and higher mitochondrial content can improve the cell’s capacity to manage a high recombinant protein load. In addition, increased lipid content associated with active secretory machinery was found in human AGE1.HN cells producing recombinant alpha (1)-antitrypsin compared to the parental cell line (Niklas et al. 2013). In yeast, ER stress/UPR induces membrane expansion through mediation of the Ino2/4 transcription factor complex. Direct activation of the Ino2/4 complex resulted in expansion of the ER membrane. ER membrane expansion decreased ER stress independent to the amount of ER chaperones, showing ER size as an important independent factor in reversing ER stress (Schuck et al. 2009). Increasing the size of ER is proposed to lower the concentration of protein folding intermediates, allowing more time for the protein to fold whilst also avoiding aggregate formation (Apetri and Horwich 2008; Schuck et al. 2009). Overall, expansion of the ER can act as a key adaptive response to decrease the amount of misfolded protein by increasing the amount of space for protein processing (Schuck et al. 2009). Further research is required to understand how membrane expansion is controlled in mammalian cells and define whether membrane content changes occur across the whole ER structure or if changes are restricted to specific ER domains. New DNA editing technologies can offer the possibility of monitoring changes in different domains of the ER. For example, creating fluorescently labelled fusion proteins to observe changes in the ER (Schuck et al. 2009). In addition, processes involved in membrane lipid production and control of ER size offers potential targets for engineering increased recombinant protein production and quality. Autophagy and the unfolded protein response (UPR) Autophagy has been recognised as a process in ‘‘bulk material’’ degradation (Levine 2005). Autophagy
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degrades damaged organelles and parts of the cell to maintain homeostasis and provides an alternative degradation process to ERAD. Autophagy is activated as a result of many of the cellular processes, such as ER stress, that activate the UPR. Linkages and interactions between autophagy and the UPR suggest these mechanisms offer an integrated response to cellular stress imposition (Fig. 4). It is thought that under conditions of ER stress, where there is an accumulation of mis-folded protein, autophagy is induced in order to overcome the stress and restore ER homeostasis (Appenzeller-Herzog and Hall 2012). The process of autophagy involves the formation of double-membrane cytosolic vesicles (autophagosomes) around the targets for degradation. The autophagosomes fuse with endosomes or lysosomes where proteases degrade the contents. The source of the membranes for the formation of autophagosomes is thought to originate from the ER (Fig. 2), although it has been shown membranes from other parts of the cell such as the Golgi, plasma membrane and mitochondria can also contribute to autophagosome formation (Axe et al. 2008; HayashiNishino et al. 2009; Ravikumar et al. 2010; Hailey et al. 2010; Ohashi and Munro 2010). The degradation products derived from lysosomal degradation may be re-used for other biosynthetic processes (Kim et al. 2013). Recently, selective types of autophagy have been defined for specific organelles (Cebollero et al. 2012); for example, ER (reticulophagy), ribosomes (ribophagy) and mitochondria (mitophagy) (Bernales et al. 2006; Kraft et al. 2008; Wang and Klionsky 2011). The precise mechanism of reticulophagy is unclear. It is has been suggested that under ER stress specific areas of the ER that contain mis-folded proteins and insoluble aggregates are removed to form autophagosomes. In addition, under certain conditions, autophagosomes do not fuse with the lysosome but the removal of aberrant protein into these autophagosome structures is sufficient to reduce ER stress (Bernales et al. 2006). Reticulophagy counterbalances the effect of ER membrane expansion by decreasing the ER size as well as removing parts of the ER carrying unfolded proteins (Bernales et al. 2006). Reticulophagy, along with ERAD, offers parallel UPR-regulated processes (Cebollero et al. 2012). Formation of the autophagosome involves the conversion of microtubule-associated protein 1 light chain
3 (LC3-I) into LC3-II. Subsequently, LC3-II is displayed on autophagosome membranes. This conversion relies on autophagy-related proteins (Atg) proteins that are up-regulated in response to PERKmediated phosphorylation of the translation initiation factor, eIF2a. Other UPR mediators, such as IRE1 and ATF6, also affect autophagy (Cebollero et al. 2012; Appenzeller-Herzog and Hall 2012). In addition, the formation and regulation of autophagosomes also involves Atg proteins and the mammalian target of rapamycin (mTOR). mTOR is a serine-threonine protein kinase that acts as a central regulator of cell growth in response to many intracellular (e.g. amino acids, ATP) and extracellular stimuli (e.g. growth factors, hormones) (Appenzeller-Herzog and Hall 2012). mTOR exists in two complexes, mTORC1 and mTORC2, and each complex is associated with different roles. mTORC1 negatively regulates autophagy. A possible mechanism for autophagy regulation via mTORC1 is under ER stress conditions; PERKmediated phosphorylation of eIF2a results in expression of UPR target genes, such as ATF4 and CHOP, and the tribbles homologue 3 (TRB3), a downstream target of this pathway, inactivates mTORC1. mTORC1 inactivation releases autophagy mediators from inhibition and subsequently activates autophagy (Salazar et al. 2009). ATF6 has been implicated in mTORC1 activation in the early phases of ER stress/ UPR activation. Under chronic ER stress/UPR activation, this stimulation of mTORC1 is reversed by the PERK-TRB3 pathway (Appenzeller-Herzog and Hall 2012). There is also evidence of cross-talk between autophagy and ERAD activity. Autophagy acts to inhibit disposal of ERAD machinery thereby increasing ERAD activity. LC3-I is used for the vesicular transport of ERAD machinery from the ER for subsequent degradation. The conversion of LC3-I to LC3-II (present on autophagosome membranes) inhibits transport of ERAD components (Bernasconi and Molinari 2011). Autophagy is induced in antibody-producing recombinant CHO cells towards the end of batch culture when nutrients are depleted causing cell death (Hwang and Lee 2008b; Han et al. 2010). In terms of recombinant protein expression, cell death in cell culture has two consequences. Firstly through limiting the viable cell density and secondly through potential changes to product quality due to the release of
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substances into the medium (Hwang and Lee 2008a). Limited studies have focused on autophagy and engineering of this pathway in recombinant cell lines (Hwang and Lee 2008b). Kim et al. (2013) have reviewed the approaches taken to control autophagy through the addition of chemical compounds either activating or inhibiting targets in the autophagy pathway in CHO cell cultures. However, further work is required as the effects of chemical compounds are pleiotropic. Oxidative stress and the unfolded protein response (UPR) Between the ER and cytosol there is a strong redox gradient, the ER being more oxidising than the cytosol (Margittai and Sitia 2011). The formation of disulphide bonds in mammalian cells requires the action of multiple enzymes such as ER oxidoreductin 1 (Ero1) and members of the protein disulphide isomerase (PDI) family (Feige and Hendershot 2011; Sato et al. 2013). Oxidised Ero1 transfers the disulphide bond to PDI which in turn oxidises the protein substrate. During the disulphide bond reaction, O2 is used as an electron acceptor (Feige and Hendershot 2011). Within the ER both oxidation and reduction reactions occur. If unbalanced or mis-regulated these complex reaction chemistries can impair oxidative folding and subsequently cause oxidative stress (Margittai and Sitia 2011). Protein folding is sensitive to oxidative stress as well as ER stress. Increased protein synthesis causes an increase in ROS (Malhotra et al. 2008) which, in turn, can act as signals for UPR activation (Fig. 4). ROS can arise during formation of disulphide bonds, when oxygen is reduced to form hydrogen peroxide via electron transfer. In some cases ROS can amplify protein mis-folding via oxidation of amino acids or by alteration of ER function (Malhotra et al. 2008), events which all act to increase the UPR. In cases where oxidative stress is severe, cell death is induced via apoptosis. Severe protein mis-folding can also cause leakage of calcium from the ER into the mitochondria where high amounts of calcium can disrupt the electron transport chain, induce autophagy and compromise cell function further (Deniaud et al. 2008; Malhotra et al. 2008). One pathway of calcium release is the CHOP-mediated induction of Ero1 which causes
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release of calcium through inositol 1,4,5-triphosphate receptors (Li et al. 2009). Specialised cells with high secretory capacities (professional secretory cells) exhibit high expression of proteins that can prevent oxidative stress (Bertolotti et al. 2010). For example, the differentiation of B lymphocytes into antibody-secreting plasma cells not only results in ER membrane expansion but increased oxidative stress. This subsequent oxidative stress triggers the activation of signalling pathways to counteract oxidative stress. This signalling results in significantly increased expression of PDI oxidation enzyme, peroxiredoxin 4, as well as the activation of the transcription factors Nrf1 and Nrf2 which, in turn, induce the expression of genes required for detoxification and ROS removal (Sato et al. 2013; Digaleh et al. 2013). B Lymphocyte differentiation shows significant reshaping of antioxidant responses to cope with oxidative stress (Bertolotti et al. 2010). Furthermore, other studies involving expression of tissue plasminogen activator (tPA) in CHO cells shows antioxidant treatment increases tPA production and supresses apoptosis (Yun et al. 2001). Also, expression of coagulation factor FVIII shows anti-oxidant treatment decreases oxidative stress and UPR signalling to increase secretion of FVIII protein (Malhotra et al. 2008). Applications of the unfolded protein response (UPR) for selection and engineering of better hosts Proteome and transcriptome profiling technologies have been used to identify the molecular characteristics of cells with different productivities or using conditions that increase the production of recombinant proteins (Seth et al. 2007; Doolan et al. 2008; Carlage et al. 2009; Charaniya et al. 2009; Yee et al. 2009; Schaub et al. 2010). These types of studies are affected by clonal variability making it hard to distinguish between different molecular characteristics that are associated with high productivity from those inherent to the heterogeneous cell population (Oh et al. 2003; Davies et al. 2013). The main conclusion from these studies is that there are a variety of cellular functions differentially regulated in the best producer clones that can lead to high productivity. In a heterogeneous cell population some clones are more suited than others to handle the burden of recombinant protein production.
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The UPR can be a possible route to select and engineer better host cells for recombinant protein production. Kober et al. (2012) developed a method for selecting highly productive, transfected clones based on the detection of ER stress markers. This method used an ER-stress sensor based on the correlation between BiP expression and recombinant protein production. A similar system was developed by Du et al. (2013) using a UPR-inducible promoter controlling the expression of the green fluorescent protein (GFP). This study showed that both timing and extent of UPR are different between clones re-enforcing the intrinsic variability of the clones. Interestingly, the UPR is also active in the host cell line without recombinant protein expression during batch culture, suggesting that under these conditions; there are other sources of stress that activate the UPR. Several groups have reported that the increase in the recombinant protein production does not proportionally increase with transgene copy number or the amount of mRNA when expression is high (Mohan et al. 2008; Barnes et al. 2003, 2007; Ku et al. 2009). These observations suggest that after a certain threshold of the amount of mRNA, the rate-limiting steps in recombinant protein secretion may be translational or post-translational processes (Lim et al. 2010). To overcome this productivity limitation, a number of ER-related proteins have been engineered into cells by overexpression, specifically a number of chaperones including BiP (Borth et al. 2005), calnexin, calreticulin (Chung et al. 2004), a number of PDIs (Mohan et al. 2008), and components of the UPR signaling pathway such as CHOP (Nishimiya et al. 2013), ATF4 (Haredy et al. 2013; Ohya et al. 2008), GADD34 (Omasa et al. 2008) and XBP-1. The effect of these proteins depends on the system employed and the recombinant protein produced. Also, the overexpression of just one chaperone is probably not enough to improve the secretory machinery; for this reason, XBP1, the master regulator of the secretory machinery is an interesting target for cell engineering. The effects of XBP1 overexpression have led to conflicting reports, Becker et al. (2009) reported and improvement in the production of a monoclonal antibody in CHO cells overexpressing XBP1S. Campos-Da-Paz et al. (2008) and Ohya et al. (2008) overexpressed XBP1S in CHO cells and found no increase in the production of both Factor VIII and anti-thrombin-III. Ku et al. (2008) found that the
effect of XBP1S overexpression in CHO cells and NS0 cells depends on the existence of a secretory bottleneck. The co-expression of XBP1 and Ero1 increased the recombinant protein expression by 5 to 6 fold in a transient gene expression system, without affecting the quality of the product (Cain et al. 2013).
Summary and outlook To improve protein production in mammalian cells there has been extensive engineering of different aspects of protein expression, such as gene expression, protein modification, secretion, cell metabolism and apoptosis. Engineering of physiologic cellular changes associated with ER stress and UPR activation may further enhance expression of recombinant proteins. This review has discussed the physiological consequences of the ER stress and UPR activation. Different stimuli can perturb the ER function producing different outputs depending on the time and intensity of the stress. Under conditions of ER stress the UPR, a complex signaling pathway, is activated to recover homeostasis. The UPR causes an increase in the protein folding capacity of the ER by up-regulating the expression of molecular chaperones and ER size (increased phospholipid synthesis). Increased protein synthesis can also lead to oxidative stress. Oxidative stress also activates the UPR which can lead to cell apoptosis. Also the UPR acts to decrease protein load by inhibiting general translation and up-regulating the transcription of genes encoding secretory proteins. Degradation processes like ERAD and autophagy are also active during ER stress conditions to remove mis-folded proteins and recycle cellular components. If these actions fail to restore ER homeostasis the UPR can induce cell death via apoptosis. Different proteins from the UPR signaling pathway have been used for cell engineering, yet our understanding of how metabolism, protein secretion, aggregation, glycosylation, apoptosis and their physiological consequences are related, is still limited. How the different UPR arms are regulated over time and how the different culture conditions affects its activation is still unknown. More research is needed to comprehend the role of the UPR for recombinant protein production especially focusing on the biology of the cell, in order to establish desirable UPR phenotypes.
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REVIEW
The endoplasmic reticulum and unfolded protein response in the control of mammalian recombinant protein production Hirra Hussain • Rodrigo Maldonado-Agurto Alan J. Dickson
•
Received: 20 February 2014 / Accepted: 10 April 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression. Keywords Autophagy Endoplasmic reticulum ER-associated degradation ER expansion Oxidative stress Recombinant protein production Unfolded protein response Introduction There is a growing demand for the production of recombinant proteins, either as biotherapeutics or
research tools and, as a result, the need for efficient protein expression has vastly increased (Walsh 2010). Mammalian cell lines, such as Chinese Hamster Ovary (CHO), remain major expression systems for production of biopharmaceuticals due to their ability to perform appropriate post-translational modifications (Walsh 2010, Kim et al. 2012). In mammalian cells, the pathway from the gene through to the production of fully processed and folded protein involves multiple steps (Fig. 1). Several studies suggest that folding and post-translational modification of proteins in the ER is the rate-limiting stage for production of recombinant proteins since the overexpression of folding regulators and machinery involved in protein processing increases the specific productivity of secreted proteins in different cell lines (Tigges and Fussenegger 2006; Campos-Da-Paz et al. 2008; Khan and Schroder 2008; Dreesen and Fussenegger 2011). The literature surrounding endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) has been reviewed previously (Cudna and Dickson 2003; Khan and Schroder 2008); this review discusses new developments, links to other cell processes and related approaches to improve recombinant protein production. The endoplasmic reticulum (ER) and ER stress
H. Hussain R. Maldonado-Agurto A. J. Dickson (&) Faculty of Life Sciences, The Michael Smith Building, University of Manchester, Oxford Road, Manchester M13 9PT, UK e-mail: [email protected]
The ER is the central site for synthesis and processing of proteins and lipids in the secretory pathway (Lynes and Simmen 2011). The ER is a complex membranous
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Fig. 1 Protein expression pathway. Recombinant protein production is controlled at multiple steps (arrow). Each step represents a possible bottleneck for recombinant protein production (blue boxes) and different strategies have been employed to overcome these. If the amount of messenger RNA and translation are not limiting factors, the nascent polypeptide chain is transported into the ER. Once in the ER, the
recombinant protein undergoes folding and processing steps to form mature protein and subsequently secreted. If the protein load exceeds the capacity of the ER the unfolded protein response is activated to increase the folding and processing capacity of the ER. In addition, degradation pathways are upregulated to decrease protein load
Fig. 2 The endoplasmic reticulum (ER) architecture and organelle biogenesis. The ER is firstly categorised as rough ER (purple) and smooth ER (blue) due to the respective presence and absence of ribosomes on the surface. The rough ER gives rise to the nuclear envelope and autophagosomes whilst the smooth ER generates lipid droplets and peroxisomes. Russell bodies (grey) can form both the smooth and rough ER.
The smooth ER can be sub-divided into domains based on their distinct functions and interactions with other organelles, such as the mitochondrial associated membrane (MAM), ER-quality control compartment (ERQC), plasma membrane-associated membrane (PAM), ER exit sites (ERES), ER-Golgi intermediate compartment (ERGIC)
structure categorised as rough ER and smooth ER. The rough ER and smooth ER are further sub-categorised into several distinct domains (Fig. 2). The domains include ER quality control compartment (ERQC), ER exit sites (ERES), mitochondrial-associated membrane (MAM) and plasma membrane-associated ER (PAM). Each domain is associated with a different function, as well as forming links with other organelles and structures (Lynes and Simmen 2011).
The ER also plays a part in the biogenesis of organelles, such as Russell bodies, peroxisomes and lipid droplets (Fig. 2). Russell bodies store insoluble immunoglobulin aggregates (Stoops et al. 2012). They can form from either the rough or smooth ER (Mattioli et al. 2006) and form in CHO and mouse myeloma (NS0) cells (Stoops et al. 2012). Lipids produced in the ER can also change the size of the ER. The ER has also been implicated in autophagosome formation and can
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change its structure and function in response to nutrient availability, energy state and demands of the cell to maintain flow of proteins and lipids through the secretory pathway (Lynes and Simmen 2011). In the secretory pathway, newly-synthesised polypeptide chains enter the ER and undergo different folding and processing steps. The proteins are then exported to the Golgi apparatus and eventually are targeted for secretion. The accumulation of unfolded or mis-folded protein in the ER lumen can induce ER stress. ER stress can be detrimental and affect multiple aspects of cellular function, including metabolism and apoptosis (Fribley et al. 2009; Wang et al. 2011). As well as sensing the presence of aberrant protein, ER stress responses can also be triggered by nutrient deprivation, infection, chemical insult, DNA mutations or altered metabolism (Moore and Hollien 2012). The ER homeostasis can potentially be affected by cell culture conditions where changes to the environment occur such as pH alteration and osmotic stress which can affect recombinant protein production (Schroder and Kaufman 2005; Chakrabarti et al. 2011). In response to ER stress, signalling pathways such as the unfolded protein response (UPR) can be activated to restore ER homeostasis. The unfolded protein response (UPR) The UPR is a co-ordinated response to restore ER homeostasis in the event of ER stress (Cao and Kaufman 2012). In the ER membrane there are three well-characterised transmembrane sensors that mediate the UPR: pancreatic ER kinase-PKR-like kinase (PERK), activating transcription factor 6 (ATF6) and Inositol-requiring enzyme 1 (IRE1; Fig. 3). Under non-stressed homeostatic conditions these three sensors are inactive and are bound to the molecular chaperone binding immunoglobulin protein (BiP). In the presence of increased mis-folded protein, BiP dissociates from the sensors and binds with greater affinity to the mis-folded protein. Upon dissociation of BiP, PERK and IRE1 homodimerise and activate their cytoplasmic kinase domains via auto-phosphorylation. Subsequently, the phosphorylated residues act as docking sites for signalling molecules leading to cell signalling pathways to be initiated in order to restore ER homeostasis (Bertolotti et al. 2000). IRE1 is one of the most studied UPR sensors due to the presence of a homologue in yeast (Cox et al. 1993; Mori et al. 1993).
It is responsible for splicing and activating X-box binding protein 1 (XBP1). The UPR acts to overcome ER stress by inhibiting protein synthesis and up-regulating the capacity of cells to fold proteins. UPR mechanisms can also activate degradation pathways to maintain homeostasis (Walter and Ron 2011). These UPR mechanisms act largely through the activation of specific transcription events (Walter and Ron 2011). If the amount of ER stress is excessive, UPR signalling pathways can induce cell death via apoptosis. PERK-mediated phosphorylation of eukaryotic translation initiation factor 2a (eIF2a), attenuates global protein translation whilst increasing translation of some mRNA species such as activating transcription factor 4 (ATF4) which in turn induces transcription of CCAAT/-enhancerbinding protein homologous protein (CHOP). Together, ATF4 and CHOP increase the expression of genes that are involved in protein synthesis such as aminoacyl-tRNA synthetases, and translation initiation factors (Han et al. 2013). If the UPR is able to adjust to the increased protein load, protein homeostasis is restored promoting cell survival. Conversely, if the protein load is too high for the cell to adjust via the initial UPR event, the increased protein synthesis causes a change to the energy balance of the cell, depletion of ATP and generation of reactive oxygen species (ROS) which can, in turn, promote cell death via apoptosis (Han et al. 2013). In recent years our understanding of the complexity of the UPR signaling pathway has increased and a wide range of ER-stress-induced microRNAs (miRNA) have been identified in mammalian cells (Fig. 4; Chitnis et al. 2012; Behrman et al. 2011). miRNAs are small non-coding RNAs (*22 nucleotides) (Klanert et al. 2013) that regulate cell physiology through posttranscriptional regulation of gene expression. The expression of these ER-stress-induced miRNAs is cell-type dependent and cellular functions regulated by these miRNAs have been covered in different reviews (Bartoszewska et al. 2013; Chitnis et al. 2013; Maurel and Chevet 2013). The application of miRNAs as a tool to improve the production of recombinant proteins in mammalian cells has been highlighted in different publications (Strotbek et al. 2013; Sanchez et al. 2013; Jadhav et al. 2012). A single miRNA can regulate the expression of multiple genes and, as a result, it offers a targeted way for regulation of gene networks. The use of miRNAs to
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Fig. 3 The unfolded protein response (UPR). ER stress is sensed by three ER-membrane bound sensors (PERK, ATF6 and IRE1). Under conditions of ER stress, mis-folded protein accumulates in the ER which causes the dissociation of Binding immunoglobulin protein (BiP) from the sensors. The dissociation of BiP leads to the initiation of a coordinated signalling
pathway, the UPR, to restore ER homeostasis. The UPR acts via different mechanisms to inhibit protein synthesis and increase the protein folding capacity of the cell to overcome ER stress. When the UPR is unable to restore ER homeostasis, UPR activation can lead to apoptosis
engineer recombinant cell lines presents an advantage over protein-coding mRNAs, as miRNAs do not represent an additional translational burden for the cell (Hackl et al. 2012). Therefore, miRNAs are an attractive tool to regulate the activity of signaling pathways important for protein secretion, like the UPR. The UPR can also regulate gene expression in a post-transcriptional manner through the endoribonuclease activity of IRE1. This process, called regulated IRE1-dependant decay (RIDD), degrades mRNAs encoding proteins that either localise to the ER or transit through the ER as part of the secretory pathway (Hollien et al. 2009; Hollien and Weissman 2006). RIDD can also regulate protein expression through the degradation of miRNAs (Fig. 3; Upton et al. 2012). Degradation of mRNA via RIDD is thought to limit the amount of protein entering the ER and decrease the ER unfolded protein load (Hollien et al. 2009; Ali et al. 2011).
ER-associated degradation (ERAD)
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Accumulation of mis-folded or improperly processed proteins in the ER also activates protein degradation through ER-associated degradation (ERAD) (Stolz and Wolf 2010). The mis-folded proteins are transported from the ER to the cytoplasm through translocons, after which they are ubiquitinated and then degraded via the proteasome (Fig. 4; Stolz and Wolf 2010). One of the mechanisms by which mis-folded proteins are targeted for degradation results from interactions of exposed hydrophobic faces which bind to chaperones such as BiP (Stolz and Wolf 2010). Under non-stressed conditions, ERAD acts to dispose any inappropriate (mis-folded) proteins and this prevents accumulation of undesirable proteins in the ER (and their potential release from the cell). To maintain basal ERAD activity under non-stressed conditions, ERAD is regulated by the removal of ERAD machinery from the ER and subsequent degradation via proteases in the
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Fig. 4 Physiologic consequences of unfolded protein response (UPR) activation. Different stimuli that perturb ER function can lead to the accumulation of mis-folded protein activating the UPR. The UPR inhibits general translation and regulates transcriptional responses to increase the protein folding capacity of the ER and restore homeostasis. Increased phospholipid synthesis and subsequent ER membrane expansion is also a
consequence of UPR activation. During protein folding, the formation of disulphide bonds produces reactive oxygen species (ROS) which activates the UPR. Other consequences of the UPR involve the up-regulation of degradation processes such as ERAD and autophagy, which act to decrease the amount of misfolded protein in the ER
lysosome (Cali et al. 2008; Bernasconi and Molinari 2011). Under ER stress, ERAD activity is increased by inhibition of degradation of ERAD machinery which, in turn, allows increased removal of unfolded or misfolded protein. UPR activation takes a more prolonged time period (e.g. several hours) than ERAD activation; therefore ERAD acts to remove aberrant proteins rapidly (Bernasconi and Molinari 2011). Regulation of ERAD in this way prevents substantial accumulation of protein in the ER. As a consequence of ER stress and the activation of the UPR, physiological changes occur within the cell in order to maintain homeostasis. These include the consequences of the UPR on the ER and the cell as a whole that then determine the efficiency of the cell to secrete heterologous proteins.
2013). Lipids can be used as a source of energy, in addition to involvement in processes such as membrane biogenesis, membrane trafficking, cell signalling and regulation of membrane protein activity (Shevchenko and Simons 2010). Membrane biogenesis involves the coordination of many metabolic and lipid synthesis pathways (Nohturfft and Zhang 2009). Phospholipids produced by the ER and Golgi are important components in all membranes in mammalian cells (Brewer and Jackowski 2012). The lipid composition of a membrane dictates its physical properties (Brewer and Jackowski 2012). Phosphatidylcholine (PC) is the most abundant phospholipid species in mammalian membranes. Newly synthesised lipids produced by the ER are inserted into the ER membrane and are rapidly transported to their target destinations within the cell (van Meer et al. 2008). The main method by which membrane lipids are transported to different organelles in the secretory pathway is via vesicular transport (van Meer et al. 2008).
Lipid synthesis and membrane expansion The ER and Golgi are the main intracellular sites for lipid synthesis and secretion (Lagace and Ridgway
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Membrane biogenesis is essential for cell growth and can be increased in response to many cellular processes such as increased protein synthesis and secretion (Nohturfft and Zhang 2009). Certain cell types with high secretory capacities respond to environmental stimulation by changing the size and organisation of organelles. This is exemplified by B lymphocytes that differentiate into antibody-secreting plasma cells where increased protein synthesis and secretion coincides with increased lipid synthesis and ER expansion (Fagone et al. 2007). Other studies have shown that the smooth ER in liver cells increased in amount in the presence of toxins. As a result there was an increase in the amount of detoxifying enzymes associated with the smooth ER to decrease ROS (Iseri et al. 1966; Lieber 2004). The UPR can influence lipid metabolism under conditions of ER stress (Fig. 4). The spliced form of XBP1 (XBP1S), a mediator within the UPR signalling pathway, increases lipid metabolism and ER membrane biogenesis. XBP1S is essential for the development of ‘‘professional’’ secretory cells (e.g. B lymphocytes, macrophages and pancreatic islets) (Lee et al. 2005, 2008). In antibody-secreting B lymphocytes, the amount of XBP1 transcript increased in response to UPR activation and the transcript underwent splicing to produce XBP1S mRNA (and, subsequently, protein). As well as activating expression of UPR target genes in the secretory pathway, XBP1S induced expression of enzymes involved in lipid synthesis. For example, XBP1S induced expression of PC via the CDP-choline pathway which is required for ER development (Sriburi et al. 2004, 2007; Shaffer et al. 2004). However, more research is needed to uncover the precise mechanisms by which XBP1S regulates lipid synthesis. In XBP1 deficient situations, the amount of membrane lipids is significantly decreased (Sriburi et al. 2004). ATF6a, another UPR mediator, also influences ER membrane biogenesis as overexpression of ATF6a increased phospholipid and fatty acid synthesis, a contributory factor in ER expansion (Bommiasamy et al. 2009). Early studies in CHO cells treated with sodium butyrate showed increased production of recombinant protein as well as an expanded ER (Dorner et al. 1989). Engineering CHO cells to express XBP1S constitutively resulted in an expanded ER and Golgi with increased recombinant protein production capacity (Tigges and Fussenegger 2006). In another
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publication, the expression of three monoclonal antibodies (mABs) in CHO DUXB11 and CHOK1 cell lines was compared (Hu et al. 2013). CHOK1 clones produced up to 10 times more mAB than CHO DUXB11 clones. Interestingly, the ER and mitochondrial content was higher in CHOK1 than CHO DUXB1 (Hu et al. 2013) suggesting that ER expansion and higher mitochondrial content can improve the cell’s capacity to manage a high recombinant protein load. In addition, increased lipid content associated with active secretory machinery was found in human AGE1.HN cells producing recombinant alpha (1)-antitrypsin compared to the parental cell line (Niklas et al. 2013). In yeast, ER stress/UPR induces membrane expansion through mediation of the Ino2/4 transcription factor complex. Direct activation of the Ino2/4 complex resulted in expansion of the ER membrane. ER membrane expansion decreased ER stress independent to the amount of ER chaperones, showing ER size as an important independent factor in reversing ER stress (Schuck et al. 2009). Increasing the size of ER is proposed to lower the concentration of protein folding intermediates, allowing more time for the protein to fold whilst also avoiding aggregate formation (Apetri and Horwich 2008; Schuck et al. 2009). Overall, expansion of the ER can act as a key adaptive response to decrease the amount of misfolded protein by increasing the amount of space for protein processing (Schuck et al. 2009). Further research is required to understand how membrane expansion is controlled in mammalian cells and define whether membrane content changes occur across the whole ER structure or if changes are restricted to specific ER domains. New DNA editing technologies can offer the possibility of monitoring changes in different domains of the ER. For example, creating fluorescently labelled fusion proteins to observe changes in the ER (Schuck et al. 2009). In addition, processes involved in membrane lipid production and control of ER size offers potential targets for engineering increased recombinant protein production and quality. Autophagy and the unfolded protein response (UPR) Autophagy has been recognised as a process in ‘‘bulk material’’ degradation (Levine 2005). Autophagy
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degrades damaged organelles and parts of the cell to maintain homeostasis and provides an alternative degradation process to ERAD. Autophagy is activated as a result of many of the cellular processes, such as ER stress, that activate the UPR. Linkages and interactions between autophagy and the UPR suggest these mechanisms offer an integrated response to cellular stress imposition (Fig. 4). It is thought that under conditions of ER stress, where there is an accumulation of mis-folded protein, autophagy is induced in order to overcome the stress and restore ER homeostasis (Appenzeller-Herzog and Hall 2012). The process of autophagy involves the formation of double-membrane cytosolic vesicles (autophagosomes) around the targets for degradation. The autophagosomes fuse with endosomes or lysosomes where proteases degrade the contents. The source of the membranes for the formation of autophagosomes is thought to originate from the ER (Fig. 2), although it has been shown membranes from other parts of the cell such as the Golgi, plasma membrane and mitochondria can also contribute to autophagosome formation (Axe et al. 2008; HayashiNishino et al. 2009; Ravikumar et al. 2010; Hailey et al. 2010; Ohashi and Munro 2010). The degradation products derived from lysosomal degradation may be re-used for other biosynthetic processes (Kim et al. 2013). Recently, selective types of autophagy have been defined for specific organelles (Cebollero et al. 2012); for example, ER (reticulophagy), ribosomes (ribophagy) and mitochondria (mitophagy) (Bernales et al. 2006; Kraft et al. 2008; Wang and Klionsky 2011). The precise mechanism of reticulophagy is unclear. It is has been suggested that under ER stress specific areas of the ER that contain mis-folded proteins and insoluble aggregates are removed to form autophagosomes. In addition, under certain conditions, autophagosomes do not fuse with the lysosome but the removal of aberrant protein into these autophagosome structures is sufficient to reduce ER stress (Bernales et al. 2006). Reticulophagy counterbalances the effect of ER membrane expansion by decreasing the ER size as well as removing parts of the ER carrying unfolded proteins (Bernales et al. 2006). Reticulophagy, along with ERAD, offers parallel UPR-regulated processes (Cebollero et al. 2012). Formation of the autophagosome involves the conversion of microtubule-associated protein 1 light chain
3 (LC3-I) into LC3-II. Subsequently, LC3-II is displayed on autophagosome membranes. This conversion relies on autophagy-related proteins (Atg) proteins that are up-regulated in response to PERKmediated phosphorylation of the translation initiation factor, eIF2a. Other UPR mediators, such as IRE1 and ATF6, also affect autophagy (Cebollero et al. 2012; Appenzeller-Herzog and Hall 2012). In addition, the formation and regulation of autophagosomes also involves Atg proteins and the mammalian target of rapamycin (mTOR). mTOR is a serine-threonine protein kinase that acts as a central regulator of cell growth in response to many intracellular (e.g. amino acids, ATP) and extracellular stimuli (e.g. growth factors, hormones) (Appenzeller-Herzog and Hall 2012). mTOR exists in two complexes, mTORC1 and mTORC2, and each complex is associated with different roles. mTORC1 negatively regulates autophagy. A possible mechanism for autophagy regulation via mTORC1 is under ER stress conditions; PERKmediated phosphorylation of eIF2a results in expression of UPR target genes, such as ATF4 and CHOP, and the tribbles homologue 3 (TRB3), a downstream target of this pathway, inactivates mTORC1. mTORC1 inactivation releases autophagy mediators from inhibition and subsequently activates autophagy (Salazar et al. 2009). ATF6 has been implicated in mTORC1 activation in the early phases of ER stress/ UPR activation. Under chronic ER stress/UPR activation, this stimulation of mTORC1 is reversed by the PERK-TRB3 pathway (Appenzeller-Herzog and Hall 2012). There is also evidence of cross-talk between autophagy and ERAD activity. Autophagy acts to inhibit disposal of ERAD machinery thereby increasing ERAD activity. LC3-I is used for the vesicular transport of ERAD machinery from the ER for subsequent degradation. The conversion of LC3-I to LC3-II (present on autophagosome membranes) inhibits transport of ERAD components (Bernasconi and Molinari 2011). Autophagy is induced in antibody-producing recombinant CHO cells towards the end of batch culture when nutrients are depleted causing cell death (Hwang and Lee 2008b; Han et al. 2010). In terms of recombinant protein expression, cell death in cell culture has two consequences. Firstly through limiting the viable cell density and secondly through potential changes to product quality due to the release of
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substances into the medium (Hwang and Lee 2008a). Limited studies have focused on autophagy and engineering of this pathway in recombinant cell lines (Hwang and Lee 2008b). Kim et al. (2013) have reviewed the approaches taken to control autophagy through the addition of chemical compounds either activating or inhibiting targets in the autophagy pathway in CHO cell cultures. However, further work is required as the effects of chemical compounds are pleiotropic. Oxidative stress and the unfolded protein response (UPR) Between the ER and cytosol there is a strong redox gradient, the ER being more oxidising than the cytosol (Margittai and Sitia 2011). The formation of disulphide bonds in mammalian cells requires the action of multiple enzymes such as ER oxidoreductin 1 (Ero1) and members of the protein disulphide isomerase (PDI) family (Feige and Hendershot 2011; Sato et al. 2013). Oxidised Ero1 transfers the disulphide bond to PDI which in turn oxidises the protein substrate. During the disulphide bond reaction, O2 is used as an electron acceptor (Feige and Hendershot 2011). Within the ER both oxidation and reduction reactions occur. If unbalanced or mis-regulated these complex reaction chemistries can impair oxidative folding and subsequently cause oxidative stress (Margittai and Sitia 2011). Protein folding is sensitive to oxidative stress as well as ER stress. Increased protein synthesis causes an increase in ROS (Malhotra et al. 2008) which, in turn, can act as signals for UPR activation (Fig. 4). ROS can arise during formation of disulphide bonds, when oxygen is reduced to form hydrogen peroxide via electron transfer. In some cases ROS can amplify protein mis-folding via oxidation of amino acids or by alteration of ER function (Malhotra et al. 2008), events which all act to increase the UPR. In cases where oxidative stress is severe, cell death is induced via apoptosis. Severe protein mis-folding can also cause leakage of calcium from the ER into the mitochondria where high amounts of calcium can disrupt the electron transport chain, induce autophagy and compromise cell function further (Deniaud et al. 2008; Malhotra et al. 2008). One pathway of calcium release is the CHOP-mediated induction of Ero1 which causes
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release of calcium through inositol 1,4,5-triphosphate receptors (Li et al. 2009). Specialised cells with high secretory capacities (professional secretory cells) exhibit high expression of proteins that can prevent oxidative stress (Bertolotti et al. 2010). For example, the differentiation of B lymphocytes into antibody-secreting plasma cells not only results in ER membrane expansion but increased oxidative stress. This subsequent oxidative stress triggers the activation of signalling pathways to counteract oxidative stress. This signalling results in significantly increased expression of PDI oxidation enzyme, peroxiredoxin 4, as well as the activation of the transcription factors Nrf1 and Nrf2 which, in turn, induce the expression of genes required for detoxification and ROS removal (Sato et al. 2013; Digaleh et al. 2013). B Lymphocyte differentiation shows significant reshaping of antioxidant responses to cope with oxidative stress (Bertolotti et al. 2010). Furthermore, other studies involving expression of tissue plasminogen activator (tPA) in CHO cells shows antioxidant treatment increases tPA production and supresses apoptosis (Yun et al. 2001). Also, expression of coagulation factor FVIII shows anti-oxidant treatment decreases oxidative stress and UPR signalling to increase secretion of FVIII protein (Malhotra et al. 2008). Applications of the unfolded protein response (UPR) for selection and engineering of better hosts Proteome and transcriptome profiling technologies have been used to identify the molecular characteristics of cells with different productivities or using conditions that increase the production of recombinant proteins (Seth et al. 2007; Doolan et al. 2008; Carlage et al. 2009; Charaniya et al. 2009; Yee et al. 2009; Schaub et al. 2010). These types of studies are affected by clonal variability making it hard to distinguish between different molecular characteristics that are associated with high productivity from those inherent to the heterogeneous cell population (Oh et al. 2003; Davies et al. 2013). The main conclusion from these studies is that there are a variety of cellular functions differentially regulated in the best producer clones that can lead to high productivity. In a heterogeneous cell population some clones are more suited than others to handle the burden of recombinant protein production.
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The UPR can be a possible route to select and engineer better host cells for recombinant protein production. Kober et al. (2012) developed a method for selecting highly productive, transfected clones based on the detection of ER stress markers. This method used an ER-stress sensor based on the correlation between BiP expression and recombinant protein production. A similar system was developed by Du et al. (2013) using a UPR-inducible promoter controlling the expression of the green fluorescent protein (GFP). This study showed that both timing and extent of UPR are different between clones re-enforcing the intrinsic variability of the clones. Interestingly, the UPR is also active in the host cell line without recombinant protein expression during batch culture, suggesting that under these conditions; there are other sources of stress that activate the UPR. Several groups have reported that the increase in the recombinant protein production does not proportionally increase with transgene copy number or the amount of mRNA when expression is high (Mohan et al. 2008; Barnes et al. 2003, 2007; Ku et al. 2009). These observations suggest that after a certain threshold of the amount of mRNA, the rate-limiting steps in recombinant protein secretion may be translational or post-translational processes (Lim et al. 2010). To overcome this productivity limitation, a number of ER-related proteins have been engineered into cells by overexpression, specifically a number of chaperones including BiP (Borth et al. 2005), calnexin, calreticulin (Chung et al. 2004), a number of PDIs (Mohan et al. 2008), and components of the UPR signaling pathway such as CHOP (Nishimiya et al. 2013), ATF4 (Haredy et al. 2013; Ohya et al. 2008), GADD34 (Omasa et al. 2008) and XBP-1. The effect of these proteins depends on the system employed and the recombinant protein produced. Also, the overexpression of just one chaperone is probably not enough to improve the secretory machinery; for this reason, XBP1, the master regulator of the secretory machinery is an interesting target for cell engineering. The effects of XBP1 overexpression have led to conflicting reports, Becker et al. (2009) reported and improvement in the production of a monoclonal antibody in CHO cells overexpressing XBP1S. Campos-Da-Paz et al. (2008) and Ohya et al. (2008) overexpressed XBP1S in CHO cells and found no increase in the production of both Factor VIII and anti-thrombin-III. Ku et al. (2008) found that the
effect of XBP1S overexpression in CHO cells and NS0 cells depends on the existence of a secretory bottleneck. The co-expression of XBP1 and Ero1 increased the recombinant protein expression by 5 to 6 fold in a transient gene expression system, without affecting the quality of the product (Cain et al. 2013).
Summary and outlook To improve protein production in mammalian cells there has been extensive engineering of different aspects of protein expression, such as gene expression, protein modification, secretion, cell metabolism and apoptosis. Engineering of physiologic cellular changes associated with ER stress and UPR activation may further enhance expression of recombinant proteins. This review has discussed the physiological consequences of the ER stress and UPR activation. Different stimuli can perturb the ER function producing different outputs depending on the time and intensity of the stress. Under conditions of ER stress the UPR, a complex signaling pathway, is activated to recover homeostasis. The UPR causes an increase in the protein folding capacity of the ER by up-regulating the expression of molecular chaperones and ER size (increased phospholipid synthesis). Increased protein synthesis can also lead to oxidative stress. Oxidative stress also activates the UPR which can lead to cell apoptosis. Also the UPR acts to decrease protein load by inhibiting general translation and up-regulating the transcription of genes encoding secretory proteins. Degradation processes like ERAD and autophagy are also active during ER stress conditions to remove mis-folded proteins and recycle cellular components. If these actions fail to restore ER homeostasis the UPR can induce cell death via apoptosis. Different proteins from the UPR signaling pathway have been used for cell engineering, yet our understanding of how metabolism, protein secretion, aggregation, glycosylation, apoptosis and their physiological consequences are related, is still limited. How the different UPR arms are regulated over time and how the different culture conditions affects its activation is still unknown. More research is needed to comprehend the role of the UPR for recombinant protein production especially focusing on the biology of the cell, in order to establish desirable UPR phenotypes.
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