© 1997 American Heart Association, Inc.
Articles |
Conformational Changes in Apolipoprotein B Modulate Intracellular Assembly and Degradation of ApoB-Containing Lipoprotein Particles in HepG2 Cells
From the Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.
Correspondence to Khosrow Adeli, Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset St, Windsor, Ontario, Canada N9B 3P4. E-mail adeli{at}uwindsor.ca
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Abstract The linkage between the conformation of apolipoprotein B100 (apoB) and the intracellular assembly and degradation of apoB-containing lipoproteins was investigated in the present study. Disruption of disulfide bond formation in newly synthesized apoB molecules through the use of the reducing agent DTT resulted in a decrease in the secretion of apoB-containing lipoproteins from HepG2 cells compared with control cells. The synthesis of total apoB (apoB100 plus nascent chains), as well as a number of control proteins, such as albumin and
1-antitrypsin, was
decreased significantly in DTT-treated cells. However, the
intracellular accumulation of full-length apoB100 molecules
was not inhibited in the presence of DTT. Subcellular fractionation
indicated that apoB molecules isolated from the microsomes of
DTT-treated cells had an increased association with the microsomal
membrane compared with apoB isolated from untreated cells.
Analysis of the distribution of apoB-containing lipoproteins
from the lumen of isolated microsomes demonstrated that in the presence
of DTT, there was a shift in the distribution, such that there was a
decrease in the formation of HDL-sized (lipid-poor) apoB-containing
lipoproteins and a decrease in the formation of LDL/VLDL apoB
particles. Alterations in apoB conformation and their impact on
degradation were also investigated by using DTT and by inhibiting
N-linked glycosylation with tunicamycin. DTT appeared to
change the rate and pattern of apoB degradation. Degradation was
accelerated in both intact and permeabilized HepG2
cells. ApoB degradation occurred in DTT-treated
permeabilized cells without the usual generation of the
70-kD and 335-kD fragments and was largely
N-acetyl-leucyl-leucyl-norleucinal (ALLN) insensitive. In
tunicamycin-treated cells, DTT further accelerated the degradation of
unglycosylated apoB. Overall, the data suggest that the misfolding of
apoB may prevent the proper association of apoB with lipids, resulting
in impairment of the assembly of mature apoB-containing lipoproteins.
Alteration in the conformation of apoB also appears to alter the
degradation pathway of apoB, such that the protein is degraded through
a pathway that is at least in part ALLN insensitive.
Key Words: apolipoprotein B • HepG2 cells • conformation • dithiothreitol • degradation
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The assembly and subsequent secretion of VLDL from liver cells is a complex process requiring the coordinate production of a number of components, including cholesterol, neutral lipids, phospholipids, and apoB. ApoB is a 512-kD protein, the production of which is essential to the secretion of VLDL. Evidence to date suggests that the hepatic production rate of apoB is posttranscriptionally modulated.1 2 3 4 5 A significant proportion of newly synthesized apoB is rapidly degraded in rat hepatocytes6 7 8 and HepG2 cells.9 ApoB that is not assembled into a secretion-competent lipoprotein particle may be sorted to a degradation pathway.10 11 12 This degradation has been demonstrated to be catalyzed by an ALLN-sensitive protease. Several studies have demonstrated that intracellular apoB degradation is a function of lipid availability. Increasing the intracellular levels of triglyceride by treatment of HepG2 cells with oleate has been shown to protect apoB from degradation,13 14 while inhibition of triglyceride synthesis resulted in increased degradation of apoB. Boren and colleagues15 16 have suggested that the lack of availability of triglycerides results in assembly of apoB into small, dense "HDL"-like particles, which are more susceptible to degradation. Recent studies in digitonin-permeabilized HepG2 cells also directly showed the intracellular degradation of nascent apoB-containing lipoprotein particles in the lumen of the ER by an ALLN-sensitive process.17
The conformational status of apoB has also been proposed to be important in the regulation of intracellular apoB degradation.18 19 As secretory proteins are transported through the secretory pathway, a number of cotranslational and posttranslational modifications occur, such as disulfide bond formation and N-linked glycosylation, which have a direct impact on protein conformation. The ER functions as a quality control mechanism for removal of abnormally synthesized and assembled proteins.20 It has been shown that misfolded proteins are retained in the ER and their secretion is prevented.21 22 Misfolding could be induced by the inhibition of glycosylation and/or disulfide bond formation. Recent experiments in HepG2 cells have shown that the addition of the reducing agent DTT causes the misfolding of nascent albumin. DTT-induced misfolding of albumin resulted in the inhibition of its secretion and caused the protein to accumulate in the ER in a reduced state.23 In addition, DTT was shown recently to induce rapid and unregulated degradation of proteins that are normally stable in the ER.24 More specifically, DTT was shown to affect the production and secretion of apoB in HepG2 cells.19 These studies indicate that DTT may be a useful tool to investigate the relationship between protein conformation and the intracellular mechanisms that control its production and secretion.
In the present study, we have investigated the linkage between the conformation of apoB and its assembly and degradation in HepG2 cells. Based on data obtained from intact and permeabilized HepG2 cells, we suggest that misfolding of nascent apoB induced by the lack of glycosylation and/or the reduction of disulfide linkages, results in unregulated degradation of the protein in the cell. Disruption of disulfide bond formation appeared to reduce the efficiency of apoB translocation from the membrane into the lumen of the ER. Treatment with DTT also altered the assembly of apoB-containing lipoproteins such that there was a decrease in the formation of apoB-containing lipoprotein particles, including both dense HDL-sized (lipid-poor) apoB particles and LDL/VLDL particles. Overall, the data suggest that misfolding of apoB may prevent the proper association of apoB with lipids, resulting in impairment of the assembly of mature apoB-containing lipoproteins.
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Materials
HepG2 cells (ATCC HB 8065) were obtained from American Type Culture Collection. Cell culture media and reagents were from Life Technologies Inc. Fetal bovine serum (certified grade) was also from Life Technologies. Culture dishes and flasks were obtained from Corning or Falcon. 35S protein labeling mixture (specific activity of >1100 Ci/mmol), Enlightening and Enhance, were purchased from DuPont. ALLN, DTT, PMSF, bovine serum albumin, brefeldin A, digitonin (50% purity), trypsin (tissue-culture grade), soybean trypsin inhibitor, leupeptin, rabbit anti-goat IgG, albumin, and
1-antitrypsin antiserum, as well as other
common laboratory reagents were from Sigma Chemical Co. Trasylol
(aprotinin) was from Bayer. Tunicamycin was obtained from
Boehringer Mannheim Biochemicals. Ultra-pure electrophoresis
reagents were from Bio-Rad. Nonradioactive molecular mass markers were
from Sigma and prestained protein standards (rainbow markers) were
purchased from Amersham International. Monospecific apoB antibodies
were obtained from Medix-Biotech and purified in the laboratory.
Monoclonal antibodies against apoB were a gift from Drs Ross Milne and
Yves Marcel and were bound to Affi-gel beads, using procedures
recommended by Bio-Rad. Immunoprecipitin was obtained from Life
Technologies. Affi-Gel 10 protein A was from Bio-Rad.
Cell Culture
Monolayer cell cultures were maintained in an alpha modification
of Eagle's MEM (-MEM) containing 10% fetal calf
serum25 Cells were grown in 35- 100-mm dishes at 37°C,
5% CO2 in complete medium (-MEM, 10% fetal bovine
serum) until about 75% to 85% confluence, at which time they were
used for the experiments.
Pulse-Chase Labeling of Intact Cells
Intact cell labeling studies were conducted at 37°C.
Near-confluent HepG2 cells were preincubated in methionine-free -MEM
for 1 hour and pulsed with 50 to 100 µCi/mL of
35S-protein–labeling mix for 10 to 20 minutes. In some
experiments, the cells were pretreated with tunicamycin (3 hours, 5
µg/mL) and/or brefeldin A (1 hour, 1 µg/mL). In such
cases, the drug was present during both the pulse and the chase. If
the cells were to be treated with DTT (2 mmol/L), it was
added 1 to 12 minutes before the pulse. Cells were collected after
either the pulse (time 0) or the chase. The cells were chased in
-MEM supplemented with 10 mmol/L methionine and 5
mmol/L cysteine for various periods of time. To prevent any
subsequent disulfide formation or rearrangement between free sulfhydryl
groups,23 1/10 volume of a 1 mol/L iodoacetamide
solution in 0.5 mol/L Tris, pH 8.7, was added 5 minutes before
solubilization. Duplicate dishes were harvested, and cells were lysed
in solubilization buffer. The lysates were centrifuged for 10
minutes in a microcentrifuge tube, and the supernatants were
collected for immunoprecipitation. Media collected at each time point
were centrifuged briefly to remove any cell debris, and the
supernatants were diluted with solubilization buffer for
immunoprecipitation.
Preparation of Permeabilized HepG2 Cells
Near-confluent HepG2 cultures grown in 35-mm dishes were
depleted of methionine by incubation in methionine-free -MEM for 60
minutes at 37°C under 5% CO2. HepG2 cells were
pulse-chased and permeabilized as described
previously18 with minor modifications. Briefly, cells were
incubated with 50 to 100 µCi/mL 35S-protein–labeling
mixture for 10 to 20 minutes at 37°C, washed in -MEM three times,
and chased in -MEM containing 10 mmol/L methionine and
5 mmol/L cysteine for 10 minutes at 37°C. After extensive
washing, the cells were incubated in CSK buffer18
containing 50 µg/mL digitonin for 10 minutes. Digitoninized
cells were washed three times in CSK buffer and were immediately used
for the degradation studies. After incubation for different periods of
time, permeabilized cells were solubilized in
solubilization buffer (PBS containing 1% NP40, 1% deoxycholate,
5 mmol/L EDTA, 1 mmol/L EGTA, 2
mmol/L PMSF, 0.1 mmol/L leupeptin, 2 µg/mL
ALLN). Cell extracts were centrifuged in a
microcentrifuge at 14 000 rpm for 10 minutes, and the
supernatants were subjected to immunoprecipitation.
Trypsin Digestion of Permeabilized HepG2
Cells
Cells were treated essentially as described
previously.26 Briefly, near-confluent cells were pulsed,
chased, then incubated in CSK containing 75 µg/mL digitonin,
10 mmol/L methionine and 5 mmol/L cysteine,
150 µmol/L puromycin, 50 µg cycloheximide, 5
µg/mL ALLN for 5 minutes at room temperature. Digitoninized
cells were washed once in CSK buffer and were then incubated in the
presence and absence of trypsin (200 µg/mL) for 10 minutes at
room temperature. An equal volume of CSK containing 2 mg/mL
soybean trypsin inhibitor, 1 mmol/L PMSF, 5
µg/mL ALLN, and 100 KIU/mL Trasylol was added to all dishes
for 10 minutes at room temperature. The cells were then incubated for
an additional 10 minutes on ice and collected. The collected cells were
then centrifuged for 2 minutes at 10 000 rpm in a
microcentrifuge. The supernatant was removed and the cells were
solubilized in a solubilization buffer containing 1 mmol/L
PMSF, 100 KIU/mL Trasylol, 0.1 mmol/L leupeptin, 2
µg/mL ALLN, and 1 mg/mL soybean trypsin
inhibitor. Cell extracts were centrifuged in a
microcentrifuge at 14 000 rpm for 10 minutes, and the
supernatant was subjected to immunoprecipitation.
Subcellular Fractionations
Isolation of the microsomal fraction and the separation of the
luminal and membrane components was performed as
described.10 11 Intact cells that had been incubated for
up to 30 minutes were permeabilized, washed once with
250 mmol/L sucrose, 3 mmol/L imidazole, pH 7.4,
and once with 50 mmol/L sucrose, 3 mmol/L
imidazole, pH 7.4. The cells were collected in 0.5 mL of 50
mmol/L sucrose solution containing a cocktail of protease
inhibitors (0.1 mmol/L leupeptin, 1
mmol/L PMSF, 100 KIU/mL Trasylol, 1 µmol/L
pepstatin A, and 5 µmol/L ALLN) and
homogenized with a glass dounce homogenizer
as described.27 28 The homogenate was
centrifuged for 10 minutes at 2200g. The supernatant
containing the crude microsomes was isolated and then treated with
sodium carbonate, pH 11, to release the luminal component. Separation
of the membrane and luminal components was achieved by
ultracentrifugation at 37 000 rpm for 60 minutes at
12°C in an SW41 rotor. The luminal component was then either
immunoprecipitated or subjected to further fractionation (described
below). The isolated microsomal membrane was resuspended in 1 mL of PBS
and either immediately immunoprecipitated or stored at -20°C.
Sucrose-Gradient Ultracentrifugation of
Luminal Lipoproteins
Fractionation of the luminal content of the isolated microsomes
was performed as described.27 28 In some experiments
luminal contents isolated from microsomes were supplemented with
protease inhibitors (0.1 mmol/L leupeptin,
1 mmol/L PMSF, 100 KIU/mL Trasylol, 1 µmol/L
pepstatin A, and 5 µmol/L ALLN) and then subjected to
ultracentrifugation on a step sucrose gradient (1.5 mL
49%/3.0 mL 25%/2.0 mL 20%/3.5 mL sample/1.9 mL 5%/0.9 mL 0%
sucrose) at 35 000 rpm in a SW41 rotor for 65 hours, at 12°C. All
solutions contained the protease inhibitor cocktail as
above. Gradients were fractionated into 1-mL fractions, and the density
of the fractions was determined to assure the linearity of the sucrose
gradient. Both the membrane and luminal fractions were then diluted
with 800 µL of a solubilization buffer containing 360 µL buffer A
(250 mmol/L Tris-HCl, pH 7.4, 750 mmol/L NaCl,
25 mmol/L EDTA, 5 mmol/L PMSF, 5%
Triton-X100), 410 µL PBS, 20 µL Trasylol (10 000 KIU/mL), and 10
µL PMSF (200 mmol/L; final concentrations of Trasylol and
PMSF were 250 KIU/mL and 4.75 mmol/L, respectively), and
subjected to immunoprecipitation.
Immunoprecipitation
Samples obtained from the experimental procedures were initially
preimmunoprecipitated by the addition of 2 µL of nonimmune serum.
After a 1-hour incubation at room temperature, 30 µL of
Immunoprecipitin was added and further incubated for 1 hour. Samples
were cleared by centrifuging in a microcentrifuge for 2
minutes, and the supernatant was subjected to immunoprecipitation with
monospecific antibodies to either apoB, albumin,
1-antitrypsin, or HMG-CoA reductase. Immunoprecipitation was
performed by first adding 10 µL of antibody or antiserum to each
sample and incubating overnight at 4°C. Immunoprecipitin (60 µL)
was then added to each sample and further incubated with constant
mixing at room temperature for 1 hour. Samples were centrifuged
for 2 minutes at 14 000 rpm to pellet the immunoprecipitates. In
procedures in which monoclonal antibodies to apoB were employed, they
were first bound to Affi-gel beads, and then incubated with the samples
overnight at 4°C and centrifuged to pellet the beads.
Immunoprecipitates were washed three times with the wash buffer
(10 mmol/L Tris-HCl, pH 7.4, 2 mmol/L EDTA,
0.1% SDS, 1% Triton X-100) and analyzed by SDS-PAGE (see
below).
SDS-PAGE and Fluorography
SDS-PAGE was performed essentially as described.29
Gels were composed of 5% (wt/vol) acrylamide stacking and
6% (wt/vol) acrylamide resolving gels, or were 4% to 12%
gradient gels with a 5% acrylamide stacking layer.
Electrophoresis was at 66 V for 16 hours. The gels were fixed, stained,
and fluorographed by incubating in Enhance or Enlightening (DuPont).
The gels were dried and exposed to Kodak X-Omat AR5 or DuPont
autoradiographic film at -80°C for 1 to 4 days.
Radiolabeled proteins visualized on the fluorographs were quantitated
by a Bio-Rad imaging densitometer by the use of the volume
analysis program. Quantitation of the radioactivity in the apoB
bands was achieved by cutting the bands from the gel, digestion of
excised bands, and scintillation counting. The method for quantitation
of radiolabeled proteins is indicated in each figure legend.
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Effect of DTT on Intracellular Accumulation and Secretion of ApoB
The synthesis and secretion of apoB in HepG2 cells pretreated for 3 or 12 minutes with DTT were investigated and compared with the pattern in control cells. Examination of the immunoprecipitable apoB in the cells demonstrated that treatment with DTT did not inhibit the production of mature apoB molecules (550 kD) (Fig 1
). However, preincubation for 12 minutes
with DTT did result in a decrease in apoB-related polypeptides ranging
in size from 200 to 550 kD. These polypeptides, which were
consistently observed after a short pulse period, appeared to
represent nascent chains of apoB and have been observed in
previous studies.18 26 The DTT-induced decrease in the
accumulation of nascent apoB chains may be the result of either a
diminished apoB synthesis rate and/or an increase in their degradation.
Interestingly, preincubation with DTT for 3 minutes resulted in the
coimmunoprecipitation of a protein species of approximately 60 kD in
size that was not recovered from either control cells or cells that had
been preincubated with DTT for 12 minutes. Numerous experiments in our
laboratory have consistently demonstrated the
immunoprecipitation of a 60-kD protein species from cells that have
been pretreated with 2 mmol/L DTT for 1 to 3 minutes. The
identity of this band is unknown and is currently under
investigation.
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The secretion rate of apoB was also investigated in intact HepG2 cells
treated with DTT. Fig 1B
shows the labeled apoB detected in media (B)
of control cells and cells pretreated with DTT for periods of 3 minutes
and 12 minutes. Preincubation with 2 mmol/L DTT for 3
minutes resulted in a substantial decrease in the amount of apoB
detected in the media compared with control cells. Increasing the
preincubation period to 12 minutes further increased the
inhibitory effect of DTT on apoB secretion. The effect of
DTT on apoB secretion was further quantitated by pulse-chase labeling
experiments and compared with the secretion efficiency of a control
protein, 1-antitrypsin. The secretion of 1-antitrypsin (a protein
devoid of disulfide bonds) has been shown to be unaffected by treatment
with DTT under similar experimental conditions.23 This
control protein was used here to confirm the integrity of the secretory
pathway of DTT-treated HepG2 cells. Fig 2
shows the percentage of apoB and 1-antitrypsin secreted from HepG2
cells in the presence and absence of DTT. ApoB secretion decreased from
30.3±2.5% in control cells to 9.9±2.6% (±SE, P<.05)
and 4.4±0.1% (±SE, P<.01) in cells preincubated with DTT
for 3 minutes and 12 minutes, respectively. In contrast, the secretion
of 1-antitrypsin was not significantly altered in DTT-treated HepG2
cells compared with control cells. After a 2-hour chase period, the
percentage of 1-antitrypsin detected in the media was 64.4±1.8% in
control cells compared with 69.7±0.9% (±SE, P>.05) and
66.2±11.1% (±SE, P>.05) in cells that had been
preincubated with DTT for 3 minutes and 12 minutes, respectively. The
uninhibited secretion of 1-antitrypsin in the presence of DTT
suggests that the secretory pathway of HepG2 cells is not significantly
affected by treatment with DTT. The apparent intactness of the
secretory system suggests that the decrease in secretion efficiency of
apoB in the presence of DTT is rather specific and may be the result of
the disruption in the formation of disulfide bonds within the apoB
molecule itself.
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Effect of DTT on ApoB Synthesis
Disruption of disulfide bond formation and its effect on the
synthesis of apoB was investigated by preincubating HepG2 cells with
2 mmol/L DTT for 1 minute followed by a brief 10-minute
pulse. To ascertain the general effect of DTT on protein synthesis,
total protein synthesis and the synthesis of 1-antitrypsin and
albumin in the presence of DTT were followed. Fig 3A demonstrates that the presence of DTT
results in a significant reduction in overall protein synthesis
compared with control cells (control, 2 798 110±22 000 versus +DTT,
1 589 471±51 000 cpm per dish, ±SE, P<.001).
Similarly, Fig 3B and 3C demonstrate that the presence of DTT also
caused a significant reduction in the synthesis of 1-antitrypsin
(control, 21 041±1800 versus +DTT, 10 070±600 cpm per dish, ±SE,
P<.001) and albumin (control, 17 221±1100 versus
+DTT, 7835±300 cpm per dish, ±SE, P<.001). The
accumulation of intact and nascent apoB chains (total
immunoprecipitable apoB radioactivity in apoB100 band plus nascent apoB
chains) was also reduced in the presence of DTT (control, 83 925±6200
versus +DTT, 69 203±2400 cpm per dish, ±SE, P<.05) (Fig 3D). In contrast, treatment of HepG2 cell with DTT did not
significantly affect the accumulation of intact 550-kD apoB100
molecules (control, 5510±930 versus +DTT 5841±970 cpm per dish, ±SE,
P=.39) (Fig 3E.). The level of synthesis of apoB100 was not
significantly affected by preincubation of HepG2 cells with DTT for
periods of up to 12 minutes (data not shown). Overall, the data suggest
that treatment of HepG2 cells with DTT results in a decrease in the
synthesis of several secretory proteins, as well as a decrease in
overall protein synthesis. However, although DTT reduced the total pool
of full-length and nascent apoB chains, the amount of full-length
apoB100 accumulated was relatively unaltered by the presence of
DTT.
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DTT Alters the Distribution of Newly Assembled ApoB-Containing
Lipoproteins
The effect of DTT on the ratio of membrane-associated apoB and
luminal apoB was determined. Microsomes were isolated from HepG2 cells
that had been briefly pulsed and chased in the presence and absence of
DTT. Fig 4A shows the percentage of
membrane associated and luminal apoB in control and DTT-treated cells
at time zero and after 30 minutes of chase. The percentage of luminal
apoB in control cells increased from 35.8±3.5% to 49.6±2.9% (±SE,
P<.05) after 30 minutes of chase. In contrast, the
percentage of luminal apoB isolated from DTT-treated cells remained
relatively unchanged (increased from 33.9±4.4% to 34.7±2.9%, ±SE,
P=.87) after the 30-minute incubation period. The luminal
lipoproteins from microsomes isolated from cells incubated in the
presence and absence of DTT were subsequently subjected to sucrose
gradient fractionation to determine the effect of DTT on the assembly
of apoB-containing lipoproteins. Fig 4B shows the density distribution
of luminal apoB-containing lipoproteins isolated from the microsomes of
cells treated in the presence and absence of DTT. Fractions 2 through 5
represent high-density apoB-containing lipoprotein particles
(apoB lipoproteins with density similar to that of HDL, peak density
1.065 to 1.170 g/mL), and fractions 6 through 12
represent the lower-density apoB lipoprotein particles
(LDL/VLDL-apoB, peak density 1.011 to 1.045
g/mL).10 11 Comparing the peak fractions from the
sucrose gradients indicates that there was a significant decrease in
the amount of HDL-sized (lipid-deficient) apoB particles isolated from
DTT-treated cells compared with control cells (fraction 2 of the
control, 2925±222 versus fraction 2 of DTT-treated, 1096±197 cpm per
dish, ±SE, P<.05). In addition, there was a corresponding
decrease in the amount of LDL/VLDL apoB particles isolated from the
microsomal lumen of DTT-treated cells compared with control cells
(fraction 7 of the control, 862±111 versus fraction 7 of the
DTT-treated, 362±76 cpm per dish, ±SE, P<.05). The data
suggest that DTT may prevent the transfer of membrane-associated apoB
into the lumen of the ER and may hinder the proper association of apoB
with core lipids in the lumen of the ER.
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Effect of DTT on ApoB Conformation
The effect of DTT on the folding of apoB was assessed by
nonreducing SDS-PAGE analysis of immunoprecipitated apoB
fragments generated by trypsin digestion of
permeabilized HepG2 cells treated in the presence and
absence of DTT. Fig 5 demonstrates the
apoB fragmentation pattern isolated from cells treated in the presence
of DTT compared with the pattern detected in control cells. The pattern
of apoB fragments observed under control conditions differed from the
fragmentation pattern isolated from DTT-treated cells. These
differences were most apparent in the regions of the
autoradiogram corresponding to apoB fragments with
molecular weights of between 110 and 150 kD and those with molecular
weights between 60 and 70 kD. Alteration in protease digestion has been
demonstrated to be indicative of an alteration in the conformation of
the digested protein. Changes in protein conformation may result in
alterations in protease-accessible sites, such that these sites may be
masked or novel sites exposed.
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To confirm DTT-induced misfolding of secretory proteins, the folding of
a control protein, albumin, was investigated. Albumin
has previously been shown to misfold with DTT treatment of HepG2 cells
of this protein.23 The electrophoretic analysis of
labeled albumin under reducing and nonreducing conditions is
shown in Fig 6. Albumin
immunoprecipitated from control cells migrated as a single diffuse band
under nonreducing conditions. A slow-migrating single albumin
band was detected under reducing condition. A similar slow-migrating
albumin species was recovered from DTT-treated cells under both
reducing and nonreducing conditions. The conversion of a diffuse and
faster migrating albumin band to a single slower migrating
species in the presence of DTT appears to indicate a folding effect.
The data suggest that DTT may have caused the misfolding of
albumin, most likely by disrupting its disulfide linkages.
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We also attempted to use electrophoretic migration under reducing and nonreducing conditions to investigate the folding status of apoB in the presence and absence of DTT. However, we were unable to demonstrate a significant change in the migration of apoB. It should be noted that due to the considerable size of apoB, it is difficult to detect changes in the folding of apoB by measuring the migration of the protein under reducing and nonreducing conditions, as was the case for albumin. In the upper part of the gel where apoB migrates, very small changes in migration represent significant differences in size. Changes in apoB folding may result in changes in migration that are too small to be detected under these electrophoretic conditions.
DTT Accelerates ApoB Degradation in Intact DTT-Treated
Cells
We further investigated whether DTT treatment of intact cells
affects the extent of apoB degradation. HepG2 cells were pulsed and
chased in the presence and absence of DTT. ApoB degradation was
determined by estimating the radiolabeled apoB recovered from cells and
media after the 2-hour chase period as a percentage of the initial apoB
radioactivity recovered from cells at 0 hours (Fig 7). In control cells, 38.8±3% of apoB
was recovered after 2 hours of chase, while in DTT-treated cells, a
lower percentage of apoB (26.8±1.1%) was detected after 2 hours of
chase (P<.05). The decrease in immunoprecipitable apoB
recovered from DTT-treated cells compared with control cells is
indicative of a higher degree of degradation in DTT-treated cells
(73.2±1.1% versus 61.7±3%, ±SE, P<.05). Overall, the
data suggest that apoB degradation may be more prominent in DTT-treated
cells. A higher degree of degradation was also noted in DTT-treated
permeabilized cells (see below).
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Time Course of the Effect of DTT on Intracellular ApoB Accumulation
and Stability
To assess the point in the production of apoB at which
DTT-induced degradation may occur, HepG2 cells were initially
preincubated in the presence and absence of DTT and subsequently pulsed
for various time periods in the presence and absence of DTT. Fig 8A demonstrates the immunoprecipitable
apoB100 (radioactivity in the intact 550-kD band) isolated from cells
pulsed for 10 to 30 minutes in the presence and absence of DTT. A
similar amount of apoB100 radioactivity was recovered from both DTT and
control cells after 10 minutes and 20 minutes of pulse. However, after
a pulse period of 30 minutes, there was significantly less apoB
recovered from DTT-treated cells than from control cells (control,
64 736±1800 versus + DTT, 32 746±1700 cpm per dish, ±SE,
P<.003). In addition, there was a linear increase in the
amount of immunoprecipitable apoB recovered from the control cells over
the 10-to-30 minute pulse period. However, the amount of apoB isolated
from DTT-treated cells decreased after 30 minutes of pulse in
comparison to the amount recovered from the 20-minute pulse period
(DTT-20 minutes, 43 461±3600 versus DTT-30 minutes, 32 746±1700 cpm
per dish, ±SE, P<.05). The data suggest that between 20
and 30 minutes of pulse, DTT significantly inhibits the accumulation of
newly synthesized apoB chains (control-30 minutes versus DTT-30
minutes) and also may enhance the degradation of apoB (DTT-20 minutes
versus DTT-30 minutes).
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We also assessed the effect of DTT on total immunoprecipitable apoB
radioactivity (including apoB100 and all nascent chains).
Analysis of the total apoB recovered under conditions identical
to that described in Fig 8A are shown in Fig 8B. Analogous to the
results shown in Fig 8A for apoB100, there was a linear increase in the
amount of total immunoprecipitable apoB isolated from control cells
over the 10- to 30-minute pulse period. In contrast, the amount of
total apoB recovered from DTT-treated cells did not parallel that
observed for apoB100. The amount of total apoB recovered at 20 minutes
did not increase significantly compared with the amount recovered at 10
minutes in the presence of DTT. In addition, there was significantly
less total apoB recovered from DTT-treated cells than from control
cells at both 20 minutes (control, 382 020±20 000 versus +DTT,
213 900±11 000 cpm per dish, ±SE, P<.02) and 30 minutes
(control, 434 610±27 000 versus +DTT, 162 760±4000 cpm per dish,
±SE, P<.01). There was also a significant decrease in the
amount of total immunoprecipitable apoB recovered from DTT-treated
cells at 30 minutes compared with that recovered at 20 minutes (DTT-20
minutes, 213 900±11 000 versus DTT-30 minutes, 162 760±4000 cpm
per dish, ±SE, P<.05). The data suggest that DTT may
enhance degradation of apoB after approximately 20 minutes.
Degradation of ApoB in DTT-Treated Intact Cells Is Largely
ALLN Insensitive
The effect of ALLN on apoB degradation was also investigated in
control and DTT-treated intact cells. HepG2 cells were initially
pretreated in the presence or absence of ALLN (40 µg/mL) and
subsequently preincubated with DTT before the pulse period. Fig 9 demonstrates that treatment of HepG2
cells with ALLN resulted in a significant increase in the amount of
immunoprecipitable apoB recovered compared with control cells
(17 288±900 versus 11 022±700 cpm per dish, ±SE,
P<.01). The presence of DTT partially negated the
inhibitory effect of ALLN, resulting in only a slight
increase (not statistically significant) in the apoB recovered from
DTT/ALLN-treated cells compared with DTT-treated cells (14 964±1300
versus 13 953±600 cpm per dish, ±SE, P=.51).
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The effect of DTT on the degradation of apoB in the presence and absence of ALLN after an extended chase period was also investigated. HepG2 cells were pretreated for 1 hour with brefeldin A only or with both brefeldin A and ALLN. Cells were then pulsed and chased in the presence or absence of DTT. Percent apoB degraded was determined by the densitometric quantitation of immunoprecipitated apoB at 0 time and after a 2-hour chase period. Percent apoB remaining under different conditions was as follows: –DTT/–ALLN (21%), –DTT/+ALLN (90%), +DTT/–ALLN (15%), +DTT/+ALLN (60%). As expected, ALLN inhibited apoB degradation in control cells. ALLN also partially inhibited apoB degradation in DTT-treated cells, but degradation still occurred in these cells. The data suggest that apoB degradation in DTT-treated cells occurs by both ALLN sensitive and ALLN insensitive pathways.
70-kD ApoB Fragment Is Not Detectable in DTT-Treated
Permeabilized Cells Despite Significant
Degradation
The effect of DTT on the pattern of apoB degradation was
investigated using the permeabilized cell degradation
assay.18 HepG2 cells were pulsed, briefly chased, and then
were permeabilized with digitonin. When DTT was added,
it was present at all steps. Control and DTT-treated
permeabilized cells were then incubated in CSK buffer
in the absence and presence of DTT, respectively. Immunoprecipitable
apoB detected at 0 time, and 1 to 2 hours' chase, are shown in Fig 10. In control
permeabilized cells, apoB degradation occurred, with
the generation of the typical 335-kD and 70-kD fragments, which
accumulated at 1 and 2 hours with the disappearance of the intact apoB.
In the presence of DTT, the typical fragmentation of apoB was not
observed, and the normal apoB fragments did not accumulate during the
chase. Degradation did, however, occur with DTT and appeared to be more
prominent. Percent intact apoB remaining after 2 hours (as a percentage
of the signal at 0 time) was 21.5% for DTT-treated
permeabilized cells compared with 34.2% for control
untreated cells. In some experiments, small amounts of the 335-kD and
70-kD fragments were observed in DTT-treated cells, but they were both
less abundant than that observed in control cells. The activity of the
ALLN-sensitive ER proteases in the presence of DTT was also
investigated. Degradation of HMG-CoA reductase has been demonstrated to
occur via an ALLN-sensitive protease.30 Using
permeabilized cells, Leonard and Chen31
demonstrated that HMG-CoA reductase degradation occurs with the
simultaneous accumulation of a 68-kD fragment. The
degradation of HMG-CoA reductase was investigated in the present
study using permeabilized HepG2 cells treated in the
presence and absence of DTT. The formation and accumulation of the
68-kD fragment occurred both in the presence and absence of DTT,
suggesting that the activity of the ALLN-sensitive enzyme was not
inhibited by the presence of DTT (data not shown). The data suggest
that DTT alters the normal fragmentation and degradation of the intact
apoB without inhibition of ALLN-sensitive proteases.
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As in intact cells, a protein species of approximately 60 kD in size was immunoprecipitated consistently by the apoB antibody from DTT-treated permeabilized HepG2 cells. This protein species, which was only detected in DTT-treated cells, was particularly abundant at time 0 and disappeared with similar kinetics to that of intact apoB. A protein of similar molecular size has also been observed in DTT-treated HepG2 cells immunoprecipitated with an apoB antibody.32 The protein species was suggested to be an apoB fragment occurring in intact cells. We attempted to identify the origin of this fragment through the use of a battery of monoclonal antibodies raised against specific regions of the apoB molecule. However, we were unable to definitively characterize this protein species as originating from intact apoB, since it appeared to be immunoprecipitated with apoB monoclonal antibodies raised against various regions of apoB (data not shown).
A DTT timed-addition experiment (Fig 11) was performed to investigate the
effect of DTT treatment on the accumulation and the stability of the
apoB 70-kD fragment. DTT was added in the pulse only, the chase only,
or in both the pulse and chase. In control, untreated cells, apoB
degradation occurred with the continual accumulation of the 70-kD
fragment during the chase (Fig 11). When present in both pulse and
chase, very little 70-kD fragment was detected in DTT-treated cells.
However, when DTT was added in the pulse but removed for the chase, a
higher amount of the fragment was detected (Fig 11), suggesting that
the removal of DTT for the chase period causes the restoration of the
normal apoB fragmentation. Furthermore, when cells were pulsed and
chased for 1 hour without DTT, followed by the addition of DTT in the
second hour, the amount of the 70-kD fragment detected was comparable
with that initially present at 1 hour of chase. This is in contrast
with the continual accumulation of the fragment in control cells during
the second hour. Numerous experiments conducted in our laboratory have
shown that the accumulation of the 70-kD fragment is essentially
complete after 2 hours' incubation in CSK. On the basis of this
observation, the stability of the 70-kD fragment in the presence of DTT
was investigated by extending the chase period to 3 hours and
determining the amount of the fragment that occurs in absence of DTT
compared with the amount that accumulates when DTT is added 2 hours
into the chase. When DTT was added 2 hours into the 3-hour chase
period, the amount of the 70-kD fragment accumulated was on average
96.5% of the amount accumulated in control cells. The data suggest
that the addition of DTT can prevent the formation and/or accumulation
of the 70-kD fragment. However, once formed, the 70-kD fragment was
stable, and its susceptibility to degradation was not increased by the
presence of DTT.
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ApoB Degradation in DTT-Treated Permeabilized Cells
Is Largely ALLN Insensitive
The permeabilized HepG2 degradation assay was also
employed to investigate the sensitivity of apoB degradation in
DTT-treated cells to ALLN. Control cells and cells pretreated with ALLN
were pulsed, chased, and permeabilized in the presence
of DTT. Permeabilized cells were then chased in CSK
buffer supplemented with or without DTT. When ALLN was added, it was
present at all steps. Fig 12 shows
the immunoprecipitable apoB recovered at 0 time and 1 to 2 hours of
chase in cells treated with DTT only, ALLN only, or both DTT and ALLN.
As observed in Fig 7, apoB was degraded in the presence of DTT alone.
In cells pretreated with ALLN, degradation appeared to be inhibited as
expected. This control experiment confirmed the effectiveness of ALLN
in inhibiting apoB degradation and the generation of the 70-kD apoB
fragment in control cells as previously shown.18 When both
DTT and ALLN were added, degradation of apoB occurred, apparently at
comparable rates to DTT-treated cells. Percent apoB remaining after 2
hours was 11.9% for DTT-treated cells compared with 10.7% for cells
treated with both DTT and ALLN. It should be noted that ALLN treatment
of DTT-treated cells did result in the recovery of a higher amount of
apoB at time 0, but most of the apoB was degraded during the chase
period. Therefore, apoB degradation appeared to occur in DTT-treated
cells at the same rate and with similar pattern whether or not ALLN was
present. The normal fragmentation of apoB was also not observed in
DTT-treated cells with or without ALLN, as the 70-kD fragment was not
generated to any appreciable extent with DTT, ALLN, or both (data not
shown).
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Degradation of Unglycosylated ApoB Is Also Altered in the Presence
of DTT
We have previously reported that tunicamycin-mediated inhibition
of apoB glycosylation results in changes in the rate and pattern of
apoB degradation.18 Since unglycosylated apoB is probably
not folded properly, the effect seen with degradation may have resulted
from misfolding of the protein. Here, we investigated whether DTT can
further affect the rate and pattern of the degradation of
unglycosylated apoB in tunicamycin-treated cells. HepG2 cells
pretreated with tunicamycin were pulsed, chased, and digitoninized in
the presence and absence of DTT. Permeabilized cells
were then chased in CSK buffer or in CSK supplemented with DTT. Fig 13A shows the effect of DTT on
degradation of unglycosylated apoB over 2 hours of chase. In
tunicamycin-treated cells, unglycosylated apoB (510 kD) and its
previously established18 degradation fragments (250 kD and
225 kD) were observed at 0 time, and the intact apoB was degraded over
the 2-hour chase. When tunicamycin-pretreated cells were also treated
with DTT, considerably less of the 510-kD apoB (by 48% at time 0) and
its fragments were detectable. Degradation of the 510-kD apoB after 2
hours was also more prominent (percent apoB remaining after 2 hours was
about fivefold lower in the presence of both tunicamycin and DTT
compared with percent apoB remaining in the presence of tunicamycin
alone). Interestingly, although the 250-kD and 225-kD fragments were
also detected in the presence of DTT, both were lower in amounts
detected and appeared to be lost over the 2-hour chase. In
tunicamycin-treated cells, these fragments typically appeared and
accumulated during the chase. The fragments were therefore less stable
in DTT-treated permeabilized cells. Overall, the data
suggest that DTT accelerates the degradation of unglycosylated apoB and
may also induce the degradation of its fragments.
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EM supplemented with 5 µg/mL tunicamycin. All the following
steps were also in the presence or absence of tunicamycin. After the
preincubation period, cells were pulsed, chased, and
permeabilized as in Fig 10
The combined effect of DTT and tunicamycin on apoB degradation was also
studied in intact cells, with similar observations to those made in
permeabilized cells. Fig 13B shows the
immunoprecipitable apoB recovered from HepG2 cells under control and
treatment conditions. Since apoB fragments are not observed in intact
cells, apoB100 is essentially the only apoB species recovered from
immunoprecipitation procedure. Thus, only this part of the gel is shown
in Fig 13B, since the rest of the gel did not contain any detectable
bands. Degradation appeared to occur earlier (considerably less apoB
detected at 0 time) and was accelerated in the presence of both DTT and
tunicamycin.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The secretion of apoB-containing lipoprotein particles by the hepatocyte appears to depend on the rate of apoB translocational efficiency and the balance of the apoB pool rescued from intracellular degradation. Our present report provides evidence that alteration in the conformation of apoB can not only alter the degradation pathway of apoB but also affect its assembly into a lipoprotein particle. The ability of the ER to assess the conformational status of a secretory protein and prevent the secretion of those proteins that are misfolded has been demonstrated in a number of studies. DTT has been recently employed by a number of investigators to study the folding of secretory proteins in the ER.21 22 23 24 33 Newly synthesized proteins containing cysteine residues cannot form proper disulfide linkages in the presence of DTT and therefore misfold under these conditions. DTT-induced misfolding of secretory proteins causes their retention in the ER.21 22 23 24 Recently, studies conducted in our laboratory,26 as well as others,19 have shown that treatment of HepG2 cells with DTT can alter the conformation and production of apoB.
The effect of reducing disulfide linkages on apoB was further
investigated in this study by treating HepG2 cells with DTT and
performing pulse-chase labeling experiments in intact and
permeabilized cells. DTT treatment of HepG2 cells
significantly reduced apoB secretion. This is in agreement with a
recent report demonstrating that apoB secretion could be partially
inhibited in the presence of DTT.19 Analysis of
the apoB fragments generated by trypsin digestion in the presence and
absence of DTT provides evidence to suggest that DTT causes
conformational changes in apoB. DTT-induced alteration in the
trypsin-generated fragmentation pattern of apoB has been shown to be
representative of a change in the conformation of this
protein.34 Recent evidence suggests that inhibition of
protein secretion in cells treated with DTT is likely to be the result
of secretory proteins being misfolded and therefore retained in the ER.
The secretory pathway of HepG2 cells is largely unaffected in
DTT-treated cells, as 1-antitrypsin, which contains no
disulfide bonds, was shown to be secreted in the presence of
DTT.23 In our studies, we also confirmed the integrity of
the secretory pathway in DTT-treated HepG2 cells by detecting the
secretion of 1-antitrypsin under conditions that reduce
apoB secretion. Retention of proteins in the ER as a result of DTT
treatment was also demonstrated by Braakman et al,21
investigating DTT-induced disruption of disulfide bond formation in
influenza hemagglutinin.
ApoB secretion was inhibited by DTT without a significant change in the
synthesis of full-length apoB100 molecules. The results of a recent
study also demonstrated that apoB synthesis was relatively unaffected
in the presence of DTT.19 Interestingly, in our study,
there was a substantial decrease in the synthesis of total radiolabeled
apoB chains in DTT-treated cells. This decrease was mainly the result
of a decline in the amount of incomplete nascent apoB chains, which has
been suggested to be due to an inhibition of protein synthesis
initiation.19 A second possibility is that treatment with
DTT induced a rapid degradation of apoB nascent polypeptide chains. In
contrast to apoB100, the synthesis of total protein and of
1-antitrypsin and albumin were significantly reduced when
cells were treated with DTT. Synthesis of influenza hemagglutinin was
shown not to be significantly affected in virus-infected Chinese
hamster ovary cells treated with DTT.21 However, the
synthesis of the secretory protein Gp80 (clusterin, apolipoprotein J)
was shown to be reduced by 50% to 70% from MDCK cells treated in the
presence of DTT.35 Inhibition of protein synthesis was
also observed by Chanat et al36 with respect to
chromogranin B and secretogranin II in PC12 cells.
In addition to inhibiting its secretion, we found that DTT altered the rate and pattern of apoB degradation. Our data demonstrated that DTT accelerated the loss of labeled apoB in both intact and digitonin-permeabilized cells. Using intact HepG2 cells, Shelness and Thornburg19 showed that DTT had an initial inhibitory effect on apoB degradation, but increased preincubation with DTT resulted in accelerated apoB degradation. Enhanced degradation of reduced apoB may result from the misfolding of the protein in the ER. Changes detected in the fragmentation of apoB appear to support this notion. Reduced apoB detected in DTT-treated cells was degraded in permeabilized cells without the generation of the usual 335-kD and 70-kD fragments. Interestingly, the degradation of apoB in DTT-treated cells was found to be largely ALLN insensitive, indicating that the degradation of reduced and possibly misfolded apoB may not proceed by the ALLN-sensitive degradative pathway. In addition, the DTT-induced degradation of apoB was determined to occur within 20 to 30 minutes of labeling, suggesting that misfolded apoB may be rapidly removed after synthesis. A recent study conducted by Benoist et al37 also suggests that early apoB degradation may involve an ALLN-insensitive protease. However, in this study, the activity of the putative protease was shown to be inhibited in the presence of DTT.
The importance of the conformation of apoB and its role in apoB biogenesis is further demonstrated by our previous and present studies investigating the inhibition of N-linked glycosylation of apoB. Intracellular degradation of unglycosylated apoB occurs earlier, is ALLN insensitive, and results in the appearance of additional fragments. The degradation of unglycosylated apoB was further enhanced when DTT was present. Significant degradation of reduced and unglycosylated apoB suggests that both these alterations have a profound effect on the stability of the apoB molecule. The reduction of disulfide linkages appears to render the unglycosylated apoB even more unstable, inducing its rapid degradation. The additive effects of DTT and tunicamycin further support the notion that significant conformational changes in nascent apoB profoundly affect its intracellular stability and viability for lipoprotein assembly.
One possible scenario that may explain the altered degradation of apoB that occurs through the disruption of disulfide bonds and/or the inhibition of glycosylation is that such conformational alterations mask the normal recognition sites on the apoB molecule for the ALLN-sensitive protease. In addition, such a conformational change may expose the nascent apoB to other cytosolic and/or ER proteases. Our observation that the removal of DTT from permeabilized cells resulted in the appearance of the 70-kD fragment further suggests that the reduced apoB may reform disulfide linkages, making it again a substrate for the ALLN-sensitive protease. The possibility exists that DTT treatment may have an effect on the activities of cysteine proteases, including the ALLN-sensitive protease. Our observation that the ALLN-sensitive degradation of HMG-CoA reductase produced the same degradation fragments in the presence and absence of DTT suggests that DTT treatment does not dramatically affect the ALLN-sensitive proteases involved. It has been suggested that in general the resident proteases of the ER may be relatively unaffected by the presence of reducing agents such as DTT. Braakman et al21 proposed that free sulfhydryl groups may be essential to ER protease activity and that a reducing environment achieved through the addition of DTT may maintain the activity of the ER proteases.
A second explanation for the altered degradation of apoB observed in the presence of DTT can be put forth based on the microsomal distribution of apoB in the presence and absence of DTT. Analysis of this distribution suggests an increased association of apoB with the microsomal membrane in the presence of DTT. In concert with the results of the present report, we have previously shown that DTT treatment can decrease the efficiency of apoB translocation across the ER membrane.26 This suggests that DTT-induced reduction of apoB can lead to a higher association with the ER membrane and thus a greater susceptibility to intracellular degradation. Recent evidence from our laboratory also suggests that membrane-associated apoB and luminal apoB are degraded with different kinetics and may involve distinct protease systems.17
The decrease in the formation of LDL/VLDL particles coupled to the decrease in the amount of HDL-sized (lipid-poor) particles suggests that the assembly of apoB into mature lipoprotein particles was inhibited in the presence of DTT. It is possible that alteration in the conformation of apoB may impair the transfer and/or association of the lipids required to form mature LDL/VLDL particles. Whether this impairment in lipid association is directly due to the conformational change in apoB or is a product of disruption of a key component(s) in the assembly of apoB-containing lipoproteins such as the microsomal triglyceride transfer protein remains to be demonstrated.
On the basis of the data presented, we suggest a strong linkage between the conformation of apoB and its secretion, degradation, and assembly within the hepatocyte. Proper apoB folding may be an important prerequisite for its regulated degradation and proper sorting for assembly and secretion. In the absence of correct conformation, apoB assembly into lipoprotein particles may be inhibited, and the protein may have an increased association with the ER membrane. This membrane-associated apoB may be rapidly degraded by an ALLN-insensitive pathway. It is postulated that the proper folding of the apoB molecule is important for the successful assembly into a lipoprotein particle. The interaction of apoB with lipids such as cholesterol esters and triglycerides may assist in the proper folding of the molecule and thus facilitate the assembly process. Future studies are needed to elucidate the role of lipid interaction in the folding and degradation of apoB.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by an operating grant from the Heart and Stroke Foundation of Ontario. Monoclonal antibodies against apoB were a gift from Drs Ross Milne and Yves Marcel. We gratefully acknowledge the excellent technical assistance of Debbie Rudy and Susan Sallach in the performance of these studies.
Received September 30, 1996; accepted July 10, 1997.
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