Báo cáo khoa học: Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino acids doc

Endoplasmic reticulum stress caused by aggregate-prone proteins containing homopolymeric amino 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 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. 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 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 Endoplasmic reticulum stress 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 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. N. Uchio et al. Endoplasmic reticulum 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 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-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 reticulum stress 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 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. N. Uchio et al. Endoplasmic reticulum 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 reticulum stress 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 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- 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. Endoplasmic reticulum 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). References 1 Alba MM & Guigo R (2004) Comparative analysis of amino acid repeats in rodents and humans. Genome Res 14, 549–554. 2 Zoghbi HY & Orr HT (2000) Glutamine repeats and neurodegeneration. 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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. Endoplasmic reticulum 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