Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
THE ENDOPLASMIC RETICULUM (ER) serves
as a way station during the biogenesis of nearly every integral
membrane and secreted protein synthesized in eukaryotic cells. As such,
its primary role is to facilitate the folding of nascent polypeptides.
Thus plentiful among the ER resident proteins are 1) enzymes
that catalyze conformational rearrangements, such as protein disulfide
isomerase and peptidylprolyl isomerase; 2) enzymes that
chemically modify the nascent polypeptide, such as signal sequence
peptidase and oligosaccharyl transferase; and 3) molecules
that retain the polypeptide in a soluble, aggregation-free state. The
factors constituting this last group are molecular chaperones, many of
which were first identified as heat shock proteins (HSPs) because their
synthesis is induced when cells are exposed to high temperatures or
other stresses. However, most HSPs have cellular homologs that are
constitutively produced, and these are named heat shock cognate
proteins (HSCs). HSC and HSP molecular chaperones are defined by their
molecular masses (in kDa), and a plentiful class found in every cell
type are those with a molecular mass of ~70 kDa (i.e., HSC70 and HSP70).
HSP70 and HSC70 hydrolyze ATP concomitant with their binding to and
release from polypeptide substrates, and their ATPase activities may be
activated by another group of chaperones (21, 33) known as
HSP40 or DnaJ homologs. In some cases, the HSP40 cochaperone delivers a
polypeptide substrate to the HSP70 or HSC70 (17, 37). With
the use of a variety of in vitro techniques, the HSC70 and HSP70
chaperones have been shown to bind preferentially to short stretches of
amino acids with overall hydrophobic character (3, 13,
31). Such motifs are normally buried within the core of a native
protein but may become solvent accessible during protein folding and
upon protein denaturation. Thus HSC70 and HSP70 chaperones are ideally
suited to aid unfolded proteins en route to their native conformations
by limiting protein aggregation.
Deletion of the phenylalanine at position 508 in the cystic fibrosis
(CF) transmembrane conductance regulator (CFTR) is the most common
mutation associated with CF (27). CFTR most likely functions as a chloride channel in the plasma membrane of airway and
other epithelial cells, but cells containing only the Because Because several human diseases arise from defects in protein folding
(4, 35), because of the devastating effects and widespread
occurrence of CF, and because a profound amount of information is
available on the mechanisms of molecular chaperones, the modulation of
protein folding and chaperone activity has been attempted with small
molecules and genetic manipulations. For example, it was demonstrated
by the Welch (5) and Kopito (32) laboratories
that Another means to alter protein folding in vivo is through the
modulation of intracellular chaperone concentration. For example, Rubenstein et al. (28) showed that sodium
4-phenylbutyrate (4PBA), an ammonia scavenger approved for the
treatment of urea cycle disorders and a known transcriptional
regulator, permits a fraction of The studies described above support the concept of using chaperone
modulation as a means to rectify the pathophysiology associated with
Building on previous studies, Rubenstein and Lyons (29)
show that the mechanism by which 4PBA downregulates HSC70 levels is
through a decrease in the stability of HSC70 mRNA. Although HSC70 mRNA
is known to be relatively unstable, 4PBA appears to further accelerate
its degradation. Coincident with an acceleration of mRNA degradation,
an ~40% reduction in the amount of HSC70 is evident. The mechanism
by which this occurs is unknown, but it has been shown that inhibiting
the initiation of yeast HSC70 translation leads to a rapid destruction
of its message (1). Because a large number of translation
poisons are known, many of which were first isolated as antibiotics,
this hypothesis can be tested.
Does 4PBA only modulate HSC70 levels? Although previous data
(30) suggested that this was so, new work from Choo-Kang
and Zeitlin (8) indicates that 4PBA raises HSP70 levels,
leading to increased HSP70- These combined studies suggest that modulating the levels and thus the
activities of HSC70 and HSP70 enhances CFTR maturation. They also point
to the complexity of HSP70 or HSC70 function in the cell. Whereas one
isoform (i.e., HSP70) facilitates protein folding, its homolog (i.e.,
HSC70) targets a misfolded protein for degradation. Previous data
suggest that multiple chaperones bind coordinately to nascent
polypeptides. Although the functions of many of these chaperones are
likely to be redundant, it seems logical that a subset is required for
folding and another subset is necessary for protein degradation. If one
group fails to fold the polypeptide, another is readily available to
target the substrate for destruction. The activities of the unique sets
of chaperones may be further defined by the action of the HSP40/DnaJ
cochaperones that regulate HSP70 and HSC70 function. In other cases,
the folding and degradation targeting activities may reside in one
chaperone complex. For example, HSP90 has been proposed to facilitate
both CFTR maturation and degradation (18).
Another means to catalyze CFTR folding is through the use of chemical
chaperones, an attack that has shown promise in in vitro model systems
as described above. To determine whether this approach is feasible in
an animal system, Fischer et al. (12) examined whether
TMAO corrects the Might chaperone-based therapies become clinically relevant and
generalized to the spectrum of diseases that arise from protein misfolding? The corrective effects of glycerol and 4-PBA on the maturation and secretion of another "conformational
disease"-causing protein, the Z variant of antitrypsin, have been
recently reported by Burrows et al. (6). In addition,
other chaperone modulators are currently being examined for their
ability to correct misfolded proteins. 15-Deoxyspergualin, a known
HSC70 effector and immunosuppressant, increases the cAMP-stimulated
plasma membrane Cl
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REFERENCES
F508 variant
either lack or harbor variable amounts of the protein at the plasma
membrane (7, 16). Both wild-type and the
F508 mutant
form of CFTR are translocated into the ER membrane, at which time
protein folding should commence. However, in vitro studies by Qu and
colleagues (25, 26) suggest that the efficiency of
F508 CFTR folding is severely impaired compared with that of the
wild-type protein. It has also been shown that the
F508 mutant
protein is unable to undergo an ATP-dependent conformational change in
the ER (20, 40). Consistent with an inability of
F508
CFTR to fold efficiently, incubation of cells expressing the mutant at
lower temperatures, a condition that may favor protein folding and/or
stability, permits some of this variant to traffic to the plasma
membrane where it is active (10), albeit with a reduced
open probability and half-life (9, 11, 19). In contrast,
if CFTR folding is retarded in the ER, the protein becomes a substrate
for ER-associated degradation, a quality control mechanism that
degrades aberrant proteins in the secretory pathway (4). Degradation of CFTR, like most other ER-associated degradation substrates, requires ubiquitinylation and delivery to the
multicatalytic cytoplasmic proteasome (14, 36, 38).
Although a significant proportion of even wild-type CFTR is degraded,
nearly all of the
F508 mutant is destroyed; therefore, cells
expressing only the mutant form of the protein are phenotypically null.
F508 CFTR is unable to attain a native conformation in the
ER, it should not come as a surprise that it interacts with molecular
chaperones. Specifically, cytoplasmic HSC70 and HSP90, the luminal
Ca2+-binding chaperone calnexin, and a cytoplasmic HSP40
homolog Hdj2 can be coprecipitated with CFTR (18, 22, 24,
39). HSC70 and Hdj2 also prevent the in vitro aggregation of the
first nucleotide-binding domain of CFTR (22, 34), the
domain in which the phenylalanine at position 508 resides. Although
wild-type, folded CFTR is released from the chaperones on its transit
from the ER to the Golgi apparatus, HSC70 and calnexin remain bound to
the mutant until degradation ensues (24, 39). A
HSC70-interacting factor, CHIP, has recently been shown to facilitate
the degradation of immature forms of CFTR (23). One
interpretation of these combined data is that the chaperone-CFTR
complexes represent folding intermediates.
F508 CFTR-expressing cells incubated in glycerol, a polyol
known to stabilize proteins in vitro, contain ER-matured forms of CFTR
and display cAMP (forskolin)-activated plasma membrane chloride
transport, a hallmark of functional CFTR. Glycerol also facilitates the
in vitro folding of the first nucleotide-binding domain lacking the
phenylalanine at position 508 by preventing off-pathway intermediates
from forming (25).
F508 CFTR-expressing cells incubated
in trimethylamine oxide (TMAO), an osmotic stabilizer found in sharks
to protect proteins from urea denaturation, exhibit similar properties
(5). Compounds such as TMAO and glycerol are now commonly
known as chemical chaperones.
F508 CFTR to mature in bronchial
epithelial cell lines and primary nasal epithelial cells from patients
with CF. Maturation of the mutant coincides with cAMP-stimulated plasma
membrane Cl
conductance in the treated cells. To
determine the molecular basis of 4PBA-mediated rescue of the
F508
CFTR phenotype, immunoprecipitations were performed to examine the
amount of CFTR associated with HSC70, and a 4PBA dose-dependent
decrease in the amount of CFTR-HSC70 complex was observed
(30). Because HSC70 was shown to facilitate ubiquitination
of several test proteins in vitro (2), the authors suggested that HSC70 targets CFTR for degradation and that 4PBA, through an unknown mechanism, reduces the amount of the complex by
which CFTR is delivered to the proteasome.
F508 CFTR. Now, three papers in this issue of the American Journal of Physiology-Lung Cellular and Molecular Physiology
(8, 12, 29) represent a significant leap in our
understanding of the molecular basis of chaperone modulation, with the
ultimate hope that such interventions may become clinically relevant
for CF.
F508 CFTR complex formation and
F508
CFTR maturation. One concern is that this phenomenon may arise from a
secondary effect of decreasing HSC70 levels (see above), which, in
turn, could induce a general heat shock or stress response. However,
the authors show that CFTR maturation is also facilitated by
selectively increasing the intracellular levels of HSP70 by transient
transfection with an HSP70 expression vector.
F508 CFTR trafficking defect in mice. Because the
severity of the defect associated with the
F508 form of CFTR is most
prevalent in the rodent intestine, measurements of rectal potential
difference (RPD) were undertaken in control mice, those lacking CFTR,
and
F508 CFTR mice. Administration of TMAO over 24 h by
subcutaneous injection reveals significant increases in
forskolin-activated RPD hyperpolarization in the control and
F508
CFTR mice, consistent with a partial rescue of the folding defects
associated with the wild-type and mutant proteins. Most promising from
a clinical perspective is the observation that administering the
flavinoid apigenin, a CFTR activator, further increases the
forskolin-activated RPD in the
F508 CFTR-expressing mouse. This
result supports the promise that combined therapeutic attacks may
present the best option to lessen the pathophysiology of CF. Combining
small-molecule activators of CFTR function is warranted, particularly
in this case, because the concentrations of TMAO used for these studies
are quite high and the physiological effects of prolonged
administration of TMAO are unknown.
channel activity of
F508
CFTR-expressing cells (15). However, because an increase
in ER-matured forms of
F508 CFTR could not be observed with
15-deoxyspergualin treatment, only a small amount of the mutant had
likely trafficked to the plasma membrane and yielded the desired
activity. Nevertheless, with the advent of combinatorial chemistry and
more defined methods to examine protein folding and chaperone action at
the molecular level, I suspect that we will see a new convergence of
chemical, cell biological, and biophysical tools geared to combat a
variety of human diseases.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. L. Brodsky, Dept. of Biological Sciences, 267 Crawford Hall, Univ. of Pittsburgh, Pittsburgh, PA 15260 (E-mail: jbrodsky{at}pitt.edu).
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