Download Heart Failure and Protein Quality Control

This review is part of a thematic series on Ubiquitination, which includes the following articles:
Regulation of G Protein and Mitogen-Activated Protein Kinase Signaling by Ubiquitination: Insights From Model
Organisms
Heart Failure and Protein Quality Control
Ubiquitin and Ubiquitin-Like Proteins in Protein Regulation
Seven-Transmembrane Receptors and Ubiquitination
Sudha K. Shenoy, Guest Editor
Heart Failure and Protein Quality Control
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Xuejun Wang, Jeffrey Robbins
Abstract—The heart is constantly under mechanical, metabolic, and thermal stress, even at baseline physiological
conditions, and cardiac stress may increase as a result of environmental or intrinsic pathological insults. Cardiomyocytes
are continuously challenged to efficiently and properly fold nascent polypeptides, traffic them to their appropriate
cellular locations, and keep them from denaturing in the face of normal and pathological stimuli. Because deployment
of misfolded or unfolded proteins can be disastrous, cells, in general, and cardiomyocytes, in particular, have developed
a multilayered protein quality control system for maintaining proper protein conformation and for reorganizing and
removing misfolded or aggregated polypeptides. Here, we examine recent data pointing to the importance of protein
quality control in cardiac homeostasis and disease. (Circ Res. 2006;99:1315-1328.)
Key Words: cardiac disease 䡲 cardiac failure 䡲 cardiac muscle 䡲 cardiomyocytes 䡲 cardiovascular disease
䡲 cardiovascular physiology
B
ecause of alternative splicing of primary transcripts,
posttranslational modifications, and the ability to assume
multiple conformations that differ in activity, the proteome,
in terms of its informational content, is considerably more
complex than the genome and transcriptome. Thus, it is not
surprising that controlling the quality of this information is
essential for cell survival and function. Multiple layers of
quality control for protein production and maintenance exist.
After their initial synthesis, proteins targeted for the membrane and secretory pathways are modified, folded, and
assembled in the endoplasmic reticulum (ER), whereas other
cellular proteins may be synthesized and processed independently of the ER in the cytosol. Accordingly, there exist both
ER-associated protein quality control (PQC) and ERindependent PQC. In both cases, molecular chaperones and
the ubiquitin/proteasome system (UPS) play essential roles.
In general, chaperones are responsible for protecting unfolded
or partially folded nascent or mature proteins, with many
chaperones participating in protein repair,1 whereas the UPS
is largely responsible for removing terminally misfolded
proteins permanently, thereby preventing misfolded proteins
from accumulating in the cell.2
Although the first lines of defense rest in the “proofreading” of the primary DNA and RNA sequences, the cell has
evolved multiple layers of control at the posttranslational
level as well, and nascent proteins are subject to rigorous
surveillance as they are synthesized on the polysomes.
Although small- and medium-sized proteins often can assume
their correct tertiary and quaternary conformations spontaneously, the majority of proteins cannot and depend on the help
of and interaction with other proteins to fold correctly. The
complete sequence is often necessary for assuming the
correct conformation, but the linear process of protein synthesis presents unfinished proteins to the cellular environ-
Original received September 11, 2006; revision received October 6, 2006; accepted October 30, 2006.
From the Division of Basic Biomedical Sciences (X.W.), Sanford School of Medicine of the University of South Dakota, Vermillion, SD; and
Children’s Hospital Research Foundation (J.R.), Cincinnati, Ohio.
Correspondence to Jeffrey Robbins, Division of Molecular Cardiovascular Biology, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail
[email protected]
© 2006 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000252342.61447.a2
1315
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Circulation Research
December 8/22, 2006
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ment. The solution lies in the surveillance of the process by a
large and diverse family of proteins, termed chaperones,
which mediate the correct folding of nascent or incomplete
peptides, preventing their misfolding and the subsequent
formation of insoluble aggregates. Misfolding is often initiated by the exposed hydrophobic surfaces of the nascent
protein, and the chaperones bind tightly to these, preventing
their interaction and subsequent aggregation. The complexity
of this process and the diversity of peptide sequence are
reflected in the large number and types of chaperones and
cochaperones,1,3 such as the chaperonin family, the nuclear
chaperones that help assemble nucleosomes, the
mitochondrial-specific chaperones for the respiratory chain
proteins, and a class of chaperones whose synthesis is
induced in response to stress, the small heat shock proteins
(HSPs).4
The small HSPs are a class of chaperones that have been
studied extensively and play important roles in normal
cellular function and disease development.5,6 The “heat-shock
response,” in which an entire group of genes is activated in
response to environmental stress, was first described in
Drosophila. The small HSPs play important chaperone and
protective roles in the heart and the nomenclature, and
classification of the members in the family has recently been
reviewed in detail.7 They are abundant in cardiomyocytes and
are upregulated during cardiac stress and disease,8,9 with a
single member, ␣-B crystallin (CryAB), making up as much
as 3% of the total soluble protein mass. CryAB binds both
desmin and cytoplasmic actin and possesses molecular chaperone function in vitro.10 All of these considerations, coupled
with genetic evidence linking an R120G mutation in CryAB
(CryABR120G) to human cardiac disease, prompted a series of
experiments demonstrating that CryABR120G expression was
sufficient to cause heart failure in vivo.11
The Ubiquitin/Proteasome System
The integrity of a protein is constantly questioned by the
PQC. Damage control is particularly important in long-lived
cells, such as cardiomyocytes because they normally do not
proliferate and thus are relatively sensitive to increasing
concentrations of misfolded protein. In this respect, they
share an important feature with the neuronal populations,
which also cannot proliferate under normal conditions. Ubiquitin/proteasome system (UPS)-mediated proteolysis constitutes a second line of defense for the PQC of a cell by
removing misfolded, oxidized, mutant, or otherwise damaged
proteins.12,13 The UPS also degrades normal proteins that are
no longer needed, providing temporal regulation of protein
activity.12,14 UPS-mediated proteolysis includes 2 major
steps: attachment of a polyubiquitin chain via isopeptide
bonds to the target protein molecule through a process known
as ubiquitination and the subsequent degradation of the
ubiquitinated proteins by the 26S proteasome. Both ubiquitination and proteasome-mediated degradation are highly regulated cellular processes.15 Although monoubiquitination
does occur, polyubiquitination is the posttranslational process
that targets specific proteins for degradation by the 26S
proteasome.13 Ubiquitination is performed by a cascade of
enzymatic reactions that are catalyzed by 3 classes of en-
zymes: ubiquitin-activating enzyme (E1), ubiquitinconjugating enzymes (Ubc, E2), and ubiquitin ligase (E3).2,12
Recent data indicate that polyubiquitin chain assembly factor
(E4) may also play an important role,16 as UFD2a, an E4
exclusively expressed in cardiac muscle during mouse embryonic development, is essential for normal heart development.17 There is 1 ubiquitin E1, approximately 50 E2s, and
more than 400 potential E3s in the human genome, depending
on how these proteins are defined.18,19 To date, 2 E4s have
been identified in mammalian cells.17
The high efficiency and exquisite specificity of ubiquitination relies mainly on the E3 ligases.12 Nearly all known E3s
use 1 of 2 catalytic domains: a HECT (Homologous to E6AP
Carboxyl Terminus) domain or a RING (Really Interesting
New Gene 1) finger domain.20,21 The U-box domain can also
execute ubiquitin ligase activity. The U-box is a distant
relative of the RING finger domain because it has a RINGlike conformation but lacks the canonical Zn-coordinating
residues.22 HECT domain E3s are typified by human E6AP.
There are approximately 40 HECT domain– encoding genes,
more than 380 RING finger protein genes, and 9 U-box genes
in the human genome.19,23 They can possess ubiquitin ligase
activity by themselves or as part of multisubunit E3s. HECT
E3s are monomeric enzymes that directly participate in the
chemistry of substrate ubiquitination reactions by forming a
ubiquitin–thioester intermediate and transferring a ubiquitin
monomer to the target protein molecule during each round of
catalysis in the ubiquitin–isopeptide bond formation. In
contrast, the RING finger domain proteins usually form a
multisubunit complex with partner proteins and bring activated E2s and substrates into close proximity, facilitating
ubiquitin–isopeptide bond formation.22
The SCF (Skp1-Cul1–F-box protein) complexes are the
prototype of a superfamily of cullin (Cul)-based RING
finger–type E3s that represent the largest family of ubiquitin
ligases and mediate the ubiquitination of a variety of regulatory and signaling proteins in diverse cellular pathways.24 The
SCF consists of 3 invariant components, Skp1, Cul1, and
Rbx1 (a RING finger protein, also known as Roc1 or Hrt1)
and an interchangeable F-box protein subunit. Cul1-Rbx1
forms the catalytic core of the complex and is responsible for
recruiting E2, whereas Skp1 serves as an adaptor that is able
to bind different F-box proteins.25 The F-box proteins, carrying a Skp1-interacting F-box motif and a protein–protein
interaction domain, can dock different protein substrates that
are often phosphorylated to the SCF complex for ubiquitination. In addition to Cul1, the cullin protein family has at least
6 additional closely related paralogs in humans (Cul2, -3,
-4A, -4B, -5, and -7).26 Cul-1 is required for cell cycle exit in
Caenorhabditis elegans and is part of a novel gene family.26
All of these cullins can bind Rbx1 and form ubiquitin ligases.
Thus, a large number of ubiquitin E3s are cullin dependent,
and cullin appears to be a focal point for regulation of
ubiquitin E3 assembly by neddylation, a posttranslational
modification similar to ubiquitination (see below).27
The carboxyl terminus of the Hsc70-interacting protein
(CHIP), originally identified as a cochaperone of Hsc70, is
also a ubiquitin E3 ligase.28 It has both a tetratricopeptide
repeat motif and a U-box domain. The tetratricopeptide repeat
Wang and Robbins
Figure 1. Proteasome complexes. Shown are the various particles and their subunit compositions.
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motif associates with Hsc70 and Hsp90, whereas the U-box
mediates ubiquitin ligation. Hence, CHIP is an ideal candidate E3 for PQC, which selectively leads the abnormal
proteins recognized by molecular chaperones to degradation
by the proteasome,29 and evidence continues to accumulate
indicating that this hypothesis is correct.30,31 Compared with
wild-type controls, mouse hearts lacking CHIP were compromised in their ability to respond to and cope with myocardial
infarction.32
Only a few ubiquitin E3 ligases responsible for ubiquitination of cardiac proteins have been described. Atrogin-1, an
F-box protein that was identified in skeletal muscle and is
involved in myofibrillar protein degradation, can inhibit
calcineurin-mediated cardiac hypertrophy by participating in
SCF complex activity.14 Muscle-specific RING finger
(MuRF) proteins are the other known major family of E3
proteins involved in muscle protein degradation,33,34 and
MuRF1 is a ubiquitin E3 for troponin I.35 Interestingly,
MuRF1 can also inhibit protein kinase C␧ activation and
prevent phenylephrine- and 4-phorbol myristate 13-acetate–
induced, but not insulin-like growth factor-1–induced, cardiomyocyte hypertrophy.36 These findings have raised the
possibility of affecting cardiac hypertrophy by activating
UPS-mediated degradation of specific cardiac proteins.37 In
support of this hypothesis, levels of connexin43, the most
abundant gap junction protein in ventricular myocardium, are
partially regulated by the proteasomal (as well as the lysosomal) pathway.38,39 The specific E3 mediating connexin43
ubiquitination has not been identified, but ubiquitination
appears to be involved in both proteasome- and lysosomemediated connexin43 degradation.40,41
Proteasomes
The 26S proteasome is the cellular machinery responsible for
degrading polyubiquitinated proteins. The 26S is composed
of a 20S proteolytic core particle (the 20S proteasome) and a
proteasome activation particle (the 19S proteasome), which is
located at 1 or both ends of the 20S proteasome. The 20S is
a hollow cylindrical structure formed by a stack of 4
heptameric rings: 2 central antiparallel identical ␤ rings
flanked by 2 identical outer ␣ rings (Figure 1). Each ␣ or ␤
ring consists of 7 unique protein molecules (␣1 to ␣7, ␤1 to
␤7). The unfolded protein is degraded in the cavity of the 20S
by the chymotrypsin-like, trypsin-like, and caspase-like (also
known as peptidylglutamyl-peptide hydrolase) activities,
Protein Quality Control
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which reside in the ␤5, ␤2, and ␤1 subunits, respectively. The
outer ␣ ring forms the gate, which regulates substrate entry
into the proteolytic chamber and also controls the exit of the
proteolytic peptide products.12
The 19S (also known as PA700) binds the 20S through
␣-ring interactions. The 19S from Saccharomyces cerevisiae
consists of at least 17 subunits: regulatory particle nonATPase (Rpn) 1 to 12 and regulatory particle tripleA⫹ATPase (Rpt) 1 to 6. The 6 tripleA⫹-ATPase subunits, along
with Rpn1 and Rpn2, form the base of the 19S, whereas
Rpn3, Rpn5 to Rpn9, Rpn11, and Rpn12 form its lid, with
Rpn10 linking the lid and base. The base directly interacts
with the ␣ ring of the 20S. In murine hearts, an alternatively
spliced isoform of Rpn10 has been described.42 The 19S
recognizes ubiquitinated protein molecules, deubiquitinates
and unfolds them, and channels the unfolded protein to the
20S where peptide cleavage takes place. The proteasome also
plays an important role in antigen processing and antigen
presentation through the class I major histocompatibility
complex. Certain conditions, such as viral infection or induction with interferon ␥ or tumor necrosis factor, remodel the
20S, resulting in the constitutive peptidase subunits ␤5, ␤2,
and ␤1 being replaced with the inducible ␤5i, ␤2i, and ␤1i
peptidase subunits, respectively. Remodeling can alter proteasome peptidase activity and proteomic analyses show that
purified murine cardiac 26S proteasomes contain both the 3
constitutive and 3 inducible peptidase subunits.42
The 20S can also associate with the 11S proteasome.43 The
11S is also known as proteasome activator 28 (PA28) or REG
because its subunits, PA28␣, PA28␤, and PA28␥, with
molecular masses of approximately 28 kDa, have the capacity
to enhance the peptidase activities of the 20S.43 Of the 3
PA28s, PA28␣ and PA28␤ assemble into heteroheptamers
(␣3␤4 or ␣4␤3) and PA28␥ forms homoheptamers. Both
types of 11S proteasomes have a molecular mass of ⬇200
kDa.43 All 3 PA28s are expressed in cardiomyocytes. PA28␣
and PA28␤ are found in the cytoplasm and the nucleus, but
PA28␥ is located predominantly in the nucleus.43 The 20S
can be capped by 19S or 11S proteasomes at both ends
(19S-20S-19S or 11S-20S-11S) or by the 19S at one end and
the 11S at the other (19S-20S-11S) (Figure 1). Because the
11S does not contain ATPase activity and it can enhance the
peptidase activities of the 20S, it has been hypothesized that
it enhances 20S proteasome peptide cleavage but does not
facilitate substrate entry. However, a recent study showed
that PA28␥ directs degradation of the steroid receptor coactivator SRC-3 by the 20S in an ATP- and ubiquitinindependent manner, indicating that entry may be affected.44,45 In contrast to data obtained from red blood cells and
liver cell preparations, Gomes et al recently reported that 11S
proteasomes were not detected in murine cardiac 20S proteasome preparations and were only occasionally detected in the
26S proteasome preparations, although PA28␣ is fairly abundant in the heart.42 It is possible that the purification procedures selectively leave out 11S-associated 20S proteasomes.
Using gel filtration, a significant amount of 20S proteasomes
coeluted with the 11S proteasome in native murine myocardial proteins. Consistent with the hypothesis that this pathway
plays an important role in cardiomyocyte function, a substan-
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December 8/22, 2006
tial subset of 20S proteasomes appears to associate with the
sarcomere in striated muscle and display a characteristic
striated pattern when immunolabeled in striated muscle.42,46
The physiological significance of this association has not
been established, but it is conceivable that the proteasome
may be involved in sarcomere genesis and maintenance. In
the future it will be important to define the (patho)physiological role of 11S proteasomes in the heart.
Regulation of Proteasomal Proteolytic Activity
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The potential of therapeutic manipulation of protein degradation in a specific organ or tissue type has been recognized47,48 but is limited by an incomplete understanding of
how proteasomal specificity and activity are regulated. Polyubiquitination is almost always required for a specific protein
to be degraded by the 26S proteasome.12 Thus, the decision
for a protein to be degraded by the UPS is made by exposure
or maturation of its ubiquitination signal (degron) to its
specific E3 ligase. Directly enhancing proteasomal activity
would unlikely increase degradation of normal proteins because posttranslational modification within or nearby the
degron of a normal protein is usually required for the binding
of a specific E3 complex and ensuing degradation of the
normal protein.20 Taking the opposite approach, inhibition of
the 20S proteasome would affect degradation of the majority
of cellular proteins, whereas targeting a specific E3 ligase
would likely affect a family of proteins. The most precise
approach would be to manipulate the ubiquitination signal of
the target protein. An example of the general approach is the
ubiquitin E3 inhibitor nutlin, which specifically binds to the
p53 binding pocket of MDM2 (Murine Double Minute 2), an
E3 ligase for p53, and prevents p53 from being degraded.47
As described earlier, the binding of 19S (PA700) or 11S
(PA28) proteasomes to the 20S activates the proteasome.
Although 11S binding does not require ATP, the association
of the 19S and 20S particles to form the 26S proteasome is
ATP dependent. Unfolding the substrate protein and channeling it into the 20S proteasome by the base of 19S proteasomes
also requires ATP hydrolysis.12 Thus, an overall negative
energy balance, which can occur in some cardiac disease,
might inhibit UPS proteolytic function directly.
The 20S proteasome appears to be able to selectively
recognize and degrade oxidized proteins in the heart in a
ubiquitin-independent manner.15,49 Protein oxidation often
leads to denaturation and/or partial unfolding, exposing
hydrophobic surfaces or domains that are normally buried in
the native polypeptide. Proteasomes have a relatively high
affinity for hydrophobic amino acids,50 and, thus, partially
denatured proteins that have undergone mild oxidation may
well be preferentially bound by the 20S proteasome and
targeted in a ubiquitin-independent manner for degradation.51
It should be emphasized, however, that this activity is
dependent on only mild oxidation, and if significant oxidative
injury takes place, such as what occurs during a major cardiac
ischemic event, proteasomal proteins themselves are significantly damaged and proteasomal activity decreased.52,53 Proteasomal dysfunction during ischemia has been extensively
discussed.13 The exact mechanism of this inhibition remains
to be determined as the ischemic cell will also be ATP
deficient, and, thus, the ATP-dependent ubiquitination pathways will also be affected.
Posttranslational modifications, including phosphorylation,
N-terminal acetylation, and N-terminal myristoylation, are
observed in proteasome subunits,15,54 but their functional
significance is largely undefined. The ␣3 and ␣7 subunits in
20S proteasomes isolated from Rat-1 fibroblasts and human
embryonic lung cells (L-132) are phosphorylated. Following
dephosphorylation by acidic phosphatase, immunoprecipitated proteasomes displayed a significantly lower activity
compared with the phosphorylated proteasomes.55 The ␣7
subunit is phosphorylated by proteasome-associated casein
kinase II (CK2) at Ser243 and Ser250.56 Phosphorylation of
␣7 may play an important role in stabilizing the 26S proteasome.57 Several subunits of the 19S particle in animal cells
are also phosphorylated, and this may be important in
mediating 26S proteasome assembly.58,59 Using a proteomic
approach, Ping and colleagues detected N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and
20S subunits (␣2, ␣5, ␣7, ␤3, and ␤4), N-terminal myristoylation of a 19S subunit (Rpt2), and phosphorylation of 20S
subunits (eg, ␣7).42 They also identified 2 previously unrecognized functional partners in the endogenous intact cardiac
20S proteasomes: protein phosphatase 2A (PP2A) and protein
kinase A (PKA).54 Multiple individual subunits of 20S (eg,
␣1 and ␤2) appear to be the targets of PP2A and PKA;
inhibition of PP2A or the addition of PKA significantly
modified both the serine- and threonine-phosphorylation
profile of proteasomes, and phosphorylation of the 20S
complex enhances the peptidase activity of the individual
subunits in a substrate-specific fashion. Taken together, these
studies show that the peptidase activities of cardiac 20S
proteasomes are modulated by associating partners and phosphorylation may be a key mechanism for regulation.15
The COP9 Signalosome
The COP9 signalosome (CSN) can regulate UPS function. In
mammalian cells, the CSN is a multimeric protein complex
consisting of 8 unique protein subunits, referred to as CSN1
through CSN8.27 The composition of the CSN complex and
the domain structure of its subunits resemble the 8 subunit–
containing lid subcomplex of the 19S proteasome.27 Its
essential role in mammalian development is confirmed by
embryonic lethality in csn2, csn3, and csn5 nulls.60 The
biochemical activities and cellular functions of CSN in
mammalian systems remain obscure, but studies in lower
organisms point to its importance in controlling overall
proteasomal activity. A major target of the CSN is the
cullin-based ubiquitin E3 ligase complex. Cullin family
members are hydrophilic proteins that were initially characterized as being involved in the control of yeast cell division
control and can be covalently modified by a ubiquitin-like
protein, Nedd8/Rub1, via a process known as neddylation,
which is similar to ubiquitination. The CSN is responsible for
the cleavage of the Nedd8 moiety (ie, deneddylation) from
cullins. CSN-associated deubiquitination activities have also
been described.61,62 These activities can be subdivided into
the ability to deconjugate ubiquitin from monoubiquitinated
substrates as well as depolarization of polyubiquitin.62 Col-
Wang and Robbins
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lectively, the CSN has both deneddylation and deubiquitination activity, either by possessing the intrinsic catalytic
activity or by selectively recruiting different enzymes under
different circumstances.
The lid of the 19S proteasome consists of 8 subunits that
are paralogs of the 8 CSN subunits. Both CSN and the lid of
19S proteasomes contain 6 subunits with a PCI (Proteasome,
COP9, eIF3) domain and 2 subunits with a MPN (MOV34,
PAD N-terminal) domain.60 CSN and the lid of the 19S also
have a similar architecture. Recent studies indicate that CSN
directly interacts with the 26S proteasome and may compete
with the lid of the 19S proteasome, thereby influencing
proteasomal activity.63 Both purified CSN and the lid of 19S
proteasomes can bind polyubiquitin chains in vitro.64 Although it is likely that CSN directly regulates the proteolytic
function of the proteasome, this has not been demonstrated
directly and it will be important to formally test CSN ability
to act cooperatively with the ubiquitination machinery to
achieve ubiquitin-dependent degradation of specific regulators important for cardiac function.
Assessment of UPS Proteolytic Function
Synthetic fluorogenic peptide substrates are widely used to
determine the chymotrypsin-like, trypsin-like, and caspaselike activities of the 20S proteasome in either tissue and cell
lysates or in purified proteasomes. However, there are a
number of technical concerns that limit this assay. Because
these substrates can also be cleaved by nonproteasomal
peptidases, only the portion of peptidase activities inhibited
by a specific proteasome inhibitor can be attributed to the
proteasome. An additional concern is that the synthetic
peptide substrates are usually very small and can diffuse into
the 20S proteolytic chamber, even in the absence of proteasome activators such as the 19S and the 11S proteasomes.65
Thus, although fluorogenic peptidase assays permit rapid
assessment of the catalytic activity of the 20S proteasome,
they may not accurately reflect UPS proteolytic function and
neither the ubiquitination step nor the highly regulated entry
of substrates into the 20S proteasome is effectively evaluated
by these assays.
To better assess UPS proteolytic function in cells, tissues,
and the whole animal, a series of fluorescence protein
reporters were developed.66 – 68 Biological reporters such as
green fluorescence protein (GFP) or luciferase were modified
such that they were targeted for ubiquitination and degradation by the UPS. These reporters use different ubiquitination
signals, such as a noncleavable ubiquitin (Ub) fusion construct (eg, UbG76V-GFP),67 a cleavable Ub fusion peptide that
permits the creation of an N-end rule substrate (eg, Ub-ArgGFP), and the CL1 degron (GFPu) (Figure 2).69,70 Reporters
carrying different ubiquitination signal sequences conceivably could detect different ubiquitin conjugation pathways
traversed en route to the proteasome. An example of this
general strategy is illustrated by the degron CL1, a peptide
sequence consisting of 16 amino acids (ACKNWFSSLSHFVIHL). Structural predictions indicate that the CL1 peptide
sequence can form an amphipathic helix with surface exposure of a patch of hydrophobic amino acids, which may be a
structural feature that is shared by misfolded proteins and
Protein Quality Control
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Figure 2. Schematic diagrams of the GFP reporters for UPS
proteolytic function. A, C-terminal fusion of the degron CL1
sequence renders an enhanced GFP (EGFP) as a specific substrate for the UPS. The EGFP signal is inversely correlated to
UPS function. GFPdgn and GFPu distribute to the cytoplasm
and the nucleus, serving as reporters for both compartments.
NES-GFPu, a reporter for cytoplasmic UPS, was engineered by
fusion of 2 EGFPs in tandem, insertion of a nuclear exclusion
sequence (NES) at the N-terminal of each EGFP, and C-terminal
fusion of the degron CL1. NLS-GFPu, a reporter for nuclear
UPS, was made similarly, but a nuclear localization sequence
(NLS) rather than NES was used. B, N-terminal fusion of a wildtype ubiquitin (Ub) to an unmodified GFP does not change GFP
protein stability but creates an N-end rule degradation signal for
the UPS if the first amino acid of GFP is mutated from Met to
Arg, rendering Ub-R-GFP a reporter for the N-end rule degradation by the UPS. N-terminal fusion of a modified Ub with a substitution of its final Gly with Val (UbG76V) creates UbG76V-GFP,
which is degraded by the UPS. Glycine (K) residues at positions
3 and 17 of GFP are potential ubiquitination sites (italics).
signals for ubiquitination. The fusion of CL1 to the carboxyl
terminus of GFP creates GFPu (or GFPu). GFPu is a specific
UPS substrate in cultured human embryonic kidney (HEK)
cells and neonatal rat ventricular myocytes.69 In HEK cells,
GFPu has a half-life of ⬇30 minutes, which is much shorter
than unmodified GFP. Cell lines stably expressing GFPu and
recombinant adenoviruses capable of delivering the GFPu
transgene have been successfully used.71,72
Transgenic (Tg) mouse lines that ubiquitously express a
similarly engineered, but a slightly different GFP referred to
as GFPdgn, have been made.73 The GFPdgn mice were used
for monitoring dynamic changes in UPS proteolytic function
in vivo. Systemic inhibition of the proteasome by pharmacological agents such as MG-262 and lactacystin resulted in
significant accumulation of GFPdgn in all organs examined,
including the heart.73 Interestingly, a similar approach directed at inhibiting the proteasome did not lead to accumulation of a different UPS reporter (UbG76V-GFP) in UbG76VGFP Tg mouse hearts, skeletal muscle, and brain, suggesting
that GFPdgn mice are better suited for monitoring UPS
proteolytic function in these organs.68 GFPu and GFPdgn are
distributed in both the cytoplasm and the nucleus. Recently,
nuclear and cytoplasmic variants of GFPu reporters were
created by inserting either a nuclear localization sequence
(NLS) or a nuclear export sequence (NES) at the N-terminus
of a tandem GFP construct. The nuclear and cytoplasmic
GFPu reporters (NLSGFPu and NESGFPu) can be used to
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December 8/22, 2006
investigate localized UPS changes that may affect nuclear or
cytoplasmic UPS function differently.70,74 Both reporter mice
have been used to investigate UPS proteolytic function in
several mouse models of human disease, and more extensive
use of these fluorescence protein reporters will facilitate the
study of UPS proteolytic function in cardiac physiology and
pathology.73,75–77
ER-Associated Quality Control
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In eukaryotic cells, the ER is the factory for processing and
assembly of the secretory pathway proteins, which include
proteins destined to the extracellular space, plasma membrane, and the exo/endocytic compartments. Nascent unfolded polypeptides are translocated into the lumen of ER,
where a specialized set of enzymes and chaperones controls
their posttranslational modification, helps the newly imported
polypeptides to assume their native structures, and mediates
their assembly into multimeric complexes. Although estimates have been made that this pathway processes ⬇25% of
the protein complement, it should be emphasized that, in the
cardiomyocyte, the myofibrillar protein complement embodies the majority of the protein complement of the cell and is
composed of proteins not processed in the ER. Thus, ERindependent PQC will play a particularly critical role in these
cells. Unfortunately, ER-independent PQC has not been
studied intensively, and little is known about the specific
pathways underlying ER-independent quality control. Because the UPS and many molecular chaperones exist in the
cytosol,30 it is reasonable to assume that ER-independent
quality control is partially mediated by the collaboration
between molecular chaperones and UPS-mediated
proteolysis.
On exposure to aqueous solvent, hydrophobic segments in
unfolded or partially folded proteins tend to aggregate.
BiP/Kar2p, a member of the Hsp70 family, and other ERresident chaperones bind to the hydrophobic patches, prevent
aggregation, and preserve the folding competence of nascent
peptide chains.1 Protein folding and maturation in the ER is
essential for their subsequent transport through the secretory
pathway. To prevent misfolded or unassembled proteins from
being secreted, the ER contains a quality control system that
recognizes and disposes of these proteins. ER-associated
degradation is an important level of quality control in most
cells78 and depends on specific retrotranslocation of aberrant
proteins across the ER membrane to the cytosol, where they
are degraded by the UPS. This subject has been the topic of
a number of recent reviews, and the reader is referred to those
for discussion and schematic depictions of the processes
involved.1,78
Alterations in homeostasis by various cellular stressors can
lead to a condition known as ER stress, which causes
accumulation of misfolded or unfolded proteins in the ER. In
response, the cell initiates a series of fundamental changes in
gene expression, protein synthesis, and protein degradation,
termed the unfolded protein response (UPR).79 Components
of the UPR include transcriptional induction of UPR target
genes (eg, ER-resident chaperones), inhibiting translational
initiation and degrading existing mRNAs, which attenuates
protein synthesis. Activation of the ER-associated degrada-
tion pathway also occurs. An increased level of unfolded
proteins in the ER lumen is sensed via the luminal portions of
3 ER resident transmembrane glycoproteins, activating transcription factor 6 (ATF6), inositol-requiring enzyme-1
(IRE1), and PERK (Protein kinase R–like ER Kinase), which
are normally associated with the ER chaperone BiP. Sequestration of BiP by misfolded proteins results in dissociation of
BiP from these ER sensor proteins, which, until recently, was
believed to be the activating mechanism.79 However, deletion
of the region of the BiP binding domain of IRE1 did not
impair its regulation,80 leading to the hypothesis that IRE1
can bind directly to misfolded polypeptides, resulting in
oligomerization and activation of IRE1.1 The endoribonuclease activity at the C terminus of activated IRE1 excises
precisely 26 bases from the XBP1 (X-box–Binding Protein 1)
transcript, which, after religation, creates XBP1(s). XBP1(s)
is a potent transcription activator that binds to the ER
stress–responsive element (ERSE) in the promoters of ER
stress–related genes, upregulating transcription of genes that
encode the ER chaperones, structural proteins of the secretory
pathway, components of the ER-associated degradation machinery, and components of membrane biosynthesis. Additionally, activation of IRE1 recruits TRAF2 and leads to Jun
N-terminal kinase (JNK) activation and caspase-12 cleavage,
both of which are linked to apoptosis.79 Accumulation of
misfolded proteins also allows ATF6 to reach the Golgi,
where it is processed such that it can enter the nucleus and
mediate the transcription of ER chaperone genes and Xbp1.79
Coincident with these processes, translation is also affected.
PERK is an eIF-2a kinase, and activated PERK phosphorylates eIF-2a and transiently inhibits cap-dependent translational initiation. Together with IRE1-mediated selective degradation of mRNA encoding secreted proteins,81 the net result
is to reduce the level of unfolded proteins in the ER and
reprogram the ER-associated translational apparatus to accommodate the changes needed in gene expression for
upregulation of the UPR.
Blockage of protein synthesis early in the UPR arrests the
cell cycle at the G1 phase and activates both pro- and
antiapoptotic factors.79 In general, the UPR allows the cell to
survive reversible environmental stresses. However, if these
persist, proapoptotic pathways are activated. The balance
between the cytoprotective and destructive arms is poorly
understood. Persistent accumulation of misfolded proteins
beyond the capacity of ER quality control causes cellular
dysfunction and cell death.82 This process has been implicated in a variety of human disorders, including diabetes
mellitus and neurodegenerative diseases such as Alzheimer’s
and Parkinson’s.83– 86 Sustained ER stress is a potential
causative factor for cardiomyocyte apoptosis in congestive
heart failure (CHF).87 The KDEL receptor is a retrieval
receptor for luminal ER chaperones that bear a carboxylterminal Lys-Asp-Glu-Leu sequence and contribute to ER
quality control. Transgenic overexpression of a mutant KDEL
receptor leads to aberrant ER quality control and results in
dilated cardiomyopathy in mice.88 Chronic use of the cancer
therapeutic agent imatinib mesylate (Gleevec), a smallmolecule inhibitor for the cytosolic tyrosine kinase Bcr-Abl,
causes severe CHF in human and left ventricular dysfunction
Wang and Robbins
in mice, and prolonged ER stress is implicated as the
underlying cardiotoxic mechanism.89
Consequences of Protein Misfolding and
Compromised PQC in the Heart
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For structures like sarcomeres, where the stoichiometry
among their constituents is highly maintained, the synthesis
and incorporation of protein must be accurately coupled with
the degradation of the existing protein. Myofibrillar protein
degradation is thought to occur via the UPS. Insufficiency in
the molecular chaperones needed to mediate folding of the
large sarcomeric components or UPS malfunction could
affect either nascent protein assembly or efficient removal of
abnormal proteins. These processes could then result in
abnormal protein accumulation and aberrant protein aggregation that could further impair the UPS and PQC. If the UPS
is compromised or overloaded, what are the consequences for
a cell that is terminally differentiated and cannot divide? In
neurons, the consequences are well defined and protein
misfolding is known to underlie a number of the neurodegenerative diseases.90,91 Very often, the common pathological
characteristic is the presence of distinct bodies in the affected
regions. These bodies can be extracellular or intracellular and
contain aggregates of misfolded proteins that are often
ubiquitinated but have failed to be targeted to the proteasome
for degradation. The intracellular aggregates, which are
generically referred to as inclusion bodies, are intractable to
refolding and dissolution. These insoluble and kinetically
stable entities contain common components such as desmin,
ubiquitin, and tubulin. In some cell types such as neurons, the
misfolded proteins are attached to dynein motors, transported
in a retrograde manner along the microtubules to a perinuclear location and coalesce into well-defined, electron dense
bodies known as aggresomes.92,93
In the heart, the direct consequence of inadequate PQC is
accumulation of misfolded proteins and aberrant protein
aggregation, which also are characteristic for the desminrelated cardiomyopathies. Desmin-related cardiomyopathy is
an important component of desmin-related myopathy (DRM),
which is a heterogeneous group of human myopathies characterized by the presence of abnormal intrasarcoplasmic
protein aggregates that are desmin positive. Although abnormal desmin-reactive material in affected muscle cells is a
hallmark of DRM, a number of other proteins also accumulate, including dystrophin, ubiquitin, nestin, vimentin,
CryAB, and lamin-B. These accumulations are associated
with a variable degree of myofibrillar degeneration; therefore,
DRM is also referred to as myofibrillar myopathy.94,95 Clinically, DRM can present as a generalized myopathy, and
restrictive, hypertrophic, and dilated cardiomyopathies have
all been reported.95 Mutations in the desmin, selenoprotein N,
myotilin, and CryAB genes have now been characterized as
causative for DRM.96 –100 The desmin and CryAB mutations
have been modeled in mice using cardiomyocyte-specific
transgenesis, and their expression led to cardiac disease.11,101
A defining characteristic of the human DRMs is the presence
of intracellular, electron-dense granulofilamentous bodies
detectable via transmission electron microscopy, and both Tg
models showed aberrant intrasarcoplasmic and electron-
Protein Quality Control
1321
dense aggregates that were desmin positive and characteristic
of human DRM morphology.
These bodies were reminiscent of aggresomes, as they
contained desmin, CryAB, and other aggresomal proteins.98,102–104 Subsequently, the electron-dense bodies present in the cardiomyocytes expressing CryABR120G were confirmed as being aggresomes, based on protein composition,
time course of formation, and the dependence of perinuclear
localization on microtubule-mediated transport.103 However,
whether these bodies were inherently cytotoxic, benign, or
even protective was unclear. In some systems, aggresomes
are clearly associated with pathogenesis,92,105 but there are
equally compelling data that argue for cytoprotective effects.106,107 Concentrations of aggregated, misfolded proteins
could easily interfere with either cell metabolism or the
inherent function of a cardiomyocyte, which depends on
repeated cycles of unimpeded contraction and relaxation. As
the aggregates can fill a significant volume of the cytosol and
deform the cell and/or nucleus, it is not difficult to imagine
these bodies could be inherently toxic. On the other hand,
aggresomal formation could represent an attempt by the cell
to sequester potentially cytotoxic, misfolded proteins from
the general cytoplasm. Consistent with the latter hypothesis,
preventing aggresome formation actually resulted in increased cytotoxicity in cardiomyocytes expressing
CryABR120G.108
Aggresomes are commonly found in neurodegenerative
disorders that are caused by aberrant protein conformation
and misfolding.109 Diseases that fall into this category include
Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Alexander’s disease, and
the prion-based disorders. Despite some commonality of
pathology, the etiologies are unique and location of the
aggregates can differ. In Huntington’s disease, expansion of
polyglutamine tracts results in large accumulations of intracellular aggregates containing the peptides, whereas in Alzheimer’s disease, A␤ peptide plaques and misfolded tau
neurofibrillar tangles are predominantly extracellular in restricted neuronal populations. Despite the unique primary
misfolded proteins responsible for these discrete neurodegenerative diseases, the pathologies are linked by the accumulation of abnormal aggregates containing a ␤-sheet structure,
which form because of either the intrinsic mutation(s) of a
protein(s) or alterations in its processing. The widespread
distribution of aggresomes in diverse neurodegenerative diseases, and the appearance of aggresomes in a cardiomyocytebased disease, prompted an examination of the possible
parallels between the 2 cell types and the pathological insults
to which they are subjected. Although the inability of neurons
and cardiomyocytes to divide has been challenged in recent
years,110,111 it is generally accepted that, for both cell types,
the majority of cells are terminally differentiated and incapable of division. Thus, both might be particularly sensitive to
a chronic primary pathogenic stimulus, leading to protein
denaturation or misfolding if they are unable to clear the
aggregates, as even a slow accumulation of misfolded proteins would eventually result in high cytoplasmic levels of
potentially cytotoxic entities.
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Aggresomes are typically thought of as part of the cellular
response to either excess protein accumulation or as a way of
sequestering misfolded proteins from the general cytoplasmic
milieu if the proteasomal pathways are compromised or
overloaded. UPS malfunction has long been hypothesized as
an important pathogenic mechanism for the protein conformational diseases. Support for this hypothesis was buttressed
by the discovery that loss-of-function mutations in genes
encoding UPS components can cause neurodegenerative diseases in humans and rodents.112–114 Using a HEK cell line
stably expressing the UPS reporter, GFPu, Kopito and colleagues first demonstrated that aberrant protein aggregation
caused by polyglutamine-expanded huntingtin (pQ-htt), or a
mutant cystic fibrosis membrane conductor protein, can
impair the UPS.69 Almost simultaneously, Jana et al showed
that cultured neuro2a expressing pQ-htt displayed aberrant
protein aggregation, depressed proteasomal activity, mitochondrial dysfunction, and activation of caspases 9 and 3.115
Expression of a truncated myosin-binding protein C mutant
linked to human familial hypertrophic cardiomyopathy also
resulted in the formation of abnormal protein aggregates and
impaired UPS function in cultured rat neonatal cardiomyocytes.116 Taken together, these reports indicate that aberrant
protein aggregation caused by expression of a mutant protein
can impair the UPS in cultured cells, including
cardiomyocytes.
PQC and the Formation of Soluble
Preamyloid Oligomers in Cardiomyocytes
In addition to aggresome accumulation, the neurodegenerative disorders are characterized by peptide accumulations that
contain amyloid, defined as a substance with distinct ultrastructural (10-nm fibrils with ␤-pleated sheets) and tinctorial
(apple green birefringence and Congo red–positive staining)
properties.117 Amyloid formation is a common theme in many
neurodegenerative and protein conformational disorders, and
their deposition has formed the framework for a unifying
theory across the diverse disease types. Amyloidoses are
associated with the formation of extracellular plaques or
tangles, or intracellular inclusion bodies with amyloid-like
characteristics.91 The neurodegenerative amyloidoses, in
which the deposits are localized to diverse or specific
populations of neurons, have been studied most intensively,
but other nonneuropathic localized amyloidoses exist.118 Systemic amyloidoses are well characterized in many tissues,
including the heart, and are generally thought of as a
heterogeneous syndrome characterized by the formation and
accumulation of extracellular proteinaceous fibrils. However,
aggregate and plaque accumulation can be site specific,
depending on the particular protein and for some of the
neurodegenerative diseases such as Parkinson’s and Huntington’s diseases, amyloid accumulation is intracellular. In the
heart, systemic nonhereditary amyloidosis is not uncommon.
The syndrome is known as AL amyloidosis, in which extracellular amyloid fibrils composed of monoclonal immunoglobulin light chains accumulate. Secondary amyloidoses,
termed AA amyloidosis, normally develop as a complication
of chronic inflammatory disease such as occurs in patients
with rheumatoid arthritis, although, for this syndrome, car-
diac involvement is less common. Finally, there are a number
of inherited forms of systemic amyloidoses, a common form
being associated with multiple mutations in the serum protein
transthyretin.119 The systemic amyloidoses have diverse effects on cardiac function and can result in dilated or restrictive cardiomyopathy or diastolic dysfunction.120 –122
These cardiac and neurodegenerative diseases are linked
by the formation of amyloid-positive deposits, and the amyloid hypothesis, which states that amyloid accumulation is
cytotoxic, has been widely accepted.123 However, recent data
have cast doubt on the hypothesis,124 as there is a poor
correlation between the concentration of amyloid plaques and
the degree of dementia in Alzheimer’s patients.125,126 Although the inherent toxicity of the amyloid-containing aggregates has been assumed in a wide range of human conditions
such as light chain amyloidosis, the spongiform encephalopathies, Alzheimer’s disease, Parkinson’s disease, and others,127,128 it is now generally thought that amyloid plaques are
a consequence of a long pathogenic process and represent
“tombstones,” rather than being directly cytotoxic.129,130 Consistent with the pathogenicity of these early events in aggregate formation, significant UPS impairment occurs before the
coalescence of aggregated proteins into inclusion bodies.74
Thus, in the search for the primary cytotoxic entity, the
focus has shifted away from extracellular accumulation of the
amyloid-positive plaques131 to the soluble amyloidogenic
peptides and the intracellular events that precede visible
plaque accretion.130,132 This shift in focus is explicitly rooted
in the observation that disease can precede the appearance of
the classically insoluble amyloid plaques and tangles. Although many different proteins can participate in, or initiate
the formation of, amyloid (see below), there are important
commonalities in the amyloidogenic event, which starts with
the production of a native soluble protein or peptide fragment
that is inherently prone to misfolding, yielding the precursor
for fibril formation. The misfolded but soluble protein can
then self-associate to form soluble preamyloid oligomers
(PAOs), protofibrils, and other intermediates in the amyloid
fibril pathway.133 For example, cleavage of amyloid ␤-protein
precursor via the action of the ␤ and ␥ secretases produces
A␤ protein, consisting of 40- to 42-aa residues. This peptide
fragment is highly amyloidogenic and can assume an ordered,
␤ sheet– containing conformation. These structures then contribute to the formation of small soluble oligomers that can go
on to develop into protofibrils, which, in turn, self-associate
into the mature amyloid fibril found in the characteristic
plaques and tangles (Figure 3).
Data that A␤ protein and other amyloidogenic proteins
exert their cellular toxicity as soluble PAO intermediates and
not as insoluble aggregates or fibrils have been gathered.127,134,135 In situ experiments, in which small soluble
prefibrillar A␤ was added to mouse brain slice cultures,
confirmed the neurotoxicity of the soluble protein.132 A␤
amyloid oligomers were found in the cerebrospinal fluid of
Alzheimer’s patients, and the soluble A␤ oligomer concentration in the human brain more accurately predicted disease
severity than plaque accumulation.126,136,137 In Huntington’s
disease, the cellular toxicity of soluble amyloid induced by
the expanded glutamine repeats present in the mutated hun-
Wang and Robbins
Protein Quality Control
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Figure 3. Consequences of protein misfolding. Shown is a schematic diagram of the temporal consequences of protein misfolding.
Nascent proteins interact with chaperones or small HSPs and fold correctly. However, because of genetic mutation or environmental
stimuli such as mechanical stress, hypoxia or ischemia, misfolded proteins, or peptide fragments accumulate. These can aggregate into
kinetically stable, insoluble entities or be recognized by the PQC, ubiquitinated, and degraded by the proteasome. Isolated regions in
some of these unfolded or cleaved proteins are able to assume a ␤-pleated sheet structure and interact with each other to form a
series of intermediate but stable structures, resulting in soluble preamyloid oligomers, which are cytotoxic. These entities can, under
certain circumstances, go on to form protofibrils and may coalesce, resulting in the classic amyloid fibrils, plaques, and tangles. The
arrangements of the ␤-strands perpendicular to the fiber axis and the ␤-sheets parallel to the axis in the mature amyloid fibrils are represented by the red arrows. The reversibility of many of the processes leading to mature amyloid fibril deposition is emphasized by the
bidirectional arrows.
tingtin protein has also been linked to neural pathogenesis. In
fact, rather than being a predictor for disease, the visible
inclusion bodies that accrete as a result of mutant huntingtin
expression were associated with improved cell survival, at the
same time, leading to decreased soluble mutant huntingtin
levels in the neurons and suggesting that inclusion body
formation is actually protective against the toxic soluble form
of the mutant protein.138 These results, along with extensive
other data,129,132,139 –142 indicate that the soluble PAO probably has a larger cytotoxic role than the insoluble fibrillar
amyloid deposits.
Hundreds, if not thousands, of peptide domains have
amyloidogenic potential,143–146 a concept underscored by the
lack of homology or any obvious sequence identity in the
amyloidogenic peptides. The commonality underlying PAO
formation is the ability of a peptide or peptide fragment
domain to form cross-␤ structures, and the A-11 antibody,
which recognizes the (presumably) toxic conformer, has
recently been characterized.134 Although these soluble oligomers are presumably intermediates in mature fibril formation,136 it became apparent that the oligomers of the amyloidogenic proteins were, in fact, long lived. Subsequently,
oligomers of A␤ were found in cell cultures transfected with
A␤1– 42, in Tg models of Alzheimer’s disease, and in postmortem Alzheimer’s brain specimens. Other amyloidogenic proteins such, as ␣-synuclein (Parkinson’s), transthyretin, insulin, or polyglutamine-containing peptides (Huntington’s) also
form these oligomers.140,147–149
These data imply that PAO has a shared conformation that
is conserved among diverse proteins, although it is also clear
that amyloidogenic peptides differ in their degree of toxicity
in a sequence-dependent manner.150 Whether these different
proteins also share a common mechanism of pathogenic
action is unknown at this time, although there are some data
indicating that the soluble PAOs, at least those recognized by
the A-11 antibody, can affect membrane integrity and cal-
cium homeostasis,139 both of which are critical parameters in
maintaining cardiomyocyte function and viability. Consistent
with this concept, PAO appears to be present in at least some
models of heart disease, particularly where protein misfolding
has occurred. Using the A-11 antibody, significant PAO
concentrations were detected in the CryABR120G heart failure
model and widespread distribution in cardiomyocytes derived
from various forms of human heart failure was noted as
well.103
Abnormal Protein Metabolism in the
Diseased Heart
Regardless of the primary cause(s), CHF is often preceded
and accompanied by increased cardiomyocyte protein synthesis and hypertrophy. Invariably, this leads to increased
production of abnormal proteins, which are cotranslationally
degraded by the UPS.151 Ischemic heart disease is the most
common cause of CHF. Ischemia/reperfusion injury, hypoxia,
and oxidative stress all can stress the heart, affect the folding
and assembly of nascent and mature polypeptides, and
increase compromised protein levels. Thus, the UPR is
activated in cardiac myocytes in response to hypoxia and
global ischemia/reperfusion,152,153 whereas sustained ER
stress inhibits UPS-mediated proteolysis of reporter proteins.154 Consistent with the physiological significance of
these data, Martindale et al recently reported that cardiomyocyte-restricted overexpression of a tamoxifen-regulated form
of ATF6 could induce ER stress–related gene expression and
protect against ischemia/reperfusion injury in ex vivo mouse
heart preparations.153 Adding to the pathological load, removal of the abnormal proteins by the proteasome can also be
decreased in heart failure. The removal of abnormal proteins
relies on the collaboration between chaperones and the UPS,
and multiple lines of evidence (see below) suggest that UPS
proteolytic function is compromised in heart failure.
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To dissect molecular mechanisms underlying cardiovascular disease induced by CryABR120G, Chen et al determined
temporal changes in UPS proteolytic function in CryABR120G
Tg mouse hearts.77 Ubiquitinated proteins in both the soluble
fraction and total protein extracts from the heart progressively
increased in CryABR120G Tg mice, but not wild-type (WT)
CryAB Tg mice, at 1 and 3 months of age, whereas free
ubiquitin remained unchanged, compared with normal littermates. Ubiquitinated proteins are normally degraded efficiently by 26S proteasomes.12 An increase in ubiquitinated
proteins indicates either proteasomal malfunction or enhanced ubiquitination. Cytosolic protein levels of both total
and phosphorylated ␤-catenin, an endogenous substrate of the
UPS, were increased in CryABR120G hearts. To demonstrate
UPS impairment, GFPdgn reporter mice were crossed with
CryABR120G or WT-CryAB Tg mice. As observed in hearts
treated with specific proteasome inhibitors,73 GFPdgn protein
was dramatically increased in CryABR120G/GFPdgn double Tg
hearts compared with GFPdgn single Tg littermates. The
increase in GFPdgn protein in CryABR120G/GFPdgn double Tg
hearts was greater at 3 months than at 1 month. The increase
was attributable to decreased degradation rather than increased synthesis of GFPdgn, as the steady-state GFPdgn
transcript levels were not increased. Furthermore, the ability
of crude protein extracts from CryABR120G Tg hearts to
degrade immunoprecipitated GFPdgn protein in vitro was
significantly compromised. These data indicate that UPS
proteolytic function in the heart is significantly impaired by
expression of CryABR120G protein, which misfolds and aggregates.104,108 The function of 20S proteasomes is unlikely the
primary cause of observed proteasomal malfunction as their
peptidase activities were actually increased, not reduced, as
the hearts entered congestive failure. Further analyses suggested that the primary defect can be attributed to the
compromised entry of ubiquitinated proteins into the 20S
proteasome and that aberrant protein aggregation is necessary
for CryABR120G to impair UPS proteolytic function.77 Similar
findings were obtained with another mouse model of desminrelated cardiomyopathy produced by cardiac expression of a
human DRM-linked desmin mutation.71,76
The proteasome is also compromised in animal models of
pressure-overload cardiomyopathy and myocardial ischemia/
reperfusion, 2 common causes of CHF. In mice with thoracic
aortic constriction–induced pressure overload, Tsukamoto et
al measured significant decreases in proteasomal
chymotrypsin-like, trypsin-like, and caspase-like peptidase
activities 2 weeks after surgery but before any discernable
cardiac dysfunction. These decreases became more substantial at 4 weeks when cardiac failure became evident. Consistent with proteasome functional depression, ubiquitinated
proteins significantly and progressively increased between 2
and 4 weeks after thoracic aortic constriction.155 Using a rat
model, Bulteau et al demonstrated that myocardial ischemia/
reperfusion resulted in decreased cytosolic proteasomal peptidase activity and oxidative modification of the components
of the 20S proteasome.52 This study, along with a previous
report by Okada et al,156 confirmed that 20S proteasomal
subunits are modified under these pathological conditions,
although the pathophysiological significance of altered UPS
function in either pressure-overloaded cardiomyopathy or
ischemia/reperfusion injury remains to be determined.
Cardiac proteasomal function decreases with ageing and
may contribute to the temporal pathogenesis of some CHF.
Although ubiquitination enzyme activity does not consistently change with age, accumulation of oxidized proteins and
highly ubiquitinated proteins are associated with ageing and
some ageing-related diseases, raising the possibility that the
proteasome degradation pathway is impaired.157,158 Proteasome peptidase activities in cytosolic proteins extracted from
the hearts of Fisher 344 rats decreased from 8 to 26 months,
and this could be partially attributed to decreases in 20S
proteasome abundance in the heart.159 The ability of cardiac
20S proteasomes purified from aged rats to degrade [14C]methyl casein in vitro was also decreased significantly,
compared with activity from younger rats.159 These decreases
in proteolytic activity of senescent proteasomes are correlated
with alterations in subunit composition and/or posttranslational modifications of the proteasomal subunits. Taken
together, these lines of evidence are consistent with the
hypothesis that the removal of abnormal proteins by the
proteasome is insufficient in failing human hearts, and, in the
future, it may be productive to examine the status of UPS
proteolytic function in the heart during progression to heart
failure.
However, the potential therapeutic manipulation of proteasomal activity is fraught with unknowns. Although acute and
short-term proteasome inhibition with pharmacological inhibitors reduced myocardial ischemia/reperfusion injury, possibly through inducing heat-shock responses and preventing
nuclear factor ␬B activation,13,160,161 pharmacological inhibition of 20S proteasomes caused necrotic and apoptotic cell
death in cardiomyocyte cultures.72,155 Interestingly, a recent
case report from a lung cancer patient suggests that chemotherapy using the proteasome inhibitor bortezomib causes
reversible severe cardiac failure.162 In a separate report on
relapsed and refractory multiple myeloma patients receiving a
regimen containing bortezomib, heart failure is described as a
cause of death unrelated to cancer progression.163 These
observation and reports are sporadic and preliminary but raise
the possibility that proteasome malfunction may be sufficient
to cause heart failure in humans.
Future Directions
The importance of these basic cellular processes in normal
cardiac function and the development of cardiovascular
disease is clearly established. However, our understanding of
the molecular details and relative importance of nascent
protein folding, maturation, aggregation, and degradation in
the healthy and diseased heart is in its infancy. PQC does
appear to be compromised in a range of cardiac disease, as
does chaperone and proteasome activity, and accumulation of
presumably toxic PAOs in failing animal and human hearts is
intriguing. Inadequate PQC can lead to the accumulation of
misfolded and damaged proteins that, in turn, further impairs
PQC in the heart, forming a vicious cycle that can lead to
compromised cardiomyocyte function and cell death. Nevertheless, the necessity and sufficiency of these processes have,
with rare exceptions, not been established in human cardio-
Wang and Robbins
vascular disease. The impact of cardiomyocyte-restricted
UPS inhibition on the development of heart failure should be
addressable in animal models using gain- and loss-offunction studies restricted to the relevant cell types. Similar
approaches can be used to address whether and how UPS
malfunction causes heart failure, the role proteasome malfunction plays in the process, and whether acute or chronic
manipulation of proteasomal activity is a viable therapeutic
approach. The provocative parallels between the neurodegenerative and cardiovascular diseases, which can be drawn
using the commonalities of protein conformation-based pathology, need to be explored in detail, and any potential
therapeutics based on these processes evaluated in both
systems.
Sources of Funding
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We gratefully acknowledge support from the National Heart, Lung,
and Blood Institute/NIH for grants R01HL072166 and
R01HL085629 (to X.W.) and NIH P01HL69779, R01 HL56370, P50
HL074728, P50 HL077101 (to J.R.).
Disclosures
None.
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Heart Failure and Protein Quality Control
Xuejun Wang and Jeffrey Robbins
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Circ Res. 2006;99:1315-1328
doi: 10.1161/01.RES.0000252342.61447.a2
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