Neurol Sci (2011) 32:1147–1152 DOI 10.1007/s10072-011-0811-1
ORIGINAL ARTICLE
Prefibrillar aggregates of yeast prion Sup35NM and its variant are toxic to mammalian cells Yingxia Liu • Haiyan Wei • Jianguo Qu Jianwei Wang • Tao Hung
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Received: 24 January 2010 / Accepted: 21 September 2011 / Published online: 7 October 2011 Ó Springer-Verlag 2011
Abstract The deposition of proteins as insoluble amyloid aggregates is a characteristic feature of more than 20 degenerative conditions. A growing body of evidence indicates that the oligomeric species formed by proteins, but not the mature fibrils, are inherently toxic and are associated with clinical diseases. The N-terminal and middle region of Sup35 (Sup35NM), a yeast prion, can assemble into oligomers and fibrils. Here we analyze the cytotoxicity of different aggregates of Sup35NM and its variant, the proteins that is not associated with clinical disease. Our results showed that prefibrillar aggregates generated from Sup35NM and its variant Sup35NM-1 were toxic to cultured mammalian cells. In addition, the activation of caspase-3, 8, and 9 were detected, suggesting that apoptosis was involved in the observed cytotoxicity. Our findings provide evidence for the underlying mechanism of
amyloid aggregate-induced cytotoxicity and suggest that it may arise from common structural features of the aggregates rather than from primary amino acid sequences. Keywords Sup35NM Yeast prion Aggregation Cytotoxicity Apoptosis Abbreviations PrD Prion domain TSE Transmission spongiform encephalopathy TEM Transmission electron microscopy PBS 10 mM sodium phosphate, 1.8 mM potassium phosphate, 140 mM NaCl, and 2.7 mM potassium chloride, pH 7.4 ThT Thioflavin T PI Propidium iodide
Y. Liu and H. Wei contributed equally to this work. Y. Liu H. Wei J. Wang (&) State Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Pathogen Biology, Chinese Academy of Medical Sciences, #9, Dong Dan San Tiao, Dong Cheng District, Beijing 100730, People’s Republic of China e-mail:
[email protected] Y. Liu H. Wei J. Qu J. Wang T. Hung National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, People’s Republic of China Y. Liu Nanjing Medical University, Nanjing 210029, People’s Republic of China H. Wei Technical Center of Beijing Entry–Exit Inspection and Quarantine Bureau, Beijing 100026, People’s Republic of China
Introduction The deposition of proteins as insoluble amyloid aggregates in tissues is a common feature in a wide range of human diseases including Alzheimer’s disease, type II diabetes, and transmissible spongiform encephalopathies (TSE) [1, 2]. Although proteins associated with amyloid diseases have diverse amino acid sequences, the amyloid fibrils that develop are highly ordered protein aggregates that are characterized by a filamentous morphology, high b-sheet content, protease resistance, and yellow-green birefringence when stained with Congo red [3]. Although the precise mechanism by which this process occurs remains unclear, protein aggregation has been recognized as a dynamic process that transitions from native protein states into intermediate and well-organized aggregates [4].
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Although still subject to debate, recent emerging evidence has suggested that prefibrillar aggregates are cytotoxic both in vivo and in vitro [5, 6]. A number of studies have demonstrated that prefibrillar aggregates of some proteins, such as Ab peptides, a-synuclein, and transthyretin, are the most toxic species [7–9]. Moreover, an increasing number of natural and de novo designed protein sequences that are not associated with known medical conditions, such as the SH3 domain from bovine phosphatidylinositol 3-kinase (PI-SH3) and the N-terminal domain of Escherichia coli HypF (HypF-N), have also been shown to aggregate into fibrils in vitro and have toxicity in mammalian cells in their prefibrillar but not fibril state [10]. These data appear to be in agreement with the conclusions drawn from studies of disease-associated proteins. Therefore, cytotoxicity that is associated with aggregation may be determined by common features of specific types of aggregate structures rather than any specific features of the amino acid sequences of monomer polypeptides [10]. The term prion was originally coined to describe the unusual nature of the infectious, misfolded protein that causes spongiform encephalopathy [11]. In 1994, Wickner expanded the prion concept to explain the inheritance of [URE3] and [PSI?]. The non-chromosomal genetic element [PSI?] is the prion form of the Saccharomyces cerevisiae protein Sup35p [12]. As a prion-like protein, Sup35p offers a useful model for studying prion-like transmission of protein conformation and the formation of amyloid aggregates. Sup35p is composed of a distinct N-terminal (N), middle (M), and C-terminal (C) domain [13]. The N domain constitutes the prion determining domain (PrD), which is required for the induction and maintenance of [PSI?]. The N domain is extremely rich in glutamine (Q) and asparagine (N) and contains five imperfect, nine-residue repeats (PQGGYQQYN) [14]. These repeats are similar to mammalian prion protein repeats [15]. In addition, Sup35p can be converted into an insoluble aggregate, a property that is shared with the mammalian prion protein [13]. Despite numerous studies on the morphological, physiological, and structural features of Sup35p and its aggregates, the toxicity of these aggregates on mammalian cells has never been addressed. Previously, we generated multiple Sup35NM variants that contain randomized PrDs and have shown that they can polymerize in vitro into amyloid fibrils under native conditions. In this study, we report that prefibrillar aggregates of Sup35NM and its variant are toxic to mammalian cells. The activation of caspase-3, 8, and 9 were detected after treatment with these prefibrillar aggregates, suggesting that apoptosis is one mechanism responsible for the observed cytotoxicity.
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Materials and methods Preparation of Sup35NM and variant Sup35NM-1 aggregates Sup35NM and its variant Sup35NM-1 were expressed in E. coli and purified as previously described [16]. To create the Sup35NM-1 variant, the sequence of the PrD domain (aa 2-123) from Sup35NM was randomized with the original amino acid composition. Concentrated protein solutions of Sup35NM and Sup35NM-1 were diluted into phosphate buffered saline (PBS; pH 7.4) to a final concentration of 15 lM. The fresh protein preparations aggregate into fibrils when incubated at room temperature [16]. Electron microscopy Negative staining transmission electron microscopy (TEM) was used to examine the aggregation of Sup35NM and its variant Sup35NM-1 [17]. Briefly, 10 ll of the protein solutions were applied to a glow-discharged, 200 mesh carbon-coated copper grid for 1 min, stained with several drops of 2% (w/v) aqueous uranyl acetate, and air dried. The samples were examined with a Philips Tecnai 12 electron microscope at an accelerating voltage of 80 kV. Thioflavin T (ThT) binding assay The ThT assay was performed as previously described in order to establish the presence of fibril formation [18]. The proteins were diluted to 0.3 lM in the presence of 20 lM ThT solution (50 mM Tris–HCl and 0.2 M NaCl, pH 7.4). At 0, 1, and 48 h after the proteins were diluted, 10 ll aliquots of each solution were taken for measurement of the ThT fluorescence spectrum. ThT fluorescence was monitored using a Shimadzu RF5301PC (Shimadzu, Kyoto, Japan) spectrofluorometer with excitation at 450 nm and emission at 482 nm (excitation slit, 10 nm; emission slit, 10 nm). Cell viability assay Vero (African green monkey kidney epithelial cells) cells were plated at a density of 19104 cells per well on 96-well plates. At 24 h post-plating, the cells were exposed to early prefibrillar aggregates or mature fibrils of Sup35NM or Sup35NM-1 at final concentrations of 5, 10, and 15 lM. The cells were incubated for an additional 24 h at 37°C and then assayed for cell viability using the CCK-8 assay (Dojindo Inc., Kumamoto, Japan) according to the manufacturer’s instructions.
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Annexin V and propidium iodide staining
Statistical analysis
Cells were treated with the prefibrillar aggregates or mature fibrils of Sup35NM or Sup35NM-1 for 24 h before harvest. The cells were washed twice with PBS and then resuspended in binding buffer (0.01 M HEPES/ NaOH, pH 7.4, 0.14 mM NaCl, 2.5 mM CaCl2) at a concentration of 19106 cells/ml. Each solution (100 ll) was transferred to a 5 ml culture tube and incubated with fluorescein isothiocyanate (FITC)-labeled annexin V (5 ll) and propidium iodide (PI) (10 ll) for 15 min in the dark. After the incubation, 400 ll of binding buffer was added to the cells tubes and the cells were analyzed by flow cytometry (BD FACScalibur, Franklin Lakes, NJ, USA) [19].
Data on cell death were analyzed by one-way ANOVA. A value of P \ 0.05 was considered statistically significant.
Hoechst staining assay Vero cells were grown on glass coverslips in 35 mm dishes and were plated at a density of 2 9 106 cells per dish. Cells were treated with prefibrillar aggregates of Sup35NM or Sup35NM-1 at a final concentration of 15 lM for 24 h and then washed twice with ice cold PBS, fixed in a methanol: acetic acid (v/v, 3:1) mixture at room temperature for 10 min, and then stained with 10 lg/ml of Hoechst 33258 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min in the dark. Finally, the cells were rinsed twice with distilled water, mounted on glass microscopic slides in 50% glycerol, and examined under a fluorescent microscope (Leica DM LS, Leica Microsystems, Bannockburn, IL, USA). Western blot Treated Vero cells were harvested with trypsin and lysed in RIPA buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, and 0.5% sodium deoxycholate, pH 7.5) on ice for 60 min. Cells were then centrifuged at 12,000g for 10 min at 4°C and the supernatants were harvested. Extracted proteins were normalized and separated on a 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane as previously described [20]. The membrane was blocked with 5% skim milk in PBS containing 0.5% Tween-20 (PBST) overnight at 4°C and then incubated with rabbit anti-human Caspase-3, Caspase-8, or Caspase-9 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Proteins were detected by using nitroblue tetrazolium/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) or chemiluminescence (Thermo Fisher Scientific, Rockford, IL, USA). The blots were stripped and re-probed with anti-actin antibodies (Santa Cruz Biotechnology) for loading controls following the manufacturer’s instructions.
Results Aggregation of Sup35NM and its variant Sup35NM-1 Incubation of Sup35NM and its variant Sup35NM-1 in PBS (pH 7.4) at room temperature led to the formation of time-dependent, morphological types of aggregates in vitro. Granular prefibrillar aggregates of 2–5 nm in diameter were observed by TEM for both Sup35NM and Sup35NM-1 after incubation for 1 h (Fig. 1a). Over time, mature fibrils increased with a concomitant diminishment of granular aggregates. After 48 h, only mature fibrils were observed. The mature fibrils consisted of long, smooth, unbranched structures with an average diameter of 8–14 nm (Fig. 1b). ThT fluorescence of Sup35NM and Sup35NM-1 increased slightly after 1 h of incubation, but increased markedly at the 48 h time point (Fig. 1c). These findings demonstrated that Sup35NM and its variant Sup35NM-1 polymerized into amyloid fibrils as previously described [16]. Cytotoxicity of the aggregates of Sup35NM and its variant Sup35NM-1 To assess if aggregates of a non-mammalian toxic protein are cytotoxic to mammalian cells, the cytotoxicity of prefibrillar aggregates and mature fibrils of Sup35NM and Sup35NM-1 were compared. The cell viability assay revealed that prefibrillar granular aggregates of Sup35NM significantly reduced cell viability in a dose-dependent manner (5–15 lM, P = 0.017 at 15 lM), while the mature fibrils appeared to be essentially non-toxic (Fig. 2). Likewise, prefibrillar aggregates of Sup35NM-1 resulted in marked decrease in cell viability. This decrease was statistically significant at the concentration of 10 and 15 lM (P = 0.035 at 10 lM; P = 0.021 at 15 lM). Induction of apoptosis by Sup35NM and its variant Sup35NM-1 To explore the mechanism that is responsible for the cytotoxicity of prefibrillar aggregates of Sup35NM and Sup35NM-1, we stained Vero cells with FITC-annexin V and PI and assayed them by flow cytometry (Fig. 3a). After cells were treated with the early prefibrillar aggregates of Sup35NM or Sup35NM-1 for 24 h, we observed an increase in annexin V positive cells, with 28.02% of the
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Fig. 1 Aggregation of Sup35NM and its variant Sup35NM-1. Electron micrographs of Sup35NM and its variant Sup35NM-1 after a 1 h (a) or 48 h (b) incubation at room temperature (Bar 100 nm). c Thioflavin T binding assays of fibrils formed by Sup35NM and its variant Sup35NM-1. The fluorescence emission intensity was measured after incubation for 0, 1, and 48 h. Data are expressed as mean ± standard deviation
Fig. 2 Effects of prefibrillar aggregates and amyloid fibrils of Sup35NM and its variant Sup35NM-1 on cell viability. Cytotoxicity, as measured by cell viability percentage, of Sup35NM (Panel a) and Sup35NM-1 (Panel b) aggregates in Vero cells. Values are relative to control cells treated with phosphate buffered saline alone. Data are expressed as mean ± standard deviation (SD) and obtained from triplicate experiments. *P \ 0.05
Sup35NM-treated cells and 44.86% of the Sup35NM-1treated cells staining positive. Furthermore, nuclear staining with Hoechst 33258 supported an apoptotic mechanism of cell death (Fig. 3b). Western blot analysis showed that cleavage of caspase-3, -8, and -9 occurred after protein aggregate exposure (Fig. 3c). These findings indicate that the cells underwent apoptosis after exposure to prefibrillar aggregates of Sup35NM and Sup35NM-1.
Discussion We have previously demonstrated that amino acids in the Sup35NM PrD can be randomized without apparent loss of prion formation ability, though the variants show differential aggregation rates [16]. In this study, we characterized the cytotoxicity of the aggregates and found that prefibrillar aggregates of Sup35NM and its variant Sup35NM-1 were toxic to cultured mammalian cells and induced cell death by apoptosis.
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Several reports have suggested that amyloid aggregates formed by a wide variety of peptides or proteins are associated with cytotoxicity [7–10]. Here, we evaluated the cytotoxicity of aggregates of Sup35NM and Sup35NM-1 in mammalian cells. Cell viability assays confirmed that prefibrillar aggregates of Sup35NM, which is a yeast protein and unrelated to mammalian amyloid diseases, and its variant Sup35NM-1, were toxic to mammalian cells in a dose-dependent manner. In contrast, the mature fibrils of these proteins were essentially non-toxic. To our knowledge, this is the first report on the cytotoxicity of aggregates of Sup35NM and its variant Sup35NM-1 in mammalian cells. These findings are in agreement with the conclusions drawn from studies on disease-associated proteins such as Ab peptides, a-synuclein, and transthyretin [7–9] as well as other proteins that are unrelated to disease pathogenesis (PI-SH3 and HypF-N) [10]. Our findings provide additional evidence that aggregates produced in vitro by proteins that are unrelated to degenerative disease can also be cytotoxic.
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Fig. 3 Apoptosis induced by prefibrillar aggregates of Sup35NM and variant Sup35NM-1 in Vero cells. Apoptosis was measured by Annexin V staining and flow cytometry (a) and Hoechst 33258 staining (b). c Expression of caspase-3, -8, and -9 in Vero cells treated with prefibrillar aggregates of Sup35NM and variant Sup35NM-1. Cell lysates were separated by 12% SDS-PAGE. The expression of
caspase-3, -8, and -9 in the cytoplasm were detected by western blot using specific antibodies indicated in the panel. b-actin was used as a loading control. Vero cells were exposed to prefibrillar aggregates of Sup35NM and its variant Sup35NM-1 for 24 h prior to analysis. Cells treated with phosphate buffered saline alone were used as controls
Apoptosis is an intrinsic cell death program that is involved in the regulation of physiological and pathological processes [21]. Both Annexin V and Hoechst 33258 staining data showed that Vero cells underwent apoptosis after treatment with prefibrillar aggregates of Sup35NM and Sup35NM-1. These results are consistent with previous studies that have shown that amyloid aggregates formed by different peptides can also induce apoptosis [22, 23]. In addition, we also observed the induction of other apoptotic markers in Vero cells treated with prefibrillar Sup35NM aggregates, including the activation of caspase-3, -8, and -9. These data indicate that the induction of apoptosis in Vero cells by prefibrillar aggregates of Sup35NM and variant Sup35NM-1 may be the result of two pathways: the mitochondrial signaling pathway and the death receptor pathway. However, the precise mechanisms for these observations need to be further elucidated. Our study has found that the cytotoxicity and apoptosis mediated by Sup35NM-1 prefibrillar aggregates were comparable to wild type Sup35NM, despite the fact that the two proteins have different PrD amino acid sequences and rates of aggregation [16]. The data indicate that cytotoxicity that is associated with aggregation may arise from common characteristics of aggregate
structures rather than from primary amino acid sequences [10]. A recent study showed that soluble, prefibrillar aggregates of amyloid polymers, but not mature fibrils, were recognized by polyclonal antibodies against prefibrillar Ab peptides [24]. In addition, the polyclonal antibodies were able to suppress the aggregate-mediated cytotoxicity [24]. Taken together, these data suggest that prefibrillar aggregates of these types of proteins may cause cytotoxicity due to their structural similarities and not their amino acid sequences. In conclusion, the prefibrillar aggregates formed by Sup35NM and its variant Sup35NM-1, but not the mature fibrils, are toxic to mammalian cells and cause apoptosismediated cell death. Our findings support the hypothesis that the toxicity of prefibrillar aggregates may arise from common characteristics of the aggregate structure rather than amino acid sequence. This study adds an additional layer of understanding to the mechanisms of pathogenesis in amyloid diseases. Acknowledgments We are grateful to Dr. S. Lindquist (Whitehead Institute, MIT) for proving the Sup35NM gene, and Dr. Chin-chen Wang and Chunjuan Huang (Institute of Biophysics, Chinese Academy of Sciences) for their assistance in ThT binding assay. This research was supported in part by grant from the Major Program of
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