J Inherit Metab Dis (2006) 29:477487

DOI 10.1007/s10545-006-0251-x

SSIEM SYMPOSIUM 2005

Therapy through chaperones: Sense or antisense? Cystic fibrosis

as a model disease
Margarida D. Amaral

Received: 26 October 2005 / Accepted: 17 February 2006

C SSIEM and Springer 2006


Summary Massive production and accumulation of a single

abnormal protein may constitute a major toxic burden for the
cell and even compromise the organisms long-term viability.
Consequently, adaptation and survival have forced evolution
to create quality control mechanisms that detect, monitor,
and often degrade such abnormally folded gene products, in
which molecular chaperones are key players. Notwithstanding this, there are numerous examples of misfolded proteins
which, in spite of being recognized as aberrant and efficiently
discarded by cellular quality control, still retain some of the
functional properties of their wild-type counterparts, so that
their maintenance in the cell would be beneficial for the organism. Herein are described the cellular roles of molecular chaperones and some new insights on the mechanisms
by which they influence the development of human diseases
caused by mutations that lead to protein misfolding. A special
emphasis is given to cystic fibrosis, a classical genetic disorder resulting from the retention and degradation of a mutant,
albeit functional, protein by the endoplasmic reticulum quality control. This particular system has been a good example
to describe the mechanisms that are likely to be shared by a
number of protein substrates, to define the common characteristics of the mutants, as well as to identify the mechanistic
intervenients in their retention and degradation. Finally, new

Communicating editor: Jean-Marie Saudubray

Competing interests: None declared
Presented at the 42nd Annual Meeting of the SSIEM, Paris, 69
September 2005
M. D. Amaral ()
Department of Chemistry and Biochemistry, Faculty of Sciences,
University of Lisboa, Lisboa, Portugal; Centre of Human
Genetics, National Institute of Health, Lisboa, Portugal
e-mail: mdamaral@fc.ul.pt

approaches aimed at correcting protein folding defects are

discussed, including the potential of molecular chaperones
(e.g., through RNA interference) as novel therapeutic targets,
and the usage of chemical or pharmacological chaperones as
new therapeutic agents.

Introduction
Massive production and accumulation of a single abnormal
protein may constitute a major toxic burden for the cell and
even compromise the organisms long-term viability. Consequently, adaptation and survival have forced evolution to
create quality control mechanisms that detect, monitor, and
degrade such abnormally folded gene products.
Molecular chaperones, individually or as cooperating
functional systems, have been identified as major players
in the cellular quality control mechanisms that are responsible for checking the conformation of newly synthesized
polypeptides as well as for disposing of those unable to acquire their native three-dimensional conformations. Molecular chaperones are also known to facilitate the folding of the
polypeptide chains that they interact with.
Recent years have seen major advances in our understanding of the basic mechanisms of chaperone-assisted protein
folding and degradation. Many studies suggest that the upregulation of chaperone systems can eliminate or at least
strongly attenuate the protein folding defects that occur in a
number of protein conformation-associated diseases. These
studies establish molecular chaperones as promising major
targets in therapeutic approaches aimed at correcting the basic defect of such disorders.
These new insights regarding the mechanisms underlying conformation disease phenotypes have stimulated even
more intensive efforts to discover new approaches aimed at
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either (1) correcting the basic protein folding defect (protein repair) or (2) overcoming the intracellular retention of
the mutant protein through the manipulation of the responsible interacting intervenients (protein rescue). The latter approach relies mostly on the manipulation of the endogenous
key players in protein folding and degradation, i.e. molecular chaperones, whereas the former relies on the discovery of the so-called chemical/pharmacological chaperones,
as discussed below. It is believed that ultimately such protein
repair/rescue strategies can be of great therapeutic value in
clinical medicine.
Here, I briefly review the cellular roles of molecular chaperones and some new insights on the mechanisms by which
they influence the development of conformation diseases, and
also their potential as novel therapeutic targets. Then, examples are provided of human diseases known to arise owing
to mutations that lead to altered protein folding, including
some inherited metabolic disorders. Special emphasis is put
on cystic fibrosis (CF), as a classical example of a genetic
disorder resulting from the retention of a mutant membrane
protein in the endoplasmic reticulum (ER), where it is recognized as misfolded by the quality control system and rapidly
discarded for proteasomal degradation. Finally, I discuss the
new approaches aimed at correcting protein folding defects,
which led to the discovery of the chemical and pharmacological chaperones as potential novel therapeutic agents.

Molecular chaperones
According to Hendrick and Hartl (1993)
We define a molecular chaperone as a protein that binds to and
stabilizes an otherwise unstable conformer of another protein,
and by controlled binding and release of the substrate protein
facilitates its correct fate in vivo: be it in folding, oligomeric
assembly, transport to a particular subcellular compartment, or
controlled switching between active/inactive conformations.

Despite the well-established premise that proteins acquire

their native conformations primarily as a consequence of their
amino acid sequence, the stages of achieving proper folding,
multidomain assembly of individual subunits and oligomerization, all necessary for optimal function, are not in general
spontaneous processes in the highly crowded macromolecular environment of the cell.
Thus, most, if not all, newly in vivo-synthesized polypeptides require the intervention of auxiliary proteins to protect
them from misfolding and aggregation (with a concomitant
input of metabolic energy) in order to reach their native states
efficiently. Members of this complex cellular machinery have
been coined molecular chaperones (Ellis et al 1989). First
identified as products of genes that are upregulated in response to a number of cellular stresses, and termed heatshock proteins (Hsps) (Hightower 1980) or stress proteins
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(Hightower 1991), molecular chaperones are grouped into

a large a set of structurally unrelated protein families, all
sharing the ability to interact with nonnative conformations
of other proteins so as to promote their folding into native
forms, while not contributing with conformational information to this process (Hartl and Martin 1995). The originally
proposed classification of this large group of proteins into
families, which still holds, was based on both their structural and functional affinities (Pardue 1988). Traditionally,
the chaperones have been grouped on the basis of their apparent molecular mass in gel electrophoresis analyses (e.g.
Hsp70, Hsp60).
While being key mediators of protein folding during biogenesis in vivo under normal and stress conditions, molecular chaperones have also recently been shown to perform
a number of other roles, including translocation of proteins
across intracellular membranes, catalysis of isomerization reactions, regulation of certain signal transduction pathways,
cell proliferation and apoptosis, prevention of luminal protein transit through the secretory pathway, and regulation
of retrotranslocation of misfolded proteins for degradation
(Caplan 1999; Frydman and Hohfeld 1997; Green and Beere
2001; Hartl and Hayer-Hartl 2002; Pandey et al 2000).
Roles of molecular chaperones
Major mammalian cytosolic molecular chaperones participate in de novo folding of client proteins mainly through
two distinct mechanisms. The first of these involves chaperones such as the ubiquitous 70 kDa heat-shock protein
(Hsp70) that acts co-translationally by binding to nascent
polypeptide chains and by holding these nascent proteins in a
state competent for folding until this is actually reached. The
other mechanism concerns the formation of a large cylindrical structure (the barrel), composed of several chaperonin
subunits, that provides a microenvironment into which the
newly synthesized unfolded polypeptides enter and, once sequestered away from the macromolecular crowding of the
cytoplasm, are able to acquire their proper folded conformation (Frydman 2001; Hartl and Hayer-Hartl 2002). Because
the latter mechanism is restricted to a number of relatively
small soluble substrates, mimicking this effect in order to
obtain therapeutic benefit for a large number of substrates is
not as straightforward to establish, nor as promising, as that
of other molecular chaperones like Hsp70. Indeed, Hsp70 is
a major ubiquitous chaperone in the mammalian cytosol and
it participates in the folding of nascent polypeptides in the
protection of proteins during cellular stress, and in intracellular protein trafficking (Bukau and Horwich 1998; Frydman
and Hohfeld 1997; Hartl 1996).
Although it has a more restricted number of client proteins than Hsp70 (Pratt and Toft 2003), the dimeric Hsp90
chaperone also plays a well-established role in stress pro-

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tection (Buchner 1999; Caplan 1999). Moreover, it has

the prominent function of keeping membrane receptors
within cytoskeleton-anchored complexes capable of binding their respective ligands (Catelli et al 1985), as well as of
maintaining metastable kinase molecules and hormone receptors in states competent for ligand binding (Pratt and Toft
2003).
Both classes of chaperones associate with nonnative protein substrates through recognition of hydrophobic patches
ultimately destined to be buried in the native structure (Hartl
and Hayer-Hartl 2002).
With multiple members localized in different cellular
compartments, one of the key roles of molecular chaperones is thus to interact with nascent polypeptides to keep
them in a folding-competent state, so that their translocation
into different cellular compartments is possible. In the past
few years, great progress has been made in understanding
the mode of action of individual chaperone proteins during
the above processes, revealing, in many cases, regulatory
co-factors as essential components of a chaperone system
(Barral et al 2004; Hartl 1996).
Through direct binding to polypeptide substrates (or
client proteins), molecular chaperones have been described
to play a double function, namely by assisting protein folding
and, if unsuccessful in that task, by also promoting the disposal of the aberrant conformations through the cell degradative machinery (Hohfeld et al 2001; McClellan and Frydman
2001). Molecular chaperones thus appear as special cellular entities that are capable of distinguishing between the
nonnative and native states of the client proteins, by transiently binding to the former and by losing affinity as they
evolve to the latter. In fact, once the substrates have reached
folded conformations, the affinity for the chaperones is lost
and the folded client proteins are released (Hartl and HayerHartl 2002). However, if the aberrant conformation persists,
molecular chaperones are able to crosstalk with the proteolytic systems and thus promote degradation of the substrates
(Hohfeld et al 2001; McClellan and Frydman 2001). This
ability to discriminate between the properly folded versus
partly folded states of the client proteins has thus placed
molecular chaperones as key players in the cellular surveillance system that monitors protein folding and consequently
as potential therapeutic targets for a wide range of misfolded
protein substrates responsible for human disease.
One remarkably tight cellular check of protein misfolding
is the endoplasmic reticulum (ER) quality control (ERQC),
which ensures that nascent proteins failing to fold or to
oligomerize correctly do not traffic to distal compartments of
the secretory pathway, to the cell membrane, or to the extracellular environment. In this particular case of proteins going
through the secretory pathway, molecular chaperones acting
on both sides of the ER membrane assist nascent polypeptides synthesized by ER-bound ribosomes to fold cotrans-

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lationally. Each cotranslational and posttranslational step of

secretory protein folding thus requires specific and sequential
interaction with distinct sets of chaperone and co-chaperone
proteins (Cabral et al 2001; Helenius and Aebi 2004). When
aberrant proteins are recognized as such by the ERQC, the
misfolded conformations are retrotransported back to the cytoplasm across the ER membrane, to be rapidly targeted for
degradation through the ubiquitin (Ub)-proteasome pathway
(UPP) in a process generally known as ER-associated degradation (or ERAD). Thus, the quality control of protein folding
in the early secretory pathway functions as one of the most
efficient posttranslational checks in eukaryote gene expression.
Notwithstanding this, there are numerous examples of
misfolded proteins which in spite of being efficiently discarded by the ERQC, still retain some of the functional
properties of their wild-type (wt) counterparts, and thus their
maintenance in the cell would still be of benefit for the cell
and the organism. This is illustrated below by the example of
cystic fibrosis transmembrane conductance regulator (CFTR)
protein.
Potential novel therapeutic targets of conformational
diseases
The impact of molecular chaperones on various cellular processes that are disrupted by the presence of mutations in proteins causing a wide range of disorders (namely, monogenic
diseases) has made them obvious candidates for therapeutic
strategies aimed at correcting the basic defect responsible for
such pathologies.
Molecular chaperones thus appear as appealing molecular targets, and great interest has been aroused in the therapeutic potential of ubiquitous molecules such as Hsp70 or
Hsp90 that exert their chaperone activity on a wide range of
substrates (Wegele et al 2004). There is a general belief in
the medical community that these are pro-folding molecules
and therefore increasing their intracellular levels will unquestionably enhance the biogenesis and yield of client proteins
bearing folding (and often trafficking) mutations.
Another approach considers that molecular chaperones
are potential targets for antisense or small interference RNA
(siRNA) therapy,1 considered by many to be easier to achieve
1

These forms of therapy consist in the downregulation of a given gene

expression, through inhibition of the respective messenger RNA, by
using nucleic acids, e.g. oligonucleotides, of complementary (or antisense) sequence with expected therapeutic benefit. Structurally, antisense therapeutics are short (1020 bases), synthetic, linear oligomers
that mimic DNA or RNA. While all use naturally occurring bases, more
recently developed types of antisense oligomers, differ in the linkage
between bases, so as to prevent degradation by nucleases while maintaining the architecture required for complementary base pairing. In
order to efficiently downregulate the expression of a given gene, these

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than classical gene therapy. In this respect, pioneering work

was carried out with the plasminogen activator (tPA) protein
variant that had previously been shown to be retained within
the ER and eventually degraded (Dorner et al 1988). Using
an antisense strategy, these authors manipulated the secretory
pathway by reducing the levels of BiP, an ER-luminal member of the Hsp70 family, and observed a significant increase
in the amount of the variant tPA protein secreted (Dorner
et al 1988).
These types of observations have led to the idea that reducing the intracellular levels of chaperones might lead to
a decrease in the stringency of the cellular control and thus
promote release of misfolded, but still functional proteins.
This concept appeared particularly attractive for the rescue of
misfolded mutant proteins that are retained by the ERQC machinery. No matter how interesting this concept may be, however, additional experience in reducing the levels of different
molecular chaperones has shown that the adverse effects for
cell growth and survival are too drastic to be disregarded
(Cohen and Kelly 2003; Welch 2004). Under very controlled
conditions, however, one might still postulate a titration effect, whereby the added chaperones preferentially bind to the
mutant protein, not providing a surplus of chaperone capacity
for routine maintenance of protein homeostasis.
Along these lines, a strategy of increasing the overall cellular levels of molecular chaperones through transcriptional
activation, so as to reduce protein misfolding and aggregation, has recently been demonstrated for celastrol, an active
component from Chinese herbal medicine shown to be a remarkably specific activator of HSF1 (Westerheide et al 2004).
HSF1 (heat shock transcription factor 1) is the transcriptional
activator of heat shock genes, mostly encoding molecular
chaperones.
Thus, whereas most researchers in the field would agree
that increasing the levels of cellular molecular chaperones
will cause a general increase in protein folding and thus can
potentially produce some benefit to the organism, there is no
consensus about the effects of the opposite strategy, that of
downregulating the levels of such ubiquitous and essential
cellular factors. Presumably, forays in this area should be
limited to targets demonstrated to have very specific clients.
Exceptions concern the application of some specific antiHsp drugs in cancer therapy (reviewed by Graner and Bigner
2005). Indeed, although molecular chaperones fulfil the evolutionarily conserved cellular role of proteome watchers
by promoting folding, intracellular sorting and proteolysis
of key cellular regulators, this essential function is alienated
during oncogenesis by favouring the proliferation phenotype

oligomers only need to reach the cellular cytoplasm to target the mRNA,
in contrast with cDNA constructs used in classical gene therapy which
are required to reach the cell nucleus so as to be transcribed.

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of malignant cells through facilitation of their rapid somatic

evolution (Whitesell and Lindquist 2005).
Therapeutic examples include disruption of Hsp90 interactions with essential proteins necessary for proliferative signalling or cell cycle progression by ansamycin drugs, thus
preventing the continued growth of tumour cells (Graner and
Bigner 2005; Prodromou et al 1997).

Human diseases of protein folding. The example of

cystic fibrosis
The umbrella of conformational diseases usually encompasses a plethora of several hundreds of disorders that may,
however, occur as a consequence of quite distinct cellular
mechanisms. The two larger groups that can be distinguished
among such diseases of protein conformation are (1) situations in which the misfolded proteins accumulate as aggregates, and (2) the group of diseases in which the misfolded
proteins fail to traffic to their respective final intracellular
or extracellular destination, in some cases because they are
rapidly disposed of by a very efficient quality control mechanism (see Table 1). The former class includes most neurodegenerative conformational disorders such as Alzheimer,
Huntington or Parkinson diseases (see Table 1), which were
recently reviewed elsewhere in the context of chaperone therapy (Barral et al 2004; Cohen and Kelly 2003; Jones and Tuite
2005; Muchowski and Wacker 2005).
Cystic fibrosis and the F508del mutation
Clinically, cystic fibrosis (CF; McKusick 219700) is characterized by chronic lung disease (the main cause of morbidity and mortality), pancreatic dysfunction, raised electrolyte levels in sweat, and male infertility (reviewed in
Collins 1992; Welsh et al 1996; Rowe et al 2005). The
disease is due to the absence or malfunction of the product of the cystic fibrosis transmembrane conductance regulator gene (CFTR; McKusick 602421), a cAMP-regulated
chloride (Cl ) channel localized at the apical membrane of
secretory epithelia. About 1300 different loss-of-function
gene variants (data from The CFTR Mutation Database
http://www.sickkids.on.ca/cftr) cause distinct cellular defects in the resulting protein. However, one single mutation,
the deletion of phenylalanine residue at position 508 (Phe505
deletion, or F508del) of CFTR, occurs in about 90% of CF
patients in at least one allele.
CFTR (or ABCC7) protein is a member of the ATPbinding cassette (ABC) transporters superfamily, the largest
class of proteins encoded by the human genome, occurring in
all organisms from bacteria throughout the Eukarya. Other
examples of human ABC transporters with clinical relevance (see Table 1) include the type-1 sulphonylurea receptor

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Table 1 Human diseases that have been associated with protein conformational defects (Bernier et al 2004; Brown
et al 1997; McCracken and Brodsky 2003; Welch and Brown 1996)
Disease or effect

Misfolded protein
Diseases of misfolding and aggregation

MachadoJoseph disease

Ataxin-3

Prion disease

Prion protein (PrP)

Transthyretin amyloid disease

Transthyretin (TTR)

Long QT syndrome

hERG (human-ether-a-go-go-related protein)

Diseases of misfolding and trafficking/degradation

Retinitis pigmentosa, congenital night blindness

Rhodopsin

Gaucher disease

-Glucosidase

GM1 gangliosidosis or Morquio B disease

-Galactosidase

Cancer

Ub-activating enzyme E1
Glucocorticoid receptor
p53
pp50
Smoothened (Smo)

Cystic fibrosis

CFTR (ABCC7)

Emphysema and liver disease (1 -antitrypsin)

1 -Antitrypsin

Fabry disease

-Galactosidase A

Maple syrup urine disease

BCKD complex

TaySachs disease

-Hexosaminidase

Menkes disease

Menke protein (MNK or ABCB7)

Nephrogenic diabetis insipidus

Aquoporin-2
V2 vasopressin receptor

Hyperinsulinaemic hypoglycaemia

Sulphonylurea receptor (SUR1 or ABCC8)

Hypogonadotrophic hypogonadism

Gonadotrophin-releasing hormone receptor (GnRHR)

Drug resistance in cancer

P glucoprotein (Pgp or ABCC1)

Immune deficiency

Transporters associated with antigen processing

(TAP1 or ABCB2 and TAP2 or ABCB3)

Pain

-Opioid receptor (OPR)

Nephrogenic diabetis insipidus

V2 vasopressin receptor (V2R)

Hypocalcaemia

Calcium-sensing receptor (CaR)

Adenoma, hyperthyroidism

Thyrotropin receptor (TSHR)

(SUR1) involved in insulin secretion; the transporters associated with antigen processing (TAP1 and 2); adrenoleukodystrophy protein (ALDp); P-glycoprotein (P-gp, the product
of the multidrug resistance gene, MDR1) and the multidrug resistance-related proteins (MRPs), both types of drugexporting transporters responsible for resistance to cancer
chemotherapy (Klein et al 1999). The multidomain structure
of CFTR (see Fig. 1) comprises two membrane-spanning
domains (MSDs), two nucleotide-binding domains (NBDs),
and a large regulatory domain (RD), usually not present in
ABC transporters (Riordan 1993).
CFTR undergoes a fairly complex biosynthetic pathway
which, probably owing to this complex multidomain structure, is rather inefficient (reviewed in Riordan 1999). Largely

Fig. 1 Schematic representation of the cystic fibrosis transmembrane

conductance regulator (CFTR) protein. CFTR domains are indicated,
namely: membrane-spanning domains (MSD 1 and 2); the regulatory
domain (RD) and the two nucleotide-binding domains (NBD 1 and
2). N, amino terminus; C, carboxy terminus. The position of the most
frequent mutation (F508del) in NBD1 is also indicated as well as the
two N-linked glycans

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the mutant protein for degradation. It can thus be designated

as a bottom-up approach (see Fig. 2).
Although we are still far from having a detailed description
on the roles of all key factors involved in the folding and
degradation of CFTR, as I will illustrate below, a clearer
picture has started to emerge through the pieces of evidence
reported in recent studies.
CFTR and molecular chaperones

Fig. 2 Schematic representation of two possible approaches to drug

discovery, namely the bottom-up approach (left panel) where a rational
action is undertaken after an understanding of the basic mechanisms
involved; and the top-down approach (right panel) as for example the
blind high-throughput screens (HTS) for drug discovery (see text for
details)

depending on the cell type, only about 25% to 60% of precursor wt-CFTR is processed into the fully glycosylated,
mature form that has passed to post-Golgi cellular compartments (Benharouga et al 2002; Varga et al 2004; and MD
Amaral lab, unpublished results). Folding of the F508delCFTR mutant into a native conformation is believed to be
an even less efficient process (Qu and Thomas 1996). Indeed, such abnormal CFTR conformation is recognized by
the ERQC, which causes it to be mostly retained at the ER as a
core-glycosylated immature intermediate (Cheng et al 1990;
Lukacs et al 1994) that is then rapidly targeted for degradation
via the UPP (Jensen et al 1995; Ward et al 1995). Thus very
little, if any F508del-CFTR protein (again depending on the
cell type analysed), reaches the cellular membrane (Mendes
et al 2004, 2005). Notwithstanding this, rescuing the mutant
protein to the cell surface would be of therapeutic relevance,
since it has been shown to retain at least some of its function
as a Cl channel when growth conditions are altered, namely
by lowering the temperature (Denning et al 1992).
Accordingly, there are numerous ongoing efforts towards
finding agents that promote the folding or block the degradation of nascent F508del-CFTR with the potential to provide
a therapeutic basis for the treatment of CF. These can be
developed without prior knowledge of the underlying mechanisms, through high-throughput screens (HTS) for drug discovery (Verkman 2004) in a so-called top-down approach
(see Fig. 2).
Another way of achieving the same objective is through
modulation of chaperone-assisted folding and degradation
pathways, which has recently emerged as a promising therapeutic strategy. This rational design of therapeutic approaches, however, relies on the basic understanding of the
mechanism for F508del-CFTR misfolding through the identification of the key factors of the ERQC machinery that select
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It is generally accepted that the folded state of proteins depends both on intrinsic structural motifs (determined by the
proteins primary sequence) and on extrinsic factors present
in the cellular environment (e.g. multiple molecular chaperones). Both of these two types of factors contributing to
folding have been recently reviewed elsewhere in the context of CFTR (Amaral 2004, 2005; Welch and Brown 1996).
Therefore, here I will focus briefly on these aspects and on
the desired therapeutic manoeuvres aimed at overcoming the
basic defect of F508del-CFTR through modulation of the
intracellular levels of molecular chaperones.
Biogenesis of CFTR begins with synthesis and folding
in the ER, where the protein is core-glycosylated, giving an
immature precursor form (also known as band B). wt-CFTR,
but not F508del-CFTR, matures through the Golgi, where it is
processed by complex glycosylation events to yield a 160
kDa mature form (band C) (Denning et al 1992). Several
molecular chaperones and co-chaperones have been shown
to participate in the biosynthesis and processing of CFTR,
namely Hsc70/Hsp70-Hdj-1/2 (Farinha et al 2002; Meacham
et al 1999; Yang et al 1993), calnexin (Farinha and Amaral
2005; Pind et al 1994); CHIP (Meacham et al 2001), Hsp90
(Loo et al 1998), and more recently, also HspBP1 (Alberti et
al 2004) and Bag-2 (Arndt et al 2005).
Altogether, these results, have led us to recently propose
a model for the ERQC in which wt-CFTR and mutant CFTR
acquire early different folding conformations that are distinctly recognized by molecular chaperones and which, in
turn, cause them to be discarded at two different ERQC
checkpoints (Farinha and Amaral 2005). According to this
model, the Hsc70/Hsp70 chaperones act at the first checkpoint of the ERQC by promoting folding of nascent CFTR
through its interaction with the folding co-chaperones Hdj2/Hdj-1 (or Hsp40). It is believed that if it takes too long
for the nascent polypeptide to reach the folding conformation (as for F508del-CFTR and probably minor amounts of
wt-CFTR), the Hsc70/Hsp70 chaperones can also mediate
retention and degradation of the nascent polypeptide. This is
probably achieved through an exchange of partners, in which
the pro-folding Hsp40 co-chaperone is replaced by the
degradative factor CHIP in its Hsp70 interaction (Meacham
et al 2001). Calnexin (an ER-specific chaperone) in turn acts
at the second checkpoint of the ERQC, mediating retention

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483

Fig. 3 Molecular chaperones

for which the respective role in
biogenesis, maturation or
degradation of wild-type (wt-)
and F508del-CFTR has been
elucidated. The desired
modulation effect (clinical
action) for each chaperone in
order to obtain clinical benefit is
indicated, namely for
upregulation and for
downregulation

and degradation of a large proportion of wt-CFTR, possibly

through its interaction with the novel ER degradation enhancing -mannosidase-like protein EDEM (Farinha and Amaral
2005). This model thus postulates that, in order to target substrate proteins towards folding or degradation, Hsp70/Hsc70
family members need to interact with distinct co-chaperones
that function as regulators of these activities, although it is
generally believed that some of the key factors in the CFTR
degradative pathway are still unidentified.
This basic understanding of the roles of these molecular
chaperones as major players in the ERQC machinery that
is responsible for the retention of F508del-CFTR within the
ER and for mediating its degradation raises novel possibilities for developing therapeutic strategies aimed at correcting
the mutant protein misfolding defect in CF, namely through
modulation of chaperone-assisted folding and degradation
pathways. Figure 3 summarizes the desired effects on the
cellular levels of these chaperones needed to obtain some
clinical benefit.
However, there are several caveats associated with this
approach. Indeed, we have previously shown that overexpressing the Hsp70-Hdj1 chaperone pair alone stabilizes wtCFTR but not F508del-CFTR (Farinha et al 2002). Moreover, we have shown (Farinha and Amaral 2005) that wtCFTR and F508del-CFTR interact differently, both with calnexin and with ER degradative factor EDEM. It must be
emphasized, however, that these insights have been acquired
through studies in heterologous expression culture systems.
Thus, if therapeutic approaches are to be pursued, testing the
effects of such overexpression at the organism level remains

to be performed. This might be achieved through targeted

gene therapy with vectors containing tissue-specific promoters upstream of the cDNA of interest to increase levels, or
through siRNA approaches for attenuation.
Protein repair/rescue strategies
As well as the above-mentioned strategies designed to manipulate extrinsic, i.e., trans-influencing, factors involved in
the ERQC, such as molecular chaperones, there are other
approaches, including those aimed at correcting the intrinsic
folding defect of a given target protein through the usage of
chemical or pharmacological chaperones. Because those two
strategies are distinct, I shall call them protein rescue (i.e.,
manipulating the ERQC) and protein repair (i.e., usage of
corrector compounds) strategies, respectively.
Correction of the folding defect by chemical chaperones
Studies from the past 10 years or so indicate that the simple
introduction of small molecules into the cell can bring about
environmental changes that promote folding (or avoid misfolding) of mutant proteins into aberrant conformations, thus
reducing the onset and/or progression of the clinical phenotypes associated with disorders due to those aberrant protein
conformations. Because protein misfolding is thought to be
central to the pathological state, understanding the common
structures and mechanisms, as illustrated above for CFTR,
holds promise for the development of broad-spectrum drugs
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that will be effective for the treatment of many of these

diseases.
In parallel, efforts to overcome these defects have led
to the development of various interventions that successfully repair protein folding while precluding the proteins
from either aggregation or degradation pathways. Certain low-molecular-weight compounds, such as glycerol,
trimethylamine N-oxide and deuterated water, can stabilize proteins against thermally induced denaturation in vitro
(Tatzelt et al 1996). These compounds, known as chemical
chaperones, have been found to stabilize some conformational mutants, promoting their proper folding and transport
to their site of action where, in many cases, they can be functional. Glycerol, in particular, and polyols in general have
been reported to be able to increase the relative hydration
around a protein, resulting in a decreased relative surface
area of the polypeptide. This could lead to tighter packing
of the protein and eventually to enhanced stability. Unfortunately, the concentrations of glycerol required for rescue of
mutant proteins are very high (up to molar values), so this
approach is unlikely to be translated into clinical therapy.
CFTR again is a good example of how conformationally
defective molecules are sensitive to functional repair by a
variety of experimental interventions. Indeed, restoring the
folding defect of F508del-CFTR was originally achieved using glycerol (Sato et al 1996) but also more recently with
myo-inositol, betaine or taurine (Zhang et al 2003a). Despite
their reduced toxicity in comparison to glycerol, the robustness and corrective efficacy of such compounds remains to
be demonstrated in whole organisms, although some in vivo
data are indicative that such strategy may still be valid (Zhang
et al 2003b).
Correction of the folding defect by pharmacological
chaperones
A further refinement of this idea of the use of molecules
aimed at repairing misfolded proteins has been proposed by
Bouvier and colleagues (Morello et al 2000), who introduced
the concept of pharmacological chaperones, also termed
pharmacoperones (Ulloa-Aguirre et al 2004). This novel therapeutic strategy, aimed at increasing the residual function of
mutant proteins by employing specific small molecules to repair misfolded and/or unstable mutant enzymes or proteins
that have residual function, has also been termed enzyme enhancement therapy (EET) by some authors (Desnick 2004).
The key aspect introduced by pharmacological versus chemical chaperones is specificity. Whereas chemical chaperones
are molecules that potentially act on every cellular protein,
pharmacological chaperones exert a specific action on a given
target molecule (Bernier et al 2004). Morello and colleagues
also provided the first proof-of-concept by carrying out work
on mutant forms of the V2 vasopressin receptor (V2R), which
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J Inherit Metab Dis (2006) 29:477487

by failing to traffic to the cell membrane causes nephrogenic

diabetes insipidus (Morello et al 2000). The essential concept is that the desired folding is achieved through specific
binding of ligands to immature receptor molecules. Thus,
any molecule capable of structural adaptation is a potential pharmacological chaperone, including inhibitors (if reversibly bound), as shown for various V2R mutants in this
pioneer study (Morello et al 2000). It should be emphasized
that repair by pharmacological chaperones implies an almost
mutation-specific mode of action, inasmuch as their binding is determined by a particular conformation possibly not
common to all mutant forms of a given receptor.
A few examples of pharmacological agents and their mode
of action in repairing proteins, including some related to endocrine and metabolic diseases, are summarized in Table 2.
Further descriptions of how the rescuing of misfolded proteins can be effectively modulated by pharmacological chaperones in a large number of conformational disorders can be
found in two excellent recent reviews (Bernier et al 2004;
Ulloa-Aguirre et al 2004).

Conclusion
As described, not only do molecular chaperones have dual
effects within the cell in terms of protein folding versus degradation (mostly due to the co-chaperone partner they interact
with), but also their effects on each particular substrate (and
possibly cell type) should be carefully analysed before one
jumps to conclusions about the potential therapeutic benefit
of chaperone manipulation.
Even if it appears relevant and appealing from the mechanistic perspective to increase the intracellular levels of one
particular molecular chaperone, in practice this may not result in any increase in productive substrate protein folding,
owing to the presence of such chaperones already in excess
within the target cell. Alternatively, even in situations where
a modest effect is observed in cell cultures, this is likely to become null once the approach is translated to whole organisms.
Moreover, increasing the levels of a single chaperone per se
may not be beneficial because of the occurrence in limiting amounts of the appropriate pro-folding co-chaperone. Indeed, chaperones and co-chaperones have been demonstrated
to be produced in highly varying amounts in different cell
types (Wang et al 1999). Thus, in this bottom up approach,
identification of the critical intervenients in the biogenesis,
folding and processing of a given substrate must undoubtedly
precede the design of any strategy aimed at correcting a protein defect through the manipulation of intracellular levels
of chaperones. Determination of the levels of these chaperones in relevant target cells/tissues should follow. Moreover,
the effects of chaperone attenuation/increase need to be carefully monitored in cellular assays, and in animal models if

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485

Table 2 Examples of pharmacochaperones experimentally tested to correct protein conformational defects associated with
human diseases (Bernier et al 2004; Bykov et al 2003; Desnick 2004; Ulloa-Aguirre et al 2004)
Disease

Pharmacological chaperone
Misfolding/aggregation

Long QT syndrome

Cisapride, E-4031, astemizole

Prion disease

IPrP13, quinacrine, chorpromazine

Transthyretin amyloid disease

Amyloidosis inhibitors

Alzheimer disease

-Sheet breaker peptides (mini-chaperones)

Misfolding/degradation

Retinitis pigmentosa

11-cis-7-ring retinal

Gaucher disease

N-(n-nonyl)deoxydeoxynorjirimycin

GM1 gangliosidosis or Morquio B disease

Galactonorjirimycin derivatives and N-octyl-4-epi--valienamine (NOEV)

Cancer (Smo)

Cyclopamine

Cancer (p53)

Ellipticiniums

Cystic fibrosis

Benzo(c)quinolizinium compounds

Fabry

1-Deoxy-galactonorjirimycin, galactose

Hyperinsulinaemic hypoglycaemia

Diazoxide

Hypogonadotropic hypogonadism

GnHR peptidomimetic antagonist

Drug resistance

Ciclosporin, capsaicin, vinblastine, verapamil

Immunoglobulin secretion

Hapten p-nitrophenylphosphocholine

Pain

Naltrexone

Menkes disease

Copper

Nephrogenic diabetis insipidus

SR121463, VPA-985

possible. The top down methodology would likely require

similar assay detail and in vivo verification of outcome and
efficacy. From a clinical viewpoint, however, the potential
benefit from manipulating chaperones or chaperone activities will surely drive continued research in this area.
Acknowledgements I am indebted to Dr Michael Graner (Duke
University Medical Center, NC, USA), who generously agreed to
critically read the manuscript of this paper, and for valuable comments and suggestions. Work in the authors laboratory was supported by research grants in this area from FCT (Portugal) and
FEDER (European Union), namely POCTI/MGI/47382/2002 and
POCTI/SAU/MMO/58425/2004.

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