Protein Unfolding in Cardiomyopathies
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Transcript of Protein Unfolding in Cardiomyopathies
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Protein Unfolding in Cardiomyopathies
Luisa Gorza, MDa , Federica del Monte, MD, PhD b,*
a University of Padova, Padova, Italy b Massachusetts General Hospital, Charlestown, MA, USA
Molecular chaperones are a heterogeneous group
of proteins involved in assisting and controlling the
folding of nascent polypeptides. Although in vitrostudies have shown that secondary and tertiary
structures of proteins are dictated by the linear poly-
peptide sequence, the auxiliary role in folding played
by chaperones becomes obligatory in the cellular
environment, where crowding of different macro-
molecules may favor unwanted intermolecular inter-
actions with nascent polypeptides. Their aggregation
with other unfolded protein species jeopardizes stable
protein structure and would be responsible for toxic
consequences. Molecular chaperones play a crucial
role against such toxicity in physiologic conditions.
Furthermore, their importance has been recognized in
several pathologic conditions in which destabiliza-
tion of protein folding may occur concomitantly to
upregulation of many chaperone genes. A large body
of evidence emphasizes the role of chaperones in
enhancing cell resistance to different stresses, al-
though the protective effect is apparently attributed
to the preferential, if not exclusive, interaction with
specific partner proteins.
The multifaceted properties of several molecular
chaperones have been recognized for simple eukary-
otic organisms and for organisms that are morecomplex. The nomenclature for the more relevant
protein homologs among bacteria, yeast, and mam-
malian cells is listed in Tables 1–3. In this article,
the authors first review the role and mechanisms of
molecular chaperones in protein folding in the dif-
ferent cellular compartments and attempt to coordi-nate the nomenclature of the proteins as they have
been described in different organisms with the cor-
responding proteins in mammals. Recent knowledge
of the relevance of the unfolding protein response
(UPR) and degradation pathways and the role of the
chaperone proteins in the development of human
diseases is explored. Although most of the disease
entities caused by protein misfolding have been de-
scribed for other organs, this article more specifically
addresses the consequences of protein misfolding for
cardiovascular diseases.
Overview of protein folding in the mammalian
heart
Folding of cytosolic proteins
Appropriate folding of cytosolic proteins is a
relevant event in cardiomyocytes in which cellular
architecture relies on myofibril assembly and align-
ment and on its anchorage by cytoskeleton to the
sarco(endo)plasmic reticulum (SR) and sarcolemmalmembranes. Three main chaperone systems operate at
this level: heat shock protein (HSP)70 and HSP90,
TCP1 ring complex (TRiC) (Fig. 1), and small HSPs
(see Table 1). Detailed references can be found in two
recent extensive reviews [1,2].
Heat shock protein 70 and heat shock protein
90 chaperone machinery
The constitutively expressed cytosolic HSP70
protein is the heat shock cognate protein (Hsc70)
and, like bacterial and yeast homologs, is a mono-
1551-7136/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.hfc.2005.03.009 heartfailure.theclinics.com
This work was supported by FIRB2001 grant RBAU01-
FYPJ and ISS grant CS45 to Dr. Gorza and by NIH grant
NIH-NHLBI 5K08HL069842 to Dr. del Monte.
* Corresponding author. Cardiovascular Research
Center and Cardiac Unit, Massachusetts General Hospital,
149 13th Street CNY-4, Charlestown, MA 02129.
E-mail address: [email protected] (F. del Monte).
Heart Failure Clin 1 (2005) 237 – 250
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meric protein with an N-terminal ATPase domain and
a C-terminal polypeptide-binding domain. Nucleotide
binding (ADP or ATP) regulates the kinetics of
Hsc70 interaction with nascent polypeptides, in that
when ATP is bound, the exchange of polypeptide
substrate is rapid, whereas polypeptide binding is
much more stable in the ADP-bound state. Hsc70
may bind directly to nascent polypeptides after they
emerge from the ribosome tunnel or after their re-
lease from the ribosome; in some cases, polypeptide binds to HSP70 chaperones after association with the
cochaperone HSP40. HSP40 is homologous to bac-
terial DnaJ and transfers the bound polypeptide to
Hsc70 after ATP hydrolysis.
Several isoforms of HSP40/DnaJ-like molecules
that differ by tissue distribution and substrate
interaction are known. In the mammalian heart, the
homolog Dj4/DjA4 isoform constitutes about the 1%
of total protein [3] and a cardiac-specific isoform of
Hdj2/DjA1, named pDJA1, has recently been iden-
tified in the pig heart [4]. It has been proposed that
HSP40 homologs, due to marked differences in theC-terminal region, may interact with HSP70 chaper-
one machinery in different subcellular compartments
and could be involved in protein targeting or as-
sembly of a specific intermediate filament [1]. In
contrast to Hsp40 and Hsc70 mRNA levels, pDJA1
transcript levels differ among heart chambers and
between the subepicardial and subendocardial layers
of the left ventricular wall [4]. Although comparable
evidence at the protein level is still lacking, a four-
fold increase in the expression of this latter cochaper-
one occurs after 1-hour reperfusion following acute
ischemia. This evidence and the transmural gra-dient in the expression of pDJA1 transcripts in the
left ventricular wall suggest sensitive oxygen- and
stretch-sensing mechanisms in upregulation of this
cochaperone gene [4].
A relevant component of the HSP70 chaperone
machinery is the HSP90 chaperone family. Hsp90
exists as a homodimer: dimerization occurs at the
C-terminus, whereas ATP binding domains localize
at the N-terminus. Binding and hydrolysis of ATP
change Hsp90 conformation and promote loading and
release of the polypeptide substrate, respectively.
Cooperation of Hsp90 with Hsc70 occurs for somesubstrates and is mediated by cochaperones that
physically link Hsc70 and Hsp90 and allow the
Table 1
Nomenclature of major cytosolic molecular chaperone families
Mammals Yeast Prokaryotic homolog
Chaperones
HSP70 Hsc70, constitutive Ssa1 DnaK
Hsp70, inducible Ssa2 Ssb
HSP90 Hsp90a/Hsp90/Hsp84 Hsp82/Hsc82 HtpG
Hsp90b/Hsc90/Hsp86
TCP1 ring complex TRiC TRiC GroEL/GroES
GimC/prefoldin GimC
Small HSPs Alpha A-/B-crystallin
Hsp25/27
HSP70 and HSP90 cochaperones
HSP40 Hdj1/Hsp40/DjB1 Djp1 DnaJ
Hdj2/DjA1
Hdj3/DjA2
Hdj4/DjA4
Tetratricopeptide repeat clamp domain
HOP Hop Sti1
UNC-45 CG-UNC45
SM-UNC45
RAR1/SGT1 Melusin? Sgt1
PPIase FKBP52
Cyclophilin Cyp40
Others
p23 p23 Sba1
CDC37 Cdc37 cdc37
Uppercase letters indicate chaperone families, whereas lowercase refer to single members.
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transfer of the polypeptide substrate (like the Hsp-organizing protein, Hop) or assist Hsp90 for the final
folding steps (like p23) [2].
Additional substrate-specific cochaperones may
be required. Hsp70/Hsp90 chaperone machinery is
involved, together with the cochaperone UNC-45,
in folding and assembly of conventional and non-
conventional myosins in a wide variety of organisms,
from yeast to humans [5]. The specific striated
muscle SM-UNC45 isoform is expressed in skeletal
muscle and in the heart [6], where it presumably
participates with Hsc70 and Hsp90 to myosin motor maturation, giving rise to complexes that act as a
checkpoint for thick filament assembly [7]. Inhibition
of Hsp90 ATPase activity by geldanamycin blocks
sarcomeric myosin maturation, resulting in the accu-
mulation of all newly synthesized myosin as a par-
tially folded intermediate [7].
Additional knowledge about proteins that interact
with Hsc70/Hsp90 folding complexes in the heart
is awaited, especially in light of the finding that
melusin, a novel cardiac protein involved in trans-
ducing mechanical stretch of myocardial cells into
physiologic hypertrophy (see the article by Selvetellaand Lembo in this issue for a review of this topic) [8],
displays more than 50% similarity with the zinc-
binding domain of resistance proteinase 1, an Hsp90
cochaperone protein family identified in plants [9].
TCP1 ring complex
Certain nascent polypeptides such as actin and
tubulin interact with GimC/prefoldin protein complexduring their translation and are then assisted in their
folding by the ATP-dependent multimeric chaperonin
TRiC. TRiC shows a double ring structure and, in
contrast to bacteria, lacks a capping cofactor (GroES
is the cap of the cavity formed by the GroEL
chaperonin complex). The two rings of TRiC each
contain eight different subunits that bind the protein
substrate with the apical domain and release it into
the enclosed central cavity where the folding reac-
tion takes place.
Other polypeptides bind Hsc70 first and becomesubstrates of TRiC later on, as occurs in bacteria.
In these cases, Hsc70 binds extended polypeptides
during translation and retains binding until TRiC-
mediated folding is completed.
Small heat shock protein chaperones
This heterogeneous group of low molecular mass
HSPs is more involved in control of the structural
integrity of the cytoskeleton than in protein folding,
despite the demonstration of in vitro chaperone ac-
tivity [10]. Nevertheless, for these reasons, they play
a central role in the preservation of cardiomyocytearchitecture and, thus, of mechanical function.
The most relevant small HSP expressed in car-
diomyocytes is the 22-kd protein alpha B-crystallin,
a member of the crystallin family of lens proteins.
The alpha crystallin domain is highly conserved
within the small HSP family and is thought to be
more important in the formation of the functional
Table 2
Nomen clature of majo r sarco(endo)plasmic reti culum
chaperones
Mammals Yeast
Chaperones
HSP70 Grp78/BiP Kar2p
Grp170 Lhs1p
HSP90 Grp94/gp96/endoplasmin
ORP150
Lectin Calnexin
Calreticulin
UDP-GT
Oxidoreductase PDI Pdi1p
ERp72
CaBP1
ERp29
Small HSPs Hsp47
Cochaperones J domain
HSP40 ERdj 1 – 5 Djp1
Calnexin– calreticulin complex
Oxidoreductase ERp57
Tetratricopeptide repeat clamp domain
PPIase Cyclophilin B
Uppercase letters indicate chaperone families, whereas
lowercase refer to single members.
Table 3
Nomenclature of major mitochondrial chaperones
Mammals Yeast
Chaperones
HSP70 Grp75/mtHsp75 Ssq
TCP1 ring complex Hsp60
Hsp10
Small HSPs Hsp22
Cochaperones J domain
HSP40 Mdj MDj1
Tetratricopeptide repeat clamp domain
TOM Tom70 Tom70
Tom34
Uppercase letters indicate chaperone families, whereas
lowercase refer to single members.
protein unfolding in cardiomyopathies 239
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oligomeric complex than in chaperone activity [11].
In the heart, alpha B-crystallin binds actin and desmin
filaments [12]. The latter require alpha B-crystallin to
remain functional and to prevent the aggregation of
abnormally folded desmin subunits [13].
Mitogen-activated protein kinase (MAPK)-
mediated phosphorylation of Hsp25/27 (Hsp25 in mice
and Hsp27 in humans), as it occurs after hypoxia-
reoxygenation, redistributes the protein from the cyto-
sol to the actin cytoskeleton, where it multimerizes andcontributes to microfilament stabilization [14]. In the
nonphosphorylated, monomeric state, Hsp25/27 inhibits
F-actin polymerization by binding to the plus end of
the filaments [15].
The endoplasmic reticulum folding machinery
Protein folding is a relevant function in the en-
doplasmic reticulum (ER): approximately one third
of all the proteins in eukaryotic cells are translocated
into the ER, where the unique oxidizing potentialsupports disulphide bond formation during protein
folding. In addition, protein concentration in the ER
is high; therefore, efficient folding requires that chap-
erones and folding enzymes outnumber the newly
synthesized polypeptides. An additional factor influ-
encing the equilibrium of such a gel-like protein ma-
trix is represented by the amount of ER-sequestered
Ca2+: most of the ER proteins involved in protein
folding bind large amounts of Ca2+, with variable
affinity [16]. This latter aspect, which has often been
neglected when generally considering the ER fold-
ing machinery, appears to be of specific relevance tocells like cardiomyocytes for which a tight control of
rapid changes of Ca2+ levels in the SR is required to
regulate the contractile activity, while stable levels of
Ca2+ are required for the protein folding function [17].
Similar to what is described for the cytosol, ER
chaperones work in complexes—the lectin complex
and the Grp78/Grp94 complex (Fig. 2) —that may
interact sequentially or alternatively with the protein
substrate (see Table 2) [18]. Nascent polypeptides,
entering the ER through the protein-conducting chan-
nel Sec61, are bound by Grp78/BiP, the ER homolog
of Hsc70, which localizes on the luminal surface of the pore. Polypeptides then recruit the ER chaperone
complex, depending on their intrinsic characteristics.
Fig. 1. Model of the chaperone-assisted folding of newly synthesized polypeptides in the cytosol. ( A) The nascent chain
polypeptide interacts with Hsc70 and its cofactor Hsp40. Folding proceeds by recruiting Hsp90 and its cochaperones, among
which is Hsp-organizing protein (Hop), or by binding to prefoldin and the multimeric chaperonin TRiC. ( B) Proteins like actin
and tubulin interact immediately with prefoldin and the chaperonin TRiC. ( Adapted from Barral JM, Broadley SA, Schaffar G,
et al. Roles of molecular chaperones in protein misfolding diseases. Semin Cell Dev Biol 2004;15:18; with permission.)
gorza & del monte240
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The lectin (calreticulin–calnexin) complex
This complex is relevant for the folding of most
secretory and membrane proteins. Binding of the na-
scent polypeptides to the lectin domain of calnexin
(a type I transmembrane protein) or calreticulin (asoluble ER luminal protein) is possible only in the
presence of monoglucosylated N-linked glycans,
which derive from glucosidase activity on mannose
residues. Despite the homology between calnexin and
calreticulin, each being monomeric Ca2+ binding ER
proteins, their folding activity and substrate specific-
ity are different [19]. Calreticulin appears to play
a major role in heart development and in cardiomyo-
cytes of the cardiac conduction system [20,21],
whereas it is expressed at very low levels in adult
cardiomyocytes [22]. The question concerning how
calreticulin chaperone function is supplied in adult cardiomyocytes remains unanswered. In the process
of newly synthesized proteins, calnexin primarily
binds folding intermediates preferentially, but not
uniquely from glycoproteins, assisting folding and
the assembly of protein subunits. Calnexin also plays
a role in quality control because it prevents the ex-
cretion of misfolded glycoproteins and helps inrefolding glycoproteins. Folding by the lectin com-
plex occurs through the interaction with several other
accessory proteins whose number and function ap-
parently depend on substrate specificity, except for
the oxidoreductase ERp57, which is specifically re-
cruited by the complex.
The function of calnexin and calreticulin is to
retain the unfolded glycoprotein, whereas the third
lectin involved in the complex, UDP-glucose:glyco-
protein glucosyltransferase (UDP-GT), represents a
folding sensor. UDP-GT adds a single glucose after
complete deglucosylation of N-glycans bound in still-unfolded or partially folded protein regions. Deglu-
cosylation determines the release of the protein
Fig. 2. Involvement of the ER lectin chaperone complex in folding and in targeting ER-associated degradation (ERAD). The
nascent chain polypeptides are translocated across the ER membrane through a pore (Sec61p), interact with Grp78, and after
incomplete deglucosylation, bind to calnexin (CNX) and calreticulin (CRT). The cochaperone ERp57 is recruited to assist in
disulphide bridge formation. UDP-GT provides the addition of one glucose to N-glycans bound to the unfolded region of the
polypeptide and favors re-entering of the partially folded molecule in the cycle again. Misfolded polypeptides are bound by
calnexin and EDEM and retrotranslocated through Sec61p into the cytosol by interaction with p97 ATPase. Here, they are
ubiquitinated and degraded by the proteasome machinery. PDI, protein disulfide isomerase. ( Adapted from Kaufman RJ,
Scheuner D, Schroder M, et al. The unfolded protein response in nutrient sensing and differentiation. Nat Rev Mol Cell Biol
2002;3:412; with permission.)
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substrate, whereas glycosylation by UDP-GT permits
subsequent interaction with calnexin or calreticulin
to complete the folding.
The Grp78/Grp94 complex
Nonglycosylated proteins are more efficientlyretained by Grp78 and subsequently folded by the
Grp78/Grp94 complex, which shows several analo-
gies to the HSP70/HSP90 folding machinery [23].
Except for the presence of ERdj3 (the ER homolog of
HSP40 cochaperone), the members of the complex
interact with each other even in the absence of the
protein substrate. The ER homolog of Hsp90, Grp94,
participates in the complex, together with Grp170,
several oxidoreductase enzymes, UDP-GT, and the
immunophilins. Despite the analogy to the cytosolic
chaperone machinery, the function of Grp94 remainsobscure. In contrast to Hsp90, which is involved in
the maturation of several substrates and requires
specific cochaperones, few substrates and almost
no specific cochaperones have been identified for
Grp94. Grp94 is abundantly expressed in cardiomyo-
cytes, although higher levels of expression were ob-
served in cardiomyocytes of the heart conduction
system and during development [24].
Special mention should be given to the ER
resident protein Hsp47, a member of the serin
protease – inhibitor protein family. Hsp47 is speci-
fically involved in procollagen processing and trans- port [25] and is highly induced in pathologic
conditions associated with increased collagen synthe-
sis, such as end-stage dilated cardiomyopathy [26].
Endoplasmic reticulum chaperone function and Ca2+
homeostasis
As mentioned earlier, Grp78, Grp94, calnexin,
calreticulin, and protein disulfide isomerase (PDI)
bind significant amounts of Ca2+ in vitro [16]. Total
ER Ca2+ is commonly estimated to range up to
millimolar concentrations; however, the free Ca
2+
concentration of the ER is much lower because the
cation is largely bound to matrix proteins of high
capacity, but of relatively low affinity [27]. Indeed,
changes in the amount of calreticulin, Grp78, or
Grp94 have been shown to affect ER Ca2+ storage or
release [22,28–32]. On the other hand, the folding
machinery of the ER may require a Ca2+- enriched
environment for activity. Ca2+ binding alters calnexin
and calreticulin conformation in vitro, and inactive
calreticulin– calnexin complexes are formed in the
Ca2+-depleted ER [17]. Calreticulin and/or ERp57
can also affect ER Ca2+ content through other mechanisms; namely, by direct interaction with the
SR Ca2+ pump SERCA and by modulation of its
activity [33]. However, the role played by calreticulin
in the maintenance of cardiomyocyte Ca2+ homeo-
stasis is relevant only during development and is
supplied by calsequestrin in the postnatal heart. An
additional contribution in the maintenance of Ca2+
homeostasis could then derive from the most abun-dant SR chaperones, Grp78 and Grp94. Although
information concerning a role for Grp78 in main-
tenance of cardiomyocyte Ca2+ homeostasis is lack-
ing, it was hypothesized that Grp94 upregulation in
chronically fibrillating atrial cardiomyocytes may
counteract the Ca2+ overload secondary to the
arrhythmia because overexpression of Grp94 delays
the rise in free intracellular Ca2+ and the necrotic
death of cardiomyocytes exposed in vitro to Ca2+
overload and simulated ischemia [22,31].
Folding of mitochondrial proteins
Chaperones localized in the mitochondrial matrix
are involved in the folding of locally synthesized
polypeptides and those arriving from the cytosol (see
Table 3).
TOM is an HSP90 cochaperone protein family
involved in post-traslational import of mitochondrial
proteins having a nonclassic internal targeting se-
quence. Tom70 is inserted in the cytosolic face of
the mitochondrial outer membrane by way of the
N-terminus. After binding to Tom70 and cyclingof ATP by the chaperones, the preprotein is trans-
ported through the outer membrane by the import
machinery [1].
In addition to the mitochondrial Hsc70 homolog
mtHsp75, which keeps mitochondrial-encoded pro-
teins in assembly-competent state, mitochondria use a
multimeric chaperonin formed by Hsp60 and Hsp10.
The Hsp60/Hsp10 chaperonin is a homolog to bac-
terial GroEL/GroES; it is involved in the prevention
of aggregation and refolding of mitochondrial pro-
teins, as shown in experimental hearts exposed tocardioplegia and ischemia-reperfusion [34].
Destiny of unfolded proteins: to rescue or to
degrade?
Protein folding is far from being a successful
event. It has been calculated that for a given poly-
peptide of 27 amino acids, the number of possible
starting configurations to achieve the native-state
configuration corresponds to 1016. This astronomic
number is reduced to 1010 by the nonsystematic,stochastic search of the best configuration, which
usually corresponds to the more stable, lower-energy
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structure [35]. Despite intervention of the chaperone
machinery, about 30% of newly synthesized proteins
never reach a fully folded conformation [36]. Because
these polypeptides with incompletely folded chains
expose regions of the molecule that are buried in the
native state, such species are prone to inappropriatecontacts with other molecules. To prevent cytotox-
icity secondary to protein aggregation, cells express
diverse arrays of chaperones to optimize protein
folding and, at the same time, promote turnover of
newly synthesized proteins.
The chaperone Hsp104, a member of the HSP100/
Clp subfamily of AAA ATPase, is able to resolubilize
insoluble protein aggregates but has been identified
only in bacteria and yeast [2]. Thus, efficient turn-
over of the proaggregating folding intermediates re-
mains the main resource for mammalian cells.
Protein degradation in the cytosol: the
ubiquitin-proteasome system
The ubiquitin-proteasome system represents the
major mechanism of intracellular protein breakdown.
Mostly underestimated until 1980 [37], the process
of protein degradation is a highly specific and timely
controlled event that is carried out by the coupling of
subsequent steps. The ubiquitin-proteasome system is
an energy-requiring process in which the C-terminus
of a polyubiquitin chain is covalently linked to ane-amino group of an internal lysine of the protein
substrate. This event requires the participation of an
enzymatic cascade involved in the synthesis of the
polyubiquitin chain (ubiquitin-activating enzyme E1
and ubiquitin carrier protein E2) and ligation to the
target protein (ubiquitin protein ligase E3). The
interaction between the E3 enzyme and the substrate
protein is dictated by the presence of more or less
destabilizing N-terminal residue (N-end rule path-
way). In some cases, the destabilizing N-terminus is
generated from the existing N-terminus by direct intervention of specific enzymes. The multiubiquitin
chain represents a signal for binding to the protea-
some. Indeed, ubiquitin protein ligase E3 itself
physically interacts with specific subunits of the
26S proteasome [11], a large (2 Md) complex of a
multicatalytic protease composed of a catalytic core
(20S) linked to two 19S regulatory complexes. The
19S components carry the recognition site for the
ubiquitinated substrate and the unfolding site for
the substrate protein to enter the 20S catalytic chan-
nel. HSP70 or chaperonelike proteins act to facilitate
the binding of specific proteins to the ubiquitincomplex. Degradation of partially folded proteins
still bound to Hsc70 may be promoted through the
interaction of the chaper one with specific cochaper-
ones (Bag-1 and CHIP) [2]. Bag-1 contains a ubiq-
uitinlike domain and CHIP promotes ubiquitination
through its type-E3 ubiquitin protein ligase activity.
Endoplasmic reticulum– associated proteindegradation
Proteins that are unfolded or that cannot fold are
removed from the folding pathway by ER-associated
degradation (ERAD) (see Fig. 2). ERAD implies ret-
rotranslocation of the misfolded or incompletely
assembled glycoprotein back in the cytosol, where
it is ubiquitinated and degraded by the protea-
some. Unfolded or partially folded monoglucosy-
lated N-glycan glycoproteins are again cycled by
calreticulin – calnexin. The proteins lose one mannoseresidue by a-mannosidase, interact with the lectin
endoplasmic reticulum degradation – enhancing 1,2
mannosidase-like protein (EDEM) and calnexin, and
are targeted to ERAD, whereas unfolded or partially
unfolded monoglucosylated N-glycan glycoproteins
are again cycled by calreticulin/calnexin [38]. The
transfer to the cytosol requires interaction with a
complex of substrate-specific ER proteins, which
accompany the protein substrate through the mem-
brane and recruit the p97ATPase at the cytosolic side.
This ATPase is a member of the AAA ATPase family
and represents the driving force that pulls theretrotranslocating protein into the cytosol [39]. Other
proteins undergo deglycosylation in the ER and
recycle between ER, intermediate, and Golgi com-
partments, where they undergo partial proteolysis
before ERAD [38].
Cellular responses to unfolded protein
accumulation
Conditions that lead to accumulation of inter-
mediate folding species, dysfunction of protein deg-
radation, or both are responsible for upregulation of
chaperone genes and increased expression of chaper-
ones. These events concern the cytosolic chaperone
machinery, the ER quality control system, or the
mitochondrial protein folding machinery. They may
occur independently and involve specific signaling
systems that correspond to the cytosolic heat shock
response (HSR), the ER unfold protein response
(UPR), or a recently described putative mitochondrial
UPR [40]. Pathways involved in the first two re-sponses have been carefully described and are the
subject of excellent reviews [41–43].
protein unfolding in cardiomyopathies 243
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Fig. 3. Schematic diagram of the ER stress response. (Upper panel ) Unstressed ER. ( Lower panel ) After misfolding of proteins
occurs, chaperones are recruited to bind the misfolded proteins. The recruitment sequesters the chaperones, which release binding
to IRE1-ATF6-PERK. IRE1 and PERK undergo dimerization and phosphorylation. Dimerized IRE1 cleaves XBP1 mRNA and
generates a transcription factor that translocates to the nucleus. An additional transcription factor derives from the release of
ATF6 cytosolic domain by proteolitic cleavage in the Golgi apparatus. Binding of the transcription factors leads to increased
translation of genes for the UPR proteins (chaperones, CHOP). Phosphorylation and dimerization of PERK results in
phosphorylation of the transcription factor elF2a and the blocking of protein translation.
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Hsc70 for the heat shock response and Grp78 for
the UPR play comparable roles in both pathways.
These proteins work as stress sensors, in that the
destabilization of Hsc70 binding to transcription
factors of the heat shock factor (HSF) family and of
Grp78 binding to IRE1, ATF6, or PERK trans-membrane transducers initiates the signaling for
activation of HSF- and UPR-responsive genes,
respectively (Fig. 3). Grp78 normally binds to
IRE1-ATF6-PERK proteins, thus inhibiting their
activation by dimerization. The interaction with
unfolded proteins leads to Grp78 sequestration and
release from the binding to the IRE1-ATF6-PERK
complexes (see Fig. 3). These factors, largely studied
in yeast, show correspondent pathways in eukaryote
cells in which a more complex sensor system
translates the UPR.Three distinct events are induced in response to
UPR activation. All of these events are directed
to reduce the unfolded protein load or, ultimately, to
reduce cell death. One event is represented by the
upregulation of genes that encode various compo-
nents of the chaperone machinery and the ERAD,
resulting in increased folding capacity of the ER
and in enhancement of protein degradation. A second
event is the general reduction of gene transcription
and shift of protein translation. Finally, activation of
apoptotic pathways involves activation of caspase 12
and increased expression of the proapoptotic tran-
scription factor CHOP.
Protein misfolding in human diseases
The process of protein folding and misfolding
has recently gained attention in the medical field with
the discovery of human disease entities that directly
or indirectly result from an aberration in the folding
process or in the folding process response (Table 4).
Although the majority of the disease entities linked to
such alterations have been investigated in other
organs (mostly in the brain), evidence is emerging
for the involvement of protein misfolding in the
pathogenesis of cardiac diseases. As a result of
protein misfolding, aberrant proteins are retained insubcellular compartments and targeted for refolding
by the chaperone machinery or for degradation by
way of the ubiquitin-proteasome pathways or ERAD.
Genetic mutations or environmental factors leading to
the misfolding of proteins result not only in the loss
of proteins (retention and degradation) or protein
function (loss of function, dominant negative) but
also in the formation of products toxic to the cells,
which thus gain toxic functions. Among the recog-
nized diseases (see Table 4) in which protein
misfolding has been demonstrated to play a patho-
Table 4
Partial list of disease entities caused by defects in protein folding affecting different tissues/organs
Disease Organ Gene/protein
Transthyretin cardiomyopathy Heart TransThyretin
Desmin-related cardiomyopathy Heart Alpha B-crystallin
Down syndrome Multiorgan a-Synuclein, Ab peptide, tau
Fabry’s disease Heart and vessels a-Galactosidase A
Cystic fibrosis Lung Cystic fibrosis transmembrane conductance receptor
Nephrogenic diabetes insipitus Kidney Aquaporin
Diabetes mellitus Pancreas Proinsulin 2Marfan syndrome Connective tissue Fibrillin
Retinitis pigmentosa Eye Rhodopsin
Osteogenesis imperfecta Bone Collagen type I
Glanzmann thrombasthenia Blood GPIIb
von Willebrand’s disease Blood von Willebrand
Familial hypercholesterolemia II Systemic Low density lipoprotein receptor
Congenital goitrous hypothyroidism Thyroid Thyroglobulin
Atherosclerosis Systemic Apolipoprotein
Alzheimer’s disease Brain Amyloid precursor protein
Huntington’s disease Brain Huntington
Creutzfeldt-Jacob disease Brain Prion protein
Parkinson’s disease Brain a-synuclein
Tauopathies Brain Tau proteinAmyotrophic lateral sclerosis Skeletal muscle Superoxide dismutase
Protein folding defects affecting the heart are in italics.
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genetic role, only a few have been described to lead
to cardiomyopathy and heart failure.
Genetic mutations of chaperone proteins
Chaperone function has been shown to be im- portant in the development of misfolding disease in
humans; however, mutations that involve the com-
plete loss of chaperone function causing human dis-
eases have been described and are limited to some
specialized chaperone molecules. Mutations in spe-
cific chaperone proteins may result in congenital
defects of various degrees. Mutations in a gene en-
coding to a protein similar to TRiC chaperonins has
been shown to be responsible for two congenital
syndromes: the McKusick-Kaufman (MKK) and the
Bardet-Biedl type 6 syndromes. Congenital cardiacdefects are part of the MKK syndrome in which a
missense mutation resides in the hMKK syndrome
gene. The mutation leads to a mild form of congenital
disease syndrome because the missense mutation
allows the maintenance of a partial function of the
protein. On the other hand, the Bardet-Biedl type 6
syndrome recognizes a frameshift mutation, whereby
the protein is completely nonfunctional, resulting in
more severe forms of congenital defects.
One of the first described cardiomyopathies
from mutations of chaperone proteins was a form
of desmin-related cardiomyopathy [44]. Described10 years before in skeletal muscle in a large French
family as an autosomal dominant disease, desmin-
related myopathies are inherited disorders in which
desmin, a type III intermediate filament protein,
accumulates over time in the muscle, leading to an
adult-onset muscle disease. In the heart, a desmin-
related cardiomyopathy results from a missense muta-
tion leading to a substitution of an arginine residue
in the protein core (R120G) of the B subunit of
alpha crystallin. As mentioned before, alpha cystal-
lin is a small HSPs that assists in the folding processof desmin. In this genetic-based disease, alpha
B-crystallin has a reduced or absent chaperone func-
tion [45]. Failure of the alpha B-crystallin to mediate
the proper folding of desmin into cytoskeleton struc-
tures leads to the precipitation of toxic aggregates
(8– 10 nm intermediate filaments) composed of
alpha B-crystallin, desmin, and ubiquitin [46]. The
toxic effect of the disaggregated fragments can
give rise to different forms of cardiomyopathy: hy-
pertrophic, dilated, and restrictive, with associated
rhythm disturbances.
A mice model of mutated alpha B-crystallincardiomyopathy has helped to dissect the mecha-
nisms of this form of chaperone-deficient cardio-
myopathy and the role of alpha B-crystallin in the
desmin aggregation. These experiments have helped
to demonstrate that mutated alpha B-crystallin is
sufficient for the development of cardiomyopathy
and heart failure [13] and support the involvement
of the ubiquitin pathways in the pathogenesis of thisdisease form [47]. Similarly, alpha B-crystallin was
shown to interact with an F-box protein (FBX4;
part of the ubiquitin pathway) to induce ubiquitin-
dependent degradation, a binding enhanced by the
mutated R120G form of alpha B-crystallin [48].
Toxic aggregates and amyloid
Ischemic injury was among the first conditions in
which protein unfolding and aggregation was recog-
nized in the pathogenesis of cardiac diseases.Following coronary occlusion and ischemic injury,
recovery is limited in terminally differentiated cells,
and protein aggregation represents a complication
after reperfusion. Reactive oxygen-free radicals are
produced following ischemia and reperfusion, induc-
ing oxidative stress and leading to, among others,
protein oxidative damage. In addition to the acute
coronary event, protein misfolding and aggregation
following oxidative injury are also part of the process
of aging; thus, in both conditions, the unraveling of
the mechanism of misfolding may also appropriately
address the role of molecular chaperones as atherapeutic tool.
As was mentioned earlier, the missense substitu-
tion of B subunit of alpha B-crystallin leads to
R120G desmin-related cardiomyopathy and heart
failure. Similarly, missense mutations of desmin
protein (Ile451Met) have been described to be
responsible for at least some cases of familial
idiopathic dilated cardiomyopathies [49], for which
the exact mechanism of disease development arising
from the mutation has not been dissected.
Accumulation of toxic aggregates can also deter-mine development of cardiac dysfunction and heart
failure. Characteristically described in the brain (ie,
in Alzheimer’s disease and other neurodegenerative
pathologies). Proteins of different origin can (under
specific, most often unknown circumstances) de-
posit as aggregates of b-sheet conformation, known
as amyloid.
Amyloidosis is a disorder of protein structure
that can recognize an acquired or inherited origin and
give rise to organ-specific and systemic multiorgan
diseases. Specific amyloidoses are named according
to the identity of the protein precursor constituting themain fibril component, and many different pro-
teins are known to deposit as amyloidotic fibrils in
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different organs. In addition to alpha B-crystallin in
the desmin-related myopathies, in the heart, a form
of amyloidotic cardiomyopathy from a folding defect
is secondary to deposits of a plasma protein: the thy-
roid hormone and retinal-binding protein (vitamin A)
blood carrier protein transthyretin (TTR). Severalmutations (more than 80) of the transthyretin gene
result in destabilization of the monomeric or the tet-
rameric structure of TTR that can assume an in-
termediate form prone to deposit in tissues. TTR
mutations, initially described as being transmitted as
an autosomal dominant trait, showed more complex
expression with incomplete penetration, anticipation
(prevalently in the endemic areas), and the occurrence
of sporadic cases, whereby environmental factors
may play an independent role. Of the more than 80
known mutations of TTR, about 50% affect the heart among other organs and 7 specifically form deposits
in the heart. The most common mutation causing
cardiomyopathy is the substitution of a valine residue
with isoleucine (TTR V122I), a variant most com-
monly recognized in the African American popula-
tion and in West Africa, giving rise to late-onset
cardiomyopathy without involvement of other or-
gans [50].
Similar to other types of amyloidotic cardiomy-
opathies, TTR cardiomyopathy is mostly character-
ized by thick hearts of a restrictive functional pattern
and by aggregate deposited in the extracellular space,leading to heart failure and fatal arrhythmias.
Of note, aging per se can lead to deposits of
TTR-originating fibrils, leading to a senile systemic
amyloidosis. The senile form of amyloidotic TTR,
however, derives from the unmodified normal se-
quence of the protein as opposed to the variant form
that leads to a less stable protein with earlier onset of
the disease. It is conceivable that other proteins can
become unstable over time or that overly low pene-
trance mutations can lead to senile or adult onset of
the amyloidotic diseases and to heart failure.
Misfolded proteins in the endoplasmic reticulum
Ca2+ dishomeostasis, perturbation of the redox
status of the cell, energy (ATP) or glucose depriva-
tion, altered protein post-translational modification
(glycosylation), the occurrence of misfolded proteins,
or increased unfolded protein load to the ER are
conditions that can induce ER ‘‘stress.’’ Failure of the
UPR response or in the ERAD over time can translate
into defects in chaperone-mediated protein folding
and diseased phenotypes.As described earlier, the ER response to stress is
mediated by the complex pathways of transcription
factor binding proteins IRE1, ATF6, and PERK. In
the mammalian system, the ER membrane proteins
IRE1a and -b undergo dimerization and phosphory-
lation upon stress, leading to cleavage of a 26-mer
(26 nucleotide oligomer) mRNA intron for the bZIP
transcription activator XBP1. XBP1 activates thetranscription of numerous genes for the UPR.
Experimentally, deletion of the XBP1 genes can
cause cardiomyopathy and cardiac cell death (see
Fig. 3). Myocyte necrosis leading to embryonic death
after deletion of an hXBP-1 gene (TREB5) in mice
was described by Masaki et al [51].
Transgenic mice models for the ER unfolding
response helped to evaluate the role of pathways of
ER stress response. Naturally occurring proteins
travel in their native form to and from the ER and
Golgi to be secreted or incorporated in the membraneor in subcellular compartments. Specific signals
control the retention of misfolded proteins in the
ER. Among these signals, the tetrapeptide KDEL
(K = lysine, D = apartate, E = glutamate, L = leucine)
is bound to the C-terminus of secreted proteins and
interacts with the KDEL receptor to signal the
retention [52,53] in the ER and Golgi apparatus
through coated vesicles called COPI-I. Membrane
proteins are signaled to retrieve by KKXX or
KXKXX sequences (X = other amino acids). With
similar mechanisms, misfolded proteins undergo
r et ro tr an sp or t i n t he E R t o b e r ef ol de d b ythe chaperone machinery. Mutation of the KDEL re-
ceptor has been shown in vitro to disturb the circu-
lation of secreted proteins between the ER and Golgi
apparatus, resulting in misfolded protein accumula-
tion in the ER in stable cell lines [54]. The per-
turbation of ER quality control by mutation of KDEL
resulted in transgenic mice developing dilated car-
diomyopathy with accumulation of misfolded pro-
teins in the ER [55], suggesting the role of ER folding
defect as a pathogenetic factor in cardiac failure. In
addition, cell-specific ER stress – induced transcrip-tion factors have been described [56], indicating
the possibility of specialized pathways for the UPR
in mammalian cell types mediating differences in
pathologic outcome.
In contrast, Fabry’s disease is an example of
ER protein quality control being too efficient.
Farbry’s disease is an inherited disease linked to the
X chromosome, whereby a deficient activity of
a-galactosidase A, a lysosomal enzyme for glyco-
sphingolipids, most often leads to accumulations of
intracytoplasmatic lamellar inclusions in the vascu-
lar endothelium, with consequent systemic organdefects. In a subset of cases, a cardiac variant results
in cardiac hypertrophy, accounting for 3% to 9% of
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hypertrophic cardiomyopathies, and over time, in
death. In this form, the enzyme acquires a defective
conformational structure that prevents its transport to
the Golgi apparatus and export to the lysosomes.
More than 160 mutations leading to Fabry’s disease
have been identified; among those, few lead to themaintenance of partial enzymatic activity in the
cardiac variant of the disease. The misfolding origin
of the mutated protein is supported by the beneficial
effect of the administration of galactose or other
reversible competitive inhibitors that act as chemical
chaperones enabling the dissociation from the chap-
erone machinery and the dimerization of the enzyme
and reducing its degradation by the proteasome.
Human diseases can result from defects in the
UPR or in defects in the ERAD pathway. Genetic
mutations leading to misfolding of proteins that bindto Grp78/BiP influence the UPR. Among those
mutations, defects in the PERK gene, for example,
lead to the accumulation of procollagen type I and the
lack of formation of mature collagen in the bone in
osteogenesis imperfecta or proinsulin in pancreatic
b-cells in diabetes mellitus. Diseases of proteins that
do not bind Grp78/BiP, on the other hand, are
unlikely to influence the UPR, and a defect in the
ubiquitin-proteasome system and ERAD is likely to
be responsible for the defect and the clinical
phenotype. Examples of misfolding diseases of
proteins that do not bind Grp78/BiP and thus result from slow degradation from ERAD are cystic fibrosis
(mutations of the chloride channel) and emphysema
(a1-antitrypsin mutation). Alterations of the ubiqui-
tin-proteasome system have also mostly been
described in the brain in relation to neurodegenerative
diseases, but it has recently been shown how defects
in the system may play an important role in the
cardiovascular system. An animal model of dilated
cardiomyopathy [57] and human dilated cardiomy-
opathy [58] have showed significant increased
expression in some of the players of the ubiquitin- proteasome pathway and an overall increase of the
total protein-ubiquitin conjugation in failing hearts,
predominantly from dilated cardiomyopathy com-
pared with ischemic cardiomyopathies.
Of note, direct involvement of the ubiquitin-
proteasome system has recently been suggested in
the pathogenesis of different stages of development
of atherosclerotic plaque and its complication in the
cardiovascular system with respect to coronary circu-
lation [59]. In addition to its role in the protein deg-
radation process, the ubiquitin-proteasome system is
involved in important aspects of cell proliferation,inflammation, and apoptosis, which are all impor-
tant aspects of the pathogenesis of atherosclerosis.
These initial observations require further investiga-
tion to better understand the role of the ubiquitin-
proteasome system in the pathogenesis of the various
aspects of the atherosclerotic process and vascular
oxidative stress.
Summary
For many years, protein misfolding was the basis
for biochemical and biophysical studies in vitro or
in microorganisms such as yeast. Recently, clinically
related studies are merging the evidence collected
from microorganisms with human diseases. Thus, a
growing body of evidence is accumulating that iden-
tifies defects in protein folding or protein degradation
as pathogenetic hallmarks for many disease entities predominantly of late onset, including cardiomyopa-
thies and heart failure. Dissecting the pathogenetic
pathways opens new opportunities for therapy aimed
to re-equilibrate the folding capacities. The devel-
opment of chemical and pharmacologic chaperones
has helped to understand the mechanisms of some
aspects of protein misfolding and may find new ap-
plications to direct target-specific therapy. Further
understanding of the mechanisms of protein for-
mation and its defects will address the important as-
pects of modern medicine of directing early diagnosisand prevention.
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