The unfolded protein response: controlling cell fate ... em PDF/The... · Nature Reviews | Molecular Cell Biology ER stress ATF6 ATF6f Folding, ERAD, quality control, ER biogenesis,

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 contrib-utes to the biogenesis of autophagosomes and peroxi-somes. 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 con-stantly adjusted through the dynamic integration of multiple environmental and cellular signals. Several feedback mechanisms ensure efficient adaptation to fluctuations in protein-folding requirements by func-tionally affecting almost every aspect of the secretory pathway1. 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 inhi-bition of folding led to the transcriptional upregulation of several key ER chaperones2. 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 fluc-tuations in unfolded protein load3,4. When cells undergo irreversible ER stress5, this pathway eliminates damaged cells by apoptosis, indicating the existence of mecha-nisms that integrate information about the duration and intensit y of stress stimuli.

Although the UPR is classically linked to protein-folding stress under both physiological and patho-logical 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 differentiation6. At the molecu-lar level, these alternative UPR outputs are attributed, in part, to the complex crosstalk between different stress and metabolic pathways. In this scenario, a dynamic sig-nalling 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

1Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile. 2Institute of Biomedical Sciences, Center for Molecular Studies of the Cell, University of Chile, Santiago, P.O. BOX 70086, Chile.3Department of Immunology and Infectious Diseases, Harvard School of Public Health, 651 Huntington Ave, Boston, Massachusetts 02115, USA.e-mails: [email protected]; [email protected]:10.1038/nrm3270 Published online 18 January 2012

The unfolded protein response: controlling cell fate decisions under ER stress and beyondClaudio Hetz1–3

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.

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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 13 | FEBRUARY 2012 | 89

© 2012 Macmillan Publishers Limited. All rights reserved

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mailto:[email protected]

Nature Reviews | Molecular Cell Biology

• Autophagy• Apoptosis• Co-translational degradation

• Quality control• Pre-emptive quality control• Protein secretion

• ERAD• Folding• Lipid synthesis

Cytosol

ER lumenER stress ER stress

IRE1α PERK

TRAF2

JNKNF-κB

NF-κB

XBP1u mRNA

mRNA

XBP1s mRNA

Intron

XBP1s

UPR target genesXBP1s UPR target genes UPR target genes

ATF4

Ribosome‘Alarm stresspathways’

mRNA degradation (RIDD)

ER stress

NRF2 β γα

β γα

eIF2α

Translation

ATF4

ATF6

ATF6f

ATF6f

Golgi

S1PS2P

COPII

a b cPhosphorylation

?

stress ‘rheostat’ to control cell fate. Special emphasis is given to unexpected regulatory checkpoints that specifi-cally 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 deathThe 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 pro-surviva 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 down-stream transcription factor, Hac1 (which is homolo-gous 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

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.

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ER stress

ATF6 ATF6f Folding,ERAD,quality control,ER biogenesis,autophagy

Adaptive responses Apoptosis phase

Caspase 2 BID

Folding,redox,autophagy

RIDD RIDD survival genes

Translation p53 BH3-only

CHOP BCL-2

GADD34

IP3R

ROS translation

Ca2+

PTP

ApoptosisBAX, BAK

XBP1s

JNKIRE1α

PERK eIF2α

ATF4

?

?

?

?

Time of exposure to stress

Intensityof stress

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 reticulum‑associate d 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)8–10. 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 pathway11. Finally, pre-emptive quality control12 and co-translational degradation13 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 gene-expression 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 genes1. 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 change14 (FIG. 1a). Active IRE1α processes the mRNA encoding the transcrip-tion factor X box-binding protein 1 (XBP1), excising a 26-nucleotide-long intron that shifts the coding readin g frame of this mRNA15–17. 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 functions18,19 (FIG. 1a). XBP1s also modulates phospholipid synthesis, which is required for ER membrane expansion under ER stress4.

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 BIP. 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.

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AutophagyA 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 transduc-ers that encode basic Leu zipper (bZIP) transcrip-tion 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 autophagy3,23 (FIG. 1b). This branch of the UPR also regulates the expres-sion of several microRNAs, which may contribute to the attenuation of protein translation or protein synthesis24.

Together, ATF4, XBP1s and ATF6f govern the expres-sion 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, pos-sibly through their interaction with other transcription factors (see below).

Chronic ER stress and apoptosis. Physiological pro-cesses 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 pathways25.

Cell death under ER stress depends on the core mitochondrial apoptosis pathway, which is regulated by the B cell lymphoma 2 (BCL-2) protein family26. In this pathway, the conformational activation of the pro-apoptotic multidomain proteins BAX and/or BH antago-nist 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 axis25. CHOP promotes both the transcription of BIM and the downregulation of BCL-2 expression, contributing to the induction of

apoptosis5,25. In addition to CHOP, ATF4 and p53 are also involved in the direct transcriptional upregulation of BH3-only proteins under ER stress5. However, the mech-anism 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 apoptosis4,25. 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 media-tors through RIDD8. As ER stress can result in distinct and contrasting outputs (FIG. 2), it is essential to under-stand 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 points27. 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 signal- regulating kinase 1 (ASK1; also known as MAP3K5) path-way 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 nutri-ent starvation by activating beclin 1 (REFS 29,30), an essen-tial autophagy regulator11. In addition, IRE1α engages alarm pathways involving p38, extracellular signal-regulated kinase (ERK) and nuclear factor-κB (NF-κB) through the binding of distinct adaptor proteins27. 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, respectively3. Under certain experimental conditions, ATF6 may also control NF-κB through AKT31; however, the connection between ATF6 and alarm stress pathways remains largely unexplored.

Dynamic regulation of the UPRRecent studies suggest that UPR sensors have funda-mental 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 ini-tiation, attenuation and fine-tuning of UPR-dependent responses are discussed below.

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BIP


IRE1α

Ire1 Clusterformation

Attenuation Buffering

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a Mammalian UPR

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b Yeast UPR

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BIP

Low stress High stress Signalling attenuation

Misfoldedprotein

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Bip Bip

Bip

Unfolded ormisfoldedproteins

XBP1 mRNAsplicing

mRNA decay

HAC1 mRNAsplicing

BIP

Bip

Phosphorylation

Time

ER lumen

ER lumen

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 signalling32. Further oligomeri-zation of IRE1 into large clusters correlates with the kinetics of its autophosphorylation and the subsequent initiation of its ability to splice XBP1 mRNA in mam-mals or HAC1 mRNA in yeast33,34. PERK signalling is also initiated by the dimerization, oligomerization and autophosphoryl ation of PERK14. 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 UPR32.

A pioneering study proposed that the binding of the ER chaperone BIP (also known as GRP78 and HSPA5) to IRE1α and PERK in mammalian cells represses their spontaneous self-dimerization and activation35. Accordingly, under ER stress conditions, BIP preferen-tially binds to misfolded proteins, which releases its inhibitory interaction with stress sensors (FIG. 3a). A sim-ilar model was described in parallel in yeast (reviewed in REF. 32) (FIG. 3b). In the case of ATF6, BIP binding to this sensor is proposed to mask its Golgi-localization signal36. BIP release allows ATF6 to interact with coat protein II (COPII), a complex of proteins that recog-nize cargoes to generate vesicles that are transported to the Golgi37. 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 Golgi38. ATF6 is expressed as a monomer and as oligomers, possibly owing to the presence of intra- and inter-disulphide bridges at its ER luminal domain39. 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 histo-compatibility complex class I (MHC-I) and which may be involved in the recognition of misfolded proteins and act in part as a stress-sensing domain40. 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 trig-ger full activation32. 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 BIP enhance the ability of these sensors to be activated, even in the absence of stress36,42. Mammalian IRE1α may not inter-act with unfolded proteins32 and, although the three-dimensional structure of its amino-terminal region is highly similar to its yeast counterpart, it has a narrow groove that is theoretically incompatible with peptide binding43. 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 PERK44. Furthermore, a systematic analysis of UPR signalling

Figure 3 | The stress-sensing mechanism and kinetics of IRE1 signalling. a | In mammalian cells, inositol-requiring protein 1α (IRE1α) is maintained in a repressed state under non-stress conditions through an association with BIP. Upon endoplasmic reticulum (ER) stress, BIP 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 BIP 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.

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Pancreatic β-cellsCells 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 stress45. For example, IRE1α and PERK were rapidly activated, as compared with ATF6, under conditions of altered ER calcium homeostasis, in which the ER calcium con-tent 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 simi-lar kinetics of activation by both ER perturbations45. A recent study also proposed that ATF6 is selectively activated by ER membrane protein load46 and, as men-tioned 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 previous ly anticipated.

UPR stress sensors may be also locally activated by specific misfolded proteins rather than by general pro-tein-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 com-plementary approaches, a hypothetical model was pro-posed 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 translated10. 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 systems8,9, and some observations suggest a physiological role for this selective output of IRE1α acti-vation. For example, insulin mRNA in pancreatic β‑cells is thought to be targeted for RIDD by IRE1α8,47,48, and the mRNA encoding microsomal triglyceride transfer protein in the intestine undergoes RIDD by the IRE1β isoform49. It is attractive to think that this selective mechanism for IRE1 activation may involve the release of BIP from local IRE1 molecules close to the trans-location point. It may also be feasible that PERK activa-tion 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 per-formed 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 interchange-able50, the temporal behaviour of their signalling differs drastically5. This is reflected by the fact that, in certain experimental systems, IRE1α signalling is turned off upon prolonged ER stress51, whereas PERK signalling can be sustained52. 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 pro-longed stress is predicted to ablate the pro-survival outcomes of XBP1s expression, whereas sustained PERK signalling favours the upregulation of many pro-apoptoti c components. By contrast, in other experimen-tal settings PERK signalling is transient (see below) and IRE1α signalling is sustained5, suggesting that the UPR regulatory network is dynamic.

Recent studies in yeast demonstrated that varia-tion 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 mitigation53–55. Unexpectedly, through a combination of genetic strategies and mathematical modelling, it was revealed that Bip binding to Ire1 has a role in buffer-ing UPR activation under low levels of stress53. 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 trans-duce. For example, although exposure to very low levels of stress agents (even 100–500-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 pro-apoptotic 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 sensor8. Specifically, the arti-ficial 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 merization8. 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 resolved54,55. Ire1 phosphoryl-ation was shown to be crucial to attenuate its RNase activity (FIG. 3b). Through the mutagenesis and pharma-cological manipulation of Ire1, the authors found that conformational changes in Ire1, rather than its phos-phorylation per se, are important for its activation. Additional phosphorylation events may subsequently

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UPRosomeA 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 survival54,55, 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 signal-ling? Furthermore, how do UPR sensors integrate the intensit y of the stress and its temporal progression?

The ‘UPRosome’: fine-tuning the UPREvidence 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 similar50, it is likely that the kinetic s and outputs of UPR signalling are determined by an intrin-sic 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 part-ners 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 dis-cusses 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 sig-nalling without affecting PERK-related events (FIG. 4A). IRE1α regulators include the pro-apoptotic proteins BAX and BAK57, 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 activa-tion domain-binding protein 1 (JAB1)62. Most of these regulators enhance IRE1α signalling, possibly as a result of enhanced or sustained activation. By contrast, BAX-inhibitor 1 (BI-1) attenuates IRE1α activity, possibly because of a physical interaction with IRE1α63–66 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 mechanism67. The composition of the UPRosome is dynamic and the association and dissocia-tion 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 apoptosis5. 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 stress63, 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 pro-apoptotic 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 flavonols68. 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 states69. Interestingly, a recent study suggested that Ire1 may be able to sense alterations in membrane composition independently of its ER luminal domain, suggesting alternative mecha-nisms for its activation that involve the cytosolic and/ or the transmembrane region70. Unexpectedly, synthetic peptides derived from the IRE1α sequence can instigate distinct IRE1α-signalling outputs, enhancing XBP1 mRNA splicing but attenuating JNK phosphorylation and RIDD71. 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 indi-cate that PERK and ATF6 can be individually modulated by specific factors. p58IPK directly interacts with PERK, inhibiting its kinase activity72,73 (FIG. 4B). As p58IPK expres-sion is upregulated under stress conditions by ATF6f and XBP1s, this feedback loop may participate in the integra-tion of UPR signalling networks. ER stress also triggers the expression of a splicing variant of BIP, which is a cytosolic form termed GRP78VA. This protein enhances PERK signalling, possibly by antagonizing p58IPK (REF. 74). The calcium-dependent phosphatase calcineurin also interacts with the cytosolic domain of PERK, promoting its autophosphorylation and downstream signalling75.

ATF6f is modulated through interactions with other factors (FIG. 4C). The protein product of the XBP1s tar-get gene Wolfram syndrome 1 (WFS1), the transmem-brane protein Wolframin, associates with and represses ATF6 signalling, possibly by inducing its proteasome-dependen t degradation in an ER stress-dependent man-ner76, suggesting the existence of a negative feedback loop from the IRE1α branch of the UPR to the ATF6

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BIP

BIP


Cytosol

ER lumen

XBP1sXBP1u

ER stress ER stress ER stress

IRE1α

UPRosome

BIPBIP

Cofactorsand adaptors:BAX, BAK, AIP1,HSP72 andMAPK-relatedproteins

Inhibitors: BI-1,RACK1 and PP2A

Acetylation

Sumoylation

p85αp38

β γα

eIF2αβ γα

eIF2α

CNCN CN

Translation Translation

GADD34PP1C

p58IPK

GRP78VA

PERK

ATF6

SS

SHSHGly

CRTCRT

WFS1

XBP1u

XBP1s

Basic ZIP HR TP

Basic ZIPTransactivationdomain

Intron

A

Da Db

B C

XBP1umRNA XBP1s

mRNA

XBP1uprotein

TPHR

Ribosome

HRTPXBP1u protein

Degradationby proteasome

IRE1αER lumen

Cytosol

Dc

Phosphorylation

PERK

BIPBIPGly

ATF4 CHOP

Golgi lumen

ATF6f

ATF6f

NF-Y, YY1,TBP andXBP1s

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)77–79 and XBP1s22. The ER luminal region of ATF6 can also associate with protein disulphide isomerase and calnexin80, although the biological function of these interaction s 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 integra-tion of information about the type and intensity of the stress stimulu s, as well as the fine-tuning of the signal-ling 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 exam-ple, the biological significance of the expression of the unspliced form of XBP1 (XBP1u) was recently revealed. Although XBP1u has an extremely short half-life15,

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 p58IPK 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.

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during its translation it drags the ribosome–mRNA-nascen t chain to the ER membrane through a highly con-served hydrophobic domain in its carboxyl terminus81. 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 IRE1α82 (FIG. 4D).

The activity of XBP1s is not only increased by its interaction with different partners, but also by post-translational modifications. The p85α regulatory sub-unit of phosphatidylinositol 3-kinase interacts with XBP1s and improves its nuclear translocation83,84. The MAPK p38 can phosphorylate XBP1s, enhancing its translocation to the nucleus85, whereas acetylation and sumoylation of XBP1s can augment or attenuate its transcriptional activity, respectively86,87. 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 degradation88. This feedback loop may con-tribute 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 syn-thesis to resume89. This regulatory loop can be modu-lated by specific drugs to alleviate ER stress, with great thera peutic potential90,91. The activity and specificity of ATF4 is also determined through its interaction with a range of transcription factors, as well as by its post-translationa l modification23. Finally, a recent report described a new regulatory connection between PERK and ATF6 in which PERK signalling facilitates the syn-thesis of ATF6 and its trafficking from the ER to the Golgi by an unknown mechanism92.

Thus, all of the recent advances reveal that the UPR cannot be considered as three linear and parallel path-ways. Instead, the signalling branches of the UPR are interconnected with each other and with additional sig-nal transduction networks, which allows them to inte-grate information for the efficient handling of cellular stress. It is important to mention that most of the regu-latory checkpoints discussed in this section have been recently described and need further characterization and confirmation in other experimental systems.

Novel outputs of the UPREmerging evidence from different experimental sys-tems 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 path-ways (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 inter-leukin-6 (IL-6), and an increasing number of reports indi-cate an important role for the UPR in pro-inflammatory responses93. For example, XBP1 deficiency in mice and Caenorhabditis elegans ablates the ability of these animals to eliminate bacterial pathogens93. Further studies indicate that pro-inflammatory stimuli that engage certain Toll-like 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 trig-gering a classical ER stress response94,95. In fact, there is some evidence suggesting that the engagement of XBP1 mRNA splicing by TLRs is independent of protein mis-folding94. 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 developed96. 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 con-centrations of glucose, which enables it to control in sulin levels96. Unexpectedly, glucose fluctuations lead to IRE1α phosphorylation on Ser724 in the absence of the clas-sical electrophoretic pattern of activation, and they do not trigger XBP1 mRNA splicing, JNK phosphorylation or BIP release from IRE1α97. At the molecular level, the stimulation of cells with low glucose concentrations decreases IRE1α Ser724 phosphorylation by promot-ing its association with the adaptor protein receptor for activated C kinase 1 (RACK1), which recruits the phos-phatase PP2A to the complex98. By contrast, ER stress or acute glucose treatment has the opposite effect, aug-menting IRE1α phosphorylation, and thus activation, through dissociation of the RACK1–PP2A complex98. These observations suggest the existence of a dynamic regulatory module that fine-tunes IRE1α phosphoryla-tion in response to ER stress inputs and mild to high increases in glucose concentration.

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LPS TLRs Adaptor(TIRAP and MYD88)

Cytokine secretion MacrophageIRE1α–XBP1

IRF4 and BLIMP1 Ig secretion B cells

IRS1

ATF6 and PERK–CHOP

Stimuli Inputs Transducers Outputs Cell types

Glucose fluctuation,obesity

Lipids,glucose

IRE1α–XBP1

IRE1α–XBP1, ATF6 and PERK

IRE1α-P–RACK1–PP2A

Lipid and/or cholesterolbiosynthesis

Liver

Insulin resistance

Insulin secretion

Liver

Pancreas

Differentiation signal B cellreceptor

BDNF

Differentiation signal

Differentiation signal

Metabolic requirements ?

? XBP1 MIST Differentiation,zymogen granulebiogenesis

Muscle,gastric cells

CXCL12 andCXCR4

Bone marrowcolonization

CHOP

? XBP1 MIST Differentiation,skeletal myotubules

Muscle

TRKB,p75

? IRE1α–XBP1 Neurite outgrowth Brain

Exocrine pancreasA type of pancreatic tissue that has ducts arranged in clusters called acini. Cells secrete into the lumen of an acinus a series of enzymes and molecules related to digestion, including trypsinogen, lipase, amylase and ribonuclease.

Endocrine pancreasThe part of the pancreas that acts as an endocrine gland, consisting of the islets of Langerhans, which contain β‑cells. Theses cells secrete insulin and other hormones.

It is becoming clear that the intersection of the UPR with inflammation, lipid metabolism and energy control pathways underlies chronic metabolic dis-eases, such as type 2 diabetes, insulin resistance and obesity96. Insulin resistance in the liver also involves the activation of IRE1α. This is possibly due to signal-ling crosstalk between the IRE1α–JNK pathway and the subsequent phosphorylation of insulin receptor substrate 1 (IRS1), which impairs insulin action99. The nature of the stimuli engaging the UPR in the liver of obese mice remained unknown until very recently. Through a proteomic and lipidomic approach, one group found drastic alterations in the lipid ER content of cells in the livers of obese mice100, resulting in the inhibition of the SERCA pump with the concomitant induction of ER stress100. Many other examples link UPR signalling with glucose homeostasis (reviewed in REF. 96). Thus, UPR components are important adjustors of energy metabolism, possibly acting as sensors that monitor and integrate information on the metaboli c state of the cell.

Cell differentiation programmes. Most of the examples of UPR’s physiological roles are related to its function in highly secretory cells. Initial studies demonstrated that XBP1 is fundamental for the differentiation of B cells into actively secreting plasma cells101. In fact, XBP1 deficiency completely ablates immunoglobulin secre-tion, leading to the speculation that the high demand of immunoglobulin synthesis generates a basal stress

condition that engages the UPR102. Then, in a dynamic and cyclic manner, the UPR might adjust the protein-folding capacity of the cell according to the need, which results in the acquisition of an efficient secretory pheno-type. The same concept is proposed for many different secretory organs, based on genetic evidence obtained by mutating UPR components in the exocrine pancreas, endocrine pancreas, salivary glands and gastric cells103–106. Although this model makes complete biological sense, studies in mice engineered to abrogate immunoglobulin synthesis demonstrated that XBP1 mRNA splicing and B cell differentiation proceed normally107. XBP1 was shown to be a crucial component of differentiation pro-grammes activated through the B cell receptor, possibly by inhibiting transcriptional repressors of plasma cell differentiation, such as interferon regulatory factor 4 (IRF4) and B lymphocyte-induced maturation protein 1 (BLIMP1)107. These results led to the proposition of a new paradigm, in which early activation of the UPR could actually prime the cell for the future demands of a high secretory activity after differentiation. IRE1α also has additional functions in the differentiation of B cells at the pre-B-cell stage, which are related to the recombi-nation of immunoglobulin genes108. In agreement with this concept, a global genomic analysis revealed an XBP1-regulated transcriptional network that involves key differentiation genes, such as muscle, intestine and stomach expression 1 (MIST1)19. Further studies indicate that the XBP1–MIST1 axis is required for the maturation of gastric zymogenic cells to ensure efficient

Figure 5 | Novel physiological outcomes of the UPR. Distinct cellular inputs activate unfolded protein response (UPR) components to transduce signals that affect a wide range of processes, including metabolism, inflammation and cell differentiation. This figure summarizes a selected list of stimuli and receptors that activate UPR signalling modules, indicating the signalling pathways that they engage and the components of the UPR involved in the process (question marks represent unknown components). The processes affected and the specific cell types involved are also indicated. ATF6, activating transcription factor 6; BDNF, brain-derived neurotrophic factor; BLIMP1, B lymphocyte-induced maturation protein 1; CHOP, C/EBP-homologous protein; CXCL12, CXC chemokine ligand 12; CXCR4, CXC chemokine receptor 4; Ig, immunoglobulin; IRE1α, inositol-requiring protein 1α; IRF4, interferon regulatory factor 4; IRS1, insulin receptor substrate 1; LPS, lipopolysaccharide; MIST, muscle, intestine and stomach expression; MYD88, myeloid differentiation primary response 88; P–RACK1, phosphorylated RACK1; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PP2A, protein phosphatase 2A; RACK1, receptor for activated C kinase 1; TIRAP, TIR domain-containing adaptor protein; TLRs, Toll-like receptors; XBP1, X box-binding protein 1.

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granule biogenesis to allow digestive enzyme secre-tion105. Remarkably, a study performed in chondro-cytes, which are specialized cells that secrete collagen, showed that chronic ER stress has a distinct conse-quence beyond apoptosis109. By analysing hypertrophic chondro cytes in transgenic mice expressing mutant collagen, the authors showed that ER stress triggers dedifferentiation into non-secreting cells109, which may contribute to the alleviation of protein-folding stress.

Other physiological outcomes of the UPR. Other stud-ies support the concept that UPR signalling modules orchestrate physiological processes that are not directly related to protein misfolding. For example, XBP1 positively controls hepatic lipogenesis at basal levels, affecting cholesterol and triglyceride levels, possibly by directly transactivating key genes in this metabolic pathway110. Similarly, ATF6 and IRE1α function in lipid

metabolism in the liver96,111,112. In addition, ER stress regulates the synthesis of the peptide hormone hep-cidine113, which is secreted by the liver to control iron homeostasis in the body.

XBP1 physically interacts with, and negatively regu-lates the levels of, forkhead box O1 (FOXO1), which is a key transcription factor involved in energy control114. Functional crosstalk between XBP1 and FOXO family members has also been proposed in the processes of ageing and longevity in C. elegans115. In addition, ER stress controls gluconeogenic programmes by activating ATF6, which negatively modulates the activity of CREB-regulated transcription co-activator 2 (CRTC2)116. Finally, brain-derived neurotrophic factor (BDNF) activates XBP1 mRNA splicing in neurons to enhance neurite outgrowth and cell differentiation117. As BDNF signals through TRKB or p75, downstream components of these receptors may regulate IRE1α.

Box 1 | Distinct phenotypes of mice deficient for UPR components

Studies of the function of components of the unfolded protein response (UPR) in vivo, using genetic manipulation, have revealed divergent and specialized functions of the pathway in distinct organs in physiology and disease4,118 (see the table). X box-binding protein 1 (XBP1) deficiency results in embryonic lethality owing to liver hypoplasia and anaemia101. Expression of XBP1 in the liver of Xbp1–/– embryos rescues viability, but these animals die shortly after birth owing to the impairment of secretory organs, that is, the exocrine pancreas and salivary glands103. Similarly, the targeted deletion of XBP1 in mice acinar gastric cells105 or B lymphocytes102,120 led to the dysfunction of these cells. XBP1 targeting in adult liver cells decreases fatty acid and sterol synthesis, without causing fatty liver110. In pancreatic β-cells, the loss of XBP1 disrupts insulin synthesis and glucose homeostasis47 and causes the constitutive hyperactivation of inositol-requiring protein 1α (IRE1α), which leads to regulated IRE1-dependent decay (RIDD)-dependent degradation of proinsulin mRNA47. IRE1α deficiency also causes embryonic lethality owing to liver failure108, a phenotype that can be rescued by the expression of IRE1α in the placenta121, however these animals show mild hypoinsulinaemia, hyperglycaemia, an altered exocrine pancreas and decreased immunoglobulin (Ig) secretion, with minor effects on their salivary glands104. Liver function is relatively normal in IRE1α-deficient mice, but they develop hepatic lipid accumulation when exposed to experimental endoplasmic reticulum (ER) stress112.

Activating transcription factor 6α (ATF6α) and ATF6β are ubiquitously expressed, and the single knockout of each gene does not cause developmental alterations. However, double ATF6α and ATF6β deficiency is embryonic lethal22. Challenging Atf6a–/– mice with an ER stress agent is also lethal, possibly owing to liver dysfunction111. Protein kinase RNA-like ER kinase (PERK)-deficient mice are normal at birth but develop drastic pancreatic β-cell degeneration and diabetes mellitus106. They also show abnormalities in the exocrine pancreas, with decreased secretion of digestive enzymes106 and impaired bone formation. PERK deficiency does not affect Ig secretion122. Animals deficient in ATF4 show defects in bone formation123 and glucose metabolism, and they are blind owing to the inefficient formation of the eye lens124.

Phenotype IRE1α XBP1 ATF6α PERK ATF4

Full knockout

Embryonic lethal Yes Yes No* No No‡

Postnatal death – – No Yes No

Tissue-specific effects

Decreased Ig secretion by B cells Yes Yes – No –

Exocrine pancreas alteration Mild Yes – Yes –

Endocrine pancreas and insulin secretion alteration Yes Yes Yes§ Yes Yes

Lipid abnormalities in liver|| Yes Yes Yes Yes –

Altered bond formation – – – Yes Yes

Blind, altered eye lens No No No No Yes

Impaired glucose metabolism Mild Yes – Yes Yes

*ATF6α and ATF6β double deficiency is embryonic lethal. ‡ATF4-deficient animals are not born on a Mendelian rate, suggesting defects during development. §Based on correlative studies in human patients with diabetes and on functional analyses of mouse models using viral-mediated manipulation. ||Only XBP1-deficient animals show a drastic alternation in basal lipid accumulation in the absence of experimental ER stress.

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ATF6

XBP1 ASK1–JNK

IRE1α PERK

UPR

eIF2α ATF4

Degeneration(autophagy)

Degeneration(apoptosis)

Protection(translation)

? ?

ER stressSporadic and familial ALS

ERAD impairment,altered traffic,PDI inactivation,protein aggregation,autophagy defects

PerspectiveThis Review discussed, in detail, the main mecha-nisms involved in UPR signalling, and provided several examples illustrating the notion that the UPR network is arranged as a nonlinear dynamic pathway in which multiple checkpoints determine the outputs of each UPR signalling branch. UPR components are part of distinct regulatory modules that orchestrate the fine-tuning of essential homeostatic processes that, in many cases, are beyond protein folding per se. This idea is supported by various examples showing that the UPR participates in cell differentiation, lipid and glucose metabolism, and inflammatory responses.

The assembly of distinct signalling platforms at the level of stress sensors, such as the IRE1α UPRosome, may modulate the amplitude and kinetics of down-stream responses by binding cofactors and by recruit-ing adaptor and signalling proteins. UPR sensors may have the intrinsic ability to orient the functional output s of the pathway, in a highly regulated and ‘custom’

manner, to favour specific downstream physiological consequences. This model may also help explain the divergent phenotypes of UPR-deficient mouse mod-els in different organs (BOX 1, reviewed in REFS 4,118). Most of the UPR-regulating events may help to adjust the functional effects of the pathway according to need in a context-dependent and integrative manner. It is still puzzlin g how the UPR provides cell type-specific effects and how the information about the nature of the stimulus, its intensity and its duration, is trans-lated into particular cell fate programmes that could be as contrasting as cell death, stress adaptation and cell differentiation, among other consequences. Based on the emerging role of the UPR in diverse disease condi-tions119, such as cance r, diabetes, ischaemia reperfusion and neuro degeneration (BOX 2), understanding how the UPR conveys information about homeostatic fluctua-tion, as well as its feedback regulation, is fundamental for the identification of future points of intervention in many important human diseases.

Box 2 | Contribution of the UPR to ALS

Endoplasmic reticulum (ER) stress is involved in the pathogenesis of several diseases, including cancer, autoimmunity, diabetes, ischaemia reperfusion and neurodegeneration (reviewed in REFS 3,4,118). In particular, ER stress is a salient feature of many neurodegenerative diseases related to protein misfolding and aggregation, including amyotrophic lateral sclerosis (ALS), Parkinson’s disease and Alzheimer’s disease125. ALS is a progressive and fatal adult-onset disease in which the selective degeneration of motor neurons leads to paralysis and premature death. Studies using genetic manipulation of the unfolded protein response (UPR) and pharmacological approaches have revealed a complex involvement of the pathway in ALS, illuminating distinct outputs of specific UPR signalling branches in the same disease125 (see the figure). Mutations in the gene encoding superoxide dismutase 1 (SOD1) cause familial ALS, and expression of mutant SOD1 in mice recapitulates most of the disease features observed in patients with this disease. Treatment of mutant SOD1 transgenic mice with salubrinal, a small molecule that induces eukaryotic translation initiator factor 2α (eIF2α) phosphorylation, leads to significant protection against disease progression126, whereas protein kinase RNA-like ER kinase (PERK) haploinsufficiency enhances disease severity 127. Conversely, X box-binding protein 1 (XBP1) deficiency in the nervous system delays ALS disease onset and prolongs life span128. These protective effects are due to increased levels of macroautophagy in motor neurons and the consequent clearance of mutant SOD1 aggregates128, which are the primary cause of the disease in this model. In addition, deficiency in the ER stress-induced pro-apoptotic genes apoptosis signal-regulating kinase 1 (Ask1) or BCL-2-interacting mediator of cell death (Bim) delays ALS, possibly by reducing motor neuron loss129,130. These studies illustrate the complex nature of the UPR and how the pathway affects certain diseases, which might depend on the outputs regulated by particular UPR signalling modules. These examples highlight the need for a systematic assessment of the contribution of each major UPR signalling component in diseases caused by protein misfolding, which will help to define optimal targets for therapeutic intervention.

Question marks indicate where the contribution of the indicated UPR component is unknown. ATF, activating transcription factor; ERAD, ER-associated degradation; IRE1α, inositol-requiring protein 1α; JNK, JUN N-terminal kinase; PDI, protein disulphide isomerase.

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AcknowledgementsI apologize to all colleagues whose work could not be cited owing to space limitations. I thank A. Couve, U. Woehlbier and A. Glavic for constructive comments, C. Wirth for editing and D. Rodriguez for input into the initial figure design. This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT), Chile, grant 1100176, Fondo de Investigación Avanzado en Areas Prioritarias (FONDAP), Chile, grant 15010006, Millennium Institute grant P09-015-F the Muscular Dystrophy Association, the Michael J. Fox Foundation for Parkinson Research, the Alzheimer’s Association and the North American Spine Society.

Competing interests statementThe author declares no competing financial interests.

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