The unfolded protein response: controlling cell fate decisions under ER stress and beyond

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1 The unfolded protein response: controlling cell fate decisions under ER stress and beyond Claudio Hetz 1 3 Abstract Protein-folding stress at the endoplasmic reticulum (ER) is a salient feature of specialized secretory cells and is also involved in the pathogenesis of many human diseases. ER stress is buffered by the activation of the unfolded protein response (UPR), a homeostatic signalling network that orchestrates the recovery of ER function, and failure to adapt to ER stress results in apoptosis. Progress in the field has provided insight into the regulatory mechanisms and signalling crosstalk of the three branches of the UPR, which are initiated by the stress sensors protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF6). In addition, novel physiological outcomes of the UPR that are not directly related to protein-folding stress, such as innate immunity, metabolism and cell differentiation, have been revealed. 1 Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile. 2 Institute of Biomedical Sciences, Center for Molecular Studies of the Cell, University of Chile, Santiago, P.O. BOX 70086, Chile. 3 Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave, Boston, Massachusetts 02115, USA. s: chetz@hsph.harvard.edu; chetz@med.uchile.cl doi: /nrm3270 Published online 18 January 2012 The endoplasmic reticulum (ER) is arranged in a dynamic tubular network involved in metabolic processes, such as gluconeogenesis and lipid synthesis. It is also the major intracellular calcium reservoir in the cell, and it contributes to the biogenesis of autophagosomes and peroxisomes. Initial protein maturation steps that take place at the ER are crucial for the proper folding of proteins that are synthesized in the secretory pathway, which amount to approximately 30% of the total proteome in most eukaryotic cells. The protein-folding machinery in the ER is particularl y challenged in specialized secretory cells owing to their high demand for protein synthesis, which constitutes a constant source of stress. The efficiency and fidelity of protein folding is constantly adjusted through the dynamic integration of multiple environmental and cellular signals. Several feedback mechanisms ensure efficient adaptation to fluctuations in protein-folding requirements by functionally affecting almost every aspect of the secretory pathway 1. The first evidence for the existence of a homeostatic pathway that overcomes perturbations in protein folding at the ER came from a pioneering study in mammalian cells, in which the pharmacological inhibition of folding led to the transcriptional upregulation of several key ER chaperones 2. This finding revealed the existence of a signal transduction feedback loop that reprogrammes gene expression under conditions of ER stress. We now know that, upon ER stress, cells activate a series of complementary adaptive mechanisms to cope with protein-folding alterations, which together are known as the unfolded protein response (UPR). The UPR transduces information about the protein-folding status in the ER lumen to the nucleus and cytosol to buffer fluctuations in unfolded protein load 3,4. When cells undergo irreversible ER stress 5, this pathway eliminates damaged cells by apoptosis, indicating the existence of mechanisms that integrate information about the duration and intensit y of stress stimuli. Although the UPR is classically linked to proteinfolding stress under both physiological and pathological conditions, it is becoming clear that it has further important functions. For example, components of the UPR regulate various processes, ranging from lipid and cholesterol metabolism and energy homeostasis, to inflammation and cell differentiation 6. At the molecular level, these alternative UPR outputs are attributed, in part, to the complex crosstalk between different stress and metabolic pathways. In this scenario, a dynamic signalling framework is integrated by the UPR to maintain organelle homeostasis in an environment of fluctuating and diversified inputs. This Review gives a comprehensive overview of UPR signalling and considers recent advances that reveal how it is tuned to orchestrate interconnected physiological events, thus operating as an unanticipated NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 13 FEBRUARY

2 a Phosphorylation b c ER lumen ER stress ER stress ER stress Cytosol IRE1α Alarm stress pathways NF-κB JNK TRAF2 XBP1u mrna mrna Ribosome mrna degradation (RIDD) Intron PERK NF-κB? NRF2 α β γ eif2α α β γ ATF6 S2P S1P COPII Golgi XBP1s mrna Translation XBP1s ATF4 ATF6f XBP1s UPR target genes ATF4 UPR target genes ATF6f UPR target genes Autophagy Apoptosis Co-translational degradation ERAD Folding Lipid synthesis Quality control Pre-emptive quality control Protein secretion Figure 1 The UPR. The unfolded protein response (UPR) stress sensors, inositol-requiring protein 1α (IRE1α), protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) and activating transcription factor 6 (ATF6), transduce information about the folding status of the ER to the cytosol and nucleus to restore protein-folding capacity. a IRE1α dimerization, followed by autotransphosphorylation, triggers its RNase activity, which processes the mrna encoding unspliced X box-binding protein 1 (XBP1u) to produce an active transcription factor, spliced XBP1 (XBP1s). XBP1s controls the transcription of genes encoding proteins involved in protein folding, ER-associated degradation (ERAD), protein quality control and phospholipid synthesis. IRE1α also degrades certain mrnas through regulated IRE1 dependent decay (RIDD) and induces alarm stress pathways, including those driven by JUN N terminal kinase (JNK) and nuclear factor-κb (NF κb), through binding to adaptor proteins. b Upon activation, PERK phosphorylates the initiation factor eukaryotic translation initiator factor 2α (eif2α) to attenuate general protein synthesis, and it may also phosphorylate nuclear factor erythroid 2 related factor 2 (NRF2), a transcription factor involved in redox metabolism. Phosphorylation of eif2α allows the translation of ATF4 mrna, which encodes a transcription factor controlling the transcription of genes involved in autophagy, apoptosis, amino acid metabolism and antioxidant responses. c ATF6 has a basic Leu zipper (bzip) transcription factor in its cytosolic domain and is localized at the ER in unstressed cells. In cells undergoing ER stress, ATF6 is transported to the Golgi apparatus through interaction with the coat protein II (COPII) complex, where it is processed by site 1 protease (S1P) and S2P, releasing its cytosolic domain fragment (ATF6f). ATF6f controls the upregulation of genes encoding ERAD components and also XBP1. At the bottom of the figure, general UPR outcomes, which may or may not require transcription, are presented. TRAF2, TNFR-associated factor 2. stress rheostat to control cell fate. Special emphasis is given to unexpected regulatory checkpoints that specifically control the signalling of individual stress sensors. Finally, novel physiological outputs of the UPR that are not directly related to protein misfolding are presented, highlighting in particular the role of the pathway in innate immunity, energy and lipid metabolism, and cell differentiation. The UPR in cell survival and cell death The mammalian UPR has evolved into a dynamic and flexible network of signalling events that responds to various inputs over a wide range of basal metabolic states. Under ER stress conditions, activation of the UPR reduces unfolded protein load through several prosurviva l mechanisms, including the expansion of the ER membrane, the selective synthesis of key components of the protein folding and quality control machinery and the attenuation of the influx of proteins into the ER. When ER stress is not mitigated and homeo stasis is not restored, the UPR triggers apoptosis. This section provides an overview of our current knowledge of the signalling mechanisms and proteins that underlie these two contrasting phases of UPR signalling. Adaptive UPR mechanisms. ER stress signalling was initially characterized in Saccharomyces cerevisiae, in which a linear pathway is governed solely by one stress sensor, inositol-requiring protein 1 (Ire1), and a downstream transcription factor, Hac1 (which is homologous to ATF CREB1 in mammals) 1. In this organism, engagement of the UPR has a clear outcome: expression of a large group of genes reinforces existing mechanisms to cope with protein-folding stress. In vertebrates, the UPR has evolved into a complex network of signalling events that target multiple cellular responses (FIG. 1), and 90 FEBRUARY 2012 VOLUME 13

3 ER stress Adaptive responses ATF6 IRE1α ATF6f XBP1s JNK Folding, ERAD, quality control, ER biogenesis, autophagy Apoptosis phase? Caspase 2 BID BAX, BAK Apoptosis RIDD RIDD survival genes? PERK eif2α ATF4 Translation? p53 BH3-only Folding, redox, autophagy CHOP BCL-2 GADD34? IP3R ROS translation Ca 2+ PTP Time of exposure to stress Intensity of stress Figure 2 Cell fate decisions under ER stress. Distinct unfolded protein response (UPR)-related responses are observed over time in cells undergoing endoplasmic reticulum (ER) stress. Early UPR responses attenuate protein synthesis at the ER by inhibiting translation (which is dependent on the protein kinase RNA-like ER kinase (PERK)-mediated phosphorylation of eukaryotic translation initiator factor 2α (eif2α)), activating mrna decay by regulated inositol-requiring protein 1 (IRE1)- dependent decay (RIDD), and activating autophagy through the IRE1α JUN N terminal kinase (JNK) pathway. In a second wave of events, the UPR transcription factors activating transcription factor 6 cytosolic fragment (ATF6f), spliced X box-binding protein 1 (XBP1s) and ATF4 promote many adaptive responses that work to restore ER function and maintain cell survival. Unmitigated ER stress induces apoptosis to eliminate irreversibly damaged cells. The B cell lymphoma 2 (BCL 2) protein family is crucial for the control of ER stress-induced apoptosis. When activated at the transcriptional or post-translational level, BCL 2 homology 3 (BH3)-only proteins regulate the activation of BAX and/or BH antagonist or killer (BAK) to trigger apoptosis. Sustained PERK signalling upregulates the pro-apoptotic transcription factor C/EBP-homologous protein (CHOP), which downregulates the anti-apoptotic protein BCL 2, induces the expression of some BH3 only proteins and upregulates growth arrest and DNA damage-inducible 34 (GADD34). The induction of GADD34 may induce the generation of reactive oxygen species (ROS) by enhancing protein synthesis through eif2α dephosphorylation, overloading cells with unfolded proteins. Altered calcium homeostasis owing to inositol 1,4,5 trisphosphate receptor (IP3R) activation, in addition to ROS, may also contribute to the opening of the mitochondrial permeability transition pore (PTP), which promotes apoptosis. CHOP, ATF4, and p53 also control the expression of a subset of BH3 only proteins. Active IRE1α may sensitize cells to apoptosis through activation of JNK and RIDD of mrna that encodes for chaperones such as. Casapse 2 may also participate in ER stress-mediated apoptosis by cleaving the BH3 only protein BH3 interacting domain death agonist (BID), which activates BAK and BAX. Dashed arrows exemplify transition steps from adaptive responses to apoptosis. Dotted arrows indicate events mediating apoptosis. Question marks indicate where the mechanism responsible for the depicted step is unclear. RIDD (Regulated IRE1 dependent decay). The degradation of a subset of mrnas encoding for proteins located in the endoplasmic reticulum, possibly through the activation of the RNase domain of inositol-requiring 1 (IRE1). ERAD (Endoplasmic reticulumassociated degradation). A pathway along which misfolded proteins are transported from the ER to the cytosol for proteasomal degradation. it is mediated by the activation of at least three major stress sensors: IRE1 (both α and β isoforms), activating transcription factor 6 (ATF6) (both α and β isoforms) and protein kinase RNA-like ER kinase (PERK) 1. Two temporally distinct waves of cellular responses are observed in vertebrate cells undergoing ER stress (FIG. 2). As an immediate reaction, the activation of PERK inhibits general protein translation through the phosphorylation of eukaryotic translation initiator factor 2α (eif2α) 7 (FIG. 1b). In addition, the selective degradation of mrna encoding for certain ER-located proteins is initiated through regulated IRE1 dependent decay (RIDD) Macroautophagy, a bulk degradation pathway, is also activated by ER stress, possibly to elimin ate damaged ER (a process termed ER phagy) and abnormal protein aggregates through the lysosomal pathway 11. Finally, pre emptive quality control 12 and cotranslational degradation 13 inhibit the translocation of a subset of proteins into the ER upon translation. Overall, these mechanisms reduce the influx of proteins into the ER to allow adaptive and repair mechanisms that re establish homeostasis. A second wave of events triggers a massive geneexpression response through the regulation of at least three distinct UPR transcription factors. Each stress senso r uses a unique mechanism to promote the activatio n of a specific transcription factor and the upregulation of a subset of UPR target genes 1. IRE1α is a kinase and endoribonuclease that, under ER stress conditions, dimerizes and autotransphosphorylates. This leads to the activation of the cytosolic RNase domain, possibly owing to a conformational change 14 (FIG. 1a). Active IRE1α processes the mrna encoding the transcription factor X box-binding protein 1 (XBP1), excising a 26 nucleotide-long intron that shifts the coding readin g frame of this mrna This results in the expression of an active and stable transcription factor, termed spliced XBP1 (XBP1s), which translocates to the nucleus to induce the upregulation of its target genes, the protein products of which operate in ER associated degradation (ERAD), the entry of proteins into the ER and protein folding, among other functions 18,19 (FIG. 1a). XBP1s also modulates phospholipid synthesis, which is required for ER membrane expansion under ER stress 4. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 13 FEBRUARY

4 Autophagy A survival pathway that is classically linked to the adaptation to nutrient starvation through the recycling of cytosolic components by lysosome-mediated degradation. In cells undergoing endoplasmic reticulum stress, autophagy may serve as a mechanism to eliminate damaged organelles and aggregated proteins. ATF6 represents a group of ER stress transducers that encode basic Leu zipper (bzip) transcription factors, including ATF6α, ATF6β, LUMAN (also known as CREB3), old astrocyte specifically-induced substance (OASIS; also known as CREB3L1), BBF2 human homologue on chromosome 7 (BBF2H7; also known as CREB3L2), cyclic AMP-responsive elemen t- binding protein hepatocyte (CREBH; also known as CREB3L3) and CREB4 (also known as CREB3L4) 20. Under ER stress conditions, ATF6 translocates to the Golgi, where it is processed by site 1 proteases in its ER luminal domain and by site 2 proteases within its region that spans the Golgi phospholipid bilayer, releasing a cytosolic fragment (ATF6f) that directly controls genes encoding ERAD components and XBP1 (REFS 16,21,22) (FIG. 1c). Finally, phosphorylation of eif2α by PERK leads to the selective translation of the mrna encoding the transcription factor ATF4, which controls the levels of pro-survival genes that are related to redox balance, amino acid metabolism, protein folding and autophagy 3,23 (FIG. 1b). This branch of the UPR also regulates the expression of several micrornas, which may contribute to the attenuation of protein translation or protein synthesis 24. Together, ATF4, XBP1s and ATF6f govern the expression of a large range of partially overlapping target genes, the protein products of which modulate adaptation to stress or the induction of cell death under conditions of chronic ER stress (see below). The target genes of each UPR transcription factor are dependent, in part, on the nature of the stimulus and the cell type affected, possibly through their interaction with other transcription factors (see below). Chronic ER stress and apoptosis. Physiological processes that demand a high rate of protein synthesis and secretion must sustain activation of the UPR s adaptive programmes without triggering cell death pathways. However, above a certain threshold, unresolved ER stress results in apoptosis (FIG. 2). The mechanisms initiating apoptosis under conditions of irreversible ER damage are now partially understood and may involve a series of complementary pathways 25. Cell death under ER stress depends on the core mitochondrial apoptosis pathway, which is regulated by the B cell lymphoma 2 (BCL 2) protein family 26. In this pathway, the conformational activation of the proapoptotic multidomain proteins BAX and/or BH antagonist or killer (BAK) is a key step in triggering caspase activation. Chronic ER stress leads to BAX- and/or BAK-dependent apoptosis through the transcriptional upregulation of BCL 2 homology 3 (BH3)-only proteins, such as BCL 2 interacting mediator of cell death (BIM) and p53 upregulated modulator of apoptosis (PUMA; also known as BBC3), which are upstream BCL 2 famil y members, as well as the cell death sensitizer NOXA (reviewed in REF. 5). The transcription of one of the key UPR pro-apoptotic players, termed C/EBP-homologous protein (CHOP; also known as GADD153), is positively controlled by the PERK ATF4 axis 25. CHOP promotes both the transcription of BIM and the downregulation of BCL 2 expression, contributing to the induction of apoptosis 5,25. In addition to CHOP, ATF4 and p53 are also involved in the direct transcriptional upregulation of BH3 only proteins under ER stress 5. However, the mechanism linking ER stress with p53 activation is unclear. Many other complementary mechanisms are proposed to induce cell death under excessive ER stress, includin g activation of the BH3 only protein BH3 interacting domain death agonist (BID) by caspase 2, as well as ER calcium release, which may sensitize mitochondria to activate apoptosis 4,25. Under certain conditions, IRE1α activation is also linked to apoptosis, possibly through its ability to activate mitogen-activated protein kinases (MAPKs; see below) and the subsequent downstream engagement of the BCL 2 family members, as well as the degradation of mrnas encoding for key folding mediators through RIDD 8. As ER stress can result in distinct and contrasting outputs (FIG. 2), it is essential to understand how UPR sensors shift their signalling output to determine divergent cell fate decisions. Control of alarm stress pathways by the UPR. UPR signalling merges with multiple components of other well-described stress responses through a series of bi directional crosstalk points 27. Engagement of alarm stress pathways by UPR sensors could modulate ER stress adaptation, apoptosis or physiological outputs that are not directly related to protein-folding stress. For example, activation of IRE1α can engage alarm genes by recruiting the adaptor protein TNFR-associated factor 2 (TRAF2), which results in the activation of the apoptosis signalregulating kinase 1 (ASK1; also known as MAP3K5) pathway and its downstream target JUN N terminal kinase (JNK) 28. JNK activation is an important pro-apoptotic signal in response to IRE1α activation, although its mechanism of action in paradigms of ER stress is not well understood. IRE1α JNK signalling can also trigger macro autophagy that is induced by ER stress and nutrient starvation by activating beclin 1 (REFS 29,30), an essential autophagy regulator 11. In addition, IRE1α engages alarm pathways involving p38, extracellular signalregulated kinase (ERK) and nuclear factor κb (NF κb) through the binding of distinct adaptor proteins 27. In a pathway that is less well understood, PERK signalling also activates the transcription factors nuclear factor erythroid 2 related factor 2 (NRF2) and NF κb, which may have consequences in regulating redox metabolism and inflammatory processes, respectively 3. Under certain experimental conditions, ATF6 may also control NF κb through AKT 31 ; however, the connection between ATF6 and alarm stress pathways remains largely unexplored. Dynamic regulation of the UPR Recent studies suggest that UPR sensors have fundamental differences in the timing of their signalling and responses to particular ER stress stimuli. Emerging evidence indicates that the amplitude and kinetics of UPR signalling are tightly regulated at different levels, which has a direct impact on cell fate decisions. Current models of the mechanisms that might underlie the initiation, attenuation and fine-tuning of UPR-dependent responses are discussed below. 92 FEBRUARY 2012 VOLUME 13

5 a Mammalian UPR Misfolded protein ER lumen? Low stress High stress Signalling attenuation Cytosol Cytosol IRE1α b Yeast UPR ER lumen Bip Ire1 Phosphorylation Bip XBP1 mrna splicing Cluster formation mrna decay Unfolded or misfolded proteins Attenuation Buffering Figure 3 The stress-sensing mechanism and kinetics Nature Reviews of IRE1 signalling. Molecular Cell a In Biology mammalian cells, inositol-requiring protein 1α (IRE1α) is maintained in a repressed state under non-stress conditions through an association with. Upon endoplasmic reticulum (ER) stress, dissociates and binds misfolded proteins. This leads to partial IRE1α phosphorylation and dimerization, which allows further IRE1α phosphorylation events and activation of the IRE1α RNase domain to catalyse X box-binding protein 1 (XBP1) mrna splicing. Under conditions of high stress, active IRE1α molecules form large clusters, which may be optimal for regulated IRE1 dependent decay (RIDD) of mrna and high levels of XBP1 mrna splicing activity. After prolonged ER stress, IRE1α clusters dissociate and the activity of this stress sensor is attenuated. It remains to be determined if binds to IRE1α upon inactivation, as indicated by the question mark. b In yeast, the dissociation of Bip from Ire1 may have an indirect role in the activation of Ire1. Oligomerization of Ire1 is essential for its autotransphosphorylation. A direct recognition model has been proposed, in which unfolded and/or misfolded proteins directly bind to the luminal domains of Ire1 through a motif that has a similar structure to the groove in major histocompatibility complex class I (MHC I). The binding of unfolded and/or misfolded proteins to Ire1 may facilitate the assembly of highly ordered Ire1 clusters between many (n) Ire1 dimers (illustrated with parentheses). The attenuation of Ire1 activity involves further phosphorylation events. Inactive Ire1 is buffered through its association with Bip. This maintains a pool of inactive Ire1 to set the threshold for its activation. UPR, unfolded protein response. n HAC1 mrna splicing Time Bip Bip Activation of UPR stress sensors. How protein-folding stress at the ER is sensed has been a central topic in the field for the past 10 years. Because of its conservation in yeast, the IRE1 signalling branch is the best studie d in terms of its molecular regulation. Dimerization of Ire1 in yeast and homodimerization of IRE1α and IRE1β in mammalian cells is central to the initiation of this branch of UPR signalling 32. Further oligomerization of IRE1 into large clusters correlates with the kinetics of its autophosphorylation and the subsequent initiation of its ability to splice XBP1 mrna in mammals or HAC1 mrna in yeast 33,34. PERK signalling is also initiated by the dimerization, oligomerization and autophosphoryl ation of PERK 14. Different models have been proposed to explain how ER stress is sensed, and these are constantly modified over time owing to new findings and to discrepancies and similarities between the yeast and mammalian UPR 32. A pioneering study proposed that the binding of the ER chaperone (also known as GRP78 and HSPA5) to IRE1α and PERK in mammalian cells represses their spontaneous self-dimerization and activation 35. Accordingly, under ER stress conditions, preferentially binds to misfolded proteins, which releases its inhibitory interaction with stress sensors (FIG. 3a). A similar model was described in parallel in yeast (reviewed in REF. 32) (FIG. 3b). In the case of ATF6, binding to this sensor is proposed to mask its Golgi-localization signal 36. release allows ATF6 to interact with coat protein II (COPII), a complex of proteins that recognize cargoes to generate vesicles that are transported to the Golgi 37. Calreticulin, an essential component of the ER quality control system, may be also involved in the retention of ATF6 at the ER. Under ER stress conditions, under-glycosylated ATF6 may not be able to interact with calreticulin, which allows its transport to the Golgi 38. ATF6 is expressed as a monomer and as oligomers, possibly owing to the presence of intra- and inter-disulphide bridges at its ER luminal domain 39. Under ER stress conditions, reduced ATF6 monomers may only reach the Golgi for further processing and activation. The crystal structure of the ER luminal domain of Ire1 revealed the presence of a groove-like structure that is similar to the peptide-loading domain in major histocompatibility complex class I (MHC I) and which may be involved in the recognition of misfolded proteins and act in part as a stress-sensing domain 40. Further studies suggested a two-step model for Ire1 activation, in which Bip release from Ire1 leads to Ire1 oligomerization (FIG. 3b), which is followed by the putative inter action of misfolded proteins with its MHC I-like groove to trigger full activation 32. Remarkably, this idea was recently validated by an elegant study in living yeast cells, in which model misfolded proteins were shown to be the ligand that activates Ire1 (REF. 41). In contrast to the yeast UPR, mutations in IRE1α or ATF6 that reduce their ability to bind enhance the ability of these sensors to be activated, even in the absence of stress 36,42. Mammalian IRE1α may not interact with unfolded proteins 32 and, although the threedimensional structure of its amino terminal region is highly similar to its yeast counterpart, it has a narrow groove that is theoretically incompatible with peptide binding 43. It remains to be determined if PERK or ATF6 activation also involves the direct recognition of unfolded proteins. These models require deeper biochemical characterization to fully understand ER stress sensing mechanisms. Selective activation of UPR stress sensors? Several studies have suggested that UPR stress sensors may respond differentially to various forms of ER stress. An early report suggested that, under certain conditions, ATF6 may be activated first, before IRE1α and PERK 44. Furthermore, a systematic analysis of UPR signalling NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 13 FEBRUARY

6 Pancreatic β-cells Cells in the pancreas that make and secrete insulin to respond to glucose fluctuations. demonstrated that stress sensors actually have distinct sensitivities to specific inducers of ER stress 45. For example, IRE1α and PERK were rapidly activated, as compared with ATF6, under conditions of altered ER calcium homeostasis, in which the ER calcium content was depleted by inhibiting the ER calcium pump sarco endoplasmic reticulum calcium ATPase (SERCA). However, IRE1α responded faster to reducing agents than calcium alterations, whereas PERK showed similar kinetics of activation by both ER perturbations 45. A recent study also proposed that ATF6 is selectively activated by ER membrane protein load 46 and, as mentioned above, perturbations of ER function related to reduced glycosylation or altered redox metabolism may favour ATF6 signalling. These findings suggest that the UPR stress-sensing process is more sophisticated than previously anticipated. UPR stress sensors may be also locally activated by specific misfolded proteins rather than by general protein-folding stress. For example, the subset of mrnas undergoing RIDD depends on the propensity of the cell to misfold particular proteins. Through a series of complementary approaches, a hypothetical model was proposed in which, upon the translation and translocation of nascent proteins into the ER, protein misfolding may trigger the local activation of adjacent IRE1α molecules, leading to the specific degradation of the mrna that is being translated 10. In this model, the range of mrnas that are degraded following IRE1α activation depends on the proteome of the cell and the cell s tendency to misfold ER folded proteins. Several reports have confirmed that RIDD occurs in mammalian systems 8,9, and some observations suggest a physiological role for this selective output of IRE1α activation. For example, insulin mrna in pancreatic β cells is thought to be targeted for RIDD by IRE1α 8,47,48, and the mrna encoding microsomal triglyceride transfer protein in the intestine undergoes RIDD by the IRE1β isoform 49. It is attractive to think that this selective mechanism for IRE1 activation may involve the release of from local IRE1 molecules close to the translocation point. It may also be feasible that PERK activation occurs in the same selective manner to inhibit the ribosom e to block local translation. The timing, intensity and attenuation of the UPR. In this section, the temporal pattern of UPR stress sensor signalling and how it controls cell fate are discussed. Most of the studies in the UPR field have been performed using high doses of pharmacological inducers of ER stress, and cells inevitably undergo apoptosis owing to the chronic and irreversible nature of the stress that is generated. This setting contrasts with cells under going physiological levels of stress, such as active secretory cells, in which UPR signalling can be perpetuated for an indefinite time. In fact, in most experiments in which the ER is pharmacologically perturbed, adaptive factors such as chaperones and ERAD components are co-expressed with apoptosis genes with virtually identical induction kinetics. This scenario has made it difficult to uncover the mechanisms underlying the distinction between adaptive versus pro-apoptotic ER stress signalling, and even more difficult to understand the transition between these two phases. Although the ER-sensing domains of PERK and IRE1α are structurally similar, and even interchangeable 50, the temporal behaviour of their signalling differs drastically 5. This is reflected by the fact that, in certain experimental systems, IRE1α signalling is turned off upon prolonged ER stress 51, whereas PERK signalling can be sustained 52. Attenuation of IRE1α signalling is one possible mechanism to explain the transition from the adaptive to the pro-apoptotic phase of the UPR, in a model in which the duration of the exposure to stress determines cell fate. Inactivation of IRE1α under prolonged stress is predicted to ablate the pro-survival outcomes of XBP1s expression, whereas sustained PERK signalling favours the upregulation of many proapoptoti c components. By contrast, in other experimental settings PERK signalling is transient (see below) and IRE1α signalling is sustained 5, suggesting that the UPR regulatory network is dynamic. Recent studies in yeast demonstrated that variation in the intensity of ER stress could also engage the UPR with distinct kinetics and outputs. For example, Ire1 signalling is deactivated only after treatment with low concentrations of stress agents, as reflected by the attenuation of HAC1 splicing, possibly owing to stress mitigation Unexpectedly, through a combination of genetic strategies and mathematical modelling, it was revealed that Bip binding to Ire1 has a role in buffering UPR activation under low levels of stress 53. Bip was found to sequester inactive Ire1, which could only be activated above a certain threshold of stress. Mammalian UPR stress sensors can also integrate the intensity of the stimulus and reflect this in the signals that they transduce. For example, although exposure to very low levels of stress agents (even fold lower than normally used in the field) triggers a full UPR response in terms of the activation of PERK, of ATF6 and of XBP1 mrna splicing, this condition does not upregulate classic proapoptotic genes, such as CHOP and growth arrest and DNA damag e-inducible 34 (GADD34; also known as PPP1R15A) 56. Furthermore, changes indicated that IRE1α signalling outputs might differ depending on the oligo merization state of the sensor 8. Specifically, the artificial dimerization of IRE1α was found to be sufficient to trigger full XBP1 mrna splicing, although optimal RIDD was only obtained upon the induction of ER stress, which may be needed for further activation events, such as IRE1α oligo merization 8. Thus, the signalling outputs of the UPR seem to mirror the intensity of the stress. Finally, two recent studies provide insight into the dynamic regulation of the yeast UPR and the possible molecular events underlying the attenuation of Ire1 activity when stress is resolved 54,55. Ire1 phosphorylation was shown to be crucial to attenuate its RNase activity (FIG. 3b). Through the mutagenesis and pharmacological manipulation of Ire1, the authors found that conformational changes in Ire1, rather than its phosphorylation per se, are important for its activation. Additional phosphorylation events may subsequently 94 FEBRUARY 2012 VOLUME 13

7 UPRosome A signalling platform assembled at the level of inositol-requiring protein 1α that controls the kinetics and amplitude of downstream unfolded protein response (UPR) signalling responses. The UPRosome also orchestrates crosstalk between the UPR and other signalling pathways through the recruitment of different adaptor proteins. trigger the destabilization of Ire1 oligomers, leading to UPR inactivation. Remarkably, failure to inactivate Ire1 prolonged UPR signalling and reduced yeast survival 54,55, indicating a critical role for phosphorylation events in the attenuation of Ire1 activity. These two studies pose new questions for the field: how are the oligomerization and sequential phosphorylation events of UPR sensor s coordinated to generate different modes of signalling? Furthermore, how do UPR sensors integrate the intensit y of the stress and its temporal progression? The UPRosome : fine-tuning the UPR Evidence is accumulating for possible mechanisms that underlie selective UPR signalling modulation and the molecular switch from pro-survival responses to cell death programmes under chronic ER stress. As the ER luminal regions of IRE1α and PERK are structurally and functionally similar 50, it is likely that the kinetic s and outputs of UPR signalling are determined by an intrinsic mechanism that involves structural changes in the cytosolic domains of the sensors and/or the association of positive and negative regulators that specifically affect their activation. Although no systematic inter actome studies have been performed for UPR stress sensors, many laboratories have identified binding partners that modulate the activity of specific UPR proximal components. Most of the studies describing UPR binding partners have been performed with IRE1α, leading to the definition of a dynamic signalling platform that has been referred to as the UPRosome (REF. 27), in which many regulatory and adaptor proteins assemble to activate and modulate downstream responses. This section discusses possible regulatory mechanisms that may control the amplitude and kinetics of individual UPR signalling branches. Differential regulation of UPR sensors by cofactors. Several proteins have been shown to physically associate with IRE1α and to modulate the amplitude of IRE1α signalling without affecting PERK-related events (FIG. 4A). IRE1α regulators include the pro-apoptotic proteins BAX and BAK 57, the cytosolic chaperone heat shock protein 72 (HSP72) 58, protein Tyr phosphatase 1B (PTP1B) 59, and the MAPK-related proteins ASK1 interacting protein 1 (AIP1) 60, JNK-inhibitory kinase (JIK) 61, and JUN activation domain-binding protein 1 (JAB1) 62. Most of these regulators enhance IRE1α signalling, possibly as a result of enhanced or sustained activation. By contrast, BAXinhibitor 1 (BI 1) attenuates IRE1α activity, possibly because of a physical interaction with IRE1α that releases BAX from the UPRosome. Finally, mammalian target of rapamycin (mtor) signalling also has crosstalk with the UPR, selectively suppressing IRE1α activation by an unknown mechanism 67. The composition of the UPRosome is dynamic and the association and dissociation of several cofactors with IRE1α is dependent on ER stress. Thus, it seems possible that the expression pattern of IRE1α cofactors may determine the thres hold of stress needed to engage downstream responses in differen t cell types. The structural and biochemical basis behind the mechanisms of action of IRE1α modulators remains largely unexplored. Do all of these regulators operate by binding to IRE1α at the same site? Of note, most IRE1α cofactors have key functions in apoptosis 5. This observation suggests an interesting scenario in which components of the UPRosome may act as sentinels with dual roles that enable them to switch and engage the core apoptosis machinery when ER damage is irreversible. Interestingly, the functional effects of BI 1, BAX and BAK on XBP1 mrna splicing are observed only when cells are exposed to moderate to low levels of ER stress 63, suggesting that the IRE1α UPRosome is tuned by the intensity and duration of the stress stimuli. Overall, these studies give interesting clues as to how the UPR network integrates information about the folding status at the ER to reprogramme cells toward an adaptive versus a proapoptotic response. The exact biochemical mechanism that explains the modulation of IRE1α activity by all of these interactors remains to be determined. A drug screen using yeast revealed the presence of an allosteric site on Ire1, in the dimer interface, that binds flavonols 68. Whether the binding of flavonols to this site regulates the yeast UPR is unknown, but this study suggests that metabolites may modulate the pathway. Similarly, small molecules that bind the kinase domain of Ire1 can enhance or reduce its activity by shifting it between two conformational states 69. Interestingly, a recent study suggested that Ire1 may be able to sense alterations in membrane composition independently of its ER luminal domain, suggesting alternative mechanisms for its activation that involve the cytosolic and/ or the transmembrane region 70. Unexpectedly, synthetic peptides derived from the IRE1α sequence can instigate distinct IRE1α signalling outputs, enhancing XBP1 mrna splicing but attenuating JNK phosphorylation and RIDD 71. This evidence suggests that independent signalling modules might exist in UPR sensors that integrate and transduce adaptive and pro-apoptotic responses. Although they are less explored, other examples indicate that PERK and ATF6 can be individually modulated by specific factors. p58 IPK directly interacts with PERK, inhibiting its kinase activity 72,73 (FIG. 4B). As p58 IPK expression is upregulated under stress conditions by ATF6f and XBP1s, this feedback loop may participate in the integration of UPR signalling networks. ER stress also triggers the expression of a splicing variant of, which is a cytosolic form termed GRP78 VA. This protein enhances PERK signalling, possibly by antagonizing p58 IPK (REF. 74). The calcium-dependent phosphatase calcineurin also interacts with the cytosolic domain of PERK, promoting its autophosphorylation and downstream signalling 75. ATF6f is modulated through interactions with other factors (FIG. 4C). The protein product of the XBP1s target gene Wolfram syndrome 1 (WFS1), the transmembrane protein Wolframin, associates with and represses ATF6 signalling, possibly by inducing its proteasomedependen t degradation in an ER stress-dependent manner 76, suggesting the existence of a negative feedback loop from the IRE1α branch of the UPR to the ATF6 NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 13 FEBRUARY

8 A ER lumen Cytosol Cofactors and adaptors: BAX, BAK, AIP1, HSP72 and MAPK-related proteins B C CRT UPRosome CRT ER stress ER stress Gly S ER stress S Gly IRE1α Phosphorylation XBP1u XBP1s Inhibitors: BI-1, RACK1 and PP2A Acetylation Sumoylation p85α p38 PERK CN α β γ eif2α Translation CN CN ATF4 α β γ eif2α Translation p58 IPK GRP78 VA GADD34 PP1C CHOP PERK NF-Y, YY1, TBP and XBP1s ATF6 ATF6f ATF6f SH SH WFS1 Golgi lumen Da XBP1u XBP1s Basic ZIP HR TP Basic ZIP Intron Transactivation domain Db XBP1u mrna TP HR Ribosome XBP1u protein Dc ER lumen Cytosol XBP1s mrna IRE1α TP HR XBP1u protein Degradation by proteasome Figure 4 Multiple checkpoints in the regulation of the UPR. A Inositol-requiring protin 1α (IRE1α) assembles into a dynamic macromolecular complex termed the unfolded protein response (UPR) osome, which modulates the kinetics and amplitude of downstream signalling though the binding of several cofactors that enhance its activity (as is the case for BAX, BH antagonist or killer (BAK), ASK1 interacting protein 1 (AIP1), heat shock protein 72 (HSP72) and mitogen-activated protein kinase (MAPK)-related proteins) or inhibit its activity (as is the case for BAX-inhibitor 1 (BI 1), receptor for activated C kinase 1 (RACK1) and protein phosphatase 2A (PP2A)). Spliced X box-binding protein 1 (XBP1s) function is controlled through post-translational modifications, including p38-mediated phosphorylation, sumoylation and acetylation. In addition, the association of XBP1s with p85α enhances its activity, whereas the interaction of XBP1s with unspliced XBP1 (XBP1u) promotes XBP1s degradation. B Protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK) signalling is attenuated through the dephosphorylation of eukaryotic translation initiator factor 2α (eif2α), via a feedback loop that involves the activating transcription factor 4 (ATF4) C/EBP-homologous protein (CHOP)-mediated upregulation of growth arrest and DNA damage-inducible 34 (GADD34) and further assembly of an active PP1C phosphatase complex. The calcium-dependent phosphatase calcineurin (CN) also interacts with PERK, enhancing its activity, whereas p58 IPK reduces PERK activity, a process that is antagonized by GPR78 VA. C Calreticulin (CRT) may retain ATF6 in the ER through interactions with its glycosylations, an inhibitory interaction that is lost under ER stress conditions, allowing ATF6 to transit to the Golgi for further processing. Alterations in the redox status of the ER may directly enhance ATF6 translocation to the Golgi by reducing Cys residues at the ER luminal domain. ATF6 is negatively regulated through an interaction with Wolfram syndrome 1 (WFS1), possibly owing to ATF6 degradation by the proteasome, whereas PERK signalling enhances ATF6 expression and its translocation to the Golgi. The activity and specificity of the ATF6 cytosolic fragment (ATF6f), in terms of its control of target genes, is modulated through physical interactions with many transcription factors, including nuclear factor-y (NF Y), YY1, TATA-binding protein (TBP) and XBP1s. D The primary structures of XBP1u and XBP1s are shown (Da). The efficiency of XBP1 mrna splicing is controlled by XBP1u, which initiates a translational pausing (TP) event via its TP domain to ensure the efficient targeting of its own mrna to the ER membrane (Db). A hydrophobic region (HR) on the nascent XBP1u peptide targets the translated XBP1u mrna to the ER membrane, enhancing its processing by active IRE1α (Dc). ZIP, Leu zipper. branch. The activity of ATF6f, and its specificity for ER stress response elements in the promoter regions of targe t genes, is determined by its direct interaction with different transcription factors, including NF Y (also known as CBF), YY1, TATA-binding protein (TBP) and XBP1s 22. The ER luminal region of ATF6 can also associate with protein disulphide isomerase and calnexin 80, although the biological function of these interactions is unknown. All of these examples reflect the highly regulated nature of the UPR, which may augment the diversity of the cellular responses controlled by this pathway. This regulatory dynamism may allow the integration of information about the type and intensity of the stress stimulu s, as well as the fine-tuning of the signalling response according to the cell s need, through the assembl y of distinct regulatory complexes. Additional checkpoints modulating ER stress signalling. Several downstream checkpoints have been identified that balance and buffer UPR activity (FIG. 4). For example, the biological significance of the expression of the unspliced form of XBP1 (XBP1u) was recently revealed. Although XBP1u has an extremely short half-life 15, 96 FEBRUARY 2012 VOLUME 13

9 during its translation it drags the ribosome mrnanascen t chain to the ER membrane through a highly conserved hydrophobic domain in its carboxyl terminus 81. Another region of XBP1u mediates translation pausing, allowing the efficient targeting of the ribosomal mrna complex to the membrane and the recognitio n of XBP1 mrna by IRE1α 82 (FIG. 4D). The activity of XBP1s is not only increased by its interaction with different partners, but also by posttranslational modifications. The p85α regulatory subunit of phosphatidylinositol 3 kinase interacts with XBP1s and improves its nuclear translocation 83,84. The MAPK p38 can phosphorylate XBP1s, enhancing its translocation to the nucleus 85, whereas acetylation and sumoylation of XBP1s can augment or attenuate its transcriptional activity, respectively 86,87. XBP1u may accumulate under conditions of prolonged ER stress in certain systems, forming a complex with XBP1s in the cytosol to prevent its nuclear translocation and induce its proteasomal degradation 88. This feedback loop may contribute to the attenuation of XBP1 dependent responses after prolonged ER stress. The PERK pathway is tuned at the level of eif2α. The PERK CHOP signalling branch stimulates the dephosphorylation of eif2α through a feedback loop that is mediated by the upregulation of GADD34, which positively regulates a phosphatase complex involving protein phosphatase 1C (PP1C), allowing protein synthesis to resume 89. This regulatory loop can be modulated by specific drugs to alleviate ER stress, with great therapeutic potential 90,91. The activity and specificity of ATF4 is also determined through its interaction with a range of transcription factors, as well as by its posttranslationa l modification 23. Finally, a recent report described a new regulatory connection between PERK and ATF6 in which PERK signalling facilitates the synthesis of ATF6 and its trafficking from the ER to the Golgi by an unknown mechanism 92. Thus, all of the recent advances reveal that the UPR cannot be considered as three linear and parallel pathways. Instead, the signalling branches of the UPR are interconnected with each other and with additional signal transduction networks, which allows them to integrate information for the efficient handling of cellular stress. It is important to mention that most of the regulatory checkpoints discussed in this section have been recently described and need further characterization and confirmation in other experimental systems. Novel outputs of the UPR Emerging evidence from different experimental systems indicates that UPR signalling modules have fundamental roles in multiple physiological processes beyond the homeostatic control of protein folding. This may reflect the complex network of interactions between the UPR branches and other signalling pathways (FIG. 5). In this section, I describe some examples that illustrate the novel physiological outputs of the UPR that have been shown by recent studies focused on innate immunity, energy and lipid metabolism, and cell differentiation. TLR signalling and XBP1. XBP1 was originally identified as one of the transcription factors upregulated after the exposure of cells to the pro-inflammatory cytokine interleukin 6 (IL 6), and an increasing number of reports indicate an important role for the UPR in pro-inflammatory responses 93. For example, XBP1 deficiency in mice and Caenorhabditis elegans ablates the ability of these animals to eliminate bacterial pathogens 93. Further studies indicate that pro-inflammatory stimuli that engage certain Tolllike receptors (TLRs), including lipopolysaccharide (LPS), specifically trigger XBP1 mrna splicing to enhance the transcription of pro-inflammatory cytokines, such as IL 6 (REF. 94). Unexpectedly, TLR stimulation represses ATF6 and PERK signalling but specifically induces XBP1 dependent IL 6 mrna upregulation without triggering a classical ER stress response 94,95. In fact, there is some evidence suggesting that the engagement of XBP1 mrna splicing by TLRs is independent of protein misfolding 94. This process is, however, IRE1α dependent and controlled through a specific signalling branch involving the adaptor proteins myeloid differentiation primary response 88 (MYD88), TIR domain-containing adaptor protein (TIRAP), TRAF6 and NADPH oxidase 2 (NOX2). The exact mechanism (or mechanisms) by which TLR stimulation represses ATF6 and PERK while activating IRE1α remains to be established. Overall, this example illustrates the complexity of UPR signalling crosstalk and shows how signalling modules of the pathway are involved in innate immunity, possibly reflecting a function for the UPR beyond protein-folding stress. Glucose metabolism. The first target genes of the UPR to be identified were chaperones and foldases of the glucose-regulated protein (GRP) family. A large body of literature now supports a crucial role for the UPR in monitoring fluctuations in glucose levels. In fact, the UPR is becoming an important target against which possible treatments for diabetes are being developed 96. These metabolic effects of the UPR are attributed only in part to its role in controlling the fidelity and efficiency of insulin folding and secretion. Several studies indicate that IRE1α is phosphorylated in response to the exposure of cells to physiological concentrations of glucose, which enables it to control in sulin levels 96. Unexpectedly, glucose fluctuations lead to IRE1α phosphorylation on Ser724 in the absence of the classical electrophoretic pattern of activation, and they do not trigger XBP1 mrna splicing, JNK phosphorylation or release from IRE1α 97. At the molecular level, the stimulation of cells with low glucose concentrations decreases IRE1α Ser724 phosphorylation by promoting its association with the adaptor protein receptor for activated C kinase 1 (RACK1), which recruits the phosphatase PP2A to the complex 98. By contrast, ER stress or acute glucose treatment has the opposite effect, augmenting IRE1α phosphorylation, and thus activation, through dissociation of the RACK1 PP2A complex 98. These observations suggest the existence of a dynamic regulatory module that fine-tunes IRE1α phosphorylation in response to ER stress inputs and mild to high increases in glucose concentration. NATURE REVIEWS MOLECULAR CELL BIOLOGY VOLUME 13 FEBRUARY