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J. Biol. Chem., Vol. 278, Issue 37, 35159-35167, September 12, 2003
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F508 Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Protein Maturation and Function*
¶¶
From the
Department of Surgery and the
||Department of Anesthesia, Surgical Research
Laboratory, University of California, San Francisco, California 94110, the
¶Children's Hospital Oakland Research Institute,
Oakland, California 94609, the **Renal Division,
Department of Medicine, Escola Paulista de Medicina, UNIFESP, 04023-900 Sao
Paulo, Brazil, the Laboratory of
Functional Genomics, Brigham and Women's Hospital and Harvard Medical School,
Boston, Massachusetts 02115, the
Department of Biology, Whitman College,
Walla Walla, Washington 99362, and the
¶¶Department of Medicine and Physiology,
University of California, San Francisco, California 94110
Received for publication, February 24, 2003 , and in revised form, June 4, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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F508 CFTR maturation and function in kidney
cells under conditions that are common to the kidney, namely osmotic stress.
Kidney cells were grown in culture and adapted to 250 mM NaCl and
250 mM urea. High performance liquid chromatography analysis of
lysates from kidney cells adapted to these conditions identified an increase
in the cellular osmolytes glycerophosphorylcholine, myo-inositol,
sorbitol, and taurine. In contrast to isoosmotic conditions, hyperosmotic
stress led to the proper folding and processing of F508 CFTR.
Furthermore, three of the cellular osmolytes, when added individually to
cells, proved effective in promoting the proper folding and processing of the
F508 CFTR protein in both epithelial and fibroblast cells. Whole-cell
patch clamping of osmolyte-treated cells showed that F508 CFTR had
trafficked to the plasma membrane and was activated by forskolin. Encouraged
by these findings, we looked at other features common to the kidney that may
impact F508 maturation and function. Interestingly, a small molecule,
S-nitrosoglutathione, which is a substrate for gamma
glutamyltranspeptidase, an abundant enzyme in the kidney, likewise promoted
F508 CFTR maturation and function.
S-Nitrosoglutathione-corrected F508 CFTR exhibited a shorter
half-life as compared with wild type CFTR. These results demonstrate the
feasibility of a small molecule approach as a therapeutic treatment in
promoting F508 CFTR maturation and function and suggest that an
additional treatment may be required to stabilize F508 CFTR protein
once present at the plasma membrane. Finally, our observations may help to
explain why F508 homozygous patients do not present with kidney
dysfunction. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The most prevalent mutation associated with the disease is the deletion of
a phenylalanine residue at position 508 (F508 CFTR)
(2). Loss of this single amino
acid results in a failure of the newly synthesized protein to move out of the
endoplasmic reticulum (ER) to its finale locale at the plasma membrane
(3). This failure is due to the
synthesis of a protein that apparently cannot fold properly. Consequently, the
protein is retained in the ER and degraded by the ubiquitin-proteosome pathway
(4,
5). Validation for the idea
that the F508 CFTR mutation leads to improper folding is supported by
the facts that: 1) misfolding of the protein can be corrected by lowering the
temperature of cells expressing the mutant protein
(6), 2) correct folding of the
protein can occur upon addition of protein stabilizing agents, such as
glycerol and TMAO (7,
8), and 3) once corrected, the
F508 protein can traffic to the plasma membrane and function as a
cAMP-regulated chloride channel
(6–9).
During osmotic stress, yeast and sharks increase the intracellular levels
of glycerol and TMAO, respectively, two cellular osmolytes known to protect
proteins from denaturation under harsh environmental conditions and as
mentioned above, effective in correcting F508 CFTR protein folding
(10,
11). Similarly, mammalian
systems have their own set of osmolytes, including sorbitol,
myo-inositol, betaine, taurine, and glycerophosphorylcholine (GPC)
(12,
13). These compounds typically
accumulate to high concentrations in the kidney medulla as part of the normal
physiological process of concentrating urine. In contrast to other organs of
epithelial origin, the kidney is considered unaffected in F508 CFTR
homozygotes. Therefore, we hypothesized that osmolytes that are synthesized
and accumulated within kidney medulla might promote the proper folding and
function of the F508 CFTR protein and therefore help explain why
F508 CF patients do not present with kidney dysfunction.
The iso-osmotic renal cortex also expresses CFTR, but unlike the renal
medulla, the absence of disease phenotype cannot be explained by elevated
osmolality. Recently, Zaman et al.
(14) reported that
S-nitrosoglutathione (GSNO) promotes the maturation of endogenous
wild type and F508 CFTR maturation, although functional data were not
presented. Interestingly, GSNO is a substrate for
-glutamyltranspeptidase,
(15) a transmembrane protein
expressed at high levels in the lumen of the renal cortical proximal kidney
(16–18).
Here we show that GSNO promotes F508 maturation and restores function
while having no effect on wild type CFTR maturation. Thus, our results point
to two possible mechanisms, intracellular osmolyte accumulation and hydrolysis
of GSNO, carried out by the kidney, as potential explanations why CF patients
exhibit little or no kidney dysfunction.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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F508 CFTR plasmid
(20) were cultured in
Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum
and 1% penicillin and streptomycin. For osmotic adaptation experiments, cells
were gradually adapted to increasing concentrations of equimolar NaCl and urea
over a 3-week period to a final concentration of 250 mM NaCl and
250 mM urea. Cells were maintained at this concentration for an
additional 2–3 weeks before the initiation of experiments. For cellular
osmolyte treatments, the compounds were added directly to the growth medium of
the cells at the concentrations indicated in the text. Expression of either
wild type or F508 CFTR using recombinant adenovirus (Ref.
21; F508 adenovirus,
purchased from Dr. J. Wilson, University of Pennsylvania) was carried out as
described previously (21).
Twenty-four hours post-infection, 2 mM sodium butyrate was added to
the cells to increase transcription of the CFTR cDNA. Eighteen hours later,
cells were either harvested for biochemical analysis or used for whole-cell
patch clamping. Immunoblotting—Cells were washed twice in calcium free phosphate-buffered saline and lysed in 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA supplemented with protease inhibitors (Calbiochem). The lysates were clarified at 14,000 x g at 4 °C. An aliquot of the supernatant was sampled for protein determination (BCA, Pierce), while the remaining sample was adjusted to 1X Laemmli sample buffer (40 mM Tris-HCl, pH 6.8, 1% SDS, 50 mM dithiothreitol, 7.5% glycerol) and heated at 37 °C for 20 min. Equal amounts of total protein were loaded on 7.5% polyacrylamide gels. Following SDS-PAGE, the proteins were transferred to nitrocellulose and probed with a monoclonal antibody specific for the CFTR protein (0.3 µg/ml M3A7, Upstate Biotechnology) and an horseradish peroxidase-conjugated goat anti-mouse secondary antibody at 1:5,000. CFTR protein was visualized by chemiluminescence.
Radiolabeling and Immunoprecipitation—Cells were incubated
in methionine-free medium for 15 min prior to the addition of 50 µCi/ml of
[35S]methionine for 4 h in either the absence or presence of 100
µM GSNO. Increasing the concentration of GSNO resulted in an
increase in F508 CFTR protein maturation and thus increased levels of
[35S]methionine incorporated into the nascent protein. This enabled
better detection of the newly synthesized F508 CFTR protein during the
pulse-chase and immunoprecipitation experiments. After 4 h of labeling, the
cells were washed and one set of plates was harvested immediately for
immunoprecipitation. To the remaining plates, the medium containing the
radiolabel was removed and the cells washed with fresh medium and then
incubated in medium containing 1 mM cold methionine for 4 h in
either the absence or presence of GSNO and then harvested as described above.
CFTR was immunoprecipitated from each sample (after being adjusted for equal
protein concentration) at 4 °C overnight with 2 µg each of M3A7 and
L12B4 (Upstate Biotechnology). Protein G-Sepharose was added to the lysate for
1 h at 4 °C, pelleted at 5,000 x g for 10 s, and washed
twice with RIPA (1% Triton X-100, 1% sodium deoxycholate in phosphate-buffered
saline) buffer. The immune complexes were incubated with 2x Laemmli
sample buffer for 30 min at 37 °C, the beads pelleted, and the supernatant
then loaded onto a 7.5% gel. Proteins were visualized by fluorography.
Patch Clamping—Cells were whole-cell patch-clamped at 37
°C as described previously
(22). On the stage of an
inverted microscope cells were bathed in (in mM): 136
N-methyl-D-glucamine, 127 HCl, 1.7 CaCl2, 1
MgCl2, 10 Hepes, 10 glucose, 500 mannitol, pH 7.3. Patch pipettes
were filled with (in mM): 130
N-methyl-D-glucamine, 130 HCl, 10 EGTA, 1
MgCl2, 2 Hepes, 1 glucose, 500 mannitol, 5 Mg-ATP, 0.1 Li-GTP, pH
7.3. Single isolated cells were randomly selected for patch clamping. Only
seals >10 G
were used. After establishing the whole cell
configuration the membrane potential (Vm) was continuously
clamped to –40 mV. The access resistance (Ra) and
the cell membrane capacitance (Cm) were measured using the
current transients caused by a 10-mV voltage pulse. Ra =
15 ± 1.2 M (n = 104) and was not affected by
the different treatments. Current-voltage (I-V) step
protocols before and after addition of 20 µM forskolin were
applied, from a resting potential of –40 mV, from –100 mV to +40
mV, and the resulting currents were recorded. Larger voltage jumps reduced the
success rate of experiments due to seal breakage and were generally not
performed. Whole cell conductance (Gc) was calculated as
the slope between –40 and –20 mV. For the calculation of the
specific membrane conductance (Gm in
picosiemens/picofarad) Gc was corrected for
Ra and normalized to Cm. Quantitative
effects of different treatments were compared with factorial ANOVAs followed
by Bonferroni-corrected t tests. p < 0.05 was considered
significant. Calculations were done with StatView, version 4.5 (Abacus
Concepts, Berkeley, CA).
Determination of Cellular Osmolyte Concentration—Control cells (mIMCD3) and hyperosmolar-adapted cells (HT500) were grown on 10-cm plates. Intracellular osmolyte concentrations were determined via HPLC analysis using a modified protocol as described by Rauchman et al. (19). Briefly, cells were lysed in 7% perchloric acid and the lysates centrifuged at 14,000 x g for 20 min. The acid insoluble pellet was resuspended in a 0.5 N NaOH solution and used for subsequent protein analysis using the BCA assay. The acid soluble material was adjusted to neutral pH and centrifuged at 10,000 x g for 30 s. The supernatant was serially filtered through a C8 Sep-Pak column and a 0.45-µm HV filter, lyophilized, and resuspended in HPLC mobile-phase solution and loaded onto an HPLC column. Osmolytes were measured by refractive index.
Chemicals and Reagents—Forskolin (Calbiochem) was made as a 20 mM stock in Me2SO and used at 20 µM. GSNO (Sigma), prepared as a fresh stock each time, was dissolved in water at 10 mM and used at 10 µM. Osmolytes (Sigma) were dissolved in medium at 1 M and used at 300 mM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Hyperosmotic-adapted Kidney Cells Exhibit Proper Maturation and
Functional F508 CFTR—Having established conditions
that increase osmolyte levels in the mIMCD3 cells, we tested whether the
accumulation of these osmolytes would have any effect on the maturation of the
F508 CFTR protein. The rationale based on previous studies showing that
osmolytes such as glycerol and TMAO, when added to cells, correct the folding
and trafficking of F508 CFTR
(7,
8). Control mIMCD3 cells
maintained in iso-osmotic growth medium, and HT500 cells maintained in
hyperosmotic growth medium were infected with a recombinant adenovirus
expressing either wild type or F508 CFTR. Neither mock-infected mIMCD3
cells nor mock-infected HT500 cells synthesized any detectable CFTR protein,
as determined by Western blotting analysis
(Fig. 1B, lanes
1 and 4, respectively). Infection of the mIMCD3 and HT500 cells
with a recombinant adenovirus expressing wild type CFTR resulted in the
synthesis and detection of both the immature core-glycosylated B band and the
mature fully glycosylated C band (Fig.
1B, lanes 2 and 5). Expression of the
F508 CFTR protein in mIMCD3 cells resulted in the appearance of only
the core-glycosylated protein (band B), as is normally seen in cells
expressing the F508 mutant (Fig.
1B, lane 3). In the F508 CFTR-infected
HT500 cells, however, an increase in the amount of the core-glycosylated B
band, as well as the processing of a portion of the protein to the mature form
(C band) was observed (Fig.
1B, lane 6). Interestingly, F508 CFTR
expressed in the HT500 cells exhibited multiple electrophoretic species as
evidenced by a smear of bands with slower mobility as compared with the
immature B band. At the present time, the exact basis for this heterogeneous
collection of CFTR species is not clear. Others, however, have similarly shown
the mature CFTR protein to exist as a "broad smear"
(5,
25,
26). Thus, it appears that
hyperosmotic conditions promote the proper processing of at least a portion of
the mutant F508 CFTR protein, but have no apparent effect on the
maturation of wild type CFTR.
To test whether the hyperosmotic stress resulted in the synthesis and
folding of a functional F508 CFTR protein, the HT500 cells were whole
cell patch-clamped to quantify forskolin-stimulated chloride transport.
Whole-cell currents were measured under conditions selective for chloride
currents in the presence of 20 µM forskolin (see
"Experimental Procedures").
Fig. 1C shows typical
examples of current step responses recorded from mock-infected and F508
CFTR-infected HT500 cells. HT500 cells expressing F508 CFTR showed
large forskolin-activated chloride currents. Measured currents were time- and
voltage-independent (Fig.
1C) and showed linear current-voltage relationships
(Fig. 1D).
Fig. 1E summarizes the
calculated specific membrane conductances (G). Thus, whole-cell
patch-clamping demonstrated that F508 CFTR expressed in HT500 cells
showed significant function, exhibiting typical CFTR-specific characteristics,
i.e. chloride selectivity, activation by forskolin, and linear
current-voltage relationships.
Individual Osmolytes in Fibroblast Cells Promote the Maturation of
Functional of F508 CFTR—Having shown that
hyperosmotic stress both increases the levels of different osmolytes and
promotes the maturation of F508 CFTR, we tested whether the individual
osmolytes themselves were effective in correcting F508 misfolding and,
thereby, would restore chloride channel function. If so, this would indicate
that the osmolytes themselves, rather than some other effect due to salt and
urea adaptation of renal cells, can help promote F508 CFTR protein
maturation. For these experiments, we utilized 3T3 fibroblasts stably
expressing F508 CFTR under the control of the cytomegalovirus promoter
(20). Using these cells, we
previously showed that two other osmolytes, glycerol and TMAO, were effective
in correcting the misfolding of the F508 CFTR mutant
(7). Furthermore, Denning et
al. (6) used these cells to
show that incubation at low temperatures (e.g. 27 °C) also
corrected the misfolding of F508 CFTR protein. Cells were incubated in
growth medium containing the individual osmolytes for 3 days at a
concentration of 300 mM, after which the cells were lysed and CFTR
expression examined by Western blot (Fig.
2A). Cells not incubated with osmolytes nor stimulated
with the general transcriptional activator, sodium butyrate, and cells
stimulated with sodium butyrate alone to increase CFTR transcription showed
little or no F508 protein in the cells maintained at 37 °C
(Fig. 2A, lanes
1 and 2). Treatment with the osmolytes myo-inositol,
sorbitol, or taurine resulted in a portion of the F508 CFTR protein
being correctly folded and processed to the mature form
(Fig. 2A, lanes
4–6). Sorbitol treatment elicited the highest levels of the mature
form of the protein (Fig.
2A, lane 5), while betaine had little or no
effect on F508 CFTR maturation (Fig.
2A, lane 3). Curiously, betaine itself proved to
be somewhat toxic to the fibroblast cells with the cells appearing rounded
after 3 days of incubation with 300 mM betaine (data not shown).
This result is consistent with a previous study showing betaine is toxic to
renal cells when added alone to the extracellular medium but is nontoxic when
added in conjunction with urea
(27).
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Incubation of the cells at 27 °C along with the osmolytes revealed a
synergistic effect on F508 maturation. When incubated at 27 °C, low
levels of the immature form of F508 CFTR were now evident and increased
upon addition of the general transcriptional activator, sodium butyrate
(Fig. 2A, lanes
1 and 2). Note as well, and consistent with previous reports
showing F508 CFTR folding to be temperature-sensitive, the appearance
of the mature form of the protein (band C) in those cells maintained at the
lower temperature. When the cells growing at 27 °C now were treated with
the various osmolytes, significant amounts of mature F508 CFTR (band C)
were observed (Fig.
2A, lanes 4–6, 27 °C). Similar to the
situation at 37 °C, addition of betaine (300 mM) to the cells
proved to be somewhat toxic after 3 days of incubation
(Fig. 2A, lane
3). Thus, F508 CFTR maturation in 3T3 cells can be rescued by
either lowering the temperature of the cells or by the addition of various
cellular osmolytes. Moreover, osmolyte treatment along with a lower growth
temperature resulted in a synergistic enhanced effect on the maturation of the
mutant protein.
To determine whether the osmolyte-corrected F508 CFTR protein was in
fact functional, the 3T3 cells were whole-cell patch-clamped. The cells were
incubated in myo-inositol, taurine, TMAO, or sorbitol (300
mM each) for 3 days, and whole cell currents were measured under
conditions selective for chloride currents (see "Experimental
Procedures"). Fig.
2B shows examples of current step responses recorded from
the unstimulated cells (top panels) or from the forskolin-stimulated
cells (bottom panels) incubated in the absence or presence of the
osmolytes. Wild type CFTR-expressing 3T3 cells showed typical CFTR-mediated
chloride currents after forskolin stimulation
(Fig. 2B, right
panels). Untreated F508 CFTR expressing cells, either without or
with forskolin stimulation, showed no chloride currents
(Fig. 2B, left
panels). In contrast, 3 days of treatment with the different osmolytes
resulted in the appearance of large forskolin-activated chloride currents. All
measured currents were time- and voltage-independent
(Fig. 2B) and showed
linear current-voltage relations (Fig.
2C).
Fig. 2D summarizes the specific conductance (Gm) from 47 patch-clamped cells. Treatment with myo-inositol, but not any of the other osmolytes, resulted in a significant increase in Gm in the absence of forskolin stimulation. As a control for the effects of myo-inositol on basal currents we used its isomer scyllo-inositol. Cells incubated with scyllo-inositol showed no increase of basal Gm, but upon forskolin stimulation did exhibit an increase in Gm (data not shown). In cells treated with myo-inositol, TMAO, or sorbitol, stimulation with forskolin (20 µM) increased the Gm to levels similar to that found for cells expressing wild type CFTR. Treatment with taurine resulted in the largest recovery of a forskolin-stimulated Gm, which was significantly greater than Gm measured in the wild type CFTR expressing cells (Fig. 2D).
Osmolytes Promote F508 Maturation in Epithelial
Cells— Individual osmolytes proved to be effective in promoting
F508 CFTR maturation in fibroblasts after 3 days of treatment. However,
because CFTR is expressed in epithelial cells, we tested whether kidney cells
that are normally exposed to osmolytes would be even more effective in
correcting F508 misfolding. mIMCD3 cells were treated with the
individual osmolytes for either 24 or 72 h. In the case of the 24-h
treatments, the cells were first infected with adenovirus expressing
F508 and then incubated in myo-inositol, sorbitol, or taurine
at a final concentration of 300 mM. For the 72-h treatments, the
mIMCD3 cells were first incubated in myo-inositol, sorbitol, or
taurine at a final concentration of 300 mM for 24 h and then
infected with the F508 CFTR adenovirus. The infected cells then were
further incubated with the individual osmolytes for an additional 48 h prior
to cell harvest. Twenty-four hours of treatment resulted in the appearance of
low levels of mature F508 CFTR (Fig.
3A, lanes 2–4). A novel electrophoretic
species of CFTR (indicated as B1) was observed in the epithelial cells treated
with the osmolytes. Following 72 h of treatment, large increases in the amount
of the F508 CFTR band C were now observed
(Fig. 3B, lanes
2–4). Thus, individual osmolyte treatment of epithelial cells
promotes F508 CFTR protein maturation and reveals a putative folding
intermediate (i.e. B1) not normally observed for wild type CFTR
maturation.
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S-Nitrosoglutathione Promotes Maturation Resulting in Functional
F508 CFTR—Osmolytes accumulate in the cells of the inner
medullary collecting duct in response to hyperosmotic stress as the kidney
concentrates the urine. As shown above, some of these osmolytes can help to
promote the folding of the F508 CFTR, resulting in functional CFTR at
the plasma membrane. However, CFTR also is expressed in other segments of the
kidney that are not subjected to high osmotic stress. As part of our effort to
explain the lack of kidney phenotype in patients homozygous for F508
CFTR, we searched for other aspects unique to the kidney that might affect
CFTR maturation. In searching the literature, we found a study by Zaman et
al. (14) reporting that
GNSO treatment promoted the maturation of both wild type and F508 CFTR.
Although data demonstrating that the corrected F508 could now function
as a chloride channel were not presented, we were intrigued since GSNO is a
substrate for -glutamyltranspeptidase (GGT), an enzyme that is
expressed at high levels in the kidney
(16–18).
GGT hydrolyzes glutathione and S-substituted GSH derivatives within
the kidney filtrate (28) for
subsequent uptake into the cells by amino acid transporters. GSNO treatment
has been shown to have modifying effects (e.g. nitrosylation) on
other cellular proteins thereby impacting their function
(29–32).
Thus, we tested whether GSNO would have any effect on F508 maturation
and if so whether the corrected F508 CFTR would exhibit cAMP-regulated
chloride channel function. Treatment of mIMCD3 cells expressing F508
CFTR with 10 µM GSNO for either 1 or 2 h resulted in the proper
maturation of the mutant protein (Fig.
4A, lanes 2–4). Treatment of the cells
expressing wild type CFTR with GSNO had no obvious effect on the amounts of
mature CFTR produced (Fig.
4B, lanes 2–4). Next, we examined whether
the GSNO-corrected F508 protein exhibited functional chloride channel
activity. Wholecell currents were recorded from forskolin-stimulated (20
µM) F508 CFTR-expressing mIMCD3 cells pretreated with 10
µM GSNO and compared with untreated or mock-infected cells
(Fig. 4C). The
GSNO-treated cells, but not the untreated F508 CFTR-infected mIMCD3
cells, showed linear time- and voltage-independent current characteristics
(Fig. 4D). In
comparison, mock-infected or untreated F508 CFTR-infected cells showed
very small Gm values that were not statistically different
from one another (Fig.
4E). Thus, short term GSNO treatment results in a portion
of F508 CFTR to mature into the C band, resulting in CFTR
forskolin-stimulated chloride transport.
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GSNO-corrected F508 CFTR Is Unstable Compared with Wild
Type CFTR—The relatively low concentration required and the fast
kinetics of GSNO-induced F508 CFTR protein maturation highlighted this
naturally occurring compound as a potential therapeutic agent. To pursue this
possibility further, we investigated the stability of GSNO-corrected
F508 CFTR protein. The impetus here being based on Sharma et
al. (26), who reported
that F508 CFTR, corrected either by low temperature or by glycerol, was
unstable when the cells expressing the corrected F508 CFTR were
returned to 37 °C. mIMCD3 cells expressing either wild type or F508
CFTR were pulse-labeled with [35S]methionine for 4 h in either the
absence or presence of 100 µM GSNO. The medium containing the
radiolabel was removed; fresh medium was added to the cells and further
incubated for 4 h (i.e. chase) either in the absence or in the
presence of GSNO prior to quantitative immunoprecipitation
(Fig. 5). Immediately following
the pulse labeling, significant amounts of the mature form (C band) of the
F508 CFTR protein were observed. The F508 CFTR protein corrected
by GSNO treatment proved to be unstable and little of the radiolabeled protein
was detected after the 4-h chase period, either in the absence or in the
presence of GSNO. Thus, while our Western blot analysis showed the corrected
F508 CFTR, presumably due to continuous maturation of nascent
F508 CFTR protein in the presence of GSNO, our pulse-chase studies
showed that once F508 CFTR protein had folded and trafficked to the
plasma membrane, the protein was unstable.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In our studies here, we adapted mIMCD3 kidney cells to hyperosmotic
conditions by the addition of 250 mM sodium chloride and 250
mM urea. By HPLC analysis, we found that the hyperosmotically
adapted mIMCD3 cells accumulated a number of intracellular osmolytes,
including GPC, myo-inositol, sorbitol, and taurine. The accumulation
of the osmolytes was accompanied by an ability of the cells to produce a
functional form of the F508 CFTR mutant. Furthermore, when these
different osmolytes were added individually to the medium of F508 CFTR
expressing cells, a portion of F508 CFTR was processed to its mature
form and resulted in the cells exhibiting cAMP-regulated CFTR chloride channel
function. Interesting, myo-inositol activated CFTR channels in the
absence of agonist. While not within the scope of this study, signaling roles
of inositol in both calcium release from the ER as well as activation of PKC
may be involved. In this regard, others have shown that PKC has a modulating
effect on CFTR channel activity
(35,
36).
The mechanisms by which cellular osmolytes impact protein folding and/or
stability have been an active area of research (for reviews, see Refs.
37 and
38). Early work by Timasheff
and colleagues (39) concluded
that osmolytes help stabilize proteins in their native conformation due to
their preferential exclusion from the immediate vicinity of the protein.
Preferential exclusion of the osmolytes leads to an increase in the local
concentration of water surrounding the protein. The resultant increase in
surface tension would then favor a decrease in surface area of the protein,
thereby promoting the folded state (i.e. one with the lowest energy
conformation). Subsequent studies by Bolen and others
(40–42)
concluded that in the presence of osmolytes, unfavorable interactions of the
peptide backbone (but not the R groups) with the osmolytes enhances proper
protein folding, thereby helping to minimize these unfavorable interactions.
In other words, the same general principles that govern protein folding events
under iso-osmotic conditions, in particular the hydrophobic effect, are likely
amplified in solutions containing high concentrations of the osmolytes. Thus,
in the case of F508 CFTR where a potentially critical hydrophobic amino
acid phenylalanine is absent, we suspect that an osmolyte enhancement of the
hydrophobic effect may help drive (and/or stabilize) an early folding
intermediate requiring hydrophobic interactions in and around position 508. As
a consequence, at least a portion of the folding intermediates continues to
fold to the native state and thereby escape the quality control pathway
operative in the endoplasmic reticulum. Subsequent maturation in the Golgi
followed by localization at the plasma membrane results in a F508 CFTR
protein that appears functional as assayed by forskolin-dependent chloride
transport.
Our results demonstrating that osmotic stress-induced osmolyte
accumulation, or the addition of individual osmolytes to the medium of cells
results in the rescue of F508 CFTR maturation, confirms and extends
earlier work. Specifically, previous studies have shown that high
concentrations of either glycerol or TMAO, when added to fibroblast cells in
culture, restored wild type-like F508 CFTR localization and chloride
channel function (7,
8). Moreover, both glycerol and
TMAO were found to be effective in promoting the proper folding of a variety
of other temperature-sensitive protein folding mutants, including the tumor
suppressor p53, the viral oncogene protein pp60src, the ubiquitin activating
enzyme E1 (43), the water
channel protein aquaporin-2
(44),
1-antitrypsin
(45), and -ketoacid
decarboxylase (46). In
addition, cellular osmolytes appeared effective in slowing and even preventing
the formation of the pathogenic form of the so-called scrapie prion protein
(47). Based on these
observations we have collectively referred to the cellular osmolytes as
"chemical chaperones" to reflect their wide range of action in
promoting the proper folding of proteins, which due to mutation, oftentimes
fail to reach the native state.
Within the kidney, the inner medullary collecting ducts are routinely
exposed to hyperosmotic stress and thus the accumulation of intracellular
osmolytes could help to explain the lack of adverse phenotype in individuals
homozygous for F508 CFTR. The renal cortex, another segment of the
kidney that expresses CFTR, is typically exposed to iso-osmotic conditions and
therefore osmolyte accumulation would not likely explain any lack of adverse
phenotype. During the course of our studies however, Zaman et al.
(14) reported that another
small molecule, GSNO, acted to promote the proper maturation of both the wild
type and F508 forms of CFTR. Consistent with their report we found that
the addition of GSNO to the medium of F508-expressing IMCD3 cells
resulted in the proper maturation of F508 CFTR. In addition, we showed
that the GSNO-treated cells expressing the F508 CFTR mutant now were
capable of carrying out forskolin-mediated chloride transport. Recently,
Andersson et al. (48)
reported similar effects of GSNO on F508 CFTR expressing cells using a
fluorescent-based assay. Our data showed that the maturation of F508
CFTR occurred using rather low concentrations (e.g. 10
µM) of GSNO and was very rapid (within 1 h the mature form of
the F508 CFTR protein was readily apparent by Western blot
analysis).
The mechanism by which F508 CFTR maturation and subsequent function
are rescued by GSNO treatment is currently unknown. GSNO (a thiol derivative
of glutathione) is a reservoir of both nitric oxide (NO) and glutathione. NO
at low concentrations can act as a signaling molecule, activating guanylate
cyclase and the subsequent formation of the second messenger cGMP (for review,
see Ref. 49). cGMP in turn is
known to activate various ion channels and kinases
(49). Higher concentrations of
NO can lead to the modification of cysteine thiols (nitrosylation) leading to
either activation or inactivation of the protein target and thus affect
various cellular functions, including transcription, ion channel function,
chaperone function, and proteolysis (reviewed in Ref.
50). Contradictory evidence of
the effect of NO on CFTR channel function and biogenesis has been reported.
For example, NO has been shown to activate CFTR in human T lymphocytes
(51), while Ruckes-Nilges
et al. (52) reported
that NO had no effect on CFTR or any other chloride channel activation in
primary nasal epithelial cells. GSNO, on the other hand, was found to activate
chloride channels in lung epithelial cells
(53). Finally, other studies
have shown that NO inhibits CFTR protein expression
(54,
55). In the case of
glutathione, this tripeptide is an important antioxidant necessary for the
maintenance of the redox state of the cell. While others have suggested that
the redox state of the cell is important for the activation of CFTR channels
(56), in our study, the low
concentration of GSNO required to promote F508 CFTR maturation likely
would not impact glutathione levels. Thus, we suspect that the operative
mechanism by which GSNO promotes F508 CFTR maturation is by
modification of either cysteine thiols of the nascent F508 CFTR protein
or cysteine thiols of a cellular protein(s) involved in the monitoring of
quality control mechanisms of protein folding.
While GSNO levels in the blood of healthy individuals has been questioned,
it may be possible that plasma GSNO is formed in vivo
(57–59)
due to inflammation as is common in CF patients. In addition, intracellularly
synthesized GSNO could be exported out of the cells by the MRP2 transporter,
which is expressed in the proximal tubule lumenal membrane of the kidney
(60). Whatever the case,
extracellular glutathione and S-substituted GSH derivatives are a
substrate for -glutamyltranspeptidase, an enzyme expressed at high
levels in the lumenal membrane of the kidney. Hydrolysis of GSNO would produce
glutamate and S-substituted cysteinylglycine, in the kidney filtrate
(15,
28). Subsequent uptake of the
S-substituted cysteinylglycine by amino acid transporters and further
protein modifications by S-nitrosylation could be one mechanism by
which F508 CFTR protein maturation is rescued. Studies are under way to
test this and other possibilities
Our pulse-chase studies demonstrated that once F508 CFTR was
corrected by GSNO, the mature form of the protein was unstable. Whether
F508 CFTR is corrected by temperature, glycerol
(26), or GSNO, the protein
appears to be unstable relative to its wild type counterpart. Thus, the
F508 CFTR protein is not a typical temperature-sensitive folding
mutant, since most temperature-sensitive protein folding mutants, once
properly folded, usually exhibit temperature-dependent stability profiles
similar to the wild type protein
(61). This then indicates that
F508, whether acquiring a functionally competent conformation via low
temperature, osmolyte, or GSNO treatments, presumably does not achieve a
conformation identical to that of the wild type protein. Our results together
with the results of Sharma and colleagues highlight the possibility that a
second drug may be required to stabilize the F508 CFTR protein once it
has exited the ER and Golgi and trafficked to the plasma membrane.
In summary, our studies point to two mechanisms that may help to explain
the absence of kidney dysfunction in CF patients; osmolyte accumulation and
GSNO catabolism. In addition, our work further demonstrates the feasibility of
a small molecule approach as a clinical therapy to correcting F508
misfolding, while at the same time revealing a second issue that needs to be
addressed in the search for therapeutic candidates, i.e. the
stability of the fully "mature" F508 CFTR protein. While
using high concentrations of osmolytes may not be clinically desirable, GSNO,
a substrate for GGT, an enzyme also expressed in the lung
(62), is one potential
therapeutic candidate. In this regard, a study of nine CF patients receiving
one dose of aerosolized GSNO showed that the compound was safely tolerated
(63). Moreover, there are
known beneficial effects of GSNO in the lung, including improved airway
ciliary motility, airway smooth muscle relaxation, inhibition of ENaC sodium
transport (64), and
antimicrobial activity (for review, see Ref.
65). Thus, investigating the
possible mechanisms by which the kidney appears to prevail over
disease-associated CFTR mutations has led to the identification of potential
therapeutic candidates for the treatment of CF.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Surgery, University of
California at San Francisco, 1001 Potrero Ave., Bldg. 1, Rm. 210, San
Francisco, CA 94110. Tel.: 415-206-6884; Fax: 415-206-6997; E-mail:
mbh1{at}itsa.ucsf.edu.
1 The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance
regulator; CF, cystic fibrosis; ER, endoplasmic reticulum; TMAO,
trimethylamine N-oxide; GPC, glycerophosphorylcholine; GSNO,
S-nitrosoglutathione; IMCD, inner medullary collecting duct; ANOVA,
analysis of variance; HPLC, high performance liquid chromatography; GGT,
-glutamyltranspeptidase.
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ACKNOWLEDGMENTS |
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REFERENCES |
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