Download The unfolded protein response: controlling cell fate

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The unfolded protein response:
controlling cell fate decisions under
ER stress and beyond
Claudio 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.
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.
e-mails:
[email protected];
[email protected]
doi:10.1038/nrm3270
Published online
18 January 2012
1
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 particularl­y 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 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 fluctuations in unfolded protein load3,4. When cells undergo
irreversible ER stress5, this pathway eliminates damaged
cells by apoptosis, indicating the existence of mechanisms that integrate information about the duration and
intensit­y of stress stimuli.
Although the UPR is classically linked to proteinfolding 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 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
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a
Phosphorylation
b
c
ER lumen
ER stress
ER stress
Cytosol
IRE1α
PERK
TRAF2
‘Alarm stress
pathways’
NF-κB
JNK
ER stress
mRNA
Ribosome
mRNA degradation (RIDD)
XBP1u mRNA
ATF6
COPII
NF-κB ?
α
β γ
eIF2α
NRF2
Intron
α
β γ
S2P
S1P
Golgi
Translation
XBP1s mRNA
ATF4
• Autophagy
• Apoptosis
• Co-translational degradation
• ERAD
• Folding
• Lipid synthesis
ATF6f
UPR target genes
4
UPR target genes
AT
F
XB
P1
s
XBP1s
ATF6f
UPR target genes
• 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
Nature
Reviews | Molecular
Cell Biology
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 prosurviva­l 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 homeo­stasis 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
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ATF6
ATF6f
XBP1s
ER stress
IRE1α
PERK
JNK
eIF2α
Adaptive responses Apoptosis phase
?
Folding,
Caspase 2
ERAD,
quality control,
ER biogenesis,
autophagy
RIDD
RIDD survival genes
Translation
?
p53
CHOP
ATF4
Folding,
redox,
autophagy
BID
BAX, BAK
Apoptosis
?
PTP
BH3-only
BCL-2
GADD34
?
IP3R
Ca2+
ROS translation
Intensity
of stress
Time of exposure to stress
Figure 2 | Cell fate decisions under ER stress. Distinct unfolded protein response (UPR)-related responses are observed
Nature Reviews | Molecular Cell Biology
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.
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)8–10. Macroautophagy, a bulk degradation
pathway, is also activated by ER stress, possibly to
elimin­ate damaged ER (a process termed ER‑phagy)
and abnormal protein aggregates through the lysosomal
pathway 11. Finally, pre‑emptive quality control12 and cotranslational 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 geneexpression response through the regulation of at least
three distinct UPR transcription factors. Each stress
senso­r uses a unique mechanism to promote the activatio­n
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 transcription factor X box-binding protein 1 (XBP1), excising a
26‑nucleotide-long intron that shifts the coding readin­g
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.
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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 elemen­tbinding 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 expression of several microRNAs, which may contribute to the
attenuation of protein translation or protein synthesis24.
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).
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.
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 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 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 famil­y
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 mechanism linking ER stress with p53 activation is unclear.
Many other complementary mechanisms are proposed
to induce cell death under excessive ER stress, includin­g
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 mediators through RIDD8. 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
bi­directional 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 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
macro­autophagy 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 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, respectively 3. 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 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.
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a Mammalian UPR
ER lumen
BIP
Misfolded
protein
BIP
Low stress
BIP
?
BIP
Signalling attenuation
High stress
Cytosol
IRE1α
XBP1 mRNA
splicing
b Yeast UPR
ER lumen
mRNA decay
Unfolded or
misfolded
proteins
Bip
Bip
Bip
Bip
Cytosol
Ire1
Phosphorylation
n
Cluster
formation
Attenuation
Buffering
HAC1 mRNA
splicing
Time
Nature Reviews
Molecular Cell
Figure 3 | The stress-sensing mechanism and kinetics
of IRE1| signalling.
a | InBiology
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.
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 studie­d
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
autophosphoryl­ation 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 activation 35.
Accordingly, under ER stress conditions, BIP preferen­
tially 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, 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 recognize 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 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 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 inter­action of
misfolded proteins with its MHC‑I-like groove to trigger 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 interact with unfolded proteins32 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 PERK44.
Furthermore, a systematic analysis of UPR signalling
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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 content was depleted by inhibiting the ER calcium pump
sarco­endoplasmic reticulum calcium ATPase (SERCA).
However, IRE1α responded faster to reducing agents
than calcium alterations, whereas PERK showed similar kinetics of activation by both ER perturbations45.
A recent study also proposed that ATF6 is selectively
activated by ER membrane protein load46 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 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 activation. 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 activation occurs in the same selective manner to inhibit the
ribosom­e to block local translation.
Pancreatic β-cells
Cells in the pancreas that make
and secrete insulin to respond
to glucose fluctuations.
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 under­going
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 interchangeable50, 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 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 prolonged stress is predicted to ablate the pro-survival
outcomes of XBP1s expression, whereas sustained
PERK signalling favours the upregulation of many proapoptoti­c components. By contrast, in other experimental 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 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
mitigation53–55. 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 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 transduce. 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 proapoptotic genes, such as CHOP and growth arrest and
DNA damag­e-inducible 34 (GADD34; also known as
PPP1R15A)56. Furthermore, changes indicated that
IRE1α signalling outputs might differ depending on the
oligo­merization 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 oligo­merization8. 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 phosphorylation per se, are important for its activation.
Additional phosphorylation events may subsequently
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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 sensor­s
coordinated to generate different modes of signalling? Furthermore, how do UPR sensors integrate the
intensit­y 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 kinetic­s 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 inter­actome
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.
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.
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 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 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 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 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 thres­hold of stress
needed to engage downstream responses in differen­t
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 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
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 mechanisms 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 indicate that PERK and ATF6 can be individually modulated
by specific factors. p58IPK directly interacts with PERK,
inhibiting its kinase activity 72,73 (FIG. 4B). As p58IPK expression 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 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 proteasomedependen­t 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
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A
B
ER lumen
C
BIP
BIP
BIP
BIP
ER stress
UPRosome
BIP
CRT
Gly
ER stress
CRT
BIP
Gly ER stress
S
S
SH
SH
WFS1
Cytosol
PERK
IRE1α
Cofactors
and adaptors:
BAX, BAK, AIP1,
HSP72 and
MAPK-related
proteins
Inhibitors: BI-1,
RACK1 and PP2A
Acetylation
Sumoylation
XBP1s
XBP1u
p58IPK
CN CN
CN
α
β γ
eIF2α
α
β γ
eIF2α
p85α
Translation
p38
GRP78VA
GADD34
PP1C
Translation
ATF4
Phosphorylation
ATF6
CHOP
PERK
Golgi lumen
NF-Y, YY1,
TBP and
XBP1s
ATF6f
ATF6f
Da
Db
Intron
XBP1u
Basic
ZIP
XBP1s
Basic
ZIP
Dc
ER lumen
HR
HR TP
TP
Transactivation
domain
XBP1u
mRNA
XBP1u
protein
Cytosol
TP
XBP1s
mRNA
Ribosome
IRE1α
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
Nature Reviews | Molecular Cell Biology
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 GPR78VA. 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 targe­t 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
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 stimulu­s, as well as the fine-tuning of the signalling response according to the cell’s need, through the
assembl­y 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,
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during its translation it drags the ribosome–mRNAnascen­t chain to the ER membrane through a highly conserved 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 recognitio­n of XBP1
mRNA by IRE1α82 (FIG. 4D).
The activity of XBP1s is not only increased by its
interaction with different partners, but also by posttranslational 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, 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 degradation88. 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 resume89. This regulatory loop can be modulated by specific drugs to alleviate ER stress, with great
thera­peutic 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 posttranslationa­l modification23. 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 mechanism92.
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
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 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 response94,95. 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 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 concentrations of glucose, which enables it to control in­sulin
levels96. 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 BIP release from IRE1α97. 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.
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Stimuli
LPS
Inputs
TLRs
Transducers
Outputs
Adaptor
IRE1α–XBP1
Cytokine secretion
(TIRAP and MYD88)
ATF6 and PERK–CHOP
Cell types
Macrophage
Glucose fluctuation,
obesity
Lipids,
glucose
IRE1α-P–RACK1–PP2A
Pancreas
Differentiation signal
B cell
receptor
IRE1α–XBP1
Differentiation signal
?
XBP1
Metabolic requirements
?
IRE1α–XBP1, ATF6 and PERK
Lipid and/or cholesterol Liver
biosynthesis
Differentiation signal
?
XBP1
MIST
Differentiation,
skeletal myotubules
Muscle
BDNF
TRKB,
p75
?
IRE1α–XBP1
CHOP
Neurite outgrowth
Brain
Insulin secretion
IRS1
Insulin resistance
Liver
IRF4 and BLIMP1
Ig secretion
B cells
CXCL12 and
CXCR4
MIST
Bone marrow
colonization
Differentiation,
zymogen granule
biogenesis
Muscle,
gastric cells
Figure 5 | Novel physiological outcomes of the UPR. Distinct cellular inputs activate unfolded protein response (UPR)
Reviews
| Molecularand
Cellcell
Biology
components to transduce signals that affect a wide range of processes, includingNature
metabolism,
inflammation
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.
Exocrine pancreas
A 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 pancreas
The 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 diseases, such as type 2 diabetes, insulin resistance and
obesity 96. Insulin resistance in the liver also involves
the activation of IRE1α. This is possibly due to signalling 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
metaboli­c 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 secretion, 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 proteinfolding 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 normally 107. XBP1 was
shown to be a crucial component of differentiation programmes 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 recombination 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
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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
Embryonic lethal
Yes
Yes
No*
No
No‡
Postnatal death
–
–
No
Yes
No
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
Full knockout
Tissue-specific effects
||
*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.
‡
granule biogenesis to allow digestive enzyme secretion105. Remarkably, a study performed in chondrocytes, which are specialized cells that secrete collagen,
showed that chronic ER stress has a distinct consequence beyond apoptosis109. By analysing hypertrophic
chondro­c ytes 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 studies 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
pathway 110. Similarly, ATF6 and IRE1α function in lipid
metabolism in the liver 96,111,112. In addition, ER stress
regulates the synthesis of the peptide hormone hepcidine113, which is secreted by the liver to control iron
homeostasis in the body.
XBP1 physically interacts with, and negatively regulates 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. elegans 115. In addition, ER
stress controls gluconeogenic programmes by activating
ATF6, which negatively modulates the activity of CREBregulated 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.
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REVIEWS
Box 2 | Contribution of the UPR to ALS
Endoplasmic reticulum (ER) stress is involved in the
ERAD impairment,
altered traffic,
pathogenesis of several diseases, including cancer,
Sporadic and
ER stress PDI inactivation,
autoimmunity, diabetes, ischaemia reperfusion and
familial ALS
protein aggregation,
neurodegeneration (reviewed in REFS 3,4,118).
autophagy defects
UPR
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
PERK
ATF6
IRE1α
adult-onset disease in which the selective degeneration
of motor neurons leads to paralysis and premature
death. Studies using genetic manipulation of the
ATF4
XBP1
ASK1–JNK
eIF2α
unfolded protein response (UPR) and pharmacological
approaches have revealed a complex involvement
of the pathway in ALS, illuminating distinct outputs of
Degeneration Degeneration Protection
?
?
specific UPR signalling branches in the same disease125
(autophagy)
(apoptosis)
(translation)
(see the figure). Mutations in the gene encoding
superoxide dismutase 1 (SOD1) cause familial ALS, and
Nature Reviews
| Molecular
Cell Biology
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.
Perspective
This Review discussed, in detail, the main mechanisms 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 downstream responses by binding cofactors and by recruiting adaptor and signalling proteins. UPR sensors may
have the intrinsic ability to orient the functional output­s
of the pathway, in a highly regulated and ‘custom’
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Most of the UPR-regulating events may help to adjust
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Acknowledgements
I 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 statement
The author declares no competing financial interests.
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