Download Surviving protein quality control catastrophes – from cells to organisms

© 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
COMMENTARY
Surviving protein quality control catastrophes – from cells to
organisms
Kim Schneider and Anne Bertolotti*
Organisms have evolved mechanisms to cope with and adapt to
unexpected challenges and harsh conditions. Unfolded or misfolded
proteins represent a threat for cells and organisms, and the deposition
of misfolded proteins is a defining feature of many age-related human
diseases, including the increasingly prevalent neurodegenerative
diseases. These protein misfolding diseases are devastating and
currently cannot be cured, but are hopefully not incurable. In fact, the
aggregation-prone and potentially harmful proteins at the origins of
protein misfolding diseases are expressed throughout life, whereas
the diseases are late onset. This reveals that cells and organisms are
normally resilient to disease-causing proteins and survive the threat
of misfolded proteins up to a point. This Commentary will outline the
limits of the cellular resilience to protein misfolding, and discuss the
possibility of pushing these limits to help cells and organisms to
survive the threat of misfolding proteins and to avoid protein quality
control catastrophes.
KEY WORDS: Protein quality control, Proteostasis, Stress
responses
Introduction
Cells are well equipped to survey and maintain the health of
their proteomes; they employ chaperones that bind to non-native
polypeptides to prevent aggregation and to facilitate the folding
of proteins, as well as degradation systems – the ubiquitinproteasome system (UPS) and autophagy – to degrade abnormal
or damaged proteins. These diverse components of the protein
quality control systems act in a concerted manner to prevent the
accumulation of damaged or misfolded proteins and/or to promote
their elimination. Because it is vital that cells avoid damage to
proteins, cells not only keep an abundant supply of chaperones and
protein degradation machineries but they have also evolved the
ability to increase the abundance of the diverse components of the
cellular defense systems against misfolded proteins when the need
arises. These protein quality control systems have overlapping
functions and are evolutionarily conserved, indicating that the
evolutionary pressure to maintain protein homeostasis justified
the cost of these elaborate pathways. The doubling of human life
expectancy in the last 200 years (Finch, 2010) has resulted in a rise
in the number of cases of age-related diseases. These diseases might
have arisen as we reached the limits of our evolutionarily optimized
protein quality control systems, because we have not had the time
required to evolve effective defense mechanisms against age-related
diseases. Therefore, identifying strategies to push the limits of
protein quality control systems could reveal pathways to prevent
MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, CB2
0QH, UK.
*Author for correspondence ([email protected])
age-related diseases. In this Commentary, we will give an overview
of the cellular protein quality control systems, discuss how their
failure might lead to disease and how we can exploit these natural
cellular defense systems against misfolded proteins to survive
experimental and pathological protein quality control catastrophes.
Resilience to the threat of misfolded proteins
Life is harsh, and misfolded proteins represent a threat to proper cell
and organism function. To survive this threat, organisms had to
acquire resilience to misfolded proteins and have evolved an ability
to fight harsh challenges (Fig. 1). An overview of the different
cellular defense systems against misfolded proteins is presented
below.
Molecular chaperones and the heat-shock response
Studying the cellular responses to the harsh stresses that cause
protein damage has revealed the mechanisms by which cells have
acquired their resilience to such damages. Exposing cells to heat
shock provides an ideal experimental system to identify protein
chaperones because heat denatures existing proteins, leading to an
overwhelming increase in the exposure of hydrophobic sequences.
As a defense mechanism against the threat of protein aggregation,
cells induce the expression of chaperones in order to neutralize the
denatured proteins. Chaperones are a group of diverse proteins that
interact with non-native intermediates to prevent their aggregation
and facilitate their folding (Bukau et al., 2006; Kim et al., 2013).
The expression levels of many chaperones upon heat shock increase
to satisfy the increased demand that is generated by the heatmediated denaturation of proteins (Lindquist and Craig, 1988;
Morimoto, 2012), and many chaperones were therefore initially
described as heat-shock proteins (HSPs). The master regulator of the
heat-shock response is HSF1, a transcription factor that is normally
in an inactive conformation and bound to molecular chaperones
(Morimoto, 2012). When folding is compromised, HSF1 is
activated and forms trimers that bind to the heat-shock element in
the promoter region of heat-shock-responsive genes (Fig. 2), which
includes those encoding chaperones, to activate their transcription
(Morimoto, 2012).
The unfolded protein response
Similar to the response in the cytosol, conditions that impair the
folding of proteins in the endoplasmic reticulum (ER) create a stress
(ER stress) to which cells adapt by mounting a response known as
the unfolded protein response (UPR), aimed at restoring protein
homeostasis in the ER (Wiseman et al., 2010) (see Fig. 2).
Components of the mammalian UPR have been discovered by
studying how cells react to ER stress, a condition elicited
experimentally by treating cells with drugs that block ER
function, such as tunicamycin and thapsigargin. Tunicamycin
blocks N-linked glycosylation and thereby prevents the folding
of a large fraction of ER proteins, whereas thapsigargin, a
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ABSTRACT
Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
Homeostasis
Stress
Adaptation to restore homeostasis
Fig. 1. Principles of cellular resilience. Organisms have evolved
mechanisms to cope with and adapt to unexpected challenges and harsh
conditions. A condition that perturbs cellular homeostasis represents a stress.
To survive stress, cells induce signaling pathways ( plain lines) to neutralize the
perturbation and restore cellular homeostasis. Once homeostasis is restored,
stress signaling is terminated (dashed lines, negative feedback).
non-competitive inhibitor of the ER Ca2+ ATPases (ATP2A1,
ATP2A2 and ATP2A3), perturbs ER function. The UPR did not
evolve to protect cells from tunicamycin or thapsigargin. However,
because these two drugs are potent inducers of the UPR, they
have been instrumental in discovering the mammalian UPR.
One component of the UPR, inositol-responsive enzyme 1 (IRE1;
encoded by ERN1), is an ER-resident transmembrane protein
that is conserved from yeast to mammals and senses stress in the
ER to increase the transcription of genes encoding ER-resident
chaperones (Mori, 2000). In addition to IRE1, metazoans have
two additional ER-stress transducers – protein kinase RNA-like
endoplasmic reticulum kinase (PERK; encoded by EIF2AK3) and
activating transcription factor 6 (ATF6) (Mori, 2000). IRE1 and
PERK sense protein misfolding in the ER lumen and convey this
signal to their effector domain that is located on the other side of the
ER membrane through oligomerization (Bertolotti et al., 2000). The
three mammalian UPR sensors IRE1, ATF6 and PERK are kept
inactive in unstressed cells in a complex with the ER chaperone
binding immunoglobulin protein (BiP or GRP78; encoded by
HSPA5), which dissociates upon stress, leading to activation of the
sensors (Bertolotti et al., 2000). A popular interpretation given to
these findings is the idea that misfolded proteins compete and
sequester BiP away from IRE1 and PERK (Liu et al., 2000;
Okamura et al., 2000). However, this interpretation disagrees with
the abundance of the ER-stress signaling proteins relative to that of
BiP (there might be 1000 more BiP than IRE1 and PERK
molecules) and with the observed stability of the BiP–IRE1 and
BiP–PERK complexes (Bertolotti et al., 2000). So how does it
work? This long-standing question has taken fifteen years to answer
and required the reconstitution of the BiP-mediated UPR-sensing
mechanism in vitro with purified components (Carrara et al., 2015).
It has been shown that BiP binds to the luminal domain of IRE1 and
PERK through its ATPase domain, leaving the substrate-binding
domain of BiP free to sample and bind to misfolded proteins
(Carrara et al., 2015), in agreement with earlier findings (ToddCorlett et al., 2007). The binding of unfolded proteins to BiP then
triggers a conformational change that is transduced from the
substrate-binding site of BiP to the ATPase domain, resulting in the
dissociation of BiP, and activation of IRE1 and PERK (Carrara
et al., 2015). Thus, UPR sensors have exploited the substratebinding site of BiP, which has a high affinity for unfolded proteins,
to survey and to respond to perturbations of folding in the ER. Like
mammalian IRE1, the luminal domain of yeast IRE1 also binds to
BiP, which dissociates upon stress (Okamura et al., 2000), but the
importance of BiP dissociation in activating the yeast UPR has been
debated (Kimata et al., 2004, 2007; Pincus et al., 2010). In addition,
the luminal domain of yeast IRE1, unlike that of mammalian IRE1
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(Oikawa et al., 2009), directly binds to unfolded proteins (Kimata
et al., 2007; Gardner and Walter, 2011). Once activated, the three
mammalian ER-stress transducers act in a coordinated manner to
restore ER homeostasis, not only by increasing the expression
of genes encoding chaperones – i.e. the ancestral branch of the
UPR – but also by reducing global protein synthesis, which
constitutes a rapid adaptive response to stress (Fig. 2). This is
mediated by PERK, which, in response to the folding perturbation
in the ER or simply when the folding supply does not match the
demand, phosphorylates the α subunit of eukaryotic translation
initiation factor 2 (eIF2α) on residue Ser51 to reduce translation
initiation (Wiseman et al., 2010; Cao and Kaufman, 2012). This
represents a first line of defense against the threat of protein
misfolding. Reducing translation, in turn, favors protein folding by
increasing the availability of chaperones (Tsaytler et al., 2011),
probably because the chaperones that normally assist in the
synthesis of new proteins become available when protein
synthesis rates are decreased. Likewise, an immediate response to
heat stress is also to reduce global protein synthesis (Richter et al.,
2010); however, surprisingly, the molecular basis for the reduced
protein synthesis in response to heat shock still remains unclear.
The integrated stress response
Phosphorylation of eIF2α and the resulting reduction in protein
synthesis is a first line of defense that is essential for survival in
response to many forms of stress. This signaling downstream of
eIF2α is therefore referred to as the integrated stress response (ISR)
(Harding et al., 2003). Viral infection and heme-deficiency signal to
the eIF2α kinases PKR (EIF2AK2) and HRI (EIF2AK1),
respectively, to result in the attenuation of protein synthesis (Ron
and Harding, 2007). The shortage of amino acids is a stress that is
sensed by the eIF2α kinase GCN2 (EIF2AK4), and activation of this
kinase also results in the attenuation of protein synthesis (Sonenberg
and Hinnebusch, 2009). When eIF2α is phosphorylated, global
protein synthesis is attenuated, thereby sparing amino acids, which is
a rapid and adaptive response conserved from yeast to mammals
(Sonenberg and Hinnebusch, 2009). However, a few selective
transcripts are preferentially translated under these conditions. One
encodes the transcription factor ATF4, which controls the expression
of genes that are involved in amino acid import and biosynthesis
(Sonenberg and Hinnebusch, 2009). The fact that many forms of
stress converge on the ISR, resulting in increased expression of genes
that are involved in amino acid metabolism, indicates that
perturbation of amino acid homeostasis might be a problem that is
common to many forms of stresses (Fig. 2). This idea was
highlighted with the finding that proteasome inhibition causes a
shortage in amino acids, a stress to which cells react to by inducing
the ISR and autophagy, in an attempt to restore amino acid
homeostasis (Suraweera et al., 2012). Downstream of ATF4 is the
transcription factor CHOP (encoded by DDIT3), which, in turn,
induces transcription of PPP1R15A. PPP1R15A is one of two
regulatory subunits of eIF2α phosphatases in mammals. PPP1R15A
recruits one of the serine/threonine protein phosphatase 1 catalytic
subunits (PP1c) in stressed cells to dephosphorylate eIF2α, thereby
terminating stress signaling (Novoa et al., 2001).
The mitochondrial UPR
Similar to the ER, mitochondria also adapt their supply of
chaperones and proteases by mounting a mitochondrial UPR
when needed (Pellegrino et al., 2013). The mitochondrial UPR
comprises a mitochondria-to-nucleus signaling pathway that senses
perturbation of homeostasis in the mitochondria, and the UPR
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COMMENTARY
COMMENTARY
Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
Stresses
BiP
Proteasome levels
and activity
BiP
ER
Amino acid levels
BiP
Autophagy
Translational
control
Translation rates
PKR
Regulated
mRNA
splicing
Regulated
proteolysis
ATF6
IRE1
PERK
HRI
mTOR
GCN2
Golgi
Splicing
Protein folding
capacity
eIF2α P
R15B
PP1c
Xbp1
eIF2B
ATF4
Ribosomal subunits
and biogenesis
Nucleus
Proteasome subunits
HSF1
HSF1
HSF1
HSP
R15A
PP1c
Chop, Ppp1r15a,
redox and metabolism
Amino acid transporter
and biosythesis
ER chaperones, ERAD and
lipid biosythesis
XBP1s
ATF6
Mitochondria
ER chaperones, Xbp1
Mitochondrial chaperones
Fig. 2. Overview of the mammalian cellular defense systems against misfolded proteins. The proteasome system and autophagy are the two cellular
degradation systems that degrade proteins and recycle amino acids, thereby contributing to both protein and amino acid homeostasis ( purple, see Fig. 1).
Signaling through mTOR and the integrated stress response (ISR) adjusts translation rates according to nutrient availability and stress. When the ISR is activated,
through GCN2, HRI, PKR or PERK, eIF2α is phosphorylated. This reduces the global rates of protein synthesis, thus sparing amino acids. Slowing down
translation increases chaperone availability and protein folding. Protein folding perturbation in the cytosol activates HSF1, a transcription factor activating
expression of HSP. Phosphorylation of eIF2α also enables the translation of ATF4, a transcription factor that controls the expression genes involved in amino
acid metabolism and transport. PPP1R15A (R15A in the figure), downstream of ATF4, is a regulatory subunit of the protein phosphatase 1 (PP1c). PPP1R15A–
PP1c dephosphorylates eIF2α to terminate stress signaling when protein and amino acid homeostasis is restored. PPP1R15B (R15B)-PP1c is the constitutive
eIF2α phosphatase. The mammalian unfolded protein response (UPR) has three branches. The PERK branch is shared with the ISR, whereas the IRE1 and
ATF6 branches are selectively activated upon perturbation of protein folding in the ER. Activated IRE1 leads to the splicing of XBP1 mRNA (XBP1s), which
encodes a bZIP transcription factor that regulates UPR target genes. Dissociation of BiP from ATF6 results in ATF6 trafficking to the Golgi where it is cleaved by
the site-1-protease (S1P) and site-2-protease (S2P), with release of the 50 kDa cytosolic domain of ATF6, a transcription factor controlling UPR genes. IRE1 and
ATF6 fight stress by increasing the transcription of genes that are involved in maintaining ER homeostasis, such as genes encoding ER chaperones. Likewise,
protein homeostasis in the mitochondria is maintained through a mitochondrial UPR. Solid line, forward signaling; dashed lines, negative feedback.
responds by adjusting the expression of mitochondrial chaperones
and proteases in the nucleus, a pathway that has been best
characterized in Caenorhabditis elegans (Pellegrino et al., 2013).
Finley, 2007). In mammals, the transcription factor erythroidderived 2-related factor 1 (Nrf1; also known as NFE2L1) adjusts the
expression of proteasome subunits to meet the cellular needs
(Radhakrishnan et al., 2010).
Adapting protein degradation to the demand
The proteasome
When the demand for protein degradation exceeds the proteolytic
capacity, cells increase the expression levels of proteasome subunits
in a concerted manner (Hanna and Finley, 2007). In yeast, this is
controlled by Rpn4, a transcription factor that regulates the
expression levels of proteasome subunits through a homeostatic
negative-feedback loop (Xie and Varshavsky, 2001). Rpn4 is an
unstable protein, which is normally rapidly degraded but
accumulates when the proteasome is overwhelmed (Hanna and
Autophagy
Autophagy is also a tightly regulated process and was initially
discovered as a response to starvation (Nakatogawa et al., 2009).
When the supply of nutrients is sufficient, non-selective autophagy
is repressed. This repression is under the control of a serine/
threonine protein kinase, mammalian target of rapamycin (mTOR)
(Zoncu et al., 2011). However, mTOR is much more than the
molecular switch for autophagy. This kinase is in fact a central
regulator of cellular metabolism and senses nutrients, growth factors
and energy levels, and adjusts metabolic processes depending on the
conditions. When the supply of nutrients is sufficient, mTOR
promotes anabolic processes, including protein synthesis as well as
ribosome biogenesis, while repressing autophagy. Under conditions
that are unfavorable for cell growth – during stress or in the presence
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In parallel to the heat-shock response and the UPR, cells also have
the ability to increase the abundance of the two cellular degradation
pathways – the proteasome system and autophagy – to match any
arising need.
of the drug rapamycin – mTOR is inhibited (Zoncu et al., 2011). As
a result, autophagy is induced and, concomitantly, translation and
ribosome biogenesis are repressed.
Cross-talk between proteasomal degradation, autophagy and amino acid
metabolism
When proteasome degradation is compromised, cells adapt by
inducing autophagy, and cross-talk events between the two cellular
degradation pathways have been identified (Korolchuk et al., 2009).
Both pathways contribute not only to protein homeostasis but also to
amino acid homeostasis. Under conditions of amino acid starvation,
both autophagy and proteasomal degradation are required to
maintain adequate amino acid levels in order to sustain protein
synthesis (Onodera and Ohsumi, 2005; Vabulas and Hartl, 2005). In
the absence of starvation, under normal conditions, proteasomal
degradation contributes an important fraction of the intracellular
pool of amino acids (Suraweera et al., 2012). Proteasome inhibition
results in a lethal amino acid shortage, and this is the signal that
leads to autophagy induction as an adaptive response that aims to
restore homeostatic levels of amino acids (Suraweera et al., 2012).
From stress to cellular homeostasis – need more, make more
Many components of the cellular defense systems have been
identified by studying how cells respond to stress. This has
conveyed, erroneously, the notion that the cellular defense systems
against misfolded proteins are only required under stress. But this is
not the case. The ensemble of sophisticated pathways (Fig. 2) that
ensure resilience to protein damage are evolutionarily conserved
and are needed at all times to maintain protein homeostasis. This is
because the damages that occur during harsh stress also exist under
non-stress conditions, albeit to a lower magnitude. As discussed
above, heat stress denatures proteins and leads to an increased
abundance of unfolded proteins; this condition also normally occurs
during the synthesis of proteins when nascent chains are unfolded.
Cells have acquired their resilience and gained their strength by
surmounting difficulties (harsh stresses). As a result, cells normally
thrive at maintaining protein homeostasis when milder stresses are
continually encountered.
According to this model, cell fitness is not defined by an absence of
errors, problems or failure but by an ability to cope and deal with
errors, problems or failure. Consequently, cell fitness is lost when
cells can no longer cope with failures. This is an important problem, at
the origin of a broad range of diseases, which will be discussed below.
Protein misfolding diseases – beyond the limits of the cellular
resilience to protein damage
A broad range of age-related human diseases, including common
and devastating neurodegenerative diseases, are caused by the
deposition of misfolded proteins. These diseases include
Alzheimer’s disease, Parkinson’s disease, Huntington’s disease,
amyotrophic lateral sclerosis (ALS) and prion diseases. Although
clinically diverse, these diseases share a common pathological
hallmark – they are caused by the progressive dysfunction and death
of specific nerve cells in selective regions of the brain or the
peripheral nervous system, which are the result of the accumulation
of specific proteins of aberrant conformations (Soto, 2003). The
major component of each disease-characteristic deposit has been
identified, in most cases over two decades ago, and this has revealed
that there are no common features amongst the different diseasecausing proteins; they have different primary sequences and their
native folds are also distinct (Chiti and Dobson, 2009). Some are
tightly folded, such as superoxide dismutase 1 (SOD1) – the faulty
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proteins in some familial forms of ALS (Valentine et al., 2005) –
whereas others are natively unstructured, such as α-synuclein – the
major component of Lewy bodies in Parkinson’s disease (Goedert
et al., 2013). However, although extremely diverse, the diseasecausing proteins share one common feature – they are usually
soluble but have a propensity to misfold and aggregate.
It is now clear that neurodegenerative diseases are caused by the
gain of toxicity that is associated with the misfolding propensity of a
disease-causing protein; however, the pathogenic cascades that lead
from the misfolding of a protein to neurodegeneration remain
largely unknown, despite years of extensive research efforts. This
represents a complex organismal problem, which, in fact, results
from a cell biology problem that arises when cells become unable to
withstand the pressure of misfolded proteins. Thus, the question
becomes why proteins, which are normally soluble, eventually
misfold and aggregate late in life. As discussed above, cells
normally strive to ensure that proteins are correctly folded and have
evolved powerful and sophisticated mechanisms to maintain protein
homeostasis ( proteostasis) (Balch et al., 2008). Protein quality
control systems are normally very efficient at maintaining protein
homeostasis over several decades of life. However, the fact that
protein aggregates build up later in life suggests that the cellular
defense systems against misfolded proteins gradually fail with age,
resulting in the accumulation of misfolded proteins with devastating
consequences for cells and organisms (Morimoto and Cuervo,
2014; Vilchez et al., 2014). If cells and organisms are able to cope
with potentially harmful proteins for decades, perhaps identifying
strategies that boost the cellular defense systems against misfolded
proteins could have some value for the development of therapeutics
against the diverse misfolding diseases.
Boosting cellular defense systems against misfolded
proteins
In the past 20–30 years, many components of protein quality control
systems have been identified, and we have learned, often in great
detail, how protein quality control operates in cells. The challenge
that remains is to use this knowledge to identify strategies to correct
the broad range of diseases that arise when protein quality control is
overwhelmed. Attempts to manipulate protein quality control
pathways with small molecules have been made on several levels.
For instance, the search for inducers of HSF1 is an actively pursued
line of research because HSF1 is a master regulator of the expression
of chaperones – key components of the cellular defense systems
against misfolded proteins. Activating HSF1 mimics stress by
inducing a heat-shock response, thereby increasing chaperone
expression. Because chaperones neutralize misfolded proteins,
induction of HSF1 could be an approach to combat protein
misfolding diseases. Geldanamycin is an inhibitor of HSP90
proteins and induces the heat-shock response by activating HSF1
(Neckers and Workman, 2012). More recently, other inducers of
HSF1 have been described (Calamini et al., 2011).
Furthermore, given that kinases are popular drug targets, the UPR
kinases IRE1 and PERK have also been targeted by small molecule
inhibitors. Because stress responses are essential for cell survival,
inhibitors of stress signaling pathways are predicted to be
deleterious, a property that could be advantageous for the
discovery of cancer drugs. This possible effect on cell survival
has motivated the development of inhibitors of PERK (Axten et al.,
2012) and IRE1 (Cross et al., 2012; Ghosh et al., 2014), and of the
integrated stress-response inhibitor ISRIB, which inhibits the
signaling downstream of eIf2α phosphorylation (Sidrauski et al.,
2013). Recent and comprehensive reviews on the pharmacological
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Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
Homeostasis
Misfolded
proteins
Protein quality
control systems
Protein quality control failure
Misfolded
proteins
Protein quality
control systems
Rescue from protein quality
control failure
Misfolded
proteins
Protein quality
control systems
Young
Late-onset protein misfolding diseases
Cure
Viable
Lethal
Viable
manipulation of stress responses are available elsewhere (Hetz
et al., 2013; Maly and Papa, 2014). The next section will only
focus on strategies to rescue cells from protein quality control
catastrophes.
Identifying strategies to survive failure of protein quality
controls
Despite our good understanding of many protein quality control
pathways, one of the problems impeding the development of
targeted therapies lies in the fact that it is currently impossible to
predict which manipulations of the protein quality control pathways
will be tolerable, detrimental or beneficial. For example, PERK
inhibitors as well as ISRIB were developed to kill cancerous and
stressed cells, respectively (Atkins et al., 2013; Sidrauski et al.,
2013). In addition to their predicted cytotoxic properties, PERK
inhibitors and ISRIB have been found to protect mice from prion
diseases (Halliday et al., 2015; Moreno et al., 2013). These results
could not be anticipated from 15 years of intensive explorations of
the pathway. Thus, pharmacological manipulations of stressresponse pathways bring surprises.
Because it is impossible to predict the outcomes of
pharmacological intervention of stress-response pathways, we
have focused on identifying unbiased approaches that can be used
to rescue cells from protein quality control catastrophes with the
view that if we manage to ‘cure’ cells from such profound defects,
this might also be feasible in an organism (Fig. 3). Such strategies
are appealing because they hold the promise to ameliorate the broad
range of diseases that arise when protein quality control systems are
overwhelmed.
In searching for an experimental system in which to identify such
strategies, we have chosen to mimic experimentally the protein
quality control failures that might arise during aging or in diseases
by blocking key components of protein quality control systems
(Fig. 3). Such perturbations are brutal and usually fatal to cells, and
thereby provide robust and unbiased experimental conditions that
can be used to search for strategies to help cells survive under such
catastrophic circumstances. Recently, we have identified a number
of approaches that have not only revealed interesting pathways that
might have therapeutic benefits but that have also helped to identify
new components of protein quality control systems.
Surviving proteasome failure
The proteasome is essential for the degradation of many cellular
proteins. Consequently, dysfunction of the ubiquitin-proteasome
system (UPS) is associated with a broad range of diseases, including
cancer and neurodegeneration (Tanaka and Matsuda, 2014). We use
inhibition of the proteasome as an experimental model of global
perturbation of protein quality control that could be relevant to
human diseases. Aiming to identify strategies to help cells survive
Fig. 3. Rescuing protein quality
control failure. Cells have efficient
protein quality control systems that
maintain protein homeostasis and cell
viability. With age, quality control
gradually seems to fail, leading to the
accumulation of misfolded proteins.
Identifying strategies to survive protein
quality control failure might help to
prevent the broad range of age-related
diseases that are associated with the
accumulation of misfolded proteins.
proteasome degradation failure, we found that the deleterious
consequences of proteasome inhibition can be rescued through
amino acid supplementation in yeast, mammalian cells and
Drosophila (Suraweera et al., 2012). This demonstrates that cells
can tolerate large amounts of undesired proteins but not the amino
acid scarcity that results from proteasome inhibition (Suraweera
et al., 2012). Thus, proteins that accumulate in cells upon
proteasome inhibition appear to be deleterious largely because
they sequester a pool of amino acids that would normally be recycled
(Suraweera et al., 2012). These findings reveal that the proteasome is
not just a waste disposer but is actually vital as an amino-acidrecycling machine (Fig. 2). Encouraged by these findings, which
reveal a previously unappreciated aspect of proteasome degradation,
we continued to search for strategies that rescue cells when the
proteasome fails. Using an unbiased screen in yeast, we asked: can
we identify genes that, when overexpressed, rescue the viability of
the yeast cells that are defective for proteasome degradation? The
screen was very stringent and led to the identification of (only) three
potent suppressors of the proteasome defects of a Rpt6
thermosensitive mutant, two of which had been identified
previously (Rpt6 and Rpn14) and one encoding a previously
uncharacterized protein that we named Adc17 (encoded by TMA17).
Because this newly identified suppressor was very potent, we could
elucidate its function and found that Adc17 is a proteasomeassembly chaperone whose expression is increased upon
proteasome stress (Hanssum et al., 2014).
As mentioned above, when the demand for protein degradation
exceeds the proteolytic capacity, cells increase the expression levels
of proteasome subunits in a concerted manner (Hanna and Finley,
2007). Although increasing the levels of proteasome subunits is
necessary to increase proteasome abundance, this is not sufficient
because the levels of functional proteasomes depend not only on the
expression of proteasome subunits but also on their precise and
correct assembly. The proteasome comprises 33 subunits, and its
assembly is an extremely complex and challenging process in the
crowded cellular environment (Beckwith et al., 2013; Murata et al.,
2009; Tomko and Hochstrasser, 2013), but it was unknown how
stressed cells overcome the challenging task of assembling
proteasomes. We found that Adc17 promotes an early and ratelimiting step in the proteasome assembly process, and that its
expression is increased upon proteasome stress to help cells
assemble more proteasomes when the need arises. As a result,
Adc17 is vital for cells so that they can cope with increased demands
for proteasome-mediated degradation (Hanssum et al., 2014).
However, how metazoans cope with overwhelming demands for
proteasome assembly is currently unclear.
The identification of Adc17 through an unbiased suppressor
screen has revealed a new component of the protein quality control
system and provided new insights into the mechanisms by which
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cells maintain proteasome homeostasis. Although this discovery
is still in its infancy, identifying such strategies that can boost
proteasomal degradation might ultimately be of therapeutic value in
order to ameliorate the large number of pathologies associated with
proteasome deficiencies. Using the same concept, pharmacological
inhibition of USP14 has been proposed as an approach to boost
proteasome degradation and reduce the accumulation of unrelated
aggregation-prone proteins in cells (Lee et al., 2010). It will be
interesting to see whether inhibitors of USP14 can prevent the
accumulation of aggregation-prone proteins in mouse models of
neurodegenerative diseases.
Surviving failure of quality control in the ER
Failure to maintain protein homeostasis in the ER is associated with a
broad range of diseases (Hetz and Mollereau, 2014). Therefore,
identifying strategies to help cells survive the deleterious treatment of
tunicamycin could help to identify therapeutic pathways. We
discovered that the small molecule guanabenz rescues HeLa cells
from the otherwise lethal accumulation of misfolded proteins in the
ER upon treatment with tunicamycin (Tsaytler et al., 2011).
Deciphering the mechanisms underlying the cytoprotective effects
of guanabenz shed light on an interesting aspect of cell biology.
Guanabenz protects cells by prolonging translation attenuation,
the first line of defense against the accumulation of misfolded
proteins (Tsaytler et al., 2011). This occurs because guanabenz
selectively binds to and inhibits the regulatory subunit of the
stress-induced eIF2α phosphatase that comprises PPP1R15A and
PP1c, whereas guanabenz does not interact with nor inhibit the
constitutive eIF2α phosphatase PPP1R15B–PP1c, thereby avoiding
persistent eIF2α phosphorylation (Tsaytler et al., 2011). This is very
important because inhibition of the two eIF2α phosphatases leads to
persistent eIF2α phosphorylation and results in persistent inhibition of
protein synthesis, which is lethal (Harding et al., 2009). As a result,
guanabenz increases the availability of chaperones to misfolded
proteins and, consequently, rescues tunicamycin-stressed HeLa cells
from collapse of proteostasis (Tsaytler and Bertolotti, 2012).
The approach of fine-tuning translation to overcome protein
misfolding defects by inhibiting PPP1R15A is very attractive as it
is straightforward, potent and selective, as well as potentially
applicable to a broad range of conditions that are caused by the
accumulation of misfolded proteins. Indeed, the misfolding of
proteins in the ER is associated with many human diseases (Kim
and Arvan, 1998). However, although guanabenz is a selective
inhibitor of PPP1R15A in non-neuronal cells, this selectivity does not
apply in an organismal context because guanabenz is a centrally active
hypotensive drug with a nanomolar affinity for the α2-adrenergic
receptor (Holmes et al., 1983). Guanabenz, initially, was marketed as
a drug for the treatment of hypertension, but its use was associated
with side effects that are a direct consequence of its activity as a α2adrenergic agonist, such as hypotension, respiratory depression,
bradycardia, drowsiness, lethargy and even coma (Hall, 1985).
From experimental to pathological protein quality control
catastrophes
Although guanabenz cannot be used to selectively inhibit
PPP1R15A in vivo, we found that its adrenergic activity is a
separate function from that of the inhibition of PPP1R15A (Tsaytler
et al., 2011), indicating that it might be possible to generate
guanabenz derivatives in which the adrenergic activity is ablated. To
that end, we searched for selective PPP1R15A inhibitors and
synthetized a range of guanabenz derivatives, and identified
Sephin1 (a selective inhibitor of a holophosphatase), which, like
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Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
guanabenz, rescues HeLa cells from cytotoxic tunicamycin
treatment (Das et al., 2015). Sephin1 selectively binds to and
inhibits PPP1R15A to safely prolong the benefit of translation
attenuation following stress. Importantly, translation attenuation
is prolonged only transiently because Sephin1 selectively inhibits
the stress-induced eIF2α phosphatase PPP1R15A–PP1c, but not
the constitutive PPP1R15B–PP1c enzyme, thereby avoiding the
deleterious effects that result from inhibiting both eIF2α
phosphatases (Harding et al., 2009). The cytoprotective activity of
Sephin1 on stressed cells is abolished in cells that lack PPP1R15A,
further confirming that the beneficial effect of Sephin1 on stressed
cells is mediated entirely through inhibition of PPP1R15A (Das
et al., 2015). Although it is closely related to guanabenz, Sephin1
has no α2-adrenergic activity. Like guanabenz, Sephin1
concentrates in the brain and nervous system, but does not cause
any of the side effects that are associated with guanabenz because it
is devoid of α2-adrenergic activity (Das et al., 2015).
Guanabenz also has another activity – it reduces the levels of
prions in yeast and in mammals (Tribouillard-Tanvier et al., 2008a).
Sephin1 is inactive in yeast; indeed, yeast lack both PPP1R15A and
α2-adrenergic receptors, so the activity of guanabenz in yeast is not
mediated by α2-adrenergic receptors or PPP1R15A, and different
mechanisms for the activity in yeast have been proposed
(Tribouillard-Tanvier et al., 2008b).
Having established that Sephin1 is a specific and selective
PPP1R15A inhibitor that could be suitable for in vivo studies,
we have tested whether Sephin1 can prevent protein-misfolding
diseases in mice. For these proof-of-principle studies, we selected
two protein-misfolding diseases for which there is robust genetic
evidence that abnormal signaling through PPP1R15A is involved in
the disease mechanism – Charcot-Marie-Tooth 1B (CMT-1B)
(D’Antonio et al., 2013; Pennuto et al., 2008) and ALS that is due to
mutation in the superoxide dismutase SOD1 (Wang et al., 2014).
We found that oral administration of Sephin1 almost completely
prevents the motor, histological and molecular defects of these two
otherwise unrelated protein-misfolding diseases in mice (Das et al.,
2015).
In humans as well as in mice, mutations in myelin protein zero
(MPZmutant), a transmembrane protein that is produced by Schwann
cells in the peripheral nervous system, causes the misfolding and
ER-retention of the affected protein (Wrabetz et al., 2006). This
results in the demyelinating neuropathy CMT-1B. At the molecular
and cellular level, the pathology at the origin of CMT-1B is
analogous to the perturbation created experimentally by treating
cells with tunicamycin – a massive accumulation of misfolded
proteins in the ER. CMT-1B is an extremely rare disease, but it is
clear that it is a disorder resulting from malfunction of the ER and is
therefore a useful model.
In the case of SOD1-ALS, the link between mutant SOD1 and ER
stress is indirect, yet robust. SOD1 is a cytosolic protein, and mutant
SOD1 proteins have been found to bind to Derlin-1 on the cytosolic
side of the ER membrane, blocking the degradation of ER proteins,
which then accumulate in the ER, thereby causing ER stress
(Nishitoh et al., 2008). Supporting the notion that mutant SOD1
causes ER stress, deletion of one allele of PERK accelerates the
disease progression in an ALS-SOD1 mouse model, whereas nontransgenic mice lacking one allele of PERK are normal (Wang et al.,
2011). These findings reveal a strong link between the ISR and
SOD1-ALS, and are consistent with the established function of
PERK in helping cells to survive protein misfolding stress.
Moreover, genetic inactivation of PPP1R15A markedly
ameliorates the disease (Wang et al., 2014), and we have
Journal of Cell Science
COMMENTARY
COMMENTARY
recapitulated these finding in a different ALS-SOD1 model through
selective pharmacological inhibition of PPP1R15A (Das et al.,
2015).
Journal of Cell Science (2015) 128, 3861-3869 doi:10.1242/jcs.173047
rational therapeutics against a devastating group of diseases that
affect an increasing number of individuals in aging populations.
Acknowledgements
Translation begins with the recognition of the initiation codon AUG
by the ternary complex comprising the GTP-bound form of
eukaryotic initiation factor 2 (eIF2; comprising three subunits, α,
β and γ), the initiator methionyl-tRNA (Met-tRNAi) and the small
(40S) ribosomal subunit to form the 43S pre-initiation complex
(PIC) (Hinnebusch and Lorsch, 2012). GTP in the ternary complex
is hydrolyzed and GDP must be exchanged for GTP before another
round of translation initiation can occur. Phosphorylation of eIF2α
renders the interaction between the guanine nucleotide exchange
factor eIF2B and eIF2 unproductive (Hinnebusch and Lorsch,
2012). Therefore a minute increase in eIF2α phosphorylation has a
profound impact on translation rates (Kaufman, 1999).
Reducing translation rates by prolonging eIF2α phosphorylation
increases chaperone availability in the ER (Tsaytler et al., 2011),
and this in turn is likely to increase protein folding both in the ER
and in the cytosol. Indeed, we found that Sephin1 completely
prevented SOD1 aggregation and slightly increased the levels of the
soluble protein. SOD1 mutants are known to have folding defects
(Bruns and Kopito, 2007), and slowing down translation might
improve their folding and prevent their aggregation (Das et al.,
2015).
Importantly, use of Sephin1 is not only beneficial but also safe in
mice (Das et al., 2015), which could be anticipated for two reasons.
First, PPP1R15A-knockout mice are largely normal (Kojima et al.,
2003), therefore selective inhibition of PPP1R15A function is not
expected to have adverse effects. Second, if Sephin1 only inhibits
PPP1R15A, then it is predicted to be safe, as we have found (Das
et al., 2015).
In addition to demonstrating that selective inhibition of
PPP1R15A can safely prevent diverse protein misfolding diseases
in mice, our work also provides the proof-of-principle that
regulatory subunits of phosphatases can be selectively and safely
inhibited in vivo.
The journey from cells to organisms
Moving from cells in culture to mice took time and effort. We now
need to apply the same rigor to exploit this knowledge for the
development of safe therapeutics. Although there is still much to do,
our work provides proof-of-principle that approaches aimed at
rescuing cells from protein quality control failure are relevant to
diseases in mammals. The physiological relevance of studying the
cellular responses of fibroblasts or HeLa cells to tunicamycin might
have been unclear for many years, but our recent work underlines
the importance of studying fundamental cellular processes for the
discovery of effective and safe therapeutic approaches.
Conclusions
The recent investigations discussed here that have aimed to identify
strategies to help cells survive when different components of protein
quality control systems are faulty have newly revealed aspects and
components of fundamental cell biology processes. It is fascinating
to realize that our knowledge of cell biology is still only partial
and that some important components of vital cellular pathways
still remain to be discovered. This research area is not only
interesting in its own right but also because we can reasonably
anticipate that, ultimately, our knowledge will serve to develop
We are grateful to members of the Bertolotti laboratory for discussions, in particular
Indrajit Das, Anna Sigurdardottir and Marta Carrara for comments on this
manuscript, as well as Lea Sistonen and Rick Morimoto for discussion on the heatshock response.
Competing interests
The authors declare no competing or financial interests.
Funding
Research in the Bertolotti laboratory is funded by the Medical Research Council
(UK); and the European Research Council (ERC) under the European Union’s
Seventh Framework Programme (FP7/2007-2013) [ERC grant number 309516].
K.S. is supported by the Swiss National Science Foundation. A.B. is an honorary
fellow of the Clinical Neurosciences Department of Cambridge University.
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