Endoplasmicreticulumstresscausedby aggregate-prone
proteins containinghomopolymericamino acids
Naohiro Uchio
1
, Yoko Oma
1
, Kazuya Toriumi
1
, Noboru Sasagawa
1
, Isei Tanida
2
, Eriko Fujita
3
,
Yoriko Kouroku
3
, Reiko Kuroda
1
, Takashi Momoi
3
and Shoichi Ishiura
1
1 Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo, Japan
2 Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan
3 Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, Japan
Homopolymeric aminoacids (HPAAs) are distinct
tracts of aminoacids comprising consecutive sequences
of the same amino acid, and are often present in native
proteins [1]. Some HPAA-containing proteins cause
genetic diseases via HPAA expansion [2–5]. At least
nine neurodegenerative diseases are known to be
caused by polyglutamine expansion (e.g. Huntington’s
disease). Expanded polyglutamine-containing proteins
form neuronal intracellular inclusions in animal models
and in the central nervous system of human patients.
Aside from expanded polyglutamine, intranuclear dis-
ease-causative proteins with polyalanine expansions
have been observed in the skeletal muscle of patients
with oculopharyngeal muscular dystrophy, a known
polyalanine disease [6,7]. Another protein that causes a
polyalanine disease is HOXD13, which also forms
aggregations in cellular models [8]. The aggregation of
causative proteins is a hallmark of all polyglutamine
diseases and of some polyalanine diseases. Moreover,
Huntington’s disease-like 2, of which the symptoms
are similar to those of Huntington’s disease, has been
described as being causedby a CTG repeat expansion
translated into either polyalanine or polyleucine
stretches [9]. Polyaspartic acid expansion in the carti-
lage oligomeric matrix protein is reported to be the
cause of pseudoachondroplasia and multiple epiphyseal
dysplasia [10].
To clarify the physiological functions and cellular
effects of HPAAs, we previously examined patterns of
the intracellular localization of HPAAs fused to yellow
fluorescent proteins (YFPs) in cultured mammalian
cells, which showed specific intracellular localization
Keywords
ER stress; polyglutamine; proteasome;
protein aggregation; ubiquitin
Correspondence
S. Ishiura, Department of Life Sciences,
Graduate School of Arts and Sciences, The
University of Tokyo, 3-8-1 Komaba,
Meguro-Ku, Tokyo 153-8902, Japan
Fax: +81 35454 6739
Tel: +81 35454 6739
E-mail: cishiura@mail.ecc.u-tokyo.ac.jp
(Received 2 May 2007, revised 1 August
2007, accepted 3 September 2007)
doi:10.1111/j.1742-4658.2007.06085.x
Many human proteins have homopolymericamino acid (HPAA) tracts,
but their physiological functions or cellular effects are not well understood.
Previously, we expressed 20 HPAAs in mammalian cells and showed char-
acteristic intracellular localization, in that hydrophobic HPAAs aggregated
strongly and caused high cytotoxicity in proportion to their hydrophobic-
ity. In the present study, we investigated the cytotoxicity of these aggre-
gate-prone hydrophobic HPAAs, assuming that the ubiquitin proteasome
system is impaired in the same manner as other well-known aggregate-
prone polyglutamine-containing proteins. Some highly hydrophobic
HPAAs caused a deficiency in the ubiquitin proteasome system and excess
endoplasmic reticulum stress, leading to apoptosis. These results indicate
that the property of causing excess endoplasmicreticulumstressby protea-
some impairment may contribute to the strong cytotoxicity of highly
hydrophobic HPAAs, and proteasome impairment and the resulting excess
endoplasmic reticulumstress is not a common cytotoxic effect of aggre-
gate-prone proteins such as polyglutamine.
Abbreviations
CHOP, C ⁄ EBP homologous protein; CFP, cyan fluorescent protein; ER, endoplasmic reticulum; ERAD, ER-associated degradation; GFP,
green fluorescent protein; HPAA, homopolymericamino acid; UPS, ubiquitin proteasome system; YFP, yellow fluorescent proteins.
FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS 5619
depending on the HPAA [11]. In particular, hydropho-
bic HPAAs formed characteristic perinuclear aggre-
gates. Moreover, the proportion of cell death and
caspase-3 activity by HPAA expression became stron-
ger in proportion to the hydrophobicity of the amino
acids composing the HPAA [12]. The aggregate-prone
hydrophobic HPAAs were thought to have cytotoxic-
ity associated with the rates of aggregation; however,
the mechanism of this strong cytotoxicity of hydropho-
bic HPAAs was not clearly determined.
Aggregate-prone proteins and peptides are associ-
ated with numerous conformational disorders, includ-
ing neurodegenerative diseases (e.g. amyloid beta
peptide in Alzheimer’s disease, huntingtin in Hunting-
ton’s disease, a-synuclein in Parkinson’s disease and
prion protein in prion diseases). These diseases show
pathological formation and accumulation of causative
proteins, indicating a general cytotoxic mechanism for
human conformational diseases [13]. One possible
mechanism is an aberration in the ubiquitin protea-
some system (UPS) [14]. Recent findings indicate that
the UPS is involved in the pathology of Parkinson’s,
Huntington’s and prion diseases, as well as amyo-
trophic lateral sclerosis. In rare cases, an aberration in
the UPS is a primary and direct contributor to the
pathogenesis although, in many cases (e.g. Hunting-
ton’s disease, amyotrophic lateral sclerosis), it appears
that inhibition of the UPS by the aggregate of disease-
causative proteins may lead to secondary neuronal
damage.
Endoplasmic reticulum (ER) stress has been attrib-
uted to the pathology of neurodegenerative diseases
such as Alzheimer’s disease [15], polyglutamine dis-
eases [16,17] and prion diseases [18]. With polygluta-
mine diseases, ER stress is causedby the inhibition of
ER-associated degradation (ERAD) resulting from
proteasome impairment. In the normal ERAD process,
misfolded or malfolded proteins in the ER lumen are
retrotranslocated to the cytosol and eliminated by the
UPS [19]. Defective ubiquitin-dependent proteolysis
with proteasome impairment therefore causes an accu-
mulation of protein in the ER and, as a consequence,
induces ER stress [17]. Proteasome impairment has
been reported in the neurodegenerative diseases
described above, suggesting that the same mechanism
may cause the pathogenesis of these conformational
disorders.
In the present study, we used hydrophobic HPAAs
as model proteins of conformational disorders, taking
advantage of different levels of solubility and cytotox-
icity for each hydrophobic HPAA, and examined
whether UPS impairment is a common phenomenon
caused byaggregate-prone proteins.
Results
Aggregation of hydrophobic HPAAs and
accumulation of ubiquitinated proteins in
mammalian cells
We previously expressed 20 different HPAAs of
approximately 30 residues each, and longer HPAAs
containing Ala70 and Glu150 fused to the C-termi-
nus of YFP in COS-7 cells [11]. In the present
study, we investigated homopolymeric Ala, Cys, Ile,
Leu, Met, Phe and Val as hydrophobic 30-residue
HPAAs, and the longer HPAAs (homopolymeric
Ala70 and Gln150) as model proteins for polyalanine
and polyglutamine diseases. The intracellular localiza-
tion and western blotting of expressed HPAAs in
C2C12 cells are shown inFig. 1A,B. Strong aggrega-
tion was observed, as previously described in COS-7
cells [11], and all HPAAs, except homopolymeric
Ala, formed cytoplasmic aggregates and made
SDS-resistant high-molecular weight proteins. The
longer homopolymeric Ala (Ala70) also made a
cytoplasmic aggregate similar to the other aggre-
gate-prone HPAAs. Because hydrophobic HPAAs
formed cytoplasmic aggregates, we performed western
blotting with anti-ubiquitin. Some hydrophobic
HPAAs, such as homopolymeric Ile and Leu,
showed notable accumulation of polyubiquitinated
protein in the upper running gels (Fig. 1B). A simi-
lar result was observed in Neuro2a cells (data not
shown). Immunostaining with anti-ubiquitin serum
also showed the accumulation of ubiquitinated pro-
tein in the cytoplasm when homopolymeric Ile was
expressed (Fig. 1C; see also Supplementary Material,
Fig. S1), and a similar result was obtained by the
expression of homopolymeric Leu (data not shown).
Homopolymeric Ile and Leu therefore caused the
accumulation of polyubiquitinated protein within the
cytoplasm.
Decreased proteasome activity by hydrophobic
HPAAs
Next, we investigated whether the accumulation of
ubiquitinated protein was causedby proteasome
impairment. We assessed proteasome chymotryptic
activity by measuring the cleavage of the fluores-
cent peptide substrate Suc–LLVY–MCA. Homopoly-
meric Ile and Leu showed a significant reduction in
proteasome activity (approximately 40% and 30%,
respectively) (Fig. 2A). Addition of proteasome inhib-
itor MG132 completely abolished the activity
(< 0.05%) (data not shown). Considering that the
Endoplasmic reticulumstress N. Uchio et al.
5620 FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS
transfection efficiency was approximately 60%, the
reduction in proteasome activity in homopolymeric
Ile- or Leu-expressing cells was expected to be
much higher. Additionally, the localization of poly-
ubiquitinated protein accumulation by MG132 was
quite similar to that with homopolymeric Ile and
Leu (Figs 1C and 2B), suggesting that the cyto-
plasmic accumulation of polyubiquitinated protein
(Fig. 1A,B) could be explained by proteasome
impairment.
ER stressby hydrophobic HPAAs
In a process known as ERAD, misfolded or malfolded
proteins generated in the ER are transported back to the
cytosol and degraded by the UPS [19]. A UPS defect
with proteasome impairment causes an accumulation of
protein in the ER, followed by ER stress [17]. Because
proteasome activity was impaired by the expression of
some hydrophobic HPAAs, we investigated whether ER
stress was induced by the expression of these HPAAs by
A
B
C
Fig. 1. Aggregation of hydrophobic HPAAs
and the accumulation of ubiquitinated pro-
tein. (A) The intracellular localization of
hydrophobic HPAAs. Cytoplasmic aggrega-
tion of hydrophobic HPAAs was observed in
all cells, with the exception of homopoly-
meric Ala. Scale bar ¼ 10 lm. (B) SDS-resis-
tant high-relative molecular mass proteins of
hydrophobic HPAAs detected by western
blotting with anti-GFP ⁄ YFP serum. The
accumulation of polyubiquitinated protein
was also detected by western blotting with
anti-ubiquitin antibody. (C) The accumulation
of polyubiquitinated protein in the cytoplasm
of homopolymeric Ile-expressing cells. Dis-
persed polyubiquitinated protein accumula-
tion in the cytoplasm was observed in the
cells in which homopolymeric Ile showed
dispersed localization (arrowhead). Perinu-
clear accumulation was also observed in the
cells in which homopolymeric Ile showed
strong aggregation near the nucleus (arrow).
Scale bar ¼ 50 lm.
N. Uchio et al. Endoplasmicreticulum stress
FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS 5621
examining C ⁄ EBP homologous protein (CHOP) expres-
sion and caspase-12 activation. CHOP is induced by ER
stress and mediates ER stress-induced apoptosis signal-
ing [20], and caspase-12 is specifically activated in ER
stress-induced apoptosis [21–23].
When we added tunicamycin or thapsigargin to
C2C12 cells (Fig. 3A), ER stress-induced activation of
caspase-12 and caspase-3 was clearly observed in our
cell system. We then investigated the effect of HPAAs
on CHOP expression. Western blotting showed a
remarkable induction of CHOP and activation of cas-
pase-12 byhomopolymeric Ile and Leu expression
(Fig. 3C). Induction of Bip ⁄ GRP78 was also observed
(data not shown). Caspase-3, a key mediator of apop-
tosis, was also activated. Moreover, by immunostain-
ing with anti-active caspase-12 serum, almost all the
homopolymeric Ile-expressing cells were strongly
stained with the antibody (Fig. 3D). Such strong stain-
ing was not observed in the YFP-expressing cells
(Fig. S2). Nuclear condensation, similar to that during
ER stress-induced apoptosis, was also observed
(Fig. 3B,E). Homopolymeric Ile and Leu therefore
induced excess ER stress, which resulted in cell death
characteristic of ER stress-induced apoptosis.
ER ⁄ Golgi protein accumulation by polyisoleucine
We then examined whether the degradation of misfold-
ed membrane proteins was inhibited in cells expressing
hydrophobic HPAAs using dysferlin as a model
ERAD substrate. Dysferlin is an ER ⁄ Golgi membrane
protein known as a causative agent of limb–girdle
muscular dystrophy type 2B [24]. Tet-dysferlin-C2C5 is
a cell line that is stably and Tet-inducibly transfected
with myc-tagged dysferlin, as shown in Tet-dysferlin
cells expressing Gln72 [25]. We observed more dysfer-
lin aggregates in cells expressing homopolymeric Cys,
Ile and Glu150 than in cells expressing cyan fluores-
cent protein (CFP)(Fig. 4A,C). In particular, homopol-
ymeric Ile showed notable accumulation of dysferlin.
By contrast, we did not detect a significant accumula-
tion in homopolymeric Leu-expressing cells. Dysferlin
did not accumulate following treatment with the ER
stress inducer thapsigargin, whereas apparent dysferlin
accumulation was observed by proteasome inhibiter
MG132 treatment (Fig. 4B,C). These results collec-
tively suggest that homopolymeric Ile causes actual
ER ⁄ Golgi protein accumulation in the ER lumen by
inhibiting ERAD.
Discussion
Effects of the expression of hydrophobic HPAAs
Aggregate-prone proteins, including disease-unrelated
proteins, are thought to have common toxic mecha-
nisms such as increasing intracellular Ca
2+
and caus-
ing oxidative stress [26]. A similar mechanism is
A
B
Fig. 2. Decreased proteasome activity by hydrophobic HPAAs. (A) Chymotryptic activity of the proteasome in hydrophobic HPAA-expressing
cells. The activity of untransfected cells presented as arbitrary units was normalized to 1. Student’s t-tests were performed versus the con-
trol (only YFP). *P<0.05; mean ± SE; n ¼ 4. (B) Intracellular localization of polyubiquitinated proteins in cells treated with 1 l
M proteasome
inhibitor MG132 for 24 h. Scale bar ¼ 50 lm.
Endoplasmic reticulumstress N. Uchio et al.
5622 FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS
suggested for hydrophobic HPAAs, based on their
cytotoxicity. Homopolymeric Ile and Leu inhibited
proteasome activity and caused excess ER stress.
Homopolymeric Ile, Leu and Val are the strongest
cytotoxic HPAAs among 20 tested HPAAs [12]. Their
notable cytotoxicity is partly explained by the special
property of inducing excess ER stress through protea-
some inhibition. The data showing that homopoly-
meric Ile had the strongest inhibitory effect on
proteasome activity are reasonable because Ile is the
most hydrophobic amino acid [27]. A possible alterna-
tive explanation is that hydrophobic HPAAs are them-
selves accumulated in the ER and cause ER stress
because the localization of hydrophobic HPAAs is
similar to that of the ER tracker dye (data not shown).
We reject this explanation, however, for two reasons.
First, hydrophobic HPAAs linked to YFP do not have
an ER transition signal. Second, at an early stage of
expression, dispersed, small aggregations are observed
in the cytoplasm, and the aggregation tends to accu-
mulate in the perinuclear region, where the ER is also
localized. Recently, it was reported that ER stress has
a general inhibitory effect on the UPS and induces the
accumulation of UPS substrates, including the ERAD
substrate CD3d[28]. In the present study, however,
dysferlin accumulation was not causedby the ER
stress inducers thapsigargin (Fig. 4B,C) or tunicamycin
[25]. We therefore concluded that ER stress was not
the cause of dysferlin accumulation by homopolymeric
Ile, but was the result of actual ER ⁄ Golgi protein
accumulation in the ER lumen, by inhibiting ERAD in
homopolymeric Ile-expressing cells. Other than homo-
polymeric Ile and Leu, the hydrophobic HPAAs did
not induce CHOP or activate caspase-12 (Fig. 3C),
which indicates an alternative pathway of cytotoxicity
not mediated by ER stress, and perhaps mediated by
mitochondrial stress.
Mechanism of proteasome impairment by
homopolymeric Ile and Leu
A decrease in proteasome activity can be causedby the
degradation of proteasome subunits by activated cas-
pase-3 [29]. However, we could not detect a decrease
in proteasome activity by apoptosis inducers, which
induced much more caspase-3 activation than hydro-
phobic HPAAs (data not shown). Therefore, the
decrease in proteasome activity was considered to be
associated with the aggregate formation (e.g. by
‘choking up’ the barrel-like proteasome as a result of
the difficulty in degrading polyglutamine sequences)
A
B
C
D
E
Fig. 3. Detection of ER stressby hydrophobic HPAAs in C2C12
cells. (A) Western blotting with anti-caspase-3, caspase-12 and
Bip ⁄ Grp78 sera of the cells treated by various apoptosis induc-
ers. (B) Nuclear condensation observed in thapsigargin-treated
cell. Scale bar ¼ 10 lm. (C) Western blotting of the ER stress
marker CHOP, ER stress-induced apoptosis marker caspase-12
and general apoptosis marker caspase-3. (D) Immunostaining with
anti-active caspase-12 serum with homopolymeric Ile-expressing
cells. Arrowheads indicate cells expressing homopolymeric Ile
that were strongly stained with the antibody. Scale bar ¼
100 lm. (E) Aberrant nuclear morphology of caspase-12-activated
cells causedbyhomopolymeric Ile expression. Scale bar ¼
10 lm.
N. Uchio et al. Endoplasmicreticulum stress
FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS 5623
[30,31]. As a result, the aggregation speed could over-
ride degradation, and the proteasome could be
involved in polyglutamine aggregation. As with homo-
polymeric Ile and Leu, the accumulation of ubiquiti-
nated protein occurred in cells where homopolymeric
Ile did not show evidence of aggregation (Fig. 1C),
and a similar mechanism may function in cells express-
ing hydrophobic HPAAs. We were unable to confirm
whether homopolymeric Ile- and Leu-containing YFP
are themselves ubiquitinated, but the degradation of
all hydrophobic HPAAs was inhibited by proteasome
inhibitors (Fig. S3), suggesting that homopolymeric
Ile- and Leu-containing proteins were targeted to the
UPS.
It is possible that dysferlin aggregation may induce
ER stress, followed by autophagy activation via
PERK-eIF2 phosphorylation [25,32,33]. Thus, hydro-
phobic HPAAs may alternatively be degraded by auto-
phagy. Homopolymeric Ile expression increased the
membrane-bound form of microtubule-associated pro-
tein light chain 3, an autophagy-specific marker (data
not shown).
Subtle effect of polyglutamine on proteasome
impairment
Expanded polyglutamine is reported to inhibit protea-
some activity [34,35]. We used homopolymeric Glu150,
but we did not detect a significant reduction in protea-
some activity (Fig. 2A). This may be explained by the
decreased expression level of Gln150 in C2C12 cells.
Aggresome-like structure
The perinuclear localizations of homopolymeric Ile,
Leu, Met, Phe and Val resemble aggresomes [11]
(Fig. 1A). Aggresomes were originally defined as
organelles that appear as a result of proteasome inhibi-
tor treatment [36]. In the present study, homopoly-
meric Ile and Leu had an inhibitory effect on
proteasome activity (Fig. 2A), and their perinuclear
aggregation may indicate aggresomes. It is interesting
that when only 30 residues were added to the C-termi-
nus of green fluorescent protein (GFP), which is nor-
mally soluble, a strong aggregation was caused that
AB
C
Fig. 4. ER ⁄ Golgi protein accumulation by
polyisoleucine expression. (A) Representa-
tive field of Tet-dysferlin C2C5 cells 24 h
after transfection with hydrophobic HPAAs.
CFP fluorescence (left panel) and anti-myc
staining (right panel). Scale bar ¼ 100 lm.
(B) Representative field of Tet-dysferlin
C2C5 cells incubated with 2 lgÆmL
)1
thapsi-
gargin (TG) or 2 l
M MG132 for 24 h. Hoe-
chst staining (left panel) and anti-myc
staining (right panel). Scale bar ¼ 100 lm.
(C) Ratio of the number of dysferlin-positive
cells to CFP fluorescence-positive cells or
Hoechst-positive cells. Student’s t-tests
were performed versus the control (only
CFP). *P<0.05; **P<0.01; ***P<0.001;
mean ± SE; n ¼ 3.
Endoplasmic reticulumstress N. Uchio et al.
5624 FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS
formed an aggresome-like structure. Moreover, aggre-
somes are reported to have a cytoprotective role in
response to the accumulation of aggregate-prone pro-
teins [37,38], even in autophagy activation [39]. It is
unclear whether the aggregation itself is cytotoxic or
cytoprotective. More analyses on this aggresome-like
structure with excess ER stresscausedby homopoly-
meric Ile and Leu may provide clues to resolve this
uncertainty.
Concluding remarks
Excess ER stress mediated by proteasome impairment
is limited to some hydrophobic HPAAs, and the cyto-
toxicity of the remaining hydrophobic HPAAs may
not be mediated by ER stress. We therefore suggest
that not all aggregate-proneproteins invariably induce
excessive ER stress. In the context of disease, many
studies have shown the apparent toxicity of protein
aggregates in cell and animal models, but the specific-
ity of the observed toxic effects remains unclear.
Hydrophobic HPAAs that are not causative proteins
of specific diseases may be suitable control aggregate-
prone proteins with which to address this issue.
Experimental procedures
Expression plasmid
YFP–HPAA plasmids have been described previously [11].
CFP–HPAA plasmids were made by subcloning the YFP–
HPAA plasmid into the ECFP–C1 vector.
Cell culture and transfection
C2C12 cells were cultured in DMEM (Sigma-Aldrich,
Tokyo, Japan) with 10% normal fetal bovine serum. Tet-
dysferlin C2C5 cells, stably expressing the Tet-inducible
myc-tagged dysferlin gene [25], were cultured in DMEM
with tetracycline-free 10% fetal bovine serum (Clontech,
Tokyo, Japan), and the medium was replaced with DMEM
containing 10% normal fetal bovine serum 24 h before
transfection. Transfection was performed using Lipofecta-
mine
TM
2000 (Invitrogen, Tokyo, Japan) or FuGENEÒ 6
(Roche Diagnostics, Tokyo, Japan) according to the manu-
facturer’s protocol.
Immunostaining
C2C12 and Tet-dysferlin C2C5 cells were transfected with
the YFP–HPAA and CFP–HPAA plasmids, respectively.
Twenty-four hours after transfection, the cells were fixed
with 3.7% formaldehyde in NaCl ⁄ Pi at room temperature
for 10 min, then incubated with anti-ubiquitin serum (Zymed
Laboratories, Inc., San Francisco, CA, USA), anti-active
caspase-12 serum [20], or anti-c-myc serum (Invitrogen)
overnight at 4 °C. They were then incubated with rho-
damine-labeled goat anti-(rabbit IgG) or anti-(mouse IgG)
(Bio-Rad Laboratories, Tokyo, Japan) for 30 min at room
temperature, and cell nuclei were labeled with Hoechst 33342
(Sigma-Aldrich). The fluorescence was visualized under a
microscope (IX70; Olympus, Tokyo, Japan).
Western blotting
C2C12 cells were plated at 2.0 · 10
5
cells per 35 mm dish
and incubated for 24 h. They were then transiently trans-
fected with 2 lg of YFP–HPAA plasmids. After incubation
for 48 h, the cells were harvested and sonicated in NaCl ⁄ Pi
with a protease inhibitor mix (Wako, Osaka, Japan). Pro-
tein concentrations were measured using a DC protein assay
kit (Bio-Rad Laboratories). Equal amounts of protein
(2–5 lg) were subjected to SDS ⁄ polyacrylamide gel electro-
phoresis on 12.5% gels and transferred to poly(vinylidene
difluoride) membranes (Finetrap NT-32; Nihon Eido,
Tokyo, Japan). The membranes were blocked at room
temperature for 30 min in 20 mm Tris ⁄ HCl, pH 7.5, 0.1 m
NaCl supplemented with 10% skimmed milk and incubated
with primary antibody. Rabbit polyclonal anti-GFP ⁄ YFP
(Santa Cruz Biotechnologies, Santa Cruz, CA, USA), mouse
monoclonal anti-ubiquitin (Zymed Laboratories Inc.), rab-
bit polyclonal anti-Bip ⁄ GRP78 (Stressgen Biotechnologies,
Victoria, Canada), mouse monoclonal anti-CHOP ⁄
GADD153 (Santa Cruz Biotechnologies), rabbit monoclo-
nal anti-caspase-3 (Cell Signaling Technology, Beverly, MA,
USA) and rat monoclonal anti-caspase-12 (Sigma-Aldrich)
primary sera were used. After subsequent washing steps and
incubation with horseradish peroxidase-conjugated anti-
(rabbit IgG), anti-(mouse IgG), or anti-(rat IgG) antibodies,
the blots were developed by enhanced chemiluminescence,
and images were visualized using the LAS-3000 imaging
system (Fujifilm, Tokyo, Japan).
Proteasome activity assay
C2C12 cells were plated at 2.0 · 10
5
cells per 35 mm dish
and incubated for 24 h. They were then transiently trans-
fected with 2 lg of YFP–HPAA plasmids. Twenty-four
hours after transfection, the cells were harvested and dis-
solved in extraction buffer (50 mm Tris ⁄ HCl, pH 7.5,
10 mm 2-mercaptoethanol, 1 mm EDTA). The samples
were subjected to three rounds of freezing in liquid nitrogen
for 60 s and thawing in a 30 °C water bath for 90 s, after
which the samples were centrifuged at 10 000 g for 5 min.
The total protein (5 lg) in the supernatant was dissolved in
200 lL of assay buffer (25 mm Tris ⁄ HCl, pH 7.5, 10 mm
2-mercaptoethanol, 1 mm EDTA). A fluorescent substrate
N. Uchio et al. Endoplasmicreticulum stress
FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS 5625
for proteasome chymotryptic activity, Suc–Leu–Leu–Val–
Tyr–MCA (Suc–LLVY–MCA; Peptide Institute, Osaka,
Japan), was added to a final concentration of 5 lm, and the
mixtures were incubated at 37 °C for 30 min. The reactions
were stopped by the addition of 100 lL of 10% SDS and
1 mL of 0.1 m NaOAc; the fluorescence was measured
using a spectrophotometer (FP-777: excitation ¼ 380 nm,
emission ¼ 460 nm; Jasco, Tokyo, Japan).
Acknowledgements
This work was supported by the Human Frontier
Science Program (RGP0024 ⁄ 2006-C).
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Supplementary material
The following supplementary material is available
online:
Fig. S1. No accumulation of polyubiquitinated protein
in the cytoplasm of the cells without Ile expression.
Fig. S2. Immunostaining with anti-active caspase-12
antibody of YFP-expressing cells.
Fig. S3. Inhibition of the degradation of hydrophobic
HPAAs by proteasome inhibitors.
This material is available as part of the online article
from http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
N. Uchio et al. Endoplasmicreticulum stress
FEBS Journal 274 (2007) 5619–5627 ª 2007 The Authors Journal compilation ª 2007 FEBS 5627
. Endoplasmic reticulum stress caused by aggregate-prone
proteins containing homopolymeric amino acids
Naohiro Uchio
1
, Yoko. Kodaira, Tokyo, Japan
Homopolymeric amino acids (HPAAs) are distinct
tracts of amino acids comprising consecutive sequences
of the same amino acid, and are