J. Biol. Chem.
Zaarur, Kenneth L. Rock and Michael Y.
Colbert, Feng Liang, Hermann Bihler, Nava
Anatoli B. Meriin, Martin Mense, Jeff D.
of Mutant Proteins by Slowing Down
A Novel Approach to Recovery of Function
Protein Synthesis and Degradation:
doi: 10.1074/jbc.M112.397307 originally published online August 17, 2012
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A Novel Approach to Recovery of Function of Mutant
Proteins by Slowing Down Translation*
Anatoli B. Meriin‡, Martin Mense§, Jeff D. Colbert¶, Feng Liang?, Hermann Bihler?, Nava Zaarur‡, Kenneth L. Rock¶,
and Michael Y. Sherman‡1
01655,andthe?FlatleyDiscoveryLab,Charlestown, Massachusetts 02129
Background: Current strategies to alleviate protein misfolding include manipulation of chaperones, proteasomes, or
Results: Mild translation inhibition disproportionally blocked production of misfolded proteins and improved mutant CFTR
Significance: Attenuation of translation could be a novel approach to treatment of protein-misfolding disorders.
Protein homeostasis depends on a balance of translation,
folding, and degradation. Here, we demonstrate that mild inhi-
bition of translation results in a dramatic and disproportional
reduction in production of misfolded polypeptides in mamma-
lian cells, suggesting an improved folding of newly synthesized
proteins. Indeed, inhibition of translation elongation, which
lation initiation had minimal effects on copepod GFP folding.
On the other hand, mild suppression of either translation elon-
gation or initiation corrected folding defects of the disease-as-
sociated cystic fibrosis transmembrane conductance regulator
be used as a novel approach to improve overall proteostasis in
mammalian cells, as well as functions of disease-associated
mutant proteins with folding deficiencies.
Maintenance of the functional cellular proteome relies on
proteins pose the main challenge to cellular protein homeosta-
sis. Molecular chaperones, the ubiquitin-proteasome system,
Because abnormal proteins cause many life-threatening dis-
eases, there has been an ongoing interest in approaches that
of these diseases could be partially alleviated by induction of
autophagy (2, 3), and some inducers of autophagy have shown
efficacy in animal models (4, 5). Additionally, certain protein-
misfolding pathologies can be alleviated by overexpression of
heat shock proteins (6, 7), and numerous attempts have been
made to develop inducers of molecular chaperones (8, 9).
This approach can be used for another group of disorders
associated not with gain of toxicity of misfolded polypeptides
but with insufficient function of mutant proteins. For example,
the adverse effects of mutant lysosomal glucocerebrosidase in
Gaucher disease (10) can be alleviated by an increase in the
chaperone capacity of cells (10). Misfolded molecules of the
mutant glucocerebrosidase are rapidly degraded via the endo-
plasmic reticulum (ER)2-associated protein degradation path-
way. However, folded species of glucocerebrosidase escape ER-
associated protein degradation, proceed to lysosomes, and
function normally. Accordingly, induction of ER chaperones
improves folding of glucocerebrosidase and increases its levels
(10). Analogous effects were seen with mutant ?-hexosamini-
dase A, the causative agent of Tay-Sachs disease (10). This
approach may be useful in the rescue of other mutant proteins
transported via the ER, such as mutant cystic fibrosis trans-
membrane conductance regulator (CFTR), which causes cystic
is F508del, which results in poor folding and rapid degradation
of the polypeptide via the ER-associated protein degradation
pathway (11). However, a fraction of molecules that have
acquired the correct fold escape ER degradation and are func-
tional (12, 13). Correcting the folding defect of the F508del-
CFTR mutant by chemical chaperones seems to be a successful
strategy in therapeutics discovery.
Here, we propose that, in addition to induction of chaper-
ones, slowing down translation may have beneficial effects on
the pool of chaperone substrates and thereby increase the
Grants R01 GM086890 (to M. Y. S.), R01 AI20248 (to K. L. R.), and DK32520
(to the Diabetes Endocrinology Research Center) and Training Grant
AI007272-25 (to J. D. C.).
1To whom correspondence should be addressed: Dept. of Biochemistry,
5971; Fax: 617-638-5339; E-mail: email@example.com.
transmembrane conductance regulator; IBMX, 3-isobutyl-1-methylxan-
thine; copGFP, copepod GFP; FRT, Fischer rat thyroid.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 41, pp. 34264–34272, October 5, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
34264 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER41•OCTOBER5,2012
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capacity of the folding system. Moreover, it may improve co-
translational folding (14). This possibility is supported by the
following circumstantial lines of evidence: (a) slow-translating
stretches of mRNA provide for proper domain folding in vitro
tion rate of E. coli ribosomes improves folding of recombinant
eukaryotic proteins, which have evolved to be translated by
comparatively slower eukaryotic ribosomes (16); and (c) it is a
common practice to improve production of correctly folded
recombinant proteins in E. coli by reducing growth tempera-
ture. However, there is no direct evidence that slowing down
translation can improve folding in eukaryotic cells.
Here, we demonstrate that mild inhibition of translation sig-
nificantly improves overall protein folding in mammalian cells.
Furthermore, slowing down translation improves folding of
MATERIALS AND METHODS
Reagents and Antibodies—MG132 was purchased from
BIOMOL (Farmingdale, NY). Emetine, forskolin, cyclohexi-
from Sigma. Hippuristanol was a kind gift of Dr. J. Pelletier.
Shield-1 from was obtained from Cheminpharma LLC (Farm-
ington, CT). Anti-p21 and anti-p53 antibodies were purchased
from Pharmingen and Santa Cruz Biotechnology (Santa Cruz,
CA). Anti-multiubiquitin antibody (FK2) from MBL Interna-
tional (Woburn, MA). Anti-HA and anti-?-actin antibodies
were from Cell Signaling (Danvers, MA). Mouse anti-GAPDH
antibody was purchased from Millipore. Anti-CFTR antibody
(clone 59) was a kind gift from Dr. Jack Riordan (University of
North Carolina, Chapel Hill, North Carolina, via the Cystic
Constructs—The retroviral expression construct with C-ter-
minally tagged synphilin-1 (synphilin-1-GFP) subcloned into
the pCXbsr vector was described previously (17).
Copepod GFP (copGFP) was amplified from the vector
pMaxGFP (Lonza, Allendale, NJ) and fused with FBP12
(FK506-binding protein 12; from the vector ppTuner, Clon-
tech, Mountain View, CA) by overlapping PCR. The product
was then cloned into the lentiviral vector pTRIPz (Open Bio-
systems, Huntsville, AL). A C-terminal hemagglutinin tag was
also inserted downstream of copGFP by PCR. copGFP expres-
sion was controlled by the upstream tetracycline-responsive
Cell Cultures and Growth—HeLa (cervix carcinoma) cells
were grown in DMEM supplemented with 10% fetal bovine
serum, and MCF-10A (human breast epithelial) cells were
grown in 50:50 DMEM/F-12 medium supplemented with 5%
horse serum, 20 ng/ml epidermal growth factor, 0.5 ?g/ml
hydrocortisone, 10 ?g/ml human insulin, and 100 ng/ml chol-
era toxin. All cultures were supplemented with L-glutamine as
well as penicillin and streptomycin and grown at 37 °C in an
1 ?g/ml doxycycline and further stabilized by the addition of 5
?M Shield-1 (18).
The Fischer rat thyroid (FRT) cell line stably expressing
F508del-CFTR was a generous gift from Prof. Luis Galietta
(University of Genoa, Genoa, Italy). Cells were grown as
described previously (19).
For production of retroviruses, HEK293T cells were
co-transfected with a lentiviral plasmid and helper plasmids
KCl, 1% Triton X-100, 2 mM DTT, 1 mM Na3VO4, 50 mM
?-glycerophosphate, 50 mM NaF, 5 mM EDTA, 5 mM EGTA, 1
mM PMSF, 1 mM benzamidine, 5 ?g/ml leupeptin, 5 ?g/ml
pepstatin A, and 5 ?g/ml aprotinin). Samples were adjusted to
have an equal concentration of total protein and subjected to
SDS PAGE, followed by immunoblotting. The immunoblots
represent a typical experiment repeated three times.
Aggresome Counting Microscopy—For analysis with a fluo-
rescence microscope, cells were grown on Lab-Tek chambered
cover glasses (Nunc) pretreated with poly-L-lysine (Sigma).
Fluorescence microscopy was performed at room temperature
with a Zeiss Axiovert 200 microscope using a 40?/0.75 or
100?/1.30 oil Objectives and AxioVision 4 software. GFP-
tagged proteins were observed with an Axio FITC filter set.
Images were obtained using a high resolution AxioCam MRm
randomly chosen fields to obtain a total of ?200 cells. Each
ducibility of the results.
copGFP Fluorescence and Levels—HeLa cell clones were
plated at a density of 2 ? 104cells/well and allowed to adhere.
copGFP expression was induced by the addition of 1 ?g/ml
puristanol was titrated by 2-fold serial dilutions as indicated.
Cells were incubated for 6 h at 37 °C and then prepared for
immunoblotting, cells were plated on 12-well plates and lysed
in buffer containing 1% Nonidet P-40. Protein was quantified
by BCA (Pierce), and equivalent total protein was loaded per
well. copGFP expression was monitored using anti-HA tag
antibody (Clontech), whereas protein loading was monitored
by ?-actin levels.
FRT Cell Conductance Assay—FRT cells were seeded at a
density of ?125,000 cells/cm2on HTS Transwell 24-well filter
inserts (catalog number 3378, Corning) and grown into epithe-
lial cell monolayers as described previously (19). Prior to the
assay, monolayers were treated on both sides with compound
or vehicle (negative control) for 24 h in a cell/tissue incubator
(37 °C, 5% CO2).
After 24 h of treatment, the cell incubation medium was
replaced with HEPES-buffered physiological saline as the sero-
position of 137 mM NaCl, 4.0 mM KCl, 1.8 mM CaCl2, 1 mM
7.4 with NaOH). The Transwell 24-well plate was then
measured using a four-channel transepithelial current clamp
(EP Design, Bertem, Belgium). The assay was carried out at a
well temperature of ?35–36 °C. Resistance values were col-
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lected at ?10–15-min intervals. Four data points were mea-
sured to determine base-line resistance, and three data points
were measured to determine transepithelial resistance after
each addition of agonist (final concentrations of 10 ?M forsko-
lin, 100 ?M IBMX, and 20 ?M genistein) and antagonist (final
concentration of 20 ?M CFTR(inh)-172). IBMX and genistein
to both the serosal and mucosal sides and prediluted to 10-fold
concentrations in HEPES-buffered physiological saline.
Transepithelial conductance (Gt) was calculated from series
resistance-subtracted transepithelial current clamp measure-
epithelial monolayer, activation/inhibition of functional CFTR
transport proteins results in a change in transepithelial con-
functional CFTR surface expression.
To study the effect of test compounds on CFTR surface
expression, the dose-response characteristic of the compound
was determined and compared with negative controls. ?Gt
mean values were calculated for each treatment condition, and
itor response was plotted for each test concentration.
trically tight monolayers on Snapwell filter supports (catalog
sal and mucosal membranes were exposed to compound or
vehicle (negative control) in a cell/tissue incubator (37 °C and
5% CO2) for 24 h prior to the assay. The inserts were then
transferred to a Ussing chamber (catalog number P2302, Phys-
ml of HEPES-buffered physiological saline as the serosal solu-
tion with a composition of 137 mM NaCl, 4.0 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose (with
of low Cl?physiological saline with a composition of 137 mM
sodium gluconate, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10
mM HEPES, and 10 mM glucose (with pH adjusted to 7.4 with
N-methyl-D-glucamine to create a transepithelial chloride ion
the short circuit current, reflecting the net ion (Cl?) transport
MC8 epithelial voltage clamp (Physiologic Instruments Inc.).
The assay was carried out at 37 °C. Base-line activity was
onist (final concentration of 20 ?M CFTR(inh)-172) were
applied sequentially and cumulatively at ?10-min intervals to
both serosal and mucosal epithelial surfaces. The agonists and
antagonist were added as 200–1000-fold stock solutions to
both the serosal and mucosal sides.
Translation Rate Influences Generation of Abnormal
the aggresome, an organelle that recruits small protein aggre-
gates to the centrosome location (20, 21), using synphilin-1-
GFP as a model (17). We found that this process is triggered by
the buildup of newly synthesized aberrant proteins (22), which
allowed assessing the levels of these species by monitoring
aggresome formation. Moreover, with HeLa cells, we observed
translation. To account for this unexpected effect, we hypoth-
esize that partial inhibition of translation disproportionally
reduces the generation of aberrant polypeptides. We explored
this possibility with another cell line, MCF-10A. Cells express-
ing synphilin-1-GFP were incubated with the proteasome
inhibitor MG132, which led to a rapid formation of an
aggresome (Fig. 1B, upper right panel). The translation inhibi-
tor emetine suppressed the aggresome formation in a dose-de-
pendent manner (Fig. 1, A and B), indicating inhibition of
the production of misfolded polypeptides. To test whether
we quantitatively compared the effects of emetine on aggre-
proteasome inhibitor, accumulation of a de novo synthesized
short-lived protein, e.g. p53, directly reflects the rate of transla-
tion. Accordingly, to assess the effects of emetine on general
(note that accumulation of a distinct short-lived protein p21
(data not shown), as well as of inducible copGFP (see below),
inhibition of p53 synthesis was markedly less sensitive to eme-
tine than inhibition of aggresome formation. For example, 80
nM emetine inhibited accumulation of p53 by ?2-fold but
decreased the fraction of cells forming detectable aggresomes
by almost 10-fold. Moreover, at low concentrations, emetine
markedly reduced the sizes of the remaining aggresomes (Fig.
1B). For example, the diameters of aggresomes formed in the
than those seen without translation inhibition, demonstrating
that even very mild inhibition of translation suppresses growth
of an aggresome. Because aggresome formation depends on
accumulation of abnormal newly synthesized polypeptides
(22), these data suggest that mild inhibition of translation dis-
proportionally reduces generation of abnormal polypeptides.
To further evaluate the effects of translation inhibitors on
generation of abnormal polypeptides, we took advantage of the
observation that the majority of proteins that are ubiquitinated
and degraded by the ubiquitin-proteasome pathway are newly
synthesized. Indeed, the levels of ubiquitinated species notably
with emetine prevented their buildup by 90–95% (Fig. 1, C and
D). A large fraction of newly synthesized species that are
degraded by proteasome and therefore accumulate upon its
inhibition are polypeptides that cannot fold normally. Accord-
ence of low doses of emetine should disproportionally inhibit
generation of ubiquitinated species compared with inhibition
of translation. To test this possibility, MCF-10A cells were
centrations of emetine on the cellular levels of ubiquitin conju-
nificantly more sensitive to emetine. This result supports our
hypothesis that low concentrations of emetine specifically
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and on the levels of ubiquitinated polypeptides indicate that
ation of broad-spectrum misfolded polypeptides.
The described effect on cellular proteostasis was not specific
to emetine but rather reflected mild translation inhibition.
Indeed, another inhibitor (cycloheximide) similar to emetine
preferentially inhibited both aggresome formation and accu-
mulation of ubiquitinated species (Fig. 2A) (22). Like most of
the available ribosome inhibitors, these two reagents inhibited
elongation of translation and thus slow downed the growth of
each polypeptide chain in ribosomes.
We next tested whether comparable reduction in the output
of translation achieved without slowing down polypeptide
growth has a similar effect on generation of abnormal species.
Accordingly, we employed hippuristanol, which reduces pro-
tein synthesis by inhibiting translation initiation and thus
reducing the number of active ribosomes (24). Hippuristanol
species and for translation output (Fig. 2B). Unexpectedly, at
low concentrations, this inhibitor appeared to slightly increase
protein synthesis, resulting in the minor accumulation of both
p53 and ubiquitinated species (Fig. 2B). Nonetheless, low con-
centrations of hippuristanol did not cause an increase in
aggresome formation, which was already close to maximum.
Therefore, the dose-dependence curves for production of p53
and ubiquitinated species are seen shifted above the curve for
aggresome formation. However, starting from 150 nM hip-
are parallel (Fig. 2B). These data suggest that the rate of trans-
lation affects production of abnormal proteins, and in this
model, the rate of polypeptide growth is more important for
generation of defective ribosome products than the number of
Rate of Translation Affects Folding of a Model Protein—To
investigate the effects of the rate of translation on protein fold-
ing, we utilized a recombinant GFP from the copepod Pon-
tellina plumata (copGFP) as a reporter. copGFP was fused to
emetine on aggresome appearance. Scale bar ? 20 ?m. C and D, effect of emetine on accumulation of ubiquitinated species. MCF-10A cells were incubated
with 5 ?M MG132 and the indicated concentrations of emetine, and the amounts of ubiquitinated species and p53 were assessed by immunoblotting.
C, quantification of immunoblotting. D, immunoblots. All experiments were reproduced three times, and data are representative. Error bars represent S.E.
OCTOBER5,2012•VOLUME287•NUMBER41 JOURNALOFBIOLOGICALCHEMISTRY 34267
by guest on May 15, 2014
FBP12, which facilitates its rapid degradation; however, the
fusion protein can be stabilized by the addition to the cells of
the small molecule Shield-1. (This system, which controls pro-
tein stability, has been described previously (18).) Both the
fluorescence (which reflects folding) and protein levels of this
polypeptide could be easily quantified, making it a useful fold-
Mutant copGFP was expressed in HeLa cells under the con-
trol of a tetracycline-regulated promoter using the retroviral
expression system. Incubation with doxycycline for 6 h in the
presence of Shield-1 led to ?10-fold induction of copGFP (Fig.
3B, compare the first two lanes). Various concentrations of
later, copGFP levels and fluorescence were measured. As
manner (Fig. 3, A and B), similar to the inhibition of p53 accu-
mulation (see Fig. 1). However, paradoxically, the fluorescence
was affected in a very different manner, showing a steady
increase that peaked at ?40 nM emetine, reaching 150% of the
control. Importantly, at this concentration, emetine already
reduced the levels of copGFP by ?20% (Fig. 3, A and B). Even
stronger divergence between the copGFP protein levels and
copGFP fluorescence was seen with 80 nM emetine (Fig. 3A).
The effect of emetine on the fluorescence of copGFP normal-
ized to its levels demonstrates that slowing down translation
improved folding of this protein by ?4-fold (Fig. 3C).
We next investigated whether inhibition of translation initi-
shown in Fig. 3 (compare C and E for the dose dependence of
specific activity), the difference between the inhibitory effects
was insignificant. Overall, these experiments demonstrate that
partial inhibition of translation elongation, while reducing
expression, promotes folding of certain polypeptides, thus
resulting in an overall increase in their activity.
Partial Inhibition of Translation Can Improve the Activity of
Mutant CFTR—The finding that mild inhibition of translation
improves the function of certain proteins suggested that this
treatment may be employed to correct folding defects of dis-
ease-related mutant proteins. To test this possibility, we chose
the CFTR mutant F508del. Normally, CFTR functions as a
the F508del mutation and certain other mutations jeopardize
To study the effect of translation inhibition on the functional
surface expression of F508del-CFTR, we used FRT cells stably
expressing recombinant F508del-CFTR. CFTR activity was
measured by two independent methods, an Ussing chamber
assay (25) and a conductance assay in a 24-well format (26),
both of which measure CFTR-dependent transepithelial Cl?
transport. The Ussing chamber assay was designed to measure
this transport in the presence of a chloride ion gradient. Cells
were exposed to various concentrations of emetine or left
untreated, and CFTR activity was measured 24 h after the
beginning of the treatment. During the measurement, CFTR
was sequentially activated by compounds that increase cAMP
levels and stimulate CFTR transport activity: 10 ?M forskolin,
100 ?M IBMX, and then 20 ?M genistein (Fig. 4A). To confirm
that the activated Cl?transport was CFTR-dependent, at the
end of the measurement, we added CFTR(inh)-172, which
blocked the CFTR-dependent component of the conductance.
In line with our conjecture, we observed a dose-dependent
increase in the activity of mutant CFTR in cells exposed to low
concentrations of emetine (Fig. 4A). An almost 2-fold increase
over vehicle in the CFTR-specific inhibitor response was
reached at 0.5 ?M emetine (Fig. 4A, right panel). The higher
effective emetine concentrations used in this experiment com-
pared with those used with HeLa or MCF-10A cells indicates a
lower susceptibility of FRT cells to emetine (data not shown).
The effect of 24 h of emetine incubation on CFTR activity at
the cell surface was independently confirmed in the FRT cell
conductance assay, in which CFTR-dependent transepithelial
(Cl?) conductance was measured in the absence of a chloride
with various doses of emetine for 24 h. During the measure-
ments, CFTR was first activated with 10 ?M forskolin and then
further stimulated with a combination of 100 ?M IBMX and 20
?M genistein. CFTR(inh)-172 was added at the end of the
FIGURE 2. Different effects of cycloheximide and hippuristanol on trans-
lation, accumulation of ubiquitinated species and aggresome forma-
tion. A, effects of cycloheximide on translation, accumulation of ubiquiti-
nated species, and aggresome formation. Experiments were performed as
described in the legend to Fig. 1. B, effects of hippuristanol on translation,
accumulation of ubiquitinated species and aggresome formation. Experi-
ments were performed as described in the legend to Fig. 1. All data were
reproduced several times and are representative. Error bars represent S.E.
34268 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER41•OCTOBER5,2012
by guest on May 15, 2014
experiment. Again, the dose dependence of the CFTR-specific
inhibitor response peaked at 0.5 ?M emetine as illustrated in
Fig. 4B (right panel). At the peak concentration, the CFTR-
with the negative control.
We further investigated the effects of low doses of emetine
on CFTR levels. Misfolded F508del-CFTR molecules are rap-
idly degraded by the ubiquitin-proteasome machinery. There-
fore, we predicted that a mild inhibition of translation could
paradoxically lead to elevated cellular levels of F508del-CFTR
shown in Fig. 4C, at low doses of emetine, we observed
increased expression levels of CFTR represented by both the
mature form (Band C) and the core-glycosylated form (Band
indicates that emetine enhances folding of this mutant protein
degradation (misfolded form) or for the membrane (folded
synthesized proteins not only in cytoplasm, as seen with
copGFP, but in the ER as well.
To assess the effects of the translation initiation inhibitor
hippuristanol on CFTR folding, dose-response experiments
were performed in FRT cell Ussing chambers. CFTR activity
peaked at 0.6 ?M inhibitor, culminating in an almost 3-fold
(Fig. 5A). As with emetine, the increase in activity was parallel
0.6 ?M (Fig. 5B). Therefore, with F508del-CFTR, inhibition of
both translation elongation and initiation can improve folding.
associated with protein misfolding.
A critical finding here is that mild inhibition of translation
can correct folding defects of mutant disease-associated pro-
may be beneficially affected by partial inhibition of translation,
e.g. Gaucher, Tay-Sachs, various conditions associated with
collagen mutations, and others. These diseases result from
insufficient folding and function of important enzymes and
structural proteins. Another class of conditions that might be
improved by this approach includes disorders associated with
sclerosis, inclusion body myositis, and others. Because all of
these conditions are chronic, it will be necessary to develop
nontoxic compounds that inhibit translation only mildly.
a significant impact on the quality of newly synthesized pro-
teins and the overall proteostasis. Recently, it was demon-
strated that newly synthesized polypeptides are most vulnera-
FIGURE 3. Inhibition of translation elongation enhances folding of mutant copGFP in HeLa cells. A, effects of emetine on the fluorescence and levels of
mutant copGFP. copGFP was induced for 6 h in the presence of the indicated concentrations of emetine, and its fluorescence and levels normalized to total
protein were measured as described under “Materials and Methods.” B, levels of copGFP in the experiment presented in A assessed by immunoblotting. The
levels of this inducible protein also reflect the degree of translation inhibition. C, effects of emetine on the ratio of copGFP fluorescence to copGFP level.
Calculation was based on data in A. D, effects of hippuristanol on the fluorescence and levels of mutant copGFP. copGFP was induced for 6 h in the presence
of the indicated concentrations of hippuristanol, and its fluorescence and levels were measured as described under “Materials and Methods.” E, effects of
are representative. Error bars represent S.E.
OCTOBER5,2012•VOLUME287•NUMBER41 JOURNALOFBIOLOGICALCHEMISTRY 34269
by guest on May 15, 2014
FIGURE 4. Inhibition of translation elongation corrects folding defects of mutant CFTR. A, left panel, effects of emetine on CFTR-mediated short-circuit
currents in the Ussing chamber assay. Cells were plated on filter supports and incubated with the indicated concentrations of emetine for 24 h. Inserts were
transferred to Ussing chambers. After acquisition of the base-line current, the agonists (10 ?M forskolin (Fsk), 100 ?M IBMX, and 20 ?M genistein (Gen)) and
antagonist (20 ?M CFTR(inh)-172) were added sequentially to both epithelial surfaces, and short-circuit currents (Isc) were recorded. To better illustrate the
emetine concentration effect on the CFTR inhibitor response, the post-inhibitor base-line current was subtracted from the raw current trace. Right panel,
emetine dose response. Shown are the means (n ? 2) ? S.D. of the CFTR-specific Cl?current normalized to CFTR(inh)-172. B, left panel, conductance assay of
additions. Traces represent averaged records (n ? 3) from FRT cell monolayers treated for 24 h with the indicated concentrations of emetine. Right panel,
described in the legend to Fig. 3C were measured by immunoblotting. Band C, mature form; Band B, core-glycosylated form. Error bars represent S.E.
by guest on May 15, 2014
ble to various stresses, e.g. heat shock or oxidative stress (27). It
proteins of high molecular weight, that fold slowly and often
with low efficiency, and these partially unfolded species are
inhibitors reduce the overall production of abnormal polypep-
tides probably via enhancing folding, we hypothesize that par-
tial inhibition of translation may significantly protect against
Several mutations that reduce translation initiation and
shown to have strong anti-aging effects (28, 29). Our data sug-
gest that these anti-aging effects may be relevant to improved
mutations have been shown to improve overall proteostasis
(30). Therefore, there is a possibility that minor suppression of
translation may provide an anti-aging effect and be beneficial
for a variety of age-related disorders.
In this work, we assessed translation rates by measuring de
novo synthesis of short-lived proteins upon inhibition of their
degradation. Independently, we measured accumulation of a
stable reporter protein upon induction of its synthesis. The
results obtained with these two distinct methods were similar.
We found that slowing down elongation of translation
improves general protein folding. These effects suggest either
that the rate of growth of the polypeptide chain influences the
kinetics of co-translational folding (e.g. allows association with
ribosome-bound chaperones prior to co-translational misfold-
output, thus increasing the number of available cytoplasmic
chaperones (for both co-translational and post-translational
folding). On the other hand, inhibitors of translation initiation
do not affect growth of the polypeptides chain, but only the
number of translating ribosomes. Therefore, these inhibitors
cannot influence the kinetics of co-translational folding, but
only the translation output.
The fact that only inhibitors of translation elongation
reduced the overall production of misfolded species, whereas
hippuristanol was not effective, indicates that, for the bulk of
polypeptides, translation influences the kinetics of co-transla-
tional folding. Indeed, in vitro studies suggested that the slow-
ing down of translation can improve co-translational folding
improvement with both emetine and hippuristanol, indicating
that the overall translation output is critical for CFTR folding
and suggesting that the availability of chaperones is limiting.
These findings may reflect the compartmental difference,
where chaperones are not limiting for cytoplasmic proteins
(which represent the bulk of the translation products in HeLa
cells), and translation affects mainly the kinetics of co-transla-
ER, ER chaperones may be limiting. Overall, the findings pre-
sented here can be considered a proof of principle that partial
inhibition of translation could be developed as a novel thera-
peutic modality for treatment of diseases associated with inef-
ficient folding of mutant proteins.
Acknowledgment—We thank Dr. J. Pelletier for reagents.
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