Cystic Fibrosis Research Center, Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
THE ABILITY OF NEWLY SYNTHESIZED PROTEINS, particularly
oligomeric or multidomain proteins, to fold into unique,
three-dimensional structures in vivo is dependent on their amino acid
sequence and the activities of enzymes and molecular chaperone proteins
that catalyze folding (9). Evidence is accumulating that many human diseases are caused by improper folding of nascent polypeptides as they
achieve a final three-dimensional structure. Such proteins are either
inactive or have altered activity as a result of inappropriate folding, as is the case in Marfan syndrome and familiar
hypercholesterolemia, or are mislocalized due to trafficking defects
arising from abnormal folding, as is the case for Tay-Sachs disease and
Mutations in the cystic fibrosis transmembrane conductance regulator
(CFTR) give rise to cystic fibrosis (CF), the most common lethal
autosomal genetic disease of Caucasians. Deletion of the codon for
phenylalanine-508 ( Attempts to pharmacologically manipulate the folding of Manipulating CFTR folding by "chemical chaperones" does not
appear to be readily feasible in the context of a CF patient. However,
preventing the recognition of misfolded CFTR as abnormal may be
amenable to pharmacological strategies. Thus the current article by
Rubenstein and Zeitlin offers exciting insight into how one might be
able to disrupt cellular chaperone-dependent retention of mutant CFTR
molecules in the ER, allowing their insertion into the plasma
membrane. Moreover, both 4PBA and deoxyspergualin are approved drugs
for urea cycle disorders and antitumor/immunosuppression, respectively.
Indeed, clinical trials using 4PBA have suggested a small but
significant improvement in nasal epithelial chloride transport in
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1-antitrypsin deficiency.
F508) accounts for ~70% of all disease causing
CF alleles (11) and yields a protein that is unable to exit from the
endoplasmic reticulum (ER) and traffic to the plasma membrane where it
functions normally as an ion channel. Available evidence suggests that
nascent
F508-CFTR is a temperature-sensitive protein (4), which
adopts a slightly altered protein conformation. As such, it is
recognized by ER quality control mechanisms as abnormal, leading to ER
retention (3) (but see also Ref. 6) and ultimately to degradation by
the ubiquitin-proteosome pathway (7). Such retention appears, at least
in part, to be due to the cellular chaperones, heat-shock protein Hsc70
and calnexin (10, 17). The question of how to shield or
dissociate mutant CFTR molecules from the ER quality control machinery
and allow its exit to the plasma membrane has been the focus of
research in several CF laboratories. Because
F508-CFTR retains some
regulated chloride channel activity (16), correcting the folding and/or preventing the ER retention of mutant CFTR by chaperones may have therapeutic benefit for the treatment of CF.
F508-CFTR
have included the use of protein stabilizing reagents, or chemical
chaperones, such as glycerol and trimethylamine N-oxide, as
well as deuterated water (2, 14). Indeed, evidence suggests that such
approaches can facilitate exit of
F508-CFTR from the ER and
insertion into the apical plasma membrane in vivo. A previous publication by Rubenstein and colleagues (12) has shown that sodium
4-phenylbutyrate (4PBA) similarly facilitates exit of
F508-CFTR from
the ER, though the mechanism of action has remained elusive. Previous
work has shown that
F508-CFTR remains associated with the cellular
chaperone Hsc70 to a much greater extent than wild-type CFTR (17), an
association that may direct mutant CFTR to the ubiquitin-dependent
degradation pathway (1). In the current article in focus (Ref. 13, see
page C259 in this issue), Rubenstein and Zeitlin
demonstrate that association of mutant CFTR with the heat-shock protein
Hsc70 can be reduced by exposing cells to 4PBA. Mechanistically, it
appears that 4PBA inhibits the interaction of
F508-CFTR with Hsc70,
allowing mutant CFTR to escape targeting for degradation and
facilitating its exit from the ER. 4PBA appears to do so by
downregulating the expression of Hsc70 at the protein and mRNA level,
thereby limiting the amount of Hsc70 that is available to bind to CFTR.
Such a hypothesis is consistent with the observations of Cheng and
colleagues (5), who utilized deoxyspergualin, a compound that does not
affect Hsc70 protein levels but competitively inhibits peptide binding
to Hsc70, to partially restore CFTR function in heterologous cells
expressing
F508-CFTR. It would appear, however, that the efficacious
effects of inhibiting CFTR chaperone interactions are limited to Hsc70,
since perturbation of CFTR-Hsc90 interactions have been shown to
accelerate CFTR degradation (8).
F508-homozygous CF patients. However, before such observations can
be fully realized as therapies, several questions must be addressed.
For example, just how much mutant CFTR must escape from the ER quality
control machinery to effect clinical benefit? The studies of Cheng and
colleagues (5) would argue that the amount of mutant CFTR required to
reach the plasma membrane to provide a measurable cAMP-sensitive
chloride conductive pathway is less than which can be detected as
mature CFTR protein by biochemical methods. However, since
F508-CFTR
does not retain full wild-type function, even after reaching the plasma
membrane, further pharmacological activation of plasmalemmal resident
mutant CFTR may be still required (15, 16). It remains to be determined
what impact perturbation of the ER quality control machinery will have
on protein folding and processing pathways for other proteins expressed
in the cell. In addition, Hsc70 has other cellular functions, including
the ATP-dependent uncoating of clathrin-coated vesicles in the
endocytic pathway and targeting of proteins for lysosomal degradation.
It is unclear what effects alterations in Hsc70 activity would have on
these intracellular processes. Nevertheless, the ability
to screen large libraries of compounds for drugs that disrupt
CFTR-Hsc70 interactions may be fruitful in providing a pharmacological
therapy for CF.
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ACKNOWLEDGEMENTS |
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This report was funded by grants from the National Institute of Diabetes and Digestive and Kidney Diseases and the Cystic Fibrosis Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: N. A. Bradbury, S306 BST South, Dept. of Cell Biology and Physiology, 3500 Terrace St., Univ. of Pittsburgh School of Medicine, Pittsburgh, PA 15261 (E-mail: nabrad{at}pitt.edu).
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