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
Trang 1proteins containing homopolymeric amino acids
Naohiro Uchio1, Yoko Oma1, Kazuya Toriumi1, Noboru Sasagawa1, Isei Tanida2, Eriko Fujita3, Yoriko Kouroku3, Reiko Kuroda1, Takashi Momoi3and Shoichi Ishiura1
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 amino acids (HPAAs) are distinct
tracts of amino acids 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 caused by 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 homopolymeric amino 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 endoplasmic reticulum stress by protea-some impairment may contribute to the strong cytotoxicity of highly hydrophobic HPAAs, and proteasome impairment and the resulting excess endoplasmic reticulum stress 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, homopolymeric amino acid; UPS, ubiquitin proteasome system; YFP, yellow fluorescent proteins.
Trang 2depending 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 disdis-eases [18] With
polygluta-mine diseases, ER stress is caused by 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 by aggregate-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 caused by 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
Trang 3transfection 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 stress by 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.
Trang 4examining 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 by homopolymeric 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-expressstain-ing 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 inhibitmisfold-ed 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 Ca2+ 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.
Trang 5suggested 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 caused by 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 caused by 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 stress by 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 caused by homopolymeric Ile expression Scale bar ¼
10 lm.
Trang 6[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
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.
Trang 7formed 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 stress caused by
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-prone proteins 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-mineTM 2000 (Invitrogen, Tokyo, Japan) or FuGENE 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 4C 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· 105cells 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 proPro-tein 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 Sigmonoclo-naling 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· 105cells 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 30C 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
Trang 8for 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 37C 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: excitation¼ 380 nm,
emission¼ 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
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