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Endocrine Reviews 26(4):479 –503
Copyright © 2005 by The Endocrine Society
doi: 10.1210/er.2004-0010
Beyond the Signal Sequence: Protein Routing in Health
and Disease
Cecilia Castro-Fernández,* Guadalupe Maya-Núñez,* and P. Michael Conn
Oregon National Primate Research Center (P.M.C., C.C-F., G.M-N.) and Departments of Physiology and Pharmacology and
Cell and Developmental Biology (P.M.C.), Oregon Health and Science University, Beaverton, Oregon 97006; and Research
Units in Developmental Biology (C.C-F., G.M-N.), and Reproductive Medicine (P.M.C.), Instituto Mexicano del Seguro
Social, Mexico Distrito Federal 06725, Mexico
Receptors, hormones, enzymes, ion channels, and structural
components of the cell are created by the act of protein synthesis. Synthesis alone is insufficient for proper function, of
course; for a cell to operate effectively, its components must
be correctly compartmentalized. The mechanism by which
proteins maintain the fidelity of localization warrants attention in light of the large number of different molecules that
must be routed to distinct subcellular loci, the potential for
error, and resultant disease. This review summarizes diseases
known to have etiologies based on defective protein folding or
failure of the cell’s quality control apparatus and presents
approaches for therapeutic intervention. (Endocrine Reviews
26: 479 –503, 2005)
I. Introduction
II. Protein Processing and the Role of Chaperones
A. ER processing and quality control (QC)
B. Golgi apparatus
C. Mitochondria
D. Chaperones: the role of folding in QC and routing
III. Diseases Caused by Defective Routing
A. G Protein-Coupled Receptors (GPCRs) in disease states
B. Wild-type protein degradation and oligomerization in
protein processing
C. Other proteins
IV. Diseases Caused by Conformational Errors
A. AD and neuropathies
B. Parkinson’s disease (PD)
C. Huntington’s disease (HD)
D. Viral prions
E. Cataracts
F. ␣1-AT deficiency
V. Rescue of Defective Proteins
A. Temperature
B. Chemical chaperones
C. Nonspecific agents
VI. Conclusions
I. Introduction
I
N 1971 GÜNTER BLOBEL presented a first version of the
“signal hypothesis.” He postulated that, in the case of
secretory proteins, there is an intrinsic signal that governs
protein movement to and across membranes. Blobel’s laboratory and others subsequently showed that other distinct
“address tags” direct proteins to intracellular organelles.
These principles are universal, operating similarly in yeast,
plant, and animal cells; errors in these tags can result in
hereditable disease. In 1999, the Nobel Prize recognized Blobel’s work.
Recent evidence, coming largely from mutations that result in human disease, led to another inescapable conclusion:
the successful intracellular routing of many proteins is also
governed by a sensitive quality control (QC) system that
recognizes particular structural motifs, then retains and degrades defective molecules (1). Abnormal proteins are extremely dangerous to cells because they interfere with normal functions and eventually may result in cell death. The
cell limits the accumulation of aberrant conformations with
a chaperone system to assist the overall folding process (2, 3).
A picture is now emerging that, for some wild-type (wt)
proteins, such as the opiate receptor and human (but not
rodent) GnRH receptor (GnRHR), only a modest percentage
of the newly synthesized protein is routed correctly; the
balance is destroyed. The reason for this apparent inefficiency is unclear but suggests the possibility of an unappre-
First Published Online November 9, 2004
* C.C-F. and G.M.-N. contributed equally to the preparation of this
manuscript and are designated cofirst authors.
Abbreviations: AD, Alzheimer’s disease; ␣1-AT, ␣1-antitrypsin; APP,
amyloid precursor protein; AQP2, aquaporin-2; BiP, Ig-binding protein;
BSE, bovine spongiform encephalopathy; CF, cystic fibrosis; CFTR, CF
transmembrane conductance regulator; CJD, Creutzfeldt-Jackob disease; COPII, coat protein complex II; DA, dopamine; ER, endoplasmic
reticulum; ERAD, ER-associated degradation; ERGIC, ER-Golgi intermediate compartment; GABABR, ␥-aminobutyric acid B receptor;
GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; GRP, glucose-regulated protein; HC, hemochromatosis; HD, Huntington’s disease; HFE, hemochromatosis gene; HH, hypogonadotrophic hypogonadism; Hsp, heat shock protein; LB, Lewy body; LDL, low-density
lipoprotein; LDLR, LDL receptor; ␤2M, ␤2-microglobulin; MHC, major
histocompatibility complex; NCCT, NaCl cotransporter; NDI, nephrogenic diabetes insipidus; PD, Parkinson’s disease; PDI, protein disulfide
isomerase; PS1, presenilin 1; QC, quality control; RAP, receptor-associated protein; rBAT, rat liver bile acid coenzyme A: amino acid Nacetyltransferase; Rh, rhodopsin; RP, retinitis pigmentosa; TSE, transmissible spongiform encephalopathy; UGT, uridine diphosphateglucose:glycoprotein glycosyltransferase; UPR, unfolded protein
response; V2R, vasopressin type 2 receptor; vCJD, variant CJD.
Endocrine Reviews is published bimonthly by The Endocrine Society
(http://www.endo-society.org), the foremost professional society serving the endocrine community.
479
480
Endocrine Reviews, June 2005, 26(4):479 –503
ciated level of regulation that may provide receptors for
up-regulation, enzymes for feedback adjustment, or ion
channels to adapt to rapidly changing conditions.
In some cases, protein oligomerization is requisite for export of the nascent protein from the endoplasmic reticulum
(ER); mutant or defective proteins may oligomerize with
and retain wt proteins, leading to a dominant-negative
phenotype.
Diseases caused by abnormal proteins include circumstances in which a specific protein, or protein complex, fails
to fold correctly [e.g., cystic fibrosis (CF)], or is not sufficiently
stable to perform its normal function (e.g., some forms of
cancer). They also include conditions in which abnormal
folding results in the failure of protein to be correctly positioned within the cell [e.g., hypogonadotrophic hypogonadism (HH), ␣1-antitrypsin (␣1-AT) deficiency, and some forms
of retinitis pigmentosa (RP)]. Other misfolding diseases,
amyloidoses, are caused by conformational changes coupled
to the aggregation and cytotoxicity of misfolded proteins
outside the cell (Table 1). This review addresses the molecular mechanisms of protein misfolding and protein aggregation and the newly appreciated role of this event in health
and disease.
II. Protein Processing and the Role of Chaperones
A. ER processing and quality control (QC)
The ER is a lamellar organelle associated with ribosomal
protein synthesis, co- and posttranslational modification,
and protein folding (Fig. 1). Proteins enter the ER in an
unfolded state via an aqueous channel, the Sec61 translocon,
to receive posttranslational modifications such as disulfide
bond formation, signal peptide cleavage, N-linked glycosylation, glycophosphatidyl inositol anchor addition, and
palmitoylation/myristoylation (3, 4). If the protein is not
properly folded/matured, it will remain in the ER and is
eventually degraded without reaching its normal cellular
site of action (5, 6).
TABLE 1. Examples of diseases caused by abnormal proteins
Disease
Protein(s)
Caused by defective routing
HH
GnRHR
RP
Rhodopsin
NDI
Arginine vasopressin
AQP2
Cancer
p53
Cystinuria
rBAT
CF
CFTR
Gitelman’s syndrome
Thiazide-sensitive NaCl transporter
HC
HFE
Hypercholesterolemia
LDLR
Caused by conformational errors
AD
Amyloid-␤ peptide
␶-Protein
PD
␣-Synuclein
Parkin
Ubiquitin-carboxyl-hydrolase L1
HD
Huntingtin
TSEs
Prion protein
Cataracts
␣B-crystallin
␣1-AT deficiency
␣1-ATZ
Castro-Fernández et al. • Protein Routing in Health and Disease
TABLE 2. Chaperones involved in QC
Family
Hsp70
Hsp90
Hsp40
Hsp60
Lectins
Oxidoreductases
Members
BiP, Grp78
Grp94
ERdj1-ERdj5,
Sec63
Hsp60
Calnexin,
calreticulin
PDI, ERp57,
ERp72, etc.
Hsp100
Hsp100
Small Hsp
Hsp27,
␣-crystallin
Function
Chaperone
Chaperone
Cofactor, can regulate
BiP in vitro
Mitochondrial
chaperone
Glycoprotein QC
Formation of disulfide
bonds and
isomerization and
reduction
Mitochondrial
chaperone
Holding denatured
proteins
The ER has QC machinery that discriminates between
properly folded proteins and misfolded ones; sometimes
aberrant forms acquire their correct conformation after several passages through the folding chaperone system. The ER
functions as a guardian that prevents the premature exit of
folding intermediates or incompletely assembled proteins
from the ER. This QC mechanism includes a wide range of
molecular chaperones and folding catalysts and appears to
have evolved to assist the folding process (7, 8) (Table 2).
All proteins are subjected to a “primary” QC that monitors
their architectural design through ubiquitous folding sensors
that are abundant in the ER and recognize common structural and biophysical features which distinguish native from
nonnative protein conformations (7, 8). The Ig-binding protein (BiP, discussed below), calnexin, calreticulin, glucoseregulated protein (GRP)-94, and the thiol-disulfide oxidoreductases protein disulfide isomerase (PDI) and Erp57
are molecular chaperones and sensors found in the ER (9 –
11). A misfolded or incompletely folded protein can be
bound by these factors, and these provide assisted folding.
Some proteins utilize mostly one system; others use them
sequentially or even simultaneously, and the chaperone systems can also serve as retention anchors for immature proteins (7, 12) or protein switches that either properly route a
protein or direct it to a degradation pathway (13, 14). The
secondary QC, which is often cell type specific, contains
selective mechanisms that regulate the export of proteins,
which are accompanied out of the ER by specialized chaperones and enzymes (12).
The effectiveness of the QC system depends, in large part,
on the ability of functional proteins to avoid capture because
their hydrophobic regions are generally buried. However,
when these hydrophobic surfaces are exposed, ER resident
chaperones interact with them, retaining them in the ER (8).
It appears that the glucosyl transferases recognize misfolded
proteins when hydrophobic residues situated on the carboxyl side of the glycosylation site are present. The QC mechanism, which involves a remarkable series of glycosylation
and deglycosylation reactions, enables correctly folded proteins to be distinguished from misfolded ones (15) (Fig. 2).
However, if the glycoprotein is misfolded it results in retention in the ER where it can undergo a new cycle of reglycosylation by a uridine diphosphate-glucose:glycoprotein
Castro-Fernández et al. • Protein Routing in Health and Disease
Endocrine Reviews, June 2005, 26(4):479 –503 481
FIG. 1. The QC process in the secretory pathway. The ER is a lamellar organelle associated with ribosomal protein synthesis, co- and
posttranslational modification, and protein folding. Proteins enter the ER in an unfolded state via the Sec61 translocon to receive posttranslational modifications; if the protein is not properly folded/matured, it will remain in the ER and is eventually degraded without reaching its
normal cellular site of action. The discrimination between properly folded proteins and misfolded ones takes place in the ER by the QC machinery;
sometimes aberrant forms acquire their correct conformation after several passages through the folding chaperone system. Terminally misfolded
proteins are retrotranslocated across the ER membrane to be degraded by proteasomes in a process known as ER-associated degradation.
Correctly folded proteins are allowed to exit the ER and enter the secretory pathway, through exit sites located at the ER membrane. Proteins
are packed into COPII-coated vesicles and leave the ER to be transported to the Golgi apparatus, where they fuse with the target membrane
elements in the ERGIC, near the Golgi. Once the proteins reach the Golgi complex, they are sorted to peripheral compartments such as vacuoles,
plasma membrane, and secretory vesicles. Retrieval of misfolded proteins from the Golgi complex to the ER occurs; also defective proteins can
be transported to the endosomal system for lysosomal or vacuolar degradation. SV, Secretory vesicle.
glycosyltransferase (UGT) (3, 16). Another protein, ER degradation-enhancing 1,2-mannosidase I (a recently discovered
lectin), binds misfolded proteins that are released from calnexin and promotes their degradation, rather than allowing
entry into another UGT-calnexin cycle (13). It was initially
believed that ER-resident proteinases and peptidases degraded misfolded and orphan proteins (17). However, terminally misfolded molecules are ”retrotranslocated“ or ”dislocated“ across the ER membrane to be degraded by cytosolic
proteasomes (Fig. 3). The misfolded or unassembled proteins
are retained; proteins that normally form associations with
other proteins cannot progress through the secretory pathway until they are bound to their partners. Instead, the misfolded proteins are recognized by ER chaperones or by other
factors; they are then again translocated to the cytosol
through the translocon Sec61 (18). This process, known as
ER-associated degradation (ERAD), selects the QC substrates
to be degraded, targets them to the translocon, and transports
them back to the cytosol, where the proteolytic components
of the protein degradation pathway such as ubiquitin, ubiquitin-conjugating enzymes, and the cytosolic 26S proteasome
are used (6). After retrotranslocation from the ER, misfolded
proteins are polyubiquitylated before degradation. Lack of
polyubiquitylation prevents proteasomal degradation and
leads to failure in transport of the protein from the ER lumen
or membrane out to the cytoplasm, causing the protein to
remain in the ER. Ubiquitination may facilitate the recruitment of factors that actively extract the polypeptide from the
ER membrane.
The proteins that undergo proteasomal degradation by the
proteolytic complex 26S proteasome are marked by the binding of a ubiquitin chain to a lysine residue (19 –21). The 26S
proteasome is composed of a core 20S particle. These proteins
are digested into short peptides and one or two 19S regulatory particles, which are responsible for substrate recognition and transport into the core particle. The entry channel
is quite narrow, and even in its open state, it allows entry of
only unfolded proteins (22, 23). Although excluding normal
globular proteins, this architecture requires complex ATPdependent mechanisms to recognize, unfold, and linearize
substrates and to inject them into the 20S proteasome (23).
After proteolysis, the polyubiquitin chain is released and
hydrolyzed into single ubiquitin moieties that go through a
new cycle of protein degradation (3).
When aberrant proteins accumulate in the ER due to ineffective ER degradation machinery, there is a different response that occurs. This “unfolded protein response” (UPR)
leads to synthesis of ER-resident chaperones and enzymes.
482
Endocrine Reviews, June 2005, 26(4):479 –503
Castro-Fernández et al. • Protein Routing in Health and Disease
FIG. 2. The calnexin/calreticulin cycle. The QC
mechanism involves a series of glycosylation and
deglycosylation reactions that enable correctly
folded proteins to be distinguished from misfolded ones. It appears that the glucosyl transferases recognize misfolded proteins when hydrophobic residues situated on the carboxyl side of
the glycosylation site are present. When proteins
translocate the ER membrane, a core of oligosaccharides (Glc3Man9-GlcNAc2) is attached to asparagine residues in the Asn-X-Ser/Thr consensus sequence (step 1). After the folding process,
two of the outermost glucose residues are eliminated by the glucosidases I and II (step 2). Disulfide bonds are transiently formed by the Erp57
(a thiol oxidoreductase chaperone), which interacts specifically with calnexin and calreticulin,
and the monoglucosylated protein (step 3). After
the last glucose residue is eliminated by glucosidase II, the complex dissociates and releases a
protein with a carbohydrate structure of
Man9GlcNAc2 (step 4). Correctly folded proteins
are then transported out of the ER to the secretory pathway. When the glycoprotein is not correctly folded, the N-glycan is reglycosylated by
the UGT (steps 5 and 6), which induces a new
round of calnexin/calreticulin binding preventing
the exit of the protein from the ER.
Drugs that block N-linked glycosylation or disulfide bond
formation or alter calcium homeostasis produce this response. If the response fails to clear the ER, apoptosis is
induced (8). The UPR and ERAD are partially overlapping
responses that lead to elimination of misfolded secretory
proteins. Induction of the UPR up-regulates multiple genes
involved in ERAD/proteasomal protein degradation. Defects in ERAD constitutively activate the UPR, and simultaneous deficiency of both pathways decreases cell viability.
In contrast, correctly folded proteins are transported out of
the ER and do not go through this QC system, which only
retains and degrades misfolded proteins (7, 24). Defective
components can cause the QC system to lead to destruction
of recoverable proteins at a great cost to energy input. A
disturbed QC leads to disease and eventually to cell death.
Proteins are allowed to exit the ER and enter the secretory
pathway when they are properly folded, through exit sites
located at the ER membrane (25–28). Misfolded proteins and
ER chaperones do not usually exit the ER; however, it has
been observed that some misfolded proteins that are exported from the ER shuttle between the ER and the Golgi
apparatus during their maturation. The UPR also induces the
expression of genes required for ER export (28, 29). Proper
function of the QC machinery in the ER is essential for a
healthy cellular metabolism.
B. Golgi apparatus
Coated vesicles mediate intracellular transport of proteins.
Correctly folded proteins are packed into coat protein com-
plex II (COPII)-coated vesicles and leave the ER at the exit
sites and are transported to the Golgi apparatus, where they
fuse with the target membrane elements in the ER-Golgi
intermediate compartment (ERGIC), near the Golgi (25–28).
ER exit can be by bulk flow or it can be facilitated by transport
receptors, such as ERGIC-53, a transmembrane lectin that
cycles between ER, ERGIC, and Golgi. ERGIC-53 possesses
an ER exit determinant in its cytosolic domain that mediates
binding to COPII-coated vesicles. Once in the Golgi, the
proteins are sorted to peripheral compartments of the celllike vacuoles, plasma membrane, and secretory vesicles
(28, 30).
Cells also have a back-up QC system, which prevents
transport of misfolded proteins beyond the ER. The Golgi
complex also makes conformation-based discrimination of
abnormal proteins, which are targeted for degradation. In
certain cases, retrieval of misfolded proteins from the Golgi
complex to the ER occurs (31). The recycling of misfolding
proteins might be required by ERAD, assisting the ER QC
machinery (7, 28, 32–34). It appears that misfolded soluble
proteins require a modification obtained in the Golgi, presumably in the cis-Golgi, to be recognized as ERAD substrates. It is possible that retrotranslocation occurs from a
specialized compartment that soluble proteins can reach only
via the Golgi apparatus.
It is not yet clear how the QC system operates in the Golgi
complex, because the chemical environment of the Golgi
differs from that in the ER. The abundant chaperones and
folding enzymes that are found in the ER are lacking in the
Castro-Fernández et al. • Protein Routing in Health and Disease
FIG. 3. Ubiquitin proteasome pathway. Terminally misfolded proteins are retrotranslocated across the ER membrane to be degraded
by proteasomes. The misfolded protein is targeted to the Sec61 translocon and then is transported back to the cytosol (step 1). After retrotranslocation from the ER, misfolded proteins are polyubiquitylated
before degradation. The lack of polyubiquitylation prevents proteasomal degradation and leads to failure in transport of the protein from
the ER lumen or membrane out to the cytoplasm, causing the protein
to remain in the ER. The ubiquitin is attached to a cysteine residue
of an ubiquitin-activating enzyme (E1), and then transferred to a
cysteine of an ubiquitin-conjugating enzyme (E2), and finally to the
substrate by the ubiquitin-protein ligase (E3, step 2). The proteins
that undergo proteasomal degradation by the proteolytic complex 26S
proteasome are marked by the binding of a ubiquitin chain to a lysine
residue. The 26S proteasome is composed of a core 20S particle and
one or two 19S regulatory particles. The entry channel is quite narrow
and, even in its open state, it allows entry of only unfolded proteins
in a complex ATP-dependent mechanisms to recognize, unfold, and
linearize substrates and to inject them into the 20S proteasome (step
3). These proteins are digested to small peptides, which in turn are
rapidly hydrolyzed to amino acids by peptidases (step 4). After proteolysis, the polyubiquitin chain is released and hydrolyzed into single
ubiquitin moieties that go through a new cycle of protein degradation.
Golgi; the Golgi has a different ionic milieu and less soluble
protein content and is more acidic than the ER. However, the
intermediate compartment has calreticulin, glucosyltransferase, and glucosidase II, indicating a potential for glycoprotein folding to occur (35, 36). A Golgi-specific endo-␣mannosidase (“endomannosidase”) has been demonstrated
to function as a trimming endoglycosidase that has been
found to work in conjunction with calreticulin. It has been
suggested that this endomannosidase has a role in the QC of
N-glycosylation. However, it is not yet clear whether the
endomannosidase will dissociate the calreticulin-glycoprotein complex of properly folded glycoproteins and/or of
improperly folded ones that have escaped the ER (37). There
is a QC mechanism that resides in the Golgi complex, which
routes defective proteins to the endosomal system to be degraded (38). When the quantity of misfolded proteins increases in the ER, some move to the intermediate compart-
Endocrine Reviews, June 2005, 26(4):479 –503 483
ment and to the cis-Golgi (30). Proteins that are targets for
lysosomal or vacuolar degradation can either be targeted
directly to the endosomal system or can be first routed to the
plasma membrane before they are delivered to the endosomal system (12). It is possible that the QC mechanism for
some misfolded or aggregated proteins is initiated in the
endosomes, thereby precluding these proteins from reaching
the cell surface (39 – 41).
Golgi complex can be a reservoir for misfolded membrane
proteins, but whether the proteins are degraded or remain in
the Golgi for a long period of time is unknown (42). Misfolded membrane proteins tend to expose polar residues,
which are recognized as defects by the QC. Retrieval of
membrane proteins from Golgi to the ER can be triggered by
the presence of these polar residues in the transmembrane
domains. These polar residues can also cause entry from the
Golgi or cell surface into the endosomal system. A multispanning membrane protein of the Golgi, Tul1, which is a
putative ubiquitin ligase, is an enzyme that recognizes polar
residues and might separate membrane proteins that are
incorrectly folded in the transmembrane region or not completely oligomerized for vacuolar targeting (7).
Evidence based on lysosomal enzyme inhibitors suggests
that the turnover of secretory pathway membrane proteins
may happen by targeting from the trans-Golgi network to the
endosome/lysosome system (43).
The QC mechanisms in the Golgi complex became particularly important for exportable proteins that achieved
their posttranslational modification in the Golgi apparatus.
Native or mutant proteins can be directed to degradation to
maintain the local environment of the secretory pathway.
C. Mitochondria
The mitochondrion is an organelle that consists of an outer
and inner membrane that separates the matrix from the intermembrane space. Mitochondria provide the majority of
the ATP to the cell by coupling the oxidative phosphorylation with respiration. The mitochondrial respiratory chain is
composed of five enzyme complexes: reduced nicotinamide
adenine dinucleotide-Coenzyme Q reductase (complex I),
succinate Coenzyme Q reductase (complex II), ubiquinolcytochrome c reductase (complex III), cytochrome c oxidase
(complex IV), and F1F0-ATPase (complex V). In the respiratory enzyme complexes these components transfer electrons
to each other and ultimately to molecular oxygen; they also
translocate protons across the inner mitochondrial membrane. This proton gradient activates the ATP synthase that
catalyzes the conversion of ADP to ATP, completing then the
process of oxidative phosphorylation (44, 45).
Studies in yeast suggest that a protein QC system exists in
mitochondria similar to that in the ER (46). Proteins that cross
the mitochondrial membranes do so in an extended conformation, being coupled to the processes of protein unfolding
in the cytosol and protein refolding in the matrix. Precursor
proteins destined for the mitochondrion are escorted to their
proper cellular location and are maintained in a conformation compatible with import by molecular chaperones within
the cytosol, such as the heat shock protein 70 (Hsp70) and
mitochondrial import stimulation factor. Both of these chap-
484
Endocrine Reviews, June 2005, 26(4):479 –503
erones exploit the inherent instability of the precursor in
unfolding the protein so that it can properly interact with the
import machinery. Once the precursor polypeptide translocation into the matrix is complete, the precursor must adopt
its mature conformation to be able to function in the organelle. Molecular chaperones in the mitochondria are involved in translocation, refolding, and assembly of imported
proteins and proteins encoded in the mitochondria, and protein degradation (47, 48).
After completion of translocation, folding routes may differ for individual proteins. Some proteins can fold directly
after release from the membrane-associated form of Hsp70,
or they can be transferred from there directly to Hsp60,
chaperonins that mediate the folding of proteins to their
native state, or into monomers that subsequently undergo
oligomeric assembly (49). There are several other matrix
proteins, in addition to the molecular chaperones, that are
also involved in protein maturation and folding, such as
peptidases and peptidyl prolylisomerases. However, the
peptidases have no access to the precursors while they are
attached to chaperones.
When there is an unfavorable condition that provokes
proteins to become unfolded and denatured, the molecular
chaperones, in addition to binding proteins that are folding
de novo, bind to (preexistent) proteins that have become unfolded and maintain them in a soluble state before the favorable conditions are restored. This situation is also observed when proteins are not able to fold correctly because
of a chemical modification, lack of assembly partners, or
because of mutation. These misfolded proteins will be targeted for degradation. The chaperones help to maintain these
misfolded proteins in a soluble state, to prevent them from
aggregation. If there is a lack of correct chaperone function,
then these proteins aggregate and cannot be degraded. The
ATP-dependent degradation of misfolded proteins in the
mitochondrial matrix is accomplished by the proteolysis in
mitochondria 1 protease (47).
D. Chaperones: the role of folding in QC and routing
Molecular chaperones are proteins that interact transiently
with unfolded or partially folded intermediates, stabilizing
exposed residues with particular physicochemical characteristics and preventing them from forming inappropriate
intra- or intermolecular contacts (50). Chaperones increase
the efficiency of the overall folding process by reducing the
probability of aggregation and by protecting proteins as they
fold and by rescuing misfolded and even aggregated proteins and enable them to have a second chance to fold correctly (1, 51, 52). The concentrations of many chaperones are
substantially increased during cellular stress. Chaperones
frequently utilize a cycle of ATP-driven conformational
changes to fold or refold their targets. Protein folding typically takes place in the cytoplasm, for cytoplasmic proteins,
or in the ER, for transmembrane and secretory proteins (53).
The Hsps have been divided into groups according to their
mol wt. They are classified in two main groups: high mol wt
ATP-dependent Hsp that requires cochaperones to modulate
their conformation and ATP binding. This group includes
four major families (Hsp70, Hsp60, Hsp90, and Hsp100). The
Castro-Fernández et al. • Protein Routing in Health and Disease
second group of chaperones includes the small ATP-independent Hsp, such as Hsp27, that trap denatured proteins on
their surfaces. The basic reactivity of all chaperones is similar,
but their structural characteristics differ; they participate in
very diverse cellular processes, and they can be targeted to
different subcellular compartments (53, 54). Misfolded proteins can interact with one or more of the Hsp70 class of
chaperones, which protects them against aggregation (55,
56). Hsp70 molecules form multichaperone complexes with
cochaperones such as the Hsp40, and they can function in an
ATP-dependent process to catalyze the refolding of denatured or partially denatured molecules into enzymatically
active forms (51, 57, 58). Certain combinations of chaperones,
e.g., Hsp70, Hsp104, and Hsp40, are capable of disassembly
of intracellular protein aggregates and acceleration of the
refolding of the insoluble molecules into soluble, native species (21, 59 – 61).
The molecular chaperones that are used in QC are abundant in the ER. Retention-based, chaperone-mediated QC in
the ER is well characterized in the case of glycoproteins. The
two homologous lectins, calnexin, which is a transmembrane
protein, and calreticulin, which is a soluble luminal protein,
operate as chaperones in the QC of newly synthesized glycoproteins, ensuring that correctly folded glycoproteins
leave the ER, and they can also mediate glycoprotein degradation of the misfolded protein (15, 62– 66). The lectinassisted glycoprotein folding involves cycling of de- and
reglycosylation until correct folding is achieved. If the protein is correctly folded, it will leave the ER. Misfolded it will
be recognized by the ER enzyme UGT and reglycosylated,
allowing it to reassociate with the QC machinery. The protein
will be able to get completely folded as long as it goes
through the calnexin-calreticulin cycle repeatedly. Both calnexin and calreticulin suppress aggregation of misfolded
proteins via a polypeptide-binding site in their globular domains. Calnexin and calreticulin form complexes with
ERp57, a member of the PDI family, which form transient
disulfide bonds with calnexin- and calreticulin-bound glycoproteins, facilitating correct disulfide bond formation. PDI
is one of the chaperones transcriptionally up-regulated when
unfolded proteins accumulate in the cell (67).
One of the most abundant ER molecular chaperones is BiP,
the main protein of the Hsp70 family. It stabilizes newly
synthesized polypeptides during folding, prevents aggregation by binding to and stabilizing exposed hydrophobic regions of unfolded proteins, mediates retention in the ER,
suppresses formation of nonnative disulfide bonds, and has
a role in the translocation of newly synthesized proteins
across the ER membrane and in degradation of misfolded
proteins. BiP is also up-regulated when misfolded proteins
accumulate in the ER (7).
Chaperone ERGIC-53 is a lectin that serves as an escort
protein carrying high-mannose N-linked glycans, which cycles between the ER and the Golgi complex; this molecule
also helps to prevent proteins from interacting with ligands
during the early secretory pathway.
When there is an excess of misfolded proteins due to
physical or chemical stresses or to mutations, there is an
increase in chaperone and proteolytic activities, to reduce the
presence of damaging nonnative proteins. Several chaper-
Castro-Fernández et al. • Protein Routing in Health and Disease
ones can stabilize proteins undergoing denaturation and allow their refolding when the conditions are back to normal.
However, when the chaperone’s function on protein stabilization is not optimal, it results in protein aggregation.
III. Diseases Caused by Defective Routing
A. G protein-coupled receptors (GPCRs) in disease states
1. HH. HH is characterized by absent or decreased gonadal
function and manifests as delayed or disrupted sexual development in affected individuals; it constitutes one of the
most common causes of hereditary hypogonadism. Mutations in the GnRHR gene are one etiology of HH. Heterologous expression of these GnRHR alleles reveals that the
majority encode misfolded receptors with an increased propensity for targeted degradation or intracellular retention.
Mutations are predominantly in transmembrane segments,
yet (somewhat surprisingly) are widely dispersed across the
entire sequence of the GnRHR (68 –70).
In HH due to GnRH resistance, affected individuals are
either compound heterozygous or homozygous for the mutation. To date, 16 inactivating mutations in the human
(h)GnRHR gene and a splice junction mutation at the intron
1-exon 2 boundary have been described as causes of HH and
were isolated from patients with phenotypes from partial to
complete hypogonadism. The majority of these misfolded
translation products do not route correctly to the cell membrane, where the mutant protein is otherwise fully functional
(71). The expression of 13 naturally occurring hGnRHR point
mutations shows that 11 of these mutants become nonfunc-
Endocrine Reviews, June 2005, 26(4):479 –503 485
tional misrouted proteins that are rescuable by genetic or
pharmacological approaches (Fig. 4; Refs. 68, 72, and 73).
The coexistence of mutant and wt receptors may yield
multimeric complexes that are impeded from attaining a
conformation consistent with cell surface transport (74, 75).
GnRHR heterooligomerization of the misrouted protein with
the wt receptor (76) may explain the dominant-negative effect of the mutant form (75). Mutant receptors are capable of
pharmacological rescue and, when rescued, these receptors
no longer exert a dominant-negative effect.
Pharmacological chaperone treatment approximately
doubles the expression of wt hGnRHR (but not rat GnRHR)
at the plasma membrane. Based on this observation, it has
been suggested that more than half of the synthesized
hGnRHR never arrives at the plasma membrane. The disparity between the human and rat sequences appears related
to specific structural differences in the human sequence that
result in instability of the human sequence (77).
One mutant that results in HH is the E90K mutation of
the hGnRHR gene; this mutant may be rescued by deleting
the amino acid K191, an “extra” amino acid in primate
GnRHR (not found in rodents) that appears to destabilize
the hGnRHR, thus precluding movement of much of the
translation product to the plasma membrane (72). Patients
bearing the E90K defect express a severe phenotype of
hypogonadotrophic hypogonadism (78). Peptidomimetic,
cell membrane-permeant GnRH antagonists from three
different chemical classes have been shown to serve as
templates (pharmacological chaperones or “pharmacoperones”) for naturally occurring and “designer” (misfolded)
GnRHR mutants and thereby effect rescue by stabilizing
FIG. 4. hGnRHR mutant rescue with a complex indole and peptidomimetics antagonist of the GnRHR “IN3” (264). HH is characterized by absent
or decreased gonadal function and manifests as delayed or disrupted sexual development in affected individuals and constitutes one of the most
common causes of HH. Mutations in the GnRHR gene are one etiology of HH. Heterologous expression of these GnRHR alleles reveals that
the majority encode misfolded receptors with an increased propensity for targeted degradation or intracellular retention. Mutations are
predominantly in transmembrane segments, yet (somewhat surprisingly) widely dispersed across the entire sequence of the GnRHR. The
majority of these misfolded translation products do not route correctly to the cell membrane, where the mutant protein is otherwise fully
functional. The expression of 13 naturally occurring hGnRHR point mutations, shows that 11 of these mutants become nonfunctional misrouted
proteins that are rescuable by a cell membrane-permeant antagonist of GnRH, IN3. Most of misfolded hGnRHR mutants are rescued by IN3,
which allows them to escape the ER and avoid a degradation pathway.
486
Endocrine Reviews, June 2005, 26(4):479 –503
the GnRHR (68, 69, 71). Unexpectedly, even the use of
green fluorescent protein or “hemagglutin-tags” on receptor mutations, has a dramatic effect on the wt receptor
and resulted in altered routing (79), a finding that, presumably, is not unique to this receptor.
2. RP. Rhodopsin (Rh) is the visual pigment protein of vertebrate rod cells and is an integral membrane glycoprotein.
Rh synthesis requires membrane insertion of the polypeptide
opsin, and it has a covalently bound reverse agonist, the
chromophore 11-cis-retinal, which interacts with amino acid
residues located in transmembrane ␣-helices (80, 81).
To date, more than 150 opsin mutants have been identified
that cause RP, a heterogeneous group of inherited disorders
that cause progressive retinal degeneration and that lead to
photoreceptor death and subsequent severe loss of peripheral and night vision. In humans, more than 25% of all RP
cases are caused by dominant mutations in the gene encoding Rh, producing misfolded Rh, which, presumably, interferes with the maturation of wt molecules in the ER (82).
The opsin mutants are divided into three different classes
depending on their distinct biochemical features. Class I
mutants that accumulate at the cell surface; Class II mutants
retained in the ER; and Class III mutants mainly retained in
the ER but also found on the cell surface (80, 81). The most
common mutation linked to RP is the P23H mutation, which
is a Class III type mutant, located in the N-terminal tail within
one of the ␤-strands in close proximity to residues that form
the retinal plug, which keeps the chromophore in its proper
position. P23H-opsin is retained intracellularly, predominantly in a perinuclear region. The P23H mutant leads to
formation of conjugated hydrogen bonding that exposes the
chromophore-binding site. Mutations located within this region result in improper folding of opsin and poor binding of
the chromophore. The P23H mutant has a structural alteration that decreases the stability of the protein, leading to
aberrant folding and subsequent aggregation. However,
there is a pharmacological chaperone, an 11-cis-retinal analog
that facilitates proper folding and stabilization of P23Hopsin (80, 81).
3. Nephrogenic diabetes insipidus (NDI). In nephrogenic diabetes insipidus (NDI), a disease that can be inherited or
acquired, the kidney is unable to concentrate urine in response to the antidiuretic hormone arginine vasopressin,
despite normal or elevated plasma concentrations of such
hormone. NDI describes only those conditions in which arginine vasopressin release fails to induce the expected increase in the permeability of the cortical and medullary collecting ducts to water. Vasopressin regulates body water,
resulting in water reabsorption from urine, by fusion of vesicles containing aquaporin-2 (AQP2) water channels with the
apical membrane of renal collecting ducts, this being the final
step in the antidiuresis-signaling pathway that leads to increased water permeability in the duct. Mutations in either
the vasopressin type 2 receptor (V2R) gene or the AQP2 gene
can cause congenital NDI. Mutations in the V2R gene cause
the X-linked form of inheritance of NDI, and mutations in the
AQP2 gene cause a rare non-X-linked form of inheritance of
NDI (83– 85).
Castro-Fernández et al. • Protein Routing in Health and Disease
There are 155 known mutations within the V2R and, as in
the case of the GnRHR, the majority are in the transmembrane segments of the V2R, but are broadly distributed in the
sequence. More than 90% of these mutations failed to properly fold and therefore have defective intracellular transport,
being retained in intracellular compartments instead of being
transported to the cell membrane. In some cases, accumulation of V2R mutants away from the plasma membrane,
possibly in the ER, occurs because there is little maturation
(sugar trimming) of the glycosyl-groups (86).
In the AQP2 gene there have been several point mutations
identified that are associated with NDI. Autosomal-recessive
NDI-causing AQP2 mutations have a significant defective
protein trafficking that results in retention of the mutant
protein in the ER, although they do not present major differences in intrinsic water permeability or protein stability.
A mutation in the AQP2 gene, in which there was a substitution of a lysine for a glutamic acid at position 258 (AQP2E258K), has been shown to result in an autosomal-dominant
form of inherited NDI. This mutant is localized mainly in the
Golgi complex region in Xenopus oocytes, and it is stable as
the wt AQP2 (85).
The AQP2-E258K mutant forms mixed oligomers with the
wt AQP2 and has a dominant-negative effect on the function
of the wt form, which might explain the dominant inheritance of NDI, whereas in the recessive inheritance of NDI
there appears to be a lack of oligomerization of the mutant
AQP2 protein with the wt AQP2 (84, 85). The AQP2-E258K
mutant exits the ER, after which it heterotetramerizes with wt
AQP2. AQP2 mutants seem to have an impaired routing and
thereby differ in their retention site. For example, the T126M
AQP2 mutant is retained in the ER and rapidly degraded by
the proteasome; in contrast, the E258K AQP2 mutant is retained in the Golgi and then degraded by proteasomes and
lysosomes (87).
B. Wild-type protein degradation and oligomerization in
protein processing
There is evidence showing that in many cells, approximately 30 – 60% of the newly synthesized proteins never
achieve their completely folded native structure and are
thereby targeted for degradation. This is documented in the
case of the GnRH and the opioid receptors, where exit from
the ER is the limiting step in processing of the newly synthesized receptors. Frequently, receptor folding intermediates are retained in the ER until the correct conformation is
attained. In the case of the opioid receptors, there is an
apparent inefficiency of the maturation process because only
40% of the precursor receptor was converted to the mature
form and reached the cell surface, whereas the other 60% is
retained in the ER and degraded (88).
For the human GnRHR, about half of the synthesized
receptor is expressed at the plasma membrane. This is not the
case for the rodent GnRHR, which is more stable and is
almost completely routed to the cell surface. It has been
shown that in the presence of pharmacological chaperones,
there is a higher expression of hGnRHR at the plasma membrane (75).
For many years the GPCRs have been believed to be mo-
Castro-Fernández et al. • Protein Routing in Health and Disease
nomeric structures, each one being independently activated
by the binding of a single ligand. These receptors then activate G proteins. However, this classical model may be oversimplified because an increasing number of studies have
demonstrated that many GPCRs exist (in the plasma membrane) as oligomers or heterooligomers (89).
The existence of a dimeric GPCR was suggested by using
bivalent antibodies directed against a peptide antagonist of
the GnRHR; this antibody converted the antagonist into an
agonist and promoted receptor activation (90). It has been
demonstrated that several GPCRs form constitutive dimers
during biosynthesis. For example, the dimerization of the
metabotropic glutamate receptor (mGluR1a) takes place in
the ER in the HEK293 cell line (91). Also it has been shown
that oxytocin, vasopressin V1a, and V2 receptors are present
as dimers in an ER-enriched fraction (92). Several studies also
suggested that some GPCRs form dimers in the presence of
agonists and that sometimes the formation of a constitutive
dimer is dependent on receptor density (93–95).
It has been assumed that GPCR mutants do not have an
effect on the function of the wt receptor. However, it has been
demonstrated that heterodimerization between the wt and
the mutant receptors contributes, at least in part, to the dominant-negative phenotypes associated with heterozygous
mutations. A loss-of-function CCR2(Y139F) mutant acts as a
dominant-negative agent, blocking signaling through the wt
CCR2 (93). Other examples include the V2R (96) and D2
receptors (97). In the case of GnRHR and the fly Frizzled
family of Wnt receptors, the mutant forms heterooligomers
between the wt and the dominant-negative receptors, trapping them both in the ER (76, 98).
Sometimes, the simultaneous presence of two different
GPCR moieties is necessary to constitute a functional receptor. For example, neither type 1 nor type 2 ␥-aminobutyric
acid B receptor (GABABR1 nor GABABR2) when expressed
individually, activates GIRK-type potassium channels. However, the combined expression of GABABR1 and GABABR2
produces a oligomer that is capable of stimulation of channel
activity upon binding agonist by the complex (99). The heterodimerization of the GABABR1 and GABABR2 in the ER is
necessary for exporting GABABR1 to the cell surface (100,
101). In the heterodimeric complex GABABR2 is required to
activate G proteins, and GABABR1 is responsible for the
binding of its ligand (100).
The retention of the wt receptor proteins as well as the
formation of oligomers seems to modify the overall processing of the newly synthesized proteins. The retention of the wt
and mutant receptors by oligomerization in the ER could be
associated with different diseases. Further analysis of such
mechanisms will likely lead to a better understanding of the
regulation of GPCRs.
C. Other proteins
1. Cancer (p53). The tumor suppressor p53 activates numerous genes and leads to cell cycle arrest or apoptosis, protecting the cell against tumor growth. Because of its growthinhibitory activity, p53 must be maintained at low levels to
ensure cell survival and proper organism development; this
is regulated by MDM2, which inhibits the function of p53 by
Endocrine Reviews, June 2005, 26(4):479 –503 487
targeting it for ubiquitin-dependent proteasomal degradation (102). The mutant form of p53 loses the ability to induce
cell cycle arrest or apoptosis. Approximately 50% of the
human cancers lose p53 function mainly by point mutations
in the core domain, which results in loss of sequence-specific
DNA binding and transcription function. These mutations
mainly affect residues that are directly involved in contacting
DNA and that are important for maintaining the structural
stability of the highly conserved DNA-binding domain by
thermodynamically destabilizing p53, which results in its
unfolding and inactivation (Fig. 5; Ref. 103). The core domain
is relatively unstable, because the majority of residues in the
p53 core domain are targets of mutation in tumors.
A structural destabilization and partial global unfolding of
the core domain of p53, or a local distortion of the conformation required for DNA binding, or both, can result in
malfunction of the p53 tumor-associated mutants in different
ways, depending on the position and nature of each mutation
(104). Misfolded mutants have a dominant-negative effect by
forming functionally inactive heterooligomers with the wt
p53, as the mutant forms expose extended hydrophobic surfaces; such surfaces aggregate with associated wt subunits
and disturb their structure. The R249S mutant form of p53,
one of the most common cancer-associated mutations in this
gene, is located in the third loop, causing local structural
changes around the mutation site (105).
Missense single-point mutations are the most common
alterations in the p53 gene; however, many cancers lose p53
activity by inhibition of the wt protein rather than by mutation. Tumor growth suppression can be achieved by reintroduction of wt p53, and intermittent threshold levels of p53
activity may be sufficient, functioning as an important target
in the development of cancer therapies. Various strategies
FIG. 5. Mutant p53 pathways that result in destabilization, unfolding, and inactivation. The p53 point mutations mainly affect residues
that are localized in the core domain. A, Unfolded p53 loses sequencespecific DNA binding and transcriptional function by blocking interaction with target DNA. B, Misfolded mutants have a dominantnegative effect by aggregating with wt p53, thus forming functionally
inactive oligomers that are incapable of entering the nucleus. C,
External small synthetic compounds can bind to p53, stabilizing the
DNA-binding conformation, allowing nuclear transport and normal
function.
488
Endocrine Reviews, June 2005, 26(4):479 –503
have been designed to restore function to mutant p53. The
restoration of p53 function, based upon molecular modeling,
introduced additional DNA contacts or enhanced the stability of the folded state. One strategy to reactivate destabilized
p53 mutants leading to thermodynamic stabilization and
activity restoration is by using a small molecule that binds
the native, but not the denatured, state and drives the conformational equilibrium toward the native state. Furthermore, the introduction of second-site suppressor mutations,
by site-directed mutagenesis of single amino acid substitutions can restore, at least partially, the conformation for DNA
binding or the stability of the protein (103, 106). Moreover,
it has been shown that specific DNA binding of mutant p53
as well as p53-dependent apoptosis in tumor cells can be
restored by synthetic peptides that have been derived from
the p53 C terminus.
2. Cystinuria. The membrane glycoprotein rat liver bile acid
coenzyme A: amino acid N-acetyltransferase (rBAT) is localized at kidney and small intestine membranes, which are
sites for high-affinity l-cystine absorption. This glycoprotein
produces a large amount of amino acid transport. Mutations
of the membrane glycoprotein rBAT have been found to
cause the inheritable disorder cystinuria type I, which is an
autosomal-recessive failure of dibasic amino acid transport
across the luminal membrane of kidney cells and also affects
intestinal absorption of cysteine and provokes a low solubility of cysteine, leading to a severe development of cysteine
“calculi,” which are abnormal stony masses in the kidney
commonly known as kidney stones (107). So far, 32 cystinuria-specific mutations of rBAT have been described in patients with cystinuria, which include missense and nonsense
mutations, deletions, and insertions. The most common
point mutation is M467T, localized in the third transmembrane domain and found in 26% of cystinuria type I genes
(108).
There is a diminution of membrane-expressed proteins
M467T compared with wt rBAT, and the intracellular
amounts of the mutant form are higher, which suggests that
there might be a defect in the delivery of the mutants to the
plasma membrane because of their retention in the ER, probably due to improper folding. rBAT is assembled with other
proteins to constitute the complete amino acid carrier complex, and this assembly has slower or impaired activity if
rBAT is improperly folded. The transport out of the ER is
accomplished after assembly of proteins into native homo- or
heterooligomers (108).
Another mutant of rBAT, R365W, is located in the extracellular domain of rBAT and was found in some cystinuria
patients. This mutant behaves as a temperature-sensitive
folding mutant. It is probable that the cystinuria phenotype
related to this mutant form of rBAT is a consequence of a
severe trafficking defect. Most cystinuria-specific rBAT mutations are trafficking mutants, supporting the proposed role
of rBAT in the routing of the holotransporter, formed by two
different subunits (the rBAT heavy chain and a rBAT light
chain), to the plasma membrane (109).
3. CF. CF is an autosomal-recessive disorder affecting the
apical membrane of epithelial cells of the pancreas, bronchial
Castro-Fernández et al. • Protein Routing in Health and Disease
glands, biliary tree, intestinal glands, reproductive organs,
and sweat glands. It is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein, which
functions as a Cl⫺ channel and regulator of other ion channels, resulting in Cl⫺-impermeable epithelial cells in the affected organs (Fig. 6; Refs. 110 and 111).
Several mutations in the CFTR gene have been identified
that exert negative effects resulting in errors in transcription
and production of unstable mRNAs that are rapidly degraded, causing failure of the cells to synthesize sufficient
quantities of the CFTR protein. Moreover, genetic errors can
also cause production of a CFTR protein that fails to mature
and fold properly, which results in being trapped in the
ERGIC or ER, thereby failing to move further the maturation
pathway and reaching the plasma membrane (112, 113).
Unlike the hGnRHR and V2R, most patients share a common CFTR mutation, this being a deletion of phenylalanine
at position 508 (⌬508). This site is normally in the cytoplasmic
domain of CFTR; the mutant remains in the ER and is ultimately degraded by the ubiquitin-proteasome system. The
disease in the homozygous patient is quite severe, even
FIG. 6. Model of the lung epithelium in CF. In CF, the apical membrane of epithelial cells of the respiratory tract, along with other
organs, lose plasma membrane CFTR channel expression. Mutations
in the CFTR channel cause the disease, the most common being the
⌬508 CFTR mutation. A, In the normal healthy state of the lung, the
CFTR is localized to the apical membrane of the lung epithelium, and
Cl⫺, and thus Na⫹ and water, pass freely from the bloodstream into
the lungs. B, In a CF patient, the mutant channel remains largely
trapped in the cell, thereby impeding the normal flux of Cl⫺, and thus
Na⫹ and water, across the epithelium; as a result the airway surface
dries out and the mucus accumulates and becomes condensed and
thick, impeding the passage of air. C, In the presence of a chemical
chaperone or a nonspecific agent such as glycerol, the mutant CFTR
is relocalized to the cell surface, recovering the Cl⫺, and thus Na⫹ and
water, flux, rehydrating the airway membrane and allowing mucus
clearing.
Castro-Fernández et al. • Protein Routing in Health and Disease
though it has been demonstrated that ⌬F508 CFTR retains
some function as a Cl⫺ channel, although less than the wt
protein. The CF phenotype associated with ⌬F508 CFTR appears to originate from mislocalization and premature degradation, rather that the altered functional properties or decreased stability of mutant CFTR (110). Misfolding CFTR
causes this protein to be targeted to the degradative apparatus before it can be translocated to the plasma membrane.
There is evidence for a temperature sensitivity of ⌬F508
CFTR maturation, although there may be no apparent decrease in thermal lability of the mutant protein (114). Calnexin binds to the newly synthesized CFTR and dissociates
from it when it reaches its native conformation. In contrast,
the ⌬F508 CFTR remains associated to calnexin therefore,
after a prolonged interaction leading the ⌬F508 CFTR to
ERAD (115).
The ⌬F508 CFTR mutant is functional once it reaches the
native state. Growth conditions that favor proper folding of
the mutant CFTR allow increased recovery of function. Identification of a compound that corrects the ⌬F508 CFTR folding defect would be of great potential therapeutic benefit,
and some studies that utilize small molecules identified from
screening techniques are underway. CFTR activators may
also correct the impaired chloride transport in cells expressing mutant CFTRs in CF patients (116).
4. Gitelman’s syndrome. Gitelman’s syndrome is an autosomal-recessive disease characterized by renal salt wasting
with hypokalemic metabolic alkalosis, hypomagnesemia,
and hypocalciuria (117). The first clinical presentation usually occurs at 6 yr of age or older and may include muscular
pain and cramping with exercise and fatigue and weakness.
The disease is generally mild, and some patients even remain
without symptoms (118, 119). This disease is caused by mutations in the SLC12A3 gene, which encodes the thiazidesensitive NaCl cotransporter (NCCT; Fig. 7). The mutation in
the NCCT gene results in salt wasting from the distal convoluted tubule, increasing aldosterone secretion, which augments Na⫹ reabsorption. Because of the indirect coupling of
Na⫹ reabsorption to K⫹ and H⫹ secretion in the distal
nephron, the augmented Na⫹ reabsorption activity occurs at
FIG. 7. Model of the NCCT. The tertiary structure of NCCT contains 12 transmembrane domains and intracellular amino- and carboxy-terminal regions. The mutations identified in
Gitelman’s syndrome are located throughout the
entire coding sequence of the protein. Thirteen
human NCCT mutations have been functionally
tested in Xenopus laevis oocytes. The missense
mutations G439S, T649R, and G741R were partially glycosylated and impaired in their route to
the plasma membrane, and instead they are retained in the ER/pre-Golgi complex. In contrast,
the rest of the mutations impair their insertions
of a functional protein into the plasma membrane.
Endocrine Reviews, June 2005, 26(4):479 –503 489
the expense of increased K⫹ and H⫹ secretion, accounting for
the observed hypokalemia and metabolic alkalosis (120, 121).
The tertiary structure of NCCT contains 12 transmembrane
domains and intracellular amino- and carboxy-terminal regions (122). The mutations identified in Gitelman’s syndrome are located throughout the entire coding sequence of
the protein and include missense, frameshift, nonsense, and
splice-site mutations (123).
There are multiple mechanisms by which mutations could
reduce or abolish NCCT activity: impaired synthesis or routing, disturbed transport activity, and increased retrieval or
degradation. Two different classes of NCCT mutations have
been distinguished based on immunocytochemical analyses
and the metolazone-sensitive Na⫹ uptake rates: the ERretained mutants and partly functional mutants. The ERretained mutants did not exhibit significant metolazonesensitive Na⫹ uptake, and the immunocytochemical analyses
demonstrate that these mutant proteins were expressed predominantly in the cytoplasm and were absent at the plasma
membrane. These mutant transporters lacked the typical
complex-glycosylated band of 130 to 140 kDa. Together,
these findings suggest that these partly glycosylated mutants
are impaired in their routing to the plasma membrane and
are retained in the ER/pre-Golgi complex (121). The C terminus of NCCT bound to chaperone Grp58, indicating that
regulatory proteins may be involved in transporting NCCT
ER to other compartments in the cell for further processing
(124). In contrast, the partly functional mutants are present
at the plasma membrane, as demonstrated in immunocytochemical analyses, but are also clearly located in the cytoplasm (unlike the wt protein). These findings indicate that
the process of routing NCCT to the cell surface is only partly
impaired, suggesting that different mechanisms are involved
in the disturbed trafficking for the two mutant classes, but the
molecular mechanisms responsible remain to be established
(121).
5. Hemochromatosis (HC). Hereditary HC is an autosomalrecessive disorder characterized by an abnormal increase in
the absorption of iron in the gastrointestinal tract. The organs
in which iron deposits primarily are liver, pancreas, heart,
490
Endocrine Reviews, June 2005, 26(4):479 –503
joints, and endocrine organs (125). Clinical consequences of
iron accumulation in these organs include cirrhosis of the
liver, hepatocellular carcinoma, diabetes, heart failure, arthritis, and hypogonadism (126). The majority of North
American patients with HC have a mutation in cysteine 282
(to tyrosine) in a major histocompatibility complex (MHC)
class I-like gene, called hemochromatosis gene (HFE) (127,
128). A second mutation (H63D) was reported to be also
present in HC patients who are heterozygous for the C282Y
mutation (129).
The human HFE protein is a membrane protein homologous to MHC class I molecules, which contain an extracellular peptide-binding region (␣1 and ␣2 loops), an Ig-like
domain (␣3), a transmembrane region, and a short cytoplasmic tail. HFE contains intramolecular disulfide bridges in the
␣2- and ␣3-domains that stabilize its tertiary structure (127).
The disulfide bridge in the ␣3-domain of MHC class I molecules is required for their association with ␤2-microglobulin
(␤2M), leading to efficient intracellular processing and transport to the plasma membrane (130, 131). The C282Y mutation
prevents the interaction between HFE with ␤2M and eliminates cell surface presentation. The role of ␤2M/heavy chain
interaction is to facilitate and stabilize the folding of the
heavy chain during biosynthesis (130, 132).
The process by which HFE may affect iron uptake is poorly
understood. Recent evidence demonstrated that HFE diminishes the binding affinity of iron-loaded transferrin to its
receptor (133, 134). In fact, the binding of HFE and transferrin
receptor is required for HFE transport to endosomes (125).
However, the physiological relevance of this finding remains
unclear.
The association of the heavy chain with the ␤2M occurs in
the ER and is then transported through the cis-Golgi network,
to the middle and trans-Golgi cisternae where the glycosyl
side chain is modified to a more complex form en route to the
plasma membrane (135, 136). Class I molecules that fail to
assemble properly are recycled between the ER and Golgi,
rather than being retained exclusively in the ER (137).
6. Hypercholesterolemia. Plasma low-density lipoproteins
(LDL) bind to the LDL receptor (LDLR), a transmembrane
glycoprotein that mediates LDL cellular uptake, internalization, and its delivery to lysosomes. Loss of LDLR function
leads to decreased LDL catabolism, and its cholesterol is
released, resulting in elevated levels of plasma cholesterol.
Mutations in the LDLR gene results in protein misfolding
and ER retention, causing familial hypercholesterolemia,
which is a common autosomal-dominant inherited disorder
(138, 139). To date, there are more than 600 mutations that
have been characterized in the LDLR; however, 50% of these
mutations in familial hypercholesterolemia patients lead to
a class 2 mutation, which are proteins that are partially or
totally retained in the ER or have an ER-Golgi-defective
transport (140). The BiP chaperone has been shown to bind
to LDLR by coimmunoprecipitation studies, where an interaction of Grp78 with either the wt or the mutant LDLR was
observed, and there was no interaction with other proteins.
Moreover, the interaction of Grp78 with mutant LDLR was
shown to be a prolonged one compared with that of the wt
LDLR, demonstrating that Grp78 is the chaperone respon-
Castro-Fernández et al. • Protein Routing in Health and Disease
sible for retention of the misfolded receptors in the ER. The
mechanisms responsible for this LDLR retention in the ER
are not yet clear (138).
A prolonged retention of misfolded or incompletely
folded proteins in the ER leads to their degradation. The
proteasomal degradation pathway mediates the LDLR mutant degradation, but the precise mechanism for the LDLR is
still unknown. The receptor-associated protein (RAP) has
been identified to function as a specialized chaperone in the
folding and trafficking of LDLR along the early secretory
pathway. RAP, which binds only to members of the LDLR
family, promotes the receptor folding and maturation by
preventing the formation of nonproductive intermolecular
disulfide bonds and premature ligand binding within the
early secretory pathway (141). It is still not known whether
RAP is also involved in ER retention and/or the proteasomal
degradation of the LDLR mutants (139).
IV. Diseases Caused by Conformational Errors
The so-called conformational diseases are disorders in
which the constituent protein involves change in size or
fluctuations in shape with resultant self-association and tissue deposition as amyloid fibrils (142). This diverse group of
diseases includes disorders such as Alzheimer’s and Parkinson’s diseases, amyloidoses, ␣1-antitrypsin deficiency, and
the prion encephalopathies. These diseases result when a
specific protein is subjected to a conformational rearrangement that results in aggregation and deposition within tissues or cellular compartments (143, 144). These disorders
have common features: they have either sporadic or inherited patterns, they appear later in life (generally after the
fourth or fifth decade), and all are characterized by neuronal
loss and synaptic abnormalities. In contrast, the clinical
symptoms and disease progression are very different among
such disorders (145, 146).
Amyloid structures can be recognized because of Congo
red and thioflavin S binding and a core structure based on
the presence of highly organized ␤-sheets. This characteristic
makes them very resistant to proteolytic degradation (147,
148). When these aggregates are formed, they are stable for
long periods, forming a bed for the progressive accumulation
of more deposits in tissue and eventual conversion into amyloid fibrils (1). The deposits in amyloidoses are extracellular
but also include some intracellular aggregates. These can
often be observed as thin fibrillar structures, sometimes assembled further into larger aggregates or plaques (Fig. 8;
Refs. 146 and 148). The capacity of polypeptide chains to
form amyloid structures seems to be a generic feature of
polypeptide chains, and it is not restricted to the relatively
small number of proteins associated with recognized clinical
disorders (149, 150). The polypeptides involved in amyloid
diseases include full-length proteins (e.g., lysozyme or Ig
light chains), biological peptides (amylin, atrial natriuretic
factor), and fragments of larger proteins produced as a result
of specific processing (e.g., the Alzheimer ␤-peptide). These
disorders may also result from fragments due to general
degradation [e.g., polyglutamine stretches cleaved from proteins with polyglutamine extensions such as huntingtin,
ataxins, and the androgen receptor (148)].
Castro-Fernández et al. • Protein Routing in Health and Disease
Endocrine Reviews, June 2005, 26(4):479 –503 491
FIG. 8. Schematic representation of the possible mechanism of formation of amyloid fibrils. The so-called conformational diseases are disorders
in which the constituent protein involves change in size or fluctuations in shape with resultant self-association and tissue deposition as amyloid
fibrils. When a specific protein is subjected to a conformational rearrangement that promotes aggregation, it induces the deposition within
tissues or cellular compartments resulting in these disorders. The core structure of amyloid fibrils contains highly organized ␤-sheets, which
makes them very resistant to proteolytic degradation. When these aggregates are formed, they are stable for long periods, forming a bed for
the progressive accumulation of more deposits in tissue and eventual conversion into amyloid fibrils. These can often be observed as thin fibrillar
structures, sometimes assembled further into larger aggregates or plaques. The impairment of cellular function is the result of direct interaction
between the aggregated proteins with some cellular components. However, it has been demonstrated that, in many cases, the prefibrillar
aggregates are more toxic to the cells than mature fibrils.
In the conformational disorders the impairment of cellular
function is usually the result of direct interactions between
the aggregated proteins with a particular cellular component
(151, 152). In addition, it has been demonstrated that in many
cases the prefibrillar aggregates (also referred to as amorphous aggregated protein micelles or protofibrils) are more
toxic to the cells than mature fibrils (for review, see Ref. 148).
The absence of a direct correlation between the density of
fibrillar plaques in the brains of victims of Alzheimer’s disease (AD) and the severity of the clinical symptoms can be
explained by the toxicity exhibited by early aggregates (153).
The molecular chaperones and other QC mechanisms are
remarkably efficient in ensuring that prefibrillar aggregates
are neutralized before they can do any damage. If the efficiency of the QC mechanisms is impaired, the probability of
pathogenic behavior is increased. Most of the amyloid diseases are associated with old age, and the explanation could
be a decrease in the efficiency of these protective mechanisms, when there is an increased tendency for protein to
become misfolded or damaged, coupled with a decreased
efficiency of the molecular chaperone and UPRs (154). The
increase in human life expectancy is associated with augmentation in the prevalence of amyloid diseases, in particular in highly developed countries; this can be explained by
the inefficiency of the QC mechanisms to prevent the protein
aggregation (148, 155).
A. AD and neuropathies
AD is, at present, an incurable neurodegenerative disease
and the most common form of dementia (156). The clinical
manifestations in AD are the gradual loss of memory, followed by a progressive deterioration of higher cognitive
functions and behavior (157), caused by a massive loss of
neurons (158). Prevalence is estimated to affect 5– 8% of persons over 65 yr of age, and the incidence of inherited forms
ranges from 1–30% (159).
Primarily, extracellular plaques and intracellular neurofibrillary tangles characterize the pathology of AD. Plaques are
composed mainly of the amyloid-␤-peptide (A␤), whereas
tangles are composed of the cytoskeletal protein ␶ (142). The
formation of tangles is considered as a secondary event, i.e.,
a consequence of the formation of the A␤-plaques (160, 161).
The diseases called “tautopathies” are different from AD,
and the brain lesions in these patients are the intracellular
aggregation of ␶-proteins, without accumulation of A␤plaques (162).
To date, mutant forms of three genes, ␤-amyloid precursor
protein (APP) gene (located in chromosome 21), presenilin 1
(PS1, on chromosome 14), and presenilin 2 (PS2, chromosome
1), have been shown to cause early-onset familial AD (158).
In addition, polymorphisms in four genes, i.e., apolipoprotein E (apo E), ␣-2 microglobulin, very low density lipoprotein receptor, and low density lipoprotein receptor-related
protein, are shown to be risk factors for AD pathogenesis
(158, 163).
Amyloid formation by the Alzheimer’s A␤-protein is the
most extensively studied. The A␤-peptides are degradation
products of a transmembrane cell surface glycoprotein APP
(Fig. 9). Three different proteases called ␣-, ␤-, and ␥-secretases can cleave APP. When APP is concomitantly hydrolyzed by ␤-secretase at the N terminus and by the ␥-secretase
within the membrane, the two main products, A␤(1– 40) and
A␤(1– 42), migrate outside the cell and produce fibrils (164).
Peptides containing the 40- or 42-residue form of A␤, and
shorter derivatives, form amyloid-like fibrils in vitro, with
characteristics similar to fibrillar aggregates extracted from
AD amyloid plaques (146, 165, 166). The amyloid cascade
hypothesis predicts that the overproduction of A␤, or the
failure to eliminate this peptide, induces AD primarily
through amyloid deposition, which is postulated to be involved in neurofibrillary tangle formation (167–169). These
lesions are then associated with cell death and consequent
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Endocrine Reviews, June 2005, 26(4):479 –503
Castro-Fernández et al. • Protein Routing in Health and Disease
FIG. 9. Hypothetical mechanism of conformational change for A␤-peptides. Three different
proteases called ␣-, ␤-, and ␥-secretases can
cleave APP. When APP is concomitantly hydrolyzed by ␤-secretase at the N terminus and by the
␥-secretase within the membrane, the two main
products, A␤(1– 40) and A␤(1– 42), migrate outside the cell and produce amyloid fibrils.
memory impairment (142, 170, 171). The precise involvement
of amyloid in sporadic cases of AD and the formation of
neurofibrillary tangles should be explained.
Presenilins (PS1 and PS2) have functions similar to ␥-secretases and cleave APP to release A␤; these proteins also cleave
the Notch receptor to release the Notch intracellular domain,
activating a signal transduction pathway by their cytoplasmic domain (172). Thus, presenilins play an important role
in AD pathogenesis. Tangle formation is also coupled by
further increase of ␶ production, hyperphosphorylation, and
loss of microtubule binding in the affected neurons. When
the C terminus of ␶ is subjected to proteolysis, it generates
cytotoxic fragments (161, 173). These and other fragments are
thought to nucleate into tangle formations (174).
B. Parkinson’s disease (PD)
PD is the second most common, late-onset neurodegenerative disorder. The degeneration of dopaminergic neurons
in the substantia nigra resulted in the typical characteristics
of PD, which include muscular rigidity, postural instability,
and resting tremor. The clinical hallmark of PD is the deposition in brain cells of intracytoplasmic inclusion bodies
called Lewy bodies (LBs) (158, 159, 175).
Inherited forms of PD are caused by mutations in three
genes, ␣-synuclein, parkin, and ubiquitin carboxyl-terminal
hydrolase L1(158, 175, 176). Autosomal-dominant PD results
in mutations in the ␣-synuclein gene, and autosomal-recessive PD is due to mutations in the parkin gene. Among other
factors, apolipoprotein E ⑀2/⑀4 genotype and exposure to
pesticides might be risk factors for sporadic PD (177, 178).
The ␣-synuclein is a highly conserved 140-amino acid protein very abundant in the central nervous system, particularly in the presynaptic terminals (179, 180), but its physiological functions are largely unknown. This protein was
originally identified as a precursor of the nonamyloid component of the senile plaques in AD (181) and is a major
constituent of LBs and other disease-specific lesions such as
the abnormally shaped neurites known as Lewy neurites
(182–184). The ␣-synuclein is also found in the intracellular
inclusions of other neurodegenerative disorders, such as dementia with LB and the LB variant of AD commonly referred
to as a “␣-synucleinopathies,” and also in the filamentous
glial and neuronal inclusions of multiple-system atrophy
(175, 185).
It has been reported that ␣-synuclein inhibits phospholipase D2 (186), a plasma membrane protein that converts the
neutral phosphatidyl choline to phosphatidic acid and promotes cytoskeletal reorganization and directed movement of
vesicles (187). It also binds protein ␶, promoting its protein
kinase A-dependent phosphorylation (188). This protein also
has been reported to act as a modulator of the activity of
tyrosine hydroxylase, the rate-limiting enzyme in dopamine
(DA) synthesis, and as a regulator of the DA transporter (162,
175). It is possible that ␣-synuclein may protect and help
neurotransmitter vesicles to transport them from cell body to
synapse, and its aggregation may exert its toxicity by creating
pores in the lipid membrane, leading to loss of DA from
vesicle to cytoplasm (158).
In a German family with autosomal-dominant form of PD
a mutation in the gene ubiquitin carboxyl-terminal hydrolase
L1 was found (189). This mutation appears to be extremely
rare and is characterized by a relative early-onset appearance
in life (⬃50 yr). No histopathological analysis is yet available,
and it is not known whether LBs form. Parkin is a highly
conserved 465-amino acid protein with a molecular mass of
52 kDa, mainly expressed in brain (especially in midbrain DA
cells) and muscle (190, 191). Parkin is reported to function as
an E3 ubiquitin ligase and disease-linked mutations that
indeed seem to compromise its enzymatic activity.
C. Huntington’s disease (HD)
The expanding CAG repeats coding for polyglutamine are
the cause of nine neurodegenerative disorders (192). They
include spinal and bulbar muscular atrophy, HD, dentatorubral and pallidoluysian atrophy, and several forms of
spinocerebellar ataxia. Each one is characterized by selective
neuronal death in specific regions of the brain (193).
HD is an autosomal dominantly inherited neurodegenerative disorder characterized by the progressive development of mood disturbances, behavioral changes, involuntary
choreiform movements, and cognitive impairments. Onset is
most commonly in adulthood, with a typical duration of
15–20 yr before premature death (194).
The genetic defect in HD is the expansion of an unstable
Castro-Fernández et al. • Protein Routing in Health and Disease
CAG repeat encoding glutamines (Q), close to the 5⬘-end of
the gene for huntingtin protein (194). The disease phenotype
is influenced by CAG repeat length: the presence of a trinucleotide stretch to 34 –39 CAG repeats in one allele confers
risk of developing HD, but the extension above 39 repeats
conditions the appearance of the disease including age of
onset and the extent of DNA fragmentation in striatal neurons (Fig. 9C; Refs. 195–197). HD also shows the anticipation
characteristic resulting from instability of the repeat size
during transmission (198).
Huntingtin is predominantly cytoplasmic; its normal function is not well understood but may involve cytoskeletal
function or vesicle recycling. It has been proposed that the
protein may be transported to the nucleus and have a role in
the regulation of gene transcription, but this is uncertain
(199). The toxicity of huntingtin in the cytoplasm may be
caused by mutant full-length protein or by cleaved protein,
and the toxic species may be soluble monomers or oligomers
or insoluble aggregates. It has been proposed that the toxicity
of this protein may involve inhibition of the proteasome or
activation of caspases directly or via mitochondrial effects. It
is possible that aggregation of huntingtin induces oxidative
and ER stress and proteosomal and mitochondrial dysfunction (200, 201). Cytoplasmic aggregates accumulate in perinuclear or neuritic regions and are ubiquitinated. The mutant protein translocates to the nucleus, where it forms
intranuclear inclusions; however, nuclear toxicity is believed
to be caused by interference with gene transcription (193,
194).
The Huntingtin gene is expressed in all cells but it affects
only a subset of neurons. This cell type specificity may suggest the requirement for another protein for its function or it
may be specifically blocking the trophic factor support for
striatal neurons (158).
D. Viral prions
Transmissible spongiform encephalopathies (TSEs), or
prion diseases, are degenerative disorders of the central nervous system that lead to motor dysfunction, dementia, and
death. Prion diseases include scrapie of sheep, bovine spongiform encephalopathy (BSE) in cattle, and such human diseases as Creutzfeldt-Jackob disease (CJD), GertstmannSträussler Scheinker syndrome, and fatal familial insomnia.
More recently, variant CJD (vCJD), attributed to consumption of BSE-contaminant products, have claimed more than
100 victims (202). Neither humoral nor cellular immunological responses have been detected in prion diseases (203).
The precursor protein PrP 33-to 35-kDa (PrPc) is coded by
a normal cellular gene and not by a putative nucleic acid
carried within the prion (204). The PrPc is found predominantly on the outer surface of neurons, attached by a glycosylphosphatidyl inositol anchor (205, 206). In prion diseases a large protease-resistant aggregated form of PrP,
designated PrPsc, that accumulates mainly in the brain, is
believed to be the principal or only constituent of the prion
(203, 207). No differences in the primary structure of PrPc and
PrPsc have been detected, suggesting that they differ only in
their conformation (208). The tertiary structure of PrPc has
been elucidated, and the ␤-sheet content is low; the structure
Endocrine Reviews, June 2005, 26(4):479 –503 493
of the PrPsc is not currently available but the ␤-sheet content
is high (209 –211).
The refolding and the nucleation models have been proposed to explain the mechanisms by which PrP aggregates.
In the refolding model, the PrPc unfolds to some extent and
refolds under the influence of a PrPsc molecule, and an activation energy barrier separates the two states. On the other
hand, the nucleation model proposes that PrPc is in equilibrium with PrPsc, which is only stable when it forms multimers. Once a multimer is present, monomer addition
follows rapidly (212, 213). PrPc has been implicated in a
chaperone function for nucleic acids, but its potential role for
aggregation of other proteins has not been studied (214).
TSE diseases are transmissible by inoculation or ingestion
of material contaminated by infected tissues. Incubation periods before the appearance of clinical symptoms range from
months to years and, in the case of some kuru patients, may
have been as long as 40 yr (215).
1. Kuru and iatrogenic CJD. In both diseases the patients are
directly exposed to the TSE agent by contact with brain
tissues or extracts contaminated by the TSE agent. Kuru is
transmitted during the cultural handling (or ingestion) of
brain tissue of relatives who died of this disease (215). Iatrogenic CJD has been induced by transplantation of corneal
or dural tissue from patients with TSE, or by neurosurgery
using instruments not completely sterilized after use on TSE
patients (216). It has also been detected after inoculation of
GH extracted from pituitary glands pooled from large
groups of individuals (217).
2. vCJD. In 1996, authorities in the United Kingdom described
vCJD, a new CJD that is now believed to be the human form
of BSE [mad cow disease (202)]. vCJD can be distinguished
from sporadic CJD, which is the usual form of human TSE
disease, by the early age on onset, absence of electroencephalogram changes, and distinct neuropathological features
(218). Because there is no association of occupational exposure of vCJD patients to cattle on farms or in abattoirs, it is
likely that spread may have occurred through consumption
of BSE-contaminated meat products (215).
3. Sporadic CJD. The age of onset is usually between 50 –70 yr,
but can range as low as 16 yr and as high as 80 yr. The primary
clinical symptom is usually dementia, which often presents
with cognitive disturbances such as confusion, memory loss,
and bizarre behavior (219, 220). The clinical symptoms are
not associated with a mutant PrP allele; there is also no
epidemiological evidence for exposure to TSE agent although contact with people or animals with TSE disease is
present (216). At present, the means of acquisition of the TSE
agent in these patients is unknown. Several hypotheses include exposure to an unidentified virus, spontaneous generation of nonviral agent through some somatic cell mutation
of PrP in each disease individual, and stochastic initiation of
spontaneous PrPsc formation without PrP mutation. However, there is no evidence for spontaneous PrPsc formation in
any animal or human disease.
4. Familial TSEs. Familial TSE is an autosomal-dominant
disease caused by a mutation in the PrP gene (221). These
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Endocrine Reviews, June 2005, 26(4):479 –503
diseases include familial CJD, Gertstmann-Sträussler
Scheinker syndrome, and fatal familial insomnia. For most
PrP mutations, all positive individuals eventually develop
the disease. In addition to point mutations in the PrP gene,
insertions in the octapeptide repeat coding region of the PrP
have been associated with degenerative brain disease (215).
5. BSE. In the past decade, the BSE epidemic in the United
Kingdom has brought international attention to the TSE family of diseases. Feeding of protein supplements contaminated
with tissues of BSE-positive cattle spread BSE. This encephalopathy has also been transmitted to other species by feeding of contaminated meat and bone meal to ungulates and
large felines in zoos and possibly to domestic cats. Transmission to humans has also been suggested by the appearance of vCJD in humans primarily in the United Kingdom.
At the molecular level, the interactions between BSE (bovine)
PrPsc, and human PrP are rather inefficient (222).
E. Cataracts
Crystallins are the major structural proteins in the lens and
form complex protein-protein interactions with each other.
␣-Crystallins act as molecular chaperones to prevent the
aggregation and precipitation of the crystallins by forming
large assemblies predicted to contain as many as 33 subunits
(223). ␤-Crystallins contain aggregates ranging in size from
dimers to octomers, whereas ␥-crystallins exist as monomers.
␣B-Crystallin is a major lens protein that has a structural role
in maintaining the transparency of the lens and confers protection to cells against thermal, osmotic, and oxidative damages (224, 225). This protein also functions as a regulator of
apoptosis in fibroblast L-929 cells (226).
The nonlenticular function of ␣B-crystallin may be related
to its chaperone-like property, which allows it to bind irreversibly to denaturing proteins, preventing the formation of
large light-scattering aggregates (227). In the lens, this function is highly important in preventing cataracts, as proteins
are subject to osmotic and oxidative stresses over the lifetime
of the organism and are very susceptible to denaturation and
aggregation (228).
Interestingly, ␣B-crystallin is overexpressed in a number
of disorders such as AD, diffuse LB disease, and in CJD
(229 –231). In many of these diseases, ␣B-crystallin is found
to be associated with intermediate filaments in cytoplasmic
inclusion bodies (232), but the role that ␣B-crystallin may
play in these diseases in currently unknown. A missense
mutation in ␣B-crystallin, R120G, has been shown to cosegregate with a desmin-related myopathy in a French family
(233). The R120G mutation in ␣B-crystallin significantly altered the secondary, tertiary, as well as quaternary structure
of the ␣B-crystallin molecule. The mutation of R120G in
␣B-crystallin may lead to imperfect interactions with proteins such as demin in their native state, which results in
aggregations of desmin with R120G ␣B-crystallin (234).
F. ␣1-AT deficiency
␣1-AT is a member of the serpentin family of protease
inhibitors and is secreted in liver cells to control the proteolytic activity of a variety of circulating enzymes. It is the most
Castro-Fernández et al. • Protein Routing in Health and Disease
abundant serine protease inhibitor in plasma (110). In homozygous protease inhibitor phenotype ZZ (PIZZ) ␣1-AT
deficiency, a misfolded but functionally active mutant ␣1ATZ molecule is characterized by a single nucleotide substitution, which results in substitution of lysine for glutamine
342. This abnormally folded mutant protein is polymerized
and retained in the ER of hepatocytes rather than secreted
into the circulation, causing a reduction in plasma concentrations of ␣1-AT to 80 –90% of normal concentrations (143,
235). The loop where the amino acid substitution takes place
in the ␣1-ATZ molecule folds in a manner that leads to
formation of large aggregates (110).
The mutant protein retains approximately 80% of the wt
functional activity, causing a decrease in number of ␣1-AT
molecules in the lung, therefore allowing neutrophil elastase
to degrade lung parenchyma and increasing the risk of deficient individuals of developing emphysema. In a subset of
patients, homozygotes are prone to develop cirrhosis and
hepatoma. In children, there is a hepatotoxic effect in liver
cells because of the mutant ␣1-AT molecule retained in the
ER that originates chronic liver disease (236, 237).
There are still doubts regarding which degradation pathway is followed by this ␣1-ATZ mutant form in the ER. One
of these pathways could be by binding of ␣1-ATZ to calnexin,
ubiquinylation of the cytoplasmic tail of complexed calnexin,
and degradation of the ␣1-ATZ-polyubiquitinated calnexin
complex by the proteasome. However, it has been suggested
that in hepatocytes there might be degradation by a nonlysosomal proteolytic system.
Partial correction of the secretory defect of ␣1-AT, the
functional activity of which is retained by the mutant ␣1ATZ, is needed to prevent liver and lung disease in ␣1-AT
deficiency. Relatively small increases in plasma ␣1-AT levels
are needed to inhibit destructive proteolysis in deficient individuals. Compounds that enhance secretion of ␣1-AT can
be of great use in patients with this deficiency (237).
V. Rescue of Defective Proteins
A. Temperature
The defect in protein maturation and ER exit of the ⌬F508
CFTR mutant appears to be temperature sensitive. It was
observed that a small population of ⌬F508 CFTR was transported to the plasma membrane and was able to confer Cl⫺
transport by lowering the temperature to 30 C or below (112).
Lowering the temperature to 27 C is associated with a
decrease in degradation of ␣1-ATZ without any changes in
secretion; in contrast, elevation of the temperature to 42 C
results in decreased degradation and increased secretion,
preventing the retention of ␣1-ATZ in the ER by correcting
the secretory defect in ␣1-AT deficiency (235).
B. Chemical chaperones
Genetic rescue of the E90K GnRHR mutant-defective receptor was affected by deleting amino acid K191, a deletion
that enhances expression of the wt human receptor at the
plasma membrane. Membrane targeting and function of the
E90K mutant were rescued as assessed by ligand binding and
Castro-Fernández et al. • Protein Routing in Health and Disease
by measurement of coupling to an effector system (72). These
observations first presented the possibility that E90K was a
misrouted receptor, but otherwise competent, and first suggested the possibility that chemical chaperones could be
useful in rescuing mutants of the GnRHR.
The peptidomimetic, cell membrane-permeant antagonist
of GnRH, IN3, rescues most of the misfolded hGnRHR mutants and allows them to escape the QC machinery that
degrades them. IN3 serves as a nucleus for proper folding of
the mutants and rescues the mutant receptor from a degradation pathway.
When mutants were coexpressed with wt hGnRHR, the wt
was retained in the ER, leading to a dominant-negative effect
by the mutants. However, there were three mutants (E90K,
C200Y, or L266R), that were functionally recovered when
complexed with the wt receptor, suggesting that the pharmacological agent could rescue both mutants and the wt
GnRHR retained in the ER (75). Additional membranepermeant, chemically distinct, GnRHR peptidomimetic
agents that allow properly folding and routing to the plasma
membrane of GnRHR mutants have been described. However, the mutants S168R and S217R could not be rescued by
any drug tested. This suggested that these were potentially
irreversibly misfolded or that loss of function resulted from
the inability to bind ligand (69).
It has been demonstrated that the RP P23H mutant can be
induced to properly fold in the presence of the pharmacological chaperone that is a seven-membered ring variant
of 11-cis-retinal, 11-cis-7-ring retinal, forming significant
amounts of pigment. The rescued 7-P23H-Rh pattern is diffuse but has a greater localization at the cell surface, in
contrast to the mutated form located intracellularly, demonstrating that the rescued P23H-Rh was correctly glycosylated and formed a pigment, and was correctly routed to the
cell membranes. The 11-cis-7-ring retinal leads to increased
amounts of the correctly folded mutant (80, 81).
The small peptidomimetic V2R antagonists, SR121463A
and VPA-985, have been shown to rescue V2R mutants because they permeate the cell and bind to incomplete folded
mutant receptors, thus acting as pharmacological chaperones
promoting the proper folding and maturation of receptors
and their targeting to the cell surface (238).
A class of small synthetic compounds stabilizes p53 core
domain. Rescue of locally distorted mutants, such as R249S,
could be effected by using the small molecule, FL-CDB3,
which binds preferentially to the native state and shifts the
conformational equilibrium toward that of the wt in a chaperone mechanism (239). Another small compound, the ellipticine alkaloid, might be acting by directly binding to
mutant p53 and stabilizing its DNA-binding conformation.
It seems that this compound provokes a modification of the
p53 mutants toward the wt by new protein synthesis, and it
might be rescuing the folding of only the newly synthesized
p53. The ellipticine could be inducing the correct folding of
p53 mutants with the assistance of molecular chaperones
(104).
The adenosine receptor antagonist 8-cyclopentyl-1,3dipropylxanthine as well as N-acetyl-l-cysteine have been
shown to activate the Cl⫺ movement in cells expressing the
⌬F508 CFTR protein; however, the mode of action of these
Endocrine Reviews, June 2005, 26(4):479 –503 495
compounds remains unclear. 8-Cyclopentyl-1,3-dipropylxanthine seems to have an action only on the mutated CFTR
protein, because it does not further increase Cl⫺ efflux of the
wt protein. In contrast, N-acetyl-l-cysteine can exert its effect
on the wt or the ⌬F508 form of CFTR. Furthermore, cells
treated with butyrate show an increase in the overall expression of CFTR, and low levels of the ⌬F508 CFTR protein
appear to reach the plasma membrane. This effect could be
due either by allowing the mutant protein to escape from the
ER and move to the plasma membrane, or by directly influencing the folding of the mutant protein and allowing it to
be properly processed (112).
Therapeutic strategies that target amyloid formation have
been proposed. The overstabilization concept might be combined with simultaneous destabilization of the pathological
␤-sheet conformation of misfolded proteins. ␤-Sheet breaking peptides have been designed to block the conformational
changes and aggregation of both A␤ and PrP (240, 241). These
peptides can inhibit and reverse protein misfolding and aggregation and might be able to be used both prophylactically
and therapeutically. 4⬘-iodo-4⬘-Deoxydoxorubicin and tetracycline have also been reported to inhibit protein misfolding
and to dissolve preformed aggregates of several proteins,
showing that the reversal of protein misfolding and aggregation is a possible target (242, 243). Peptidomimetics, based
on the peptide LVFFA from A␤, block both A␤ seeding and
growth. Unfortunately, the pharmacological profile of these
compounds is far from ideal (244). These compounds are
typically inhibiting the aggregation of A␤ and several other
amyloidogenic polypeptides. Their lack of specificity might
be a problem because it has been shown that mammalian
melanocytes and related cell types make amyloid aggregation for functional purposes (245).
Decreasing the concentration of A␤ below the critical concentration also prevents aggregation (246). Several ␤-secretase inhibitors are now available but none are therapeutically
useful. Some ␥-secretase inhibitors are certain nonsteroidal
antiinflammatory drugs such as ibuprofen and indomethacin that shift ␥-secretase activity toward the production of
shorter less amyloidogenic A␤ forms such as A␤(1–38). However, the actual mechanism by which these compounds partially inhibit ␥-secretase is unknown (159, 247, 248).
When APP transgenic mice overexpressing A␤ are actively
immunized with A␤ peptides, deposits that would ordinarily accumulate are cleared (249). Passive immunotherapy
with iv A␤ antibody also prevents the formation of A␤ deposits in animals that would otherwise be deposit laden.
Unfortunately, active immunization of patients with AD using A␤(1– 42) revealed antibodies crossing the blood-brain
barrier causing meningoencephalitis-like inflammation in
4% of the trial participants, presumably originating from
microglial activation (250). This event resulted in the discontinuation of the clinical trial.
Monoclonal antibodies that specifically recognize the elongated polyglutamine stretch in the Huntington protein suppressed the in vitro aggregation of this protein (251). It has
also been observed that some testified aggregation inhibitors
such as Congo red, thioflavine S, chrysamine G, and Direct
fast yellow inhibits Huntington protein aggregation; however, some other A␤ aggregation inhibitors such as melato-
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Endocrine Reviews, June 2005, 26(4):479 –503
nin and rifampicin were not effective at inhibiting this
protein aggregation, indicating some protein selectivity.
Huntington protein is cleaved by caspases with the liberation
of toxic fragments (252). Caspase inhibitors have been shown
to inhibit the proteolysis of the Huntington protein and to
decrease its toxicity (253). A recent study in a transgenic
mouse model of HD indicated that the caspase inhibitor
minocycline slows disease progression (254). The efficacy of
caspase inhibitors in humans has yet to be determined.
A variety of compounds, including anthracyclines, porphyrins, and diazo dyes, block prion replication when administered with PrPsc into animal models (255, 256). Unfortunately, this is not a clinically relevant model for therapeutic
intervention, because subclinical disease exists for months in
mice and years in humans. Because quinacrine is an approved pharmaceutical, clinical trials are being carried out to
test the usefulness of this molecule in patients with CJD.
Medicinal chemistry efforts are underway to identify more
potent agents. For example, some bisacridines are 10- to
20-fold more potent than quinacrine. Recently, Zahn and
colleagues (257) have used nuclear magnetic resonance spectroscopy and chemical-shift tritiation to show that quinacrine
binds specifically to PrPc. Antibodies that bind to the PrPc
and either stabilize the normal protein conformation or inhibit aggregation were shown to clear prions in cultured
mouse neurons, indicating that aggregation inhibitors have
potential in the treatment of prion disease (258).
Another potential therapeutic strategy for the treatment of
amyloidogenic diseases is the inhibition of associated oxidative stress. A variety of antioxidants have shown clinical
benefit especially for the treatment of AD. Melatonin and
indole-3-propionic acid are potent antioxidants that appear
to both inhibit amyloid formation and decrease oxidative
stress (259). Vitamins A, E, C, and ␤-carotene all appear to
have some potential clinical utility for the treatment of AD
(260). Vitamin E has been shown to prevent A␤-induced
oxidative damage in cell culture and delays memory deficits
in animal models (261). Although there has been less success
in using antioxidant for the treatment of neurodegenerative
diseases other than AD, there is potential therapeutic utility
in this strategy. If oxidative stress is central to amyloidinduced neurodegeneration, then antioxidants should be a
benefit to multiple diseases. Coenzyme Q10, a potent antioxidant and mitochondrial support agent, has shown some
clinical benefit in HD (262).
The chemical chaperone 4-phenylbutyric acid mediates
increases in secretion of ␣1-ATZ that are retained in the ER
without affecting its degradation, although this compound
does not bind to ␣1-AT specifically (235). Similarly, the glucosidase inhibitor castanospermine mediates increased secretion of ␣1-ATZ. These compounds have the potential to
prevent lung injury, because they restore the secretory defect
that causes liver and lung disease in ␣1-AT deficiency (237,
263).
C. Nonspecific agents
Incubation of AQP2-transfected mammalian cells with
glycerol and related compounds cause a remarkable redistribution of some AQP2 mutants from ER to plasma mem-
Castro-Fernández et al. • Protein Routing in Health and Disease
brane and endosomes, conferring increased cellular water
permeability (83).
Second-site suppressor mutants have been identified by
genetic screen or site-directed mutagenesis that restores mutant p53 activity by stabilizing the global fold of the p53 core,
or by restoring wt stability and recovering the DNA conformation for DNA binding (103). Other compounds such as
glycerol and trimethylamine N-oxide can restore the function
of mutant p53 by interacting with the molecule and promoting its proper folding by still unknown mechanisms (104).
Furthermore, glycerol, deuterated water, and trimethylamine N-oxide were found to partially correct the processing
defect associated with the ⌬F508 CFTR protein. Dimethylsulfoxide can restore cAMP-dependent Cl⫺ transport to the
cells, by probably allowing the correct maturation of ⌬F508
CFTR. These compounds might be influencing a population
of ⌬F508 CFTR protein to adopt its properly folded state
(112).
Glycerol mediates increases in secretion of ␣1-ATZ without affecting the degradation, possibly up-regulating the
chaperone system. This is accomplished without influencing
the secretion efficiency of other proteins or by decreasing
proteasome activity (235, 263).
VI. Conclusions
The ER is the first intracellular organelle in which newly
synthesized proteins begin the folding process that leads to
formation of mature proteins; such mature proteins properly
route within the cell and become functional as receptors,
hormones, enzymes, ion channels, and structural components. Retention and QC systems are also found in other
compartments of the early secretory pathway, including the
cis-Golgi; these pathways serve as a back-up QC mechanism
for misfolded proteins that were not retained in the ER.
Surprisingly, given the rigor of this extensive network of QC
mechanisms along the secretory pathway, incompletely
folded proteins are sometimes transported to the plasma
membrane or even secreted. It is not yet clear whether these
proteins have special characteristics that allow them to escape the QC apparatus, or whether their (apparently undetected) transport results from saturation of the QC system.
Proteins that first enter the ER are exposed to chaperones;
such molecules stabilize the unfolded or partially folded
intermediates, retrotranslocating them to the cytosol for proteasomal degradation. Degradation of properly folded proteins is avoided because the hydrophobic regions of these
proteins, which are recognized by the regulatory components of the degradative machinery, are generally buried
within folded proteins and because the proteolytic sites
themselves are sequestered within internal chambers that are
not directly accessible to proteins in the surrounding
medium.
The stability of the proteins that are processed in the ER
is enhanced by disulfide-bond formation, N-glycosylation,
and other posttranslational modifications. If the protein folding and maturation process fails, improperly folded molecules are either retained/degraded/corrected in the ER or
may lead to buildup of toxic products. Particular diseases can
Castro-Fernández et al. • Protein Routing in Health and Disease
be explained in terms of buildup of toxins, loss of function
of (misrouted) proteins, and defects in the QC/chaperone
machinery itself.
Understanding the underlying basis of folding and the QC
system is proving useful for the design of new therapeutic
interventions in human and animal disease. This approach
has already led to the use of pharmacological chaperones,
small molecules, and temperature-shift techniques to rescue
proteins that are potentially functional but are retained intracellularly or by forming aggregates. Chemical chaperones
and ligand-induced transport presents options for designing
specific drugs to control protein misfolding or transport.
Although most studies have been done in vitro, understanding this process and the expectation of increased access to
small molecule libraries (promised by the NIH Roadmap
plan), presents the reasonable expectation of agents that will
be active in vivo and thereby of considerable therapeutic
utility.
A companion review that focuses more narrowly on rescue
of disease-associated mutants of the GnRH receptor is available (264).
Endocrine Reviews, June 2005, 26(4):479 –503 497
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
Acknowledgments
We appreciate the title suggestion from Dr. Don Pfaff of the Rockefeller University. We thank Eliot Spindel, from the Oregon National
Primate Research Center, OHSU, for commenting on a draft of the
manuscript. We also thank Shaun Brothers for reading and commenting
on drafts of the manuscript.
Address all correspondence and requests for reprints to: P. Michael
Conn, Oregon National Primate Research Center/Oregon Health and
Science University, 505 NW 185th Avenue, Beaverton, Oregon 97006.
E-mail: [email protected]
This work was supported by National Institutes of Health Grants
HD-19899, RR-00163, HD-18185, and TW/HD-00668.
22.
23.
24.
25.
26.
27.
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