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Protein Misfolding
and Human Disease
Niels Gregersen, Peter Bross, Søren Vang,
and Jane H. Christensen
Research Unit for Molecular Medicine, Institute of Clinical Medicine, Aarhus
University Hospital and Faculty of Health Sciences, University of Aarhus, Skejby
Sygehus, 8200 Aarhus N, Denmark; email: [email protected], [email protected],
[email protected], [email protected]
Annu. Rev. Genomics Hum. Genet. 2006.
7:103–24
First published online as a Review in
Advance on May 24, 2006
The Annual Review of Genomics and Human
Genetics is online at
genom.annualreviews.org
This article’s doi:
10.1146/annurev.genom.7.080505.115737
c 2006 by Annual Reviews.
Copyright All rights reserved
1527-8204/06/0922-0103$20.00
Key Words
conformational disease, protein quality control system, chaperone,
inherited disorder, protein aggregates
Abstract
Protein misfolding is a common event in living cells. In young and
healthy cells, the misfolded protein load is disposed of by protein
quality control (PQC) systems. In aging cells and in cells from certain individuals with genetic diseases, the load may overwhelm the
PQC capacity, resulting in accumulation of misfolded proteins. Dependent on the properties of the protein and the efficiency of the
PQC systems, the accumulated protein may be degraded or assembled into toxic oligomers and aggregates. To illustrate this concept,
we discuss a number of very different protein misfolding diseases
including phenylketonuria, Parkinson’s disease, α-1-antitrypsin deficiency, familial neurohypophyseal diabetes insipidus, and shortchain acyl-CoA dehydrogenase deficiency. Despite the differences,
an emerging paradigm suggests that the cellular effects of protein
misfolding provide a common framework that may contribute to the
elucidation of the cell pathology and guide intervention and treatment strategies of many genetic and age-dependent diseases.
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INTRODUCTION
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Gene variation:
nucleotide alteration
in a gene, which may
be disease-causing,
confer susceptibility
to disease, or be
neutral in its effect
Protein misfolding
disease: disease due
to misfolding of a
protein, which is
either degraded or
accumulated/
aggregated in the cell
PQC: protein
quality control
Molecular
chaperone: cellular
protein that assists in
folding to and
maintenance of the
native protein
structure
ATP: adenosine
triphosphate
Protein folding:
the process by which
a protein acquires its
native structure
The function of most cellular proteins is dependent on their three-dimensional structure, which is acquired through folding of
the polypeptide chain coded from the nuclear genome and—for 13 mitochondrial
proteins—the mitochondrial DNA. Changes
in the polypeptide chain, either resulting
from inherited or acquired gene variations or
from abnormal amino acid modifications, may
change the folding process and give rise to
misfolding of the protein. Depending on the
nature of the protein itself, the cellular compartment in which the misfolding occurs, the
activity of the folding and degradation machineries as well as interacting genetic factors
and the cell and environmental conditions,
the consequences of the misfolding may be
quite different. However, despite this diversity, a large number of very different diseases,
from early-onset inborn errors of metabolism
to late-onset neurodegenerative diseases, can
be viewed as protein misfolding diseases, often called conformational diseases (39, 88).
On this basis, a common framework related
to the molecular pathogenesis and cell pathologic mechanisms in these very different diseases is emerging.
In this review we introduce the theory of
protein folding and misfolding in general and
mention the emerging ways to predict consequences of amino acid alterations. We introduce the concept of protein quality control
(PQC) and discuss the various compartmentspecific PQC systems as well as the molecular
pathogenesis of some representative misfolding diseases originating in the various compartments. In the last sections we discuss some
of the cellular consequences of accumulated
misfolded proteins.
PROTEIN FOLDING AND
MISFOLDING: THE ROLE
OF GENE VARIATIONS
As Anfinsen showed in 1973, the amino acid
sequence of a polypeptide chain contains all
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Gregersen et al.
necessary information determining the threedimensional functional structure of a given
protein (2). The question is how this structure is achieved in the living cell. Statistically,
there are an astronomical number of possible conformations for a given polypeptide.
The Levinthal paradox states that a random
search for the final conformation would take
millions of years (56). To overcome this obstacle, nature uses a number of biochemical
“rules” and has evolved some assisting components facilitating the folding processes, the socalled molecular chaperones (28). Molecular
chaperones assist the folding by protecting the
protein during the folding process and keeping it away from misfolding, which may lead
to aggregation. The target for chaperones
is unfolded and partially folded polypeptide
chains with exposed stretches of hydrophobic
amino acids that are usually sequestered in the
core of folded proteins. Proteins with exposed
hydrophobic stretches are reversibly bound
to and released from the chaperones. Many
chaperones have an ATPase domain that orchestrates conformational changes, switching
the molecule between high and low binding
affinity states (35).
The driving force of protein folding is the
search for a conformation with lower free energy than the previous one. The various lower
energy states can be separated by barriers of
higher energy, and to overcome these barriers chaperone assistance may be required.
A certain hierarchy of interactions seems to
exist between residues for the first part of
the folding process, the so-called nucleationcondensation process, which speeds up the
folding through a number of transition states
characterized by the presence of interatomic
interactions also present in the native protein
(33, 60).
To picture protein folding, a topological
landscape representing different energy levels
has been suggested. In this model the process
of folding is described as a constant striving
toward minimal free energy with the native
structure of the protein being the conformation with the lowest energy level, the global
minimum of the landscape (24). The landscape is drawn as a rugged funnel shape in
a three-dimensional system with the free energy on the y axis and the conformational
space or entropy as a two-dimensional projection on the x and z axes. Indeed, a computer
simulation based on experimental structural
information of the acylphosphatase protein
has demonstrated the usability of the model
(25).
Because the free energy of a protein is a
function of its conformation defined by the
interactions between the amino acid residues,
even small alterations in the amino acid chain
may change the surface of the landscape, leading to the possible formation of new local freeenergy minima or, in the extreme case, formation of a new global free-energy minimum
resulting in a different stable structure of the
protein, which may be prone to aggregation.
This implies that the native state of a protein is
not always the global energy minimum. To refine the concept of energy folding landscapes
to include the aggregation tendency, which
may be aggravated by extrinsic factors, such as
high protein concentration and elevated temperature, in the living cell, one can imagine a
landscape with two deep valleys (23). The idea
is visualized in Figure 1.
Protein aggregates can be extremely stable,
even more stable than the native protein, suggesting that the aggregated states are trapped
in the kinetically deepest valleys of the landscape. It is therefore rationalized that the decision of folding toward an aggregated or native
state is a battle between conformations stabilized by intermolecular and intramolecular
interactions, respectively (23).
Furthermore, amino acid substitutions in
the protein tend to raise the local minimum
around its native state, rendering the stabilization more cumbersome and increasing the
risk of assuming misfolded conformers with
enhanced aggregation tendency. Molecular
chaperones play a vital role in this battle by rearranging structures forming interactions to
other proteins or by exposing hydrophobic
regions and thereby lifting them to a higher
Chaperone holding
Chaperone folding
Free ene
rgy
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Native protein
Aggregates
Figure 1
Free-energy folding landscape for chaperone-mediated protein folding:
hypothetical landscape of all possible protein conformations pictured with
higher altitude in the airspace symbolizing higher free energy and entropy.
Chaperones iteratively bind and release their substrates, each time raising
the free energy and enabling escapes from wrong folding pathways,
indicated by the “Unfolding” arrow. Eliminating protein by degradation
can, in principle, take place from any location in the landscape, but occurs
predominantly in areas with trapped misfolding or on pathways toward
aggregated structures, as indicated in the aggregation funnel.
level of free energy and entropy, from which
a new folding path can start. Some chaperones are even able to unfold proteins or rescue aggregated proteins, thereby lifting them
to a higher energy level for a second round of
folding (59).
In human genetic diseases, missense variations account for approximately half of the
known gene lesions (89). A pertinent question, therefore, is: Will a given variant lead to
misfolding of the protein? Because of a lack
of knowledge about the folding pathway of
proteins, there is not yet a satisfactory answer
to this question. However, a number of resources are available through the Internet, allowing the user to test different protein variants to produce an “educated guess” related
www.annualreviews.org • Protein Misfolding Diseases
Folding landscape:
a model describing
the concept that the
folding path for a
given protein strive
toward lower energy
and entropy
Protein aggregate:
structured or
amorphous
collections of protein
molecules bound
together
by—primarily—
hydrophobic
forces
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Protein quality
control system:
cellular machinery of
molecular
chaperones and
intracellular
proteases that
supervise the folding
and maintenance of
protein structures
and degrade
misfolded proteins
Hsp: heat-shock
protein
AAA: ATPases
associated with
various cellular
activities
8:30
to the pathogenic effect of the given variant.
The most promising resources today include
the PolyPhen (71), Fold-X (43), SIFT (67),
and PMUT (32) programs. The predictions
can be done based on sequence alignment with
orthologs and paralogs in addition to structural homology modeling based on experimentally determined structures. These programs can be used to indicate the effect of
given amino acid substitutions for further
evaluation. They can also be used to pinpoint regions of a protein more interesting
than others to be included in experimental
analysis or to give an overall evaluation of a
larger number of variations, revealing a tendency in their damaging effect. However, although these programs are powerful tools,
they should be used with caution, especially
in the molecular genetic evaluation of “new”
gene variations identified in patients with genetic diseases.
PROTEIN QUALITY CONTROL
SYSTEMS
In the test tube, protein folding is performed
most efficiently at low protein concentrations
and low temperature. In contrast, protein
folding in human cells takes place at 37◦ C—or
higher during fever—with very high concentrations of proteins (29). To sustain a reasonable efficiency of protein folding in this challenging environment and to protect against
the negative consequences of environmental
changes, PQC systems have evolved that supervise folding, counteract aggregation, and
eliminate misfolded and damaged polypeptide chains before they can exert toxic effects.
As discussed above, important components
of these systems are comprised by molecular
chaperones. Additionally, the PQC systems
contain specialized intracellular proteases and
accessory factors that regulate the activity of
chaperones and proteases or provide communication between the various components.
Figure 2 illustrates the principle of PQC.
During or after synthesis or upon unfolding,
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Gregersen et al.
proteins interact sequentially with different
chaperones on their path to the native conformation. Some chaperones only interact with a
subset of proteins. As long as binding sites on
chaperones are available, folding intermediates can cycle between chaperone-bound and
free states attempting to fold. However, they
may also be captured by the degradative part
of the PQC system and eliminated.
Different chaperones have preferences for
different partially folded conformations. For
example, the Hsp70 chaperone binds extended unfolded protein chains, whereas the
Hsp60 chaperone binds compact folding intermediates (35). Although there is some overlap and certain chaperones are able to fulfill
several functions, one can distinguish between
folding chaperones, holding chaperones, and
unfolding chaperones. Folding chaperones
promote folding, whereas holding chaperones, like the small heat-shock proteins lacking an ATPase activity, reversibly bind misfolded proteins. They need to transfer their
substrates to folding chaperones to accomplish folding. Unfolding chaperones typically
contain one or two so-called AAA+ domains
that harbor an ATPase, and are able to unfold
misfolded proteins, either for degradation or
to give them a new start for folding (59).
The proteases of PQC systems have a particular structure secluding the proteolytically
active sites inside cavities that are not accessible for folded proteins (41) (see Figure 2).
To be degraded, proteins must first be fully
unfolded before they are injected into the
proteolytic cavities, where they are processively degraded into small peptides. This is
typically accomplished by AAA+ domain containing “unfolding chaperones.” With respect
to the proteasome, the most prominent protease of the cytosol, proteins to be degraded
are selected by an ubiquitin-tagging system
(see below). Other PQC proteases, e.g., those
present in mitochondria, directly select their
substrates using their chaperone domains or
subunits (48). The compartmentation of the
cellular PQC systems is indicated in Figure 3.
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Holding
chaperones
Folding
chaperones
Soluble
oligomers
Proteases
Unfolding
chaperones
Amyloid
fibril
Damage/
unfolding
Insoluble
aggregate
Native
protein
Degraded polypeptide
Figure 2
Functioning of protein quality control (PQC) systems. PQC systems manage the pool of unfolded and
partially folded conformations (center). Folding chaperones promote folding, holding chaperones
maintain solubility, and unfolding chaperones disaggregate aggregates or unfold misfolded proteins and
inject them into proteolytic chambers of PQC proteases.
The production and maintenance of functional proteins, their turnover, and the
removal of damaged and aberrant proteins are
central tasks of PQC systems. Therefore, it is
not surprising that interference with these cellular functions can lead to disease. Under normal conditions the PQC system can eliminate
defective ribosomal products due to errors
in translation or post-translational processes
(81), as well as aberrant proteins originating
from the load of gene variations present in the
respective individual or from damage by covalent protein modifications like those elicited
by reactive oxygen species (ROS) (27). Disturbances of the PQC by overload with misfolding variant proteins, environmental challenges, or gene variations in PQC components may thus trigger disease processes.
PQC systems are highly dynamic, consist
of components with often redundant func-
tion, and react sensitively to the demands
by increasing or decreasing the transcription and translation of their various components. A number of PQC genes are constitutively expressed at high level, e.g., the
Hsc70 and Hsp60 chaperones and proteasome subunits; others are only expressed at
a very low level under physiological conditions, but are strongly induced upon stresses
of the folding system. Furthermore, it is increasingly recognized that additional factors
link the folding and degradation machineries together and help in tuning PQC to the
diverse demands (61). There may be tissuespecific differences in the ability to elicit
the stress response and tune PQC, which
may explain the fact that variations in proteins that are expressed in all tissues only
give rise to cellular dysfunction in certain
tissues.
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ROS: reactive
oxygen species
Hsc: heat-shock
cognate
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Cytosol
Aggregation
Misfolding
Degradation
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Synthesis
Poisonous
peptide
Chaperone
interaction
Misfolding
Chaperone
interaction
Aggregation
Folding
Folding
Chaperone
interaction
Folding
Misfolding
Degradation
Endoplasmic
reticulum
Transport
Aggregation
Mitochondrion
Figure 3
Schematic representation of the protein quality control systems in the
cytosol, endoplasmic reticulum, and mitochondria.
TRiC: TCP-1 ring
complex
ORGANELLE-SPECIFIC
PROTEIN QUALITY CONTROL
SYSTEMS AND PROTEIN
MISFOLDING DISEASES
Protein Folding and Quality Control
Systems in the Cytosol
The protein folding and PQC system of
the cytosol comprises a large number of
components constantly assembling and disassembling with their substrate. Upon emerging from the ribosome, nascent polypeptides are protected by chaperones, such
as nascent-polypeptide-associated complex
(NAC), Hsp40, Hsp70, prefoldin, and TCP-1
ring complex (TRiC), and held in a foldingcompetent state until released from the ribosome. Subsequently, most small proteins
complete their folding in the cytosol without assistance, whereas a fraction of the cytosolic proteins requires further assistance
from chaperones, e.g., Hsp90 and TRiC (45).
TRiC, which is the most complex cytosolic chaperone, is composed of a double-ring
structure, each with eight different subunits
forming a large cavity in which the polypeptide is folded to a native or near-native form,
and later released into the cytosol (87). If
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Gregersen et al.
the folding to the native structure cannot be
completed the chaperones assess whether misfolded conformers should be refolded or degraded by the ubiquitin-proteasome pathway
in order to eliminate toxic conformations (61).
Targeting a polypeptide for degradation
requires a multistep pathway to covalently attach ubiquitin monomers. Ubiquitin is activated by the ubiquitin-activating enzyme
(E1), and transferred to an ubiquitin carrier protein (E2). The E2 enzyme and the
polypeptide both bind to a specific ubiquitinprotein ligase (E3), and ubiquitin is covalently attached to the substrate. Further
steps generate a polyubiquitin chain, targeting the polypeptide substrate to the proteasome for degradation (37). A typical mammalian cell contains one or a few different E1
enzymes, some tens of E2 enzymes, and several hundred different E3 enzymes, reflecting the specificity of targeted degradation in
the mammalian organism. The integrated system of chaperones and the components of the
ubiquitin-proteasome pathway comprise the
most important cytosolic PQC system.
Failure of the PQC system to degrade misfolded proteins may lead to formation of protein aggregates. Aggregates in the cytosol may
accumulate at a single, juxtanuclear site called
an aggresome, or as soluble monomers and
oligomers, which later may precipitate into
long amyloid fibrils. Aggresomes are large
globular deposits formed by retrograde transport of aggregated material along microtubular tracks in a highly ordered transportation
system (52), whereas amyloid fibrils are long
protein aggregates with a tube-like core region formed by the inherent properties of circular β-sheet structures (25). Accumulating
data suggest that the fibrillar or aggresomal
deposits may not be the primary toxic agent,
but rather that the inherent pathogenicity resides in the soluble oligomers (13, 18, 49, 66).
By contrast, larger accumulations of aggregates may be a way to control the damage from
the cytotoxic substances. In mammalian cells,
larger aggregates are unspecifically degraded
by the lysosomal autophagic pathway (55).
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Cytosol-Associated Protein
Misfolding Diseases
Defective folding caused by amino acid substitutions that result in rapid degradation of
the variant protein is exemplified by phenylketonuria (PKU), an inborn error of phenylalanine metabolism. Amino acid substitutions in
the phenylalanine hydroxylase (PAH) protein
far from the enzyme’s active site may cause
misfolding of the protein, hinder formation of
the active tetramer, and trigger rapid degradation. In many cases PKU is thus due to
loss-of-function pathogenesis. An increase in
the chaperone concentration or lowering of
the temperature facilitates the protein folding
and, at least in cell models, rescues the enzymatic activity of PAH (36). Aggregates containing the variant PAH protein have been
found in bacterial lysate and in vitro mammalian studies (101). Whether these aggregates are an epiphenomenon, or they perform
a toxic gain-of-function effect in patients is
unknown.
Only a few well examined gain-of-function
diseases exist where the gene product of a
mutated allele interacts with and inhibits the
function of the normal allele product and
thereby acts like a poisonous peptide in a
dominant negative fashion. These disorders
include certain cardiomyopathies as well as
keratin and collagen diseases. For the skin disease epidermolysis bullosa simplex, diseaseassociated missense variations in the keratin-5
and keratin-14 genes lead to incorporation of
misfolded keratin monomers into the keratin
filament network, causing a mechanical instability. Ideally, the PQC system should detect
and eliminate the misfolded monomers. However, external stress factors may reduce its efficiency and thereby trigger the manifestation
of the phenotype. In addition to the poisonous
peptide effect, accumulation of misfolded proteins may lead to aggregate formation, as observed in cardiomyopathies, which may add to
the pathology (77, 96).
A special case of protein misfolding diseases are those caused by variations in the fold-
ing machinery itself, leading to reduced PQC
efficiency. One example is desmin-related myopathy, where αB-crystallin, a small heatshock protein, plays a role in the folding of
desmin, which has its function in the intermediate filaments of cardiomyocytes. This function, however, is compromised by a single
amino acid substitution that leads to the formation of aggregates containing both desmin
and αB-crystallin (98). A similar effect is seen
in patients with amino acid alterations in
desmin itself, rendering the pathology of a
direct protein misfolding pathogenesis most
likely.
In Parkinson’s disease (PD), protein aggregates are formed in the brain, leading to neurodegeneration (64). Point mutations or increased dosage of the α-synuclein gene lead
to a dominant form of the familial disease by
a presumed toxic gain-of-function pathogenesis due to cytosolic aggregates consisting of either wild-type or variant α-synuclein, as well
as components of the ubiquitin-proteasome
system and other cellular proteins. Earlyonset recessive forms of PD are associated
with variations in the PARKIN, UCH-L1, DJ1, or PINK1 genes. These genes code for
components involved in the ubiquitination
and turnover of α-synuclein, and it is speculated that the pathogenesis includes a loss
of PQC function, leading to α-synuclein aggregation in addition to general oxidative
stress causing mitochondrial dysfunction (7).
Post-mortem studies of sporadic PD samples
reveal a general loss of activity in the
ubiquitin-proteasomal system as well as signs
of increased oxidative stress, implying that
sporadic and familial PD evolve through the
same fundamental mechanisms (64).
The pathogenesis of amyotrophic lateral sclerosis (ALS) mediated by Cu,Znsuperoxide dismutase gene (SOD1) variations
is believed to be gain-of-function through aggregation of the misfolded variant SOD1 protein. SOD1 protects the cell from oxidative
damage by catalyzing the dismutation of the
superoxide radicals to hydrogen peroxide and
oxygen (11). The disease therefore seems to
www.annualreviews.org • Protein Misfolding Diseases
PKU:
phenylketonuria
PAH: phenylalanine
hydroxylase
Loss-of-function
pathogenesis:
pathogenesis
resulting from
insufficient protein
function due to
inadequate protein
synthesis, functional
site amino acid
alterations, or
inability to achieve
the functional
protein structure
because of
misfolding
PD: Parkinson’s
disease
Gain-of-function
pathogenesis: the
misfolded protein
accumulates/
aggregates in the
cell, giving rise to
new toxic functions
related to
physico-chemical
properties
SOD1:
Cu,Zn-superoxide
dismutase
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ER: endoplasmic
reticulum
BiP:
immunoglobulin
binding protein
Grp94: Glucose
regulated protein
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PDI: protein
disulphide isomerase
ERAD:
ER-associated
degradation
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be a case of increased oxidative damage from
enzymatic haploinsufficiency. However, artificial reduction of the enzymatic SOD1 activity does not mimic the ALS phenotype in
animal models (10, 72). In fact, several of the
SOD1 variants remain fully active (8). More
than 100 disease-associated SOD1 gene variations are known, accounting for 25% of the
familial ALS cases, which for the most part
are transmitted in a dominant fashion. Several gained functions have been proposed for
the variant proteins, such as aberrant chemistry of the Cu and Zn sites, loss of protein
function through coaggregation with the aggregates, depletion of molecular chaperones,
dysfunction of the proteasome overwhelmed
with misfolded proteins, as well as disturbance
of mitochondrion and peroxisome functions
(11).
A combined gain-of-function and loss-offunction may be a more widespread pathogenesis than presently acknowledged, and a
dysfunctional effect from accumulation of
aberrant proteins may in fact be present in
many protein misfolding diseases (see below).
Protein Folding and Quality Control
in the Endoplasmic Reticulum
The endoplasmic reticulum (ER) is the first
compartment of the secretory pathway. It is
engaged with ribosomal protein synthesis, coand post-translational modification, and protein folding. Proteins enter the organelle in
an unfolded state and begin to fold cotranslationally. The ER lumen contains high concentrations of a specialized set of chaperones and
folding enzymes, which assist protein folding
in conjunction with post-translational modifications, e.g., signal peptide cleavage, disulfide bond formation, and N-linked glycosylation. In this respect, the ER plays a crucial
role in the PQC, regulating the transport of
proteins from the ER to the Golgi apparatus, as only proteins that have attained their
native structure in the ER are exported efficiently (54). The primary PQC is applied to all
proteins and relies on the recognition of hy110
Gregersen et al.
drophobic sequences as well as the content of
unpaired cysteine residues, immature glycans,
and aggregation tendency. Interactions with
components of the primary PQC, i.e., BiP,
calnexin, calreticulin, glucose-regulated protein Grp94 and the thiol-disulphide oxidoreductases, protein disulphide isomerase (PDI),
and ERp57 on one hand, assist protein folding, and on the other, serve as ER retention
anchors. Misfolded or unassembled proteins
initially display a prolonged association with
the PQC components, but may accumulate in
the absence of efficient ER-associated degradation (ERAD) (80). Substrates for ERAD are
selected by the PQC system and translocated
to the cytosol, where they normally are degraded by the ubiquitin-proteasome system
(63, 94).
The secondary PQC often relies on cellspecific factors and facilitates secretion of
individual proteins or classes of proteins.
It was demonstrated that the ER of mammalian cells allows the secretion of several
pathogenic transthyretin variants with wildtype efficiency in spite of compromised folding and that only highly destabilized variants are subjected to ERAD and then only in
certain tissues (82). These observations indicate that ER-assisted folding—in competition
with ERAD—defines the unique secretory capacity of each tissue. The presence of such
tissue-specific PQC could provide a possible
general explanation for the development of
tissue-selective protein misfolding diseases.
A substantial number of cellular proteins
are processed and transported through the
ER. These include receptors and ion channels to be expressed on the cell surface, enzymes and hormones to be secreted, as well as
proteins with a specialized function within the
organelles of the secretory pathway. Because
many of these proteins are essential and indispensable in many physiological processes,
a variety of disease phenotypes may result
from impairment of their ER-mediated transport. A bioinformatics analysis showed that
genes encoding proteins with ER-targeting
signals are overrepresented in a large database
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of disease susceptibility genes compared with
all genes predicting proteins in the human
genome (45% versus 30%), suggesting that
defective ER processing of proteins may contribute to numerous diseases (19).
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Endoplasmic Reticulum–Associated
Misfolding Diseases
ER-associated misfolding and rapid degradation by ERAD are hallmarks of cystic fibrosis
(CF), which is a lethal autosomal recessive disease caused by mutations in the CF transmembrane conductance regulator (CFTR ) gene
encoding a chloride channel. The disease results from loss of chloride regulation in epithelia expressing the gene. More than 1000
disease-associated CFTR gene variations have
been described. However, one single variation, coding for an one-amino acid deletion
variant, F508, is by far the most common,
accounting for about 66% of all diseaseassociated variant CFTR alleles worldwide
(6).
CFTR maturation in heterologous expression systems seems to be a rather inefficient process by which only 25–30% of the
newly synthesized wild-type protein reaches
the plasma membrane (99). In addition, certain amino acid substitutions appear to stabilize the protein but impair ion transport
in such systems, thus it has been suggested
that protein stability has been compromised
for biological function during evolution (92).
Recently, however, it was observed that epithelial cells endogenously expressing CFTR
actually efficiently process the protein to postGolgi compartments (97), an observation that
questions the structural instability model of
wild-type CFTR and supports that epithelialspecific factors regulate both CFTR biogenesis and function (5). For the F508 CFTR
protein the maturation process is very inefficient and virtually all of the protein (>99%)
undergoes rapid ERAD (58, 99).
Synthesis of CFTR occurs with its concomitant insertion in the ER membrane and
attachment of Hsc70/Hsp70 to nascent cy-
tosolic domains. The cell seems to use this
Hsc70/Hsp70 control as the first checkpoint
to assess CFTR conformation, and it has been
proposed that it is the major mechanism to
discard F508 CFTR (31). Prolonged retention of unfolded F508 CFTR by Hsc70
causes the variant protein to be degraded
through ERAD. In contrast, wild-type CFTR
proceeds in the folding pathway through interaction of its N-glycosyl residues with calnexin (31). Subsequently, it acquires its native
conformation for ER export through successive rounds of de- and reglucosylation binding
to calnexin. However, prolonged presence in
the calnexin cycle may cause misfolded CFTR
to become a target for ERAD.
Like CF, hereditary emphysema due to α1-antitrypsin deficiency seems to involve ERassociated misfolding and rapid degradation.
However, in certain individuals under particular conditions, accumulation of the variant protein causes cell damage in the liver
(70). α-1-antitrypsin is a major plasma serine protease inhibitor secreted by hepatocytes to regulate the proteolytic activity of
various circulating enzymes. α-1-antitrypsin
shows considerable genetic variability, having
more than 90 naturally occurring variants (Pi
types). Severe α-1-antitrypsin deficiency affects approximately 1 in 1800 newborns and
95% of these individuals are homozygous for
the PiZ allele (E342K). E342K homozygotes
are predisposed to premature development
of pulmonary emphysema in adult life by a
loss-of-function mechanism, i.e., lack of α1-antitrypsin in the lung permits uninhibited proteolytic damage of the connective tissue matrix by neutrophil elastase (57). The
E342K substitution reduces the stability of the
monomeric form of the protein and increases
its tendency to form polymers in vitro by the
“loop-sheet” insertion mechanism. The abnormally folded and polymerized PiZ α-1antitrypsin variant is retained in the ER of
hepatocytes rather than being secreted into
the circulation, thereby causing plasma deficiency of α-1-antitrypsin. It has been shown
that the PiZ variant interacts with specific
www.annualreviews.org • Protein Misfolding Diseases
CFTR: cystic
fibrosis
transmembrane
conductance
regulator
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FNDI: familial
neurohypophyseal
diabetes insipidus
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AVP: arginine
vasopressin
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components of the ER PQC system, i.e., calnexin, BiP, Grp94, and Grp170 (78), and it is
known that degradation of the variant protein
is mediated by several pathways including the
proteosome, autophagy, as well as one or several nonproteasomal mechanisms (69).
A subset of patients with homozygous PiZ
α-1-antitrypsin deficiency develops chronic
liver disease, usually during childhood. This
condition appears to involve a toxic gain-offunction mechanism whereby retention and
accumulation of the PiZ variant protein in the
ER of the hepatocytes damage the cells by
activating specific signal transduction pathways indicating apoptosis, proinflammation,
and autophagy (46, 74, 91).
A toxic gain-of-function pathogenesis related to misfolded proteins in the ER is also
likely responsible for the development of autosomal dominant familial neurohypophyseal
diabetes insipidus (FNDI). FNDI is characterized by polyuria caused by a deficient
neurosecretion of the antidiuretic hormone,
arginine vasopressin (AVP) (22). The AVP deficiency seems to develop gradually during the
first years of life and is probably due to damaging of the AVP-producing magnocellular
neurons in the supraoptic and paraventricular nuclei of the hypothalamus. FNDI has
been linked to more than 50 different variations in the AVP gene (20, 84). It has been
proposed that the variations lead to the production of a variant hormone precursor that
fails to fold and/or dimerize efficiently in the
ER and thus is retained by the ER PQC, resulting in cytotoxic accumulation of protein in
the neurons. Heterologous expression in cell
cultures and transgenic animals has showed
that some disease-associated AVP gene variations may lead to the production of a variant
AVP hormone precursor that, compared with
the wild-type, is processed inefficiently into
AVP, probably as a result of its retention in
the ER (84). In contrast, a variant of the protein causing a recessive form of FNDI, does
not seem to affect the ER-mediated transport
of the hormone precursor, but rather its final
processing downstream of the ER (21). This
Gregersen et al.
observation may provide a relative control in
support of the hypothesis that the dominant
form of FNDI is caused by protein misfolding
and accumulation of variant AVP precursor
protein in the ER.
Variant AVP precursor proteins expressed
in cell culture have been shown to be translocated to the cytosol and degraded by the
proteasome, but ubiquitination could not be
demonstrated (34). Actually, a significant portion of the primary translation products failed
to enter the ER lumen at all. Both pathways
of degradation, via the ER lumen and directly
from the cytosol, were additionally found to
some extent for the wild-type protein. It is not
known whether cytosolic forms of the variant proteins trigger cytotoxicity in vivo. In a
murine knock-in model of an FNDI mutation
the progressive development of a typical diabetes insipidus phenotype was accompanied
by an induction of BiP, as well as a specific
and progressive loss of the AVP-producing
neurons (75). Furthermore, heterologous expression of an AVP variant protein in cell
culture and transgenic animals has indicated
that autophagy is involved in the pathogenesis
(15, 16).
Protein Folding and Quality Control
in Mitochondria
Mitochondria represent a separate cellular compartment where—in humans—
approximately 1500 proteins fold and are
degraded (90). Only 13 of the mitochondrial proteins are encoded by the mitochondrial DNA, the bulk is nuclear encoded,
synthesized in the cytosol and subsequently
imported into mitochondria. Import of mitochondrial proteins occurs mainly posttranslationally in an unfolded conformation
through pores in the outer and inner mitochondrial membrane (102). Many mitochondrial proteins, especially those of the matrix
space, contain amino terminal extensions that
counteract premature folding in the cytosol,
direct the protein along the mitochondrial
import machinery, and are cleaved off upon
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arrival in the mitochondrial matrix. Many
proteins localized in the outer and inner membrane or the intermembrane space contain internal targeting signals that are not cleaved
off.
Molecular chaperones in the cytosol like
Hsp70 and Hsp90 keep newly synthesized
mitochondrial proteins in an unfolded,
import-competent conformation (104). A mitochondrial Hsp70 homolog, the first representative of the mitochondrial PQC system,
binds to the incoming polypeptide chain and
is involved in the translocation process (102).
The mitochondrial PQC system comprises
many orthologs to bacterial and yeast PQC
systems including molecular chaperones like
the mitochondrial Hsp70, the Hsp60/Hsp10
system, and a set of proteases with AAA+
domains—resembling the proteasome—that
are localized in the matrix or the inner membrane (48).
Mitochondria-Associated Protein
Misfolding Diseases
A large number of recessively inherited genetic diseases resulting in loss-of-function due
to variations in genes encoding mitochondrial
metabolic enzymes have been described. In
many cases, variations are of the missense type
and affect the folding propensity of the protein and/or the stability of the native conformation. A typical and extensively studied
example is medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (38). The MCAD
enzyme, a homotetramer with FAD as cofactor, is involved in mitochondrial fatty acid
β-oxidation. One prevalent amino acid substitution, K304E, is responsible for approximately 90% of the disease-associated alleles in patients with clinical symptoms, and
its effects have been studied in detail (38).
The K304E variant protein is synthesized and
imported into mitochondria at normal levels, but is strongly impaired in folding and
assembly. Folding of the MCAD protein occurs through successive interaction with mitochondrial Hsp70 and the mitochondrial chap-
erone Hsp60. The K304E variant protein has
been shown to remain associated with Hsp60
for prolonged time periods (76). An important
finding was that the residual level of the natively folded K304E variant enzyme could be
strongly modulated by environmental conditions like temperature and availability of chaperones (9). Analysis of the purified fraction of
the K304E variant protein that folded to the
native state after expression under permissive
conditions revealed only slightly altered enzymatic parameters and a decreased thermal
stability (51). Similar studies with other
disease-causing MCAD variations and other
mitochondrial enzyme deficiencies due to
missense variations (47) underline that impaired folding is a major effect of diseaseassociated missense variations.
Deficiency of short-chain acyl-CoA dehydrogenase (SCAD), a mitochondrial homolog
of MCAD with specificity for shorter chain
length is due to variations in the SCAD gene.
Mostly missense disease-associated variations have been identified, and experimental
analysis of a set of variant proteins showed
that all display prolonged interaction with the
Hsp60 chaperone (68). Like that observed for
MCAD, some of the variant proteins are unable to form active enzyme whereas others
form active tetramers with efficiency depending on the folding conditions. An interesting observation of the analysis was that some
of the variant proteins have a strong tendency to aggregate inside the mitochondria
and that this tendency was increased at elevated temperature. This phenomenon raises
the possibility that aggregation may play a
role in the pathogenesis of SCAD deficiency.
In this and other instances one may envisage that both loss-of-function due to degradation of misfolding variant proteins and a
gain-of-function pathogenesis due to formation of toxic accumulated misfolded proteins
may coexist, and that secondary genetic or environmental conditions determine the balance
between these.
In hereditary spastic paraplegia, a lateonset degenerating disease resulting from
www.annualreviews.org • Protein Misfolding Diseases
MCAD:
medium-chain
acyl-CoA
dehydrogenase
SCAD: short-chain
acyl-CoA
dehydrogenase
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dysfunction of the very long axons of the
motor neurons that control voluntary movements of the legs, disease-associated variations have been detected in two genes encoding mitochondrial PQC components. One is
paraplegin, a subunit of the membrane-bound
AAA+ domain containing protease, and the
other is Hsp60, a chaperone of the mitochondrial matrix (14, 44).
Paraplegin coassembles with AFG3L2 to
a protease complex in the inner mitochondrial membrane. In the absence of a functional paraplegin gene, like in some spastic
paraplegia patients, the AFG3L2 subunit assembles to homo-oligomeric complexes that
apparently have residual function providing
sufficient protease and PQC function under
most circumstances (3), but apparently not
under those present in very long axons.
Only one disease-associated variation has
so far been identified in the HSP60 gene. It is a
missense variation that abolishes the capacity
of Hsp60 to functionally replace the bacterial
homolog GroEL in an Escherichia coli experimental system (44). Investigations with the
bacterial homolog GroEL indicate that a relatively small portion of E. coli’s proteins require
interaction with GroEL for folding and that a
number of other proteins are partially dependent (50). Hsp60 is an essential gene in flies
and yeast. Considering the oligomeric, sevenmer ring architecture of the Hsp60 chaperone
complex, incorporating defective mutant subunits that have a dominant negative effect on
the function would be expected to totally abolish Hsp60 chaperone function. The HSP60
gene variation associated with spastic paraplegia thus appears to disturb Hsp60 chaperone
function in a subtle way that only manifests in
certain cells.
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CELLULAR CONSEQUENCES
OF PROTEIN MISFOLDING
The conceptional framework of protein misfolding diseases comprising loss-of-function
and gain-of-function pathogenic mechanisms
is a practical one in the process of understand114
Gregersen et al.
ing the biochemistry and cell biology as well as
in developing strategies for treating these diseases. However, the effect of gene variations
and damaging modifications in a given protein is very complex and variable over time
in the same individual or between individuals. Indeed, with special relevance for genetic diseases, gene variations may, in addition to creating an insufficiency of protein
function, at certain times, e.g., late in life
or under cell stress conditions, give rise to a
gain-of-function pathogenesis due to insufficient elimination of misfolded proteins. Although the specific biochemistry and cellular pathology—caused by a loss of protein
function—is important for diagnosing and
treating the individual diseases, the contribution from the cellular disturbances, which are
elicited by accumulated and aggregated cellular proteins, may thus be as important. It
may vary from insignificant to being the determinant pathogenetic factor, depending on
the balance between correct folding, misfolding, degradation, and accumulation of a given
variant or damaged protein. In turn, this balance is determined by the nature of the protein in question, the cellular compartment in
which the misfolding occurs, the efficiency of
the relevant PQC system, the interacting genetic and chemical factors, as well as the cell
and environmental stress conditions.
Despite these variables, the cellular
consequences—mild or severe—may be discussed within a common framework defined
by simple physico-chemical properties of the
proteins, such as hydrophobicity and charge,
which may engage the misfolded proteins in
unproductive and toxic protein-protein interactions (88).
Within this common framework it is possible to distinguish between four levels of
cellular reactions. The first is the immediate reaction and effort of the cell to clear
it from misfolded proteins. The second involves cellular perturbations elicited by noncleared misfolded proteins, which may have
assembled into oligomeric and polymeric
forms. The third is the induction of further
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protection mechanisms against these perturbations, and the fourth is elimination of the
cell. The various mechanisms, as they are indicated in Figure 4, are not exclusive, but may
be interconnected in sequences of events depending on the load of insults as well as other
factors, such as type and age of the affected
cells.
As discussed previously, PQC systems have
evolved to protect the cells from unwanted
translation products, as well as damaged proteins, all of which may misfold. If the load
of misfolded proteins increases, a set of protective response mechanisms induces the expression of chaperones and proteases, as well
as reduces the general protein synthesis to
alleviate the load (73, 80, 93, 105). In all
cases, the mechanisms—at least partly—are
governed by an imbalance between occupied
chaperones (Hsp70s and Hsp90s) and regulatory proteins, such as inositol requiring kinase
1 (IRE1) and protein kinase-like ER kinase
(PERK) in the ER (80), heat-shock factors in
the cytosol (73), and yet unknown factors in
the mitochondria (105).
In the young and healthy cell these
mechanisms can cope with the load. But if the
misfolded protein is slowly degradable or aggregation prone, or if cell stress is intense and
long lasting or the cell has decreased degradation capacity, the misfolded protein may accumulate and affect a large number of cellular
functions.
Because the ubiquitin-proteasome system
is involved in the turnover of many cellular
proteins and degradation of aberrant polypeptides, an efficient function of this system is
very important for the clearing function of the
ER and cytosolic PQC systems. Inhibitions
of the proteasomal activity by misfolded proteins have been shown (4, 42), as has decline
of the efficiency by accumulation of aberrant
ubiquitin (62). In addition to an inefficient
degradation capacity to cope with the misfolding load, a sequestration of components
of the ubiquitin-proteasome system, including ubiquitinated proteins, as well as other
chaperones, proteases, and a number of other
Stress response
Accumulation
Loss-offunction
Toxic gain-of-function
Proteasome inhibition
Chaperone sequestration
Transcription/cell cycle
factor sequestration
Fibril pore formation
Calcium overload
Oxidative stress
Degradation
Loss-of-function
Functional insufficiency
Glutamate overload
Mitochondrial dysfunction
Cell death
Figure 4
The fate of misfolded proteins and some important cellular consequences
of accumulated proteins.
cellular constituents, such as transcription factors, has been observed in a number of protein misfolding diseases (11, 26, 52, 65, 83).
This indicates that sequestration of chaperones and other vital factors may be a pathological mechanism, at least in some cases.
The fact that we and other researchers have
shown that misfolded variant metabolic enzymes during their processing in mitochondria or the cytosol are more dependent on
chaperone assistance than the wild-type protein (1, 9, 36, 40, 68), as well as the indication
that increased oxidation and impaired protein
degradation in old age may result in so-called
chaperone overload (86), strengthen the case.
Another type of pathogenesis leading to
cell dysfunction is fibril formation, which
has been suggested to account for many of
the mechanisms leading to cell dysfunction
and death in the traditional neurodegenerative conformational diseases (17). Oligomeric
misfolded precursors to amyloid plaques have
been shown to build into membranes (18)
and form pores with devastating consequences
for membrane-associated functions, such as
ion transport, glutamate homeostasis, oxidative metabolism, and cell viability (12, 13,
17, 18). Additionally, it has been shown
that α-synuclein may form oligomers, which
www.annualreviews.org • Protein Misfolding Diseases
IRE1: inositol
requiring kinase 1
PERK: protein
kinase-like ER kinase
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OXIDATIVE CELL STRESS
Misfolded proteins are only one internal stressor which induces cell stress. Another related stressor is aging, where PQC
systems lose efficiency and misfolding occur. A characteristic
for misfolding stress is the creation of oxidative stress. Since
it has been suggested that the pathogenesis in diseases like
diabetes, cardiovascular diseases, depression, and cancer, is
related to oxidative stress, it is reasonable to suggest that protein overload/oxidative stress is a very general mechanism,
which normally is counteracted by induction of PQC systems
and antioxidant/autophagy systems. The mechanistic link between misfolded proteins and oxidative stress is therefore of
great scientific and practical interest.
fragment the Golgi apparatus (17). Whether
intramitochondrial misfolded proteins can
form membrane-inserted fibrils is not known,
but accumulated misfolded variant SOD1,
which is located—at least partly—in the intermembrane mitochondrial space, has been
shown to elicit a whole range of damaging
mechanisms, including production of ROS
and damage to several respiratory chain enzyme complexes (11). This is probably initiated by calcium overload, an important
pathologic mechanism in neurodegeneration
(53). Calcium overload stimulates oxidative
metabolism and induces the creation of oxidative stress as well as disturbs the glutamate metabolism resulting in excitotoxicity
(30). A consequence of oxidative stress is the
creation of oxidatively modified proteins (95),
which are prone to misfolding and blockage
of the degradation systems, most notably the
ubiquitin-proteasome system, which through
the mechanisms mentioned will create additional oxidative stress, initiating a vicious cycle
(see also Oxidative Cell Stress).
To suppress the development of cell damage, due in particular to oxidative stress, the
cells possess a number of defense systems,
which in the present context are the antioxidant systems (100, 103) and autophagic system (55), the functions of which are, respectively, to detoxify ROS and eliminate damaged
116
Gregersen et al.
cell domains, e.g., dysfunctional mitochondria (85). When the defense systems fail to
sustain cell health the final mechanism where
all dysfunctions meet is cell death, either as
apoptosis or necrosis (79).
Although a great deal about the effects
of misfolded proteins on various cellular
functions is known, there are still many
unanswered questions. A subject that was
only touched on here, and on which there
is an extensive literature, is the signal transduction pathways, which mediate the various
effects and the relation between them. Space
limitations have not allowed a discussion
of these molecular mechanisms, but many
are mentioned and referred to in the review
papers cited here.
CONCLUDING REMARKS
Broadening the concept of protein misfolding diseases from the traditional conformational or aggregation diseases to encompassing many inherited disorders where missense
gene variations render the variant protein unstable, either as folding intermediate or in
the native state, is changing the scientific
approach to elucidating the pathogenic and
cell pathologic mechanisms of many diseases.
From approaching the diseases from a purely
biochemical and genetic perspective, where
protein deficiency and substrate/ligand accumulation are viewed as the sole causes of the
cell pathology and clinical symptoms, we are
beginning to understand some of these disorders from a cell biological viewpoint. Ten
to twenty years ago, when disease-associated
gene variations were discovered in many genetic diseases, it was believed that genotypephenotype relationships could be established
in most genetic diseases. This turned out to
be too optimistic. In most genetic diseases
no clear genotype-phenotype relationship can
be established. There are many reasons for
this, but one of them seems to be that missense variations give rise to misfolded variant
proteins, the fates of which depend on many
factors that may vary with time in different
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cells and from patient to patient. It has been
demonstrated—at least in model systems—
that the efficiency of the PQC systems is of
prime importance in this respect. It is also
reasonable to suppose—as has been shown
in a number of diseases—that common gene
variations in other factors implicated in processing variant or damaged proteins may con-
tribute to disease expression and development. In this respect, using transcriptomic
and proteomic technologies should help in
dissecting the cell pathology of many genetic disorders as well as elucidating the sequence of pathogenic and pathological mechanisms in the age-related protein misfolding
diseases.
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SUMMARY POINTS
1. Protein folding is accomplished by intramolecular forces and passes through intermediates with decreasing energy and entropy striving toward an energy minimum.
In vitro, folding intermediates may go off the pathway and use intermolecular forces,
resulting in aggregation. In vivo, the folding process is assisted by molecular chaperones, which shield the proteins and guide them to the native structure.
2. Aberration in the amino acid chain, either by inherited gene variations or from damage
to amino acids, such as oxidative modifications, may compromise folding, even in the
presence of chaperones. Intracellular proteases, which together with the chaperones
comprise the cellular protein quality control systems, try to eliminate misfolded proteins. For certain proteins and under certain circumstances elimination is inefficient
and the misfolded protein accumulates as aggregates.
3. Aberrant proteins, which are prematurely eliminated by proteases, may give rise to
loss-of-function pathogenesis and result in protein deficiency disease. Aberrant proteins, which are not eliminated but accumulated, result in gain-of-function pathogenesis and disease pathology. Some diseases show both loss-of-function and gain-offunction pathogenic mechanisms.
4. Typical diseases due to predominantly loss-of-function pathogenesis are phenylketonuria, cystic fibrosis, the pulmonary form of α-1-antitrypsin deficiency, and
medium-chain acyl-CoA dehydrogenase deficiency. Typical diseases with predominantly gain-of-function pathogenesis are cardiomyopathies, keratin and collagen
disease, Parkinson’s disease due to α-synuclein gene variations, familial neurohypophyseal diabetes insipidus, and the liver form of α-1-antitrypsin deficiency. Mixed
pathogenesis is seen in Parkinson’s disease due to deficiency of the elimination systems, and indicated in short-chain acyl-CoA dehydrogenase deficiency.
5. Diseases with loss-of-function protein misfolding pathogenesis are typically autosomal recessively inherited, such as many metabolic disorders. In contrast, gain-of
function pathogenesis most often results in dominant diseases, either due to poisonous peptides, such as in keratin and collagen diseases, or due to the cellular effects
of the accumulated misfolded protein, such as those seen in Parkinson’s disease with
α-synuclein accumulation.
6. The effect of loss-of-function protein misfolding pathogenesis is typically insufficient of a metabolic or transport reaction as well as toxic effects of an accumulated
substrate, which is unique for the particular cellular reaction. The effect of accumulated/aggregated proteins is more generally elicited by the physico-chemical properties and not by the specific function of the proteins.
www.annualreviews.org • Protein Misfolding Diseases
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7. Although the effect may be similar for different accumulated proteins, there are probably numerous factors, including cell types affected, which determine the pathology
and severity of the insult. In particular, the expression level and the efficiency of the
protein quality control systems in the specific cell type may be important. This renders aging cells in nondividing tissues, like muscle and brain, especially vulnerable to
the consequences of protein misfolding.
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FUTURE ISSUES TO BE RESOLVED
1. There is a need to develop quantitative bioinformatic tools to predict the effects of
amino acid substitutions on the folding of a given protein.
2. There is a need for methods to determine the balance between elimination and accumulation of misfolded proteins, i.e., the efficiency of protein quality control systems,
in order to elucidate the cell pathology and decide strategies for intervention.
3. Oxidative cell stress is a common phenomenon in protein misfolding diseases as well
as in many other pathological conditions. Therefore, there is a need to elucidate
the mechanistic links between accumulated misfolded proteins and mitochondrial
dysfunction, which is the prime cause of oxidative stress.
ACKNOWLEDGMENTS
The studies concerning protein misfolding diseases were supported by the Institute of Clinical
Medicine, Aarhus University; The Danish Medical Research Council; the Danish National
Research Foundation; the Aarhus University Hospital Research Initiative; The Danish Heart
Foundation; the Aarhus University Research Foundation; the Karen Elise Jensen Foundation;
the Lundbeck Foundation; the Novo Nordisk Foundation; and the European Union (sixth
framework program).
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23. Takes the
concept of energy
landscapes to the
next level by
including the
folding of
aggregation prone
proteins and the
difficulties of
protein folding in
the crowded space
of the cytosol.
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structural
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their particular
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and
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RELATED RESOURCES
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Contents
Volume 7, 2006
A 60-Year Tale of Spots, Maps, and Genes
Victor A. McKusick p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Transcriptional Regulatory Elements in the Human Genome
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Predicting the Effects of Amino Acid Substitutions on Protein
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Protein Misfolding and Human Disease
Niels Gregersen, Peter Bross, Søren Vang, and Jane H. Christensen p p p p p p p p p p p p p p p p p p p p p 103
The Ciliopathies: An Emerging Class of Human Genetic Disorders
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The Evolutionary Dynamics of Human Endogenous Retroviral
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Genetic Disorders of Adipose Tissue Development, Differentiation,
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Pharmacogenetics and Pharmacogenomics: Development, Science,
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Mouse Chromosome Engineering for Modeling Human Disease
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The Killer Immunoglobulin-Like Receptor Gene Cluster: Tuning the
Genome for Defense
Arman A. Bashirova, Maureen P. Martin, Daniel W. McVicar,
and Mary Carrington p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 277
Structural and Functional Dynamics of Human Centromeric
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Of Flies and Man: Drosophila as a Model for Human Complex Traits
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The Laminopathies: The Functional Architecture of the Nucleus and
Its Contribution to Disease
Brian Burke and Colin L. Stewart p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 369
Structural Variation of the Human Genome
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Resources for Genetic Variation Studies
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Cumulative Index of Contributing Authors, Volumes 1–7 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 477
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Errata
An online log of corrections to Annual Review of Genomics and Human Genetics chapters
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