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Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106–126
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Biochemical and Biophysical Research Communications 365 (2008) 808–813 www.elsevier.com/locate/ybbrc
Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106–126 Mathumai Kanapathipillai a, Sook Hee Ku b, Koyeli Girigoswami b, Chan Beum Park b
a,b,*
a Department of Chemical and Materials Engineering, Arizona State University, P.O. Box 876006, Tempe, AZ 85287, USA Institute for the Bio-Century and Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 6 November 2007 Available online 26 November 2007
Abstract In prion diseases, the posttranslational modification of host-encoded prion protein PrPc yields a high b-sheet content modified protein PrPsc, which further polymerizes into amyloid fibrils. PrP106–126 initiates the conformational changes leading to the conversion of PrPc to PrPsc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106–126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106–126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity. 2007 Elsevier Inc. All rights reserved. Keywords: Prion; Small stress molecules; Ectoine; Mannosylglyceramide; Cytotoxicity
Prions are infectious particles that cause transmissible spongiform encephalopathies in animals and humans [1]. The infectious prions are composed of PrPsc, a conformationally altered form of a host-encoded glycoprotein PrPc [2,3]. PrPc is modified posttranslationally to yield a high b-sheet content structure that further polymerizes into amyloid fibrils. Although the two isoforms are chemically identical, they possess very different physicochemical properties. The mechanism by which PrPc conAbbreviations: HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; SSM, small stress molecule; MG, mannosylglycerate; MGA, mannosylglyceramide; AFM, atomic force microscopy; PrPc, normal cellular prion protein; PrPsc, infectious isoform of PrPc; PB, phosphate buffer. * Corresponding author. Address: Institute for the Bio-Century and Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Daejeon 305-701, Republic of Korea. Fax: +82 42 869 3310. E-mail address: [email protected] (C.B. Park). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.074
tributes to prion-induced neurotoxicity still remains unclear. One hypothesis is that, PrPc normally possesses a neuroprotective function [4] that is abolished or subverted by interaction with PrPsc [5]. Due to the toxicity and handling difficulties of the whole prion protein, several PrP peptide fragments were used for in vitro studies. Among those, PrP106–126 is conserved among various species and is suggested to be one of the important regions of conformational change initiation, leading to the conversion of PrPc to PrPsc [6,7]. PrP106–126 also exhibited a combination of b-sheet and random coil structures under different experimental conditions [8,9]. It aggregates into protease-resistant amyloid fibrils thereby inducing neuronal cell death by apoptosis, and causing proliferation and hypertrophy of cultured glia [10,11]. Furthermore, the toxicity of PrP106–126 is similar to that of PrPsc as both require the expression of PrPc to cause cell death [12,13].
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Molecules, which can stabilize the structure of PrPc or convert PrPsc into the normal form, can be potential drug candidates for prion related diseases. Small stress molecules (SSMs) are formed in microorganisms during stressed conditions to prevent protein denaturation and hence to maintain protein stability and function [14,15]. Furthermore, these naturally occurring solutes are known to be non-toxic to cellular environment even at concentrations as high as 1 M, which makes them more promising for drug design [16,17]. Recently, we reported that several SSMs could successfully inhibit the amyloid formation of bovine insulin and Alzheimer’s Ab42 peptides, and thereby reduce the neurotoxicity of Ab42 aggregates [18,19]. Here, our study explores, initially, the role of four SSMs—ectoine, hydroxyectoine, mannosylglyceramide (MGA), and mannosylglycerate (MG) during PrP106– 126 aggregation. The ectoines (ectoine and hydroxyectoine) are highly water-soluble, zwitterionic low-molecular weight molecules. They are biologically inert, do not interfere with most enzymatic and binding reactions, and are also highly compatible with cell metabolism. Ectoine demonstrates its protective effect on proteins and enzymes through protection from thermal stress, proteolysis, and change of pH or the salt concentration. They also exert their stabilizing effect on different proteins, nucleic acids, membranes, and whole cells [14,20–24]. MG and MGA, also known as Firoin and Firoin-A, respectively, are the examples of carbohydrate extremolytes which play a role in the high temperature and osmotic adaptation of thermophilic and hyperthermophilic organisms [23,25]. The chemically reactive end of the sugar of this group of extremolytes forms a glycosidic bond with a hydroxyl group of glyceric acid or glyceramide. In microorganisms, the anionic MG is accumulated in response to heat stress, whereas uncharged MGA increases with elevated NaCl levels [26]. Here we report that the small stress molecules, especially MGA and ectoine, inhibit amyloid formation of PrP106–126 in vitro. In this study, the aggregation kinetics of the PrP106–126 fragment, which was thought to be responsible for its toxicity, was observed by turbidity assay. It was evinced that ectoine and MGA could inhibit aggregation of PrP106–126, whereas hydroxyectoine and MG hardly had any effect on the aggregation of this peptide fragment. The AFM analysis of the samples containing these two compounds also corroborated our findings of turbidity assay showing lower amount of aggregates. Cell viability assay was done to study whether these solutes have any protective effect on neuronal cells treated with the prion peptide aggregate. The results depicted that with the increase in concentration of ectoine and MGA, the cell viability also increased interpreting the protective role of these compounds. Thus, our findings can be useful for designing these naturally occurring compounds as inhibitors of prion amyloid formation, thereby yielding a protective tool against prion-induced cell death.
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Materials and methods In vitro PrP106–126 amyloid formation. Human PrP106–126 was purchased from Bachem (Athens, GA, USA) and ectoine, hydroxyectoine, MGA, and MG were provided by bitop AG (Witten, Germany). All other chemicals and reagents were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Stock solution of PrP106–126 was prepared as described earlier [19]. Briefly, PrP106–126 was dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), sonicated in water bath, and was then aliquoted in sterile eppendorf tubes. After initial drying at room temperature and further drying with nitrogen gas, it was stored at 20 C. PrP106–126 peptide was further dissolved in appropriate buffers at the desired concentrations as per experimental conditions. Turbidity assay. Samples with PrP106–126 (20 lM) in 200 mM phosphate buffer (PB), pH 5.5, at 37 C were co-incubated with or without any one of these solutes, namely—ectoine, hydroxyectoine, MG or MGA (each 100 mM), in a 1 ml cuvette. Aggregation was measured by turbidity at 600 nm vs. PB as a blank [7]. Samples were thoroughly mixed before each reading to suspend the fibrils and aggregates uniformly. Optical densities were measured after 2 and 5 days at ambient conditions. Aggregation kinetics study. Turbidity was measured for kinetics study of ectoine and MGA at different concentrations (25, 50, 75, and 100 mM). Samples containing PrP106–126 (20 lM) in 200 mM PB, pH 5.5 was coincubated with different concentrations of ectoine and MGA, at 37 C in a 1 ml cuvette. The absorbance measurements were carried out for 7 days, by recording the absorbance of the samples every 24 h, using UV–VIS spectrophotometer (Schimadzu Co., Kyoto, Japan). Atomic force microscopy (AFM). Samples were imaged using a multimode AFM equipped with a Nanoscope III controller (Digital Instruments Inc., CA, USA) according to [27]. For AFM analysis, PrP106–126 aggregates were formed in a 200 mM PB, pH 5.5 at a concentration of 400 lM at 37 C in 3 days with or without either ectoine (1 M) or MGA (1 M). Incubation was done in sealed glass vials to prevent any possible evaporation. Samples were prepared by placing 5 ll of the pre-incubated solution on freshly cleaved mica (Ted Pella Inc., Redding, CA) at room temperature for 60 s, washed twice with 50 ll of deionized water, and blown dried with nitrogen gas. Image data were obtained in tapping mode under ambient conditions at a scan frequency of 1–2 Hz with etched silicon TESP nanoprobes (Veeco Metrology, LLC, Santa Barbara, CA) operating at a resonant frequency of 306–444 kHz. A scan area of 5 · 5 lm was imaged for two different samples in each case. MTT assay. Human neuroblastoma cells (SH-SY5Y), provided by Dr. Michael Sierks at Arizona State University, were maintained in growth medium with 40% minimal essential medium (MEM), 40% Ham’s modified F-12, 18% fetal bovine serum (FBS), 1% L-glutamine (3.6 mM), and 1% penicillin/streptomycin antibiotics in 5% CO2 at 37 C. FBS, penicillin/ streptomycin antibiotics, and L-glutamine were purchased from Invitrogen (Carlsbad, CA, USA). MEM and Ham’s F-12 were purchased from Irvine Scientific (Santa Ana, CA, USA). The cells were cultured in flasks and were plated at a concentration of 104 cells/100 ll medium per well in a 96well polystyrene plate. The cells were incubated at 37 C for 24 h for attachment to the bottom of the wells. PrP106–126 (200 lM in 200 mM PB, pH 5.5) were 24 h pre-incubated with/without ectoine or MGA (at different concentrations) and further diluted 10 times in 200 mM PB, pH 7.4, yielding the concentrations as—PrP106–126, 20 lM; ectoine or MGA, 25 lM, 1 mM, 10 mM, and 100 mM. Upon addition to the cell cultures they were further diluted 10 times with final concentrations of PrP106–126 as 2 lM. The same volume of 200 mM PB, pH 7.4, was added to the control cultures. The final concentrations of the SSMs in the culture medium were 2.5 lM, 0.1 mM, 1.0 mM, and 10 mM, respectively. Then, the treated cells were incubated for an additional 48 h at 37 C. For the cell viability assay, 10 ll of 5 mg/ml MTT [(3-4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide] in 1· RPMI 1640 was added to each well, and cell survival was determined according to Shearman et al. [28]. The plates were centrifuged and the medium was aspirated after 3 h incubation at 37 C and the blue formazan crystals were dissolved with a solution of 2-propanol containing 0.1 N HCl (100 ll). The absorbance of
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each sample was measured at 560 nm using a multiwell Victor3 microplate reader (Perkin-Elmer Inc.). Four replicate wells were used for each sample and control. Each experiment was repeated twice, and the average value was taken. Cell viability was found by dividing the sample absorbance values by the control sample absorbance.
Results and discussion Molecules that stabilize the conformation of PrPc or destabilize the infected PrPsc conformation could be effective targets for therapeutics related to prion disorders. Increasing evidences reveal that there exist some stabilization effects of SSMs on proteins against heating as well as freeze-drying [14,20]. Therefore, it is conceivable that such SSMs can inhibit the amyloid formation of amyloidogenic proteins via stabilizing their monomers. In the present study we have selected four SSMs—ectoine, hydroxyectoine, MG, and MGA due to their unique properties. The chemical structures of these SSMs are shown in Fig. 1A. Due to the relation of PrP106–126 aggregation with toxicity, the aggregation kinetics of PrP106–126 was observed using turbidometry in this work. Absorbance was measured under ambient conditions at 48 h and at 120 h for samples containing PrP106–126 peptide in PB, co-incubated with or without ectoine, hydroxyectoine, MG, and MGA (Fig. 1B). The absorbance of samples of the prion peptide co-incubated with ectoine and MGA indicated a decrease in aggregation, whereas aggregation of the peptide fragment in the presence of hydroxyectoine and MG was similar in amount to that of control containing PrP106–126 only. According to initial turbidity data, ectoine and MGA were selected as possible inhibitors of prion aggregation and concentration effect on the aggre-
gate formation was further monitored for these compounds only (Fig. 2). The prion amyloid formation followed a typical sigmoidal curve with an initial lag phase, followed by elongation and saturation phase. The lag time for each sample increased with concentration of solutes except the sample containing PrP106–126 only. In amyloid aggregate formation process, the lag time indicated the formation of stable nuclei whereas the elongation phase implicated the growth of fibrils [29]. AFM analysis of the PrP106–126 samples co-incubated with or without either ectoine or MGA were done to visualize their morphology. PrP106–126 samples with or without either ectoine or MGA was incubated for 3 days and the representative images were taken for each sample (Fig. 3). It was clear from these images that in control samples higher amount of aggregates were found whereas the samples co-incubated with ectoine or MGA showed scarce amount of aggregation. Thus, from the turbidity assay (Fig. 2) and AFM analysis (Fig. 3) we can adumbrate that ectoine and MGA could be potential inhibitors of prion aggregation. As ectoine and MGA were found to exhibit the property of decreasing the cytotoxic aggregates of prion peptide, it becomes necessary to excavate whether they can be used safely for the treatment of prion disorders. As revealed from the earlier studies that these solutes were non-toxic at even high concentrations [16,17], we wanted to check whether they had any protective role for the cells treated with prion peptide. For this purpose, the viabilities of human neuroblastoma cells (SH-SY5Y) were found using MTT assay with PrP106–126 (2 lM) alone and samples co-incubated with ectoine or MGA at different sets of concentrations. To see the effect of only solutes, cells were also incubated with ectoine or MGA only in absence of
Fig. 1. (A) Chemical structures of SSMs—ectoine, hydroxyectoine, MG, and MGA. (B) Effect of small stress molecules (SSMs)—ectoine, hydroxyectoine, MG, and MGA on prion amyloid formation. Bar graph depicting turbidity of PrP106–126 (20 lM) amyloid formation when co-incubated with the SSMs (100 mM each) at 37 C.
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Fig. 2. Effect of concentration of (A) ectoine and (B) MGA on prion amyloid formation. Data shown are turbidity time profile for PrP106–126 (20 lM) co-incubated with these two solutes at concentrations of 25, 50, 75, and 100 mM, respectively.
PrP106–126. Cells co-incubated with ectoine or MGA together with PrP106–126 showed high survival rate compared to control samples (Fig. 4A and B). The sample con-
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taining 2 lM PrP106–126 showed about a 34% decrease in cell viability compared to the control cells, while the samples co-incubated with ectoine or MGA showed decrease in the viability ranging from 8% to 26% and 12% to 30% depending on the concentration of solute, respectively. These results illustrate that ectoine and MGA could be a potential inhibitor for prion-induced toxicity. The focus of our study was to find out the efficacy of small stress solutes as anti-amyloidogenic therapeutics for prion disorders. Our results indicated that ectoine and MGA partially inhibited the aggregation and neurotoxicity of PrP106–126, but hydroxyectoine and MG did not. Hydroxyectoine is more hydrophilic than ectoine because of hydroxyl group, and MG has negative charge on its carboxyl group. PrP106–126 consists of N-terminal polar head (KTNMKHM) and long hydrophobic tail (AGAAAAGAVVGGLG) and the latter is believed to be responsible for amyloid formation [6,8,30]. Considering the sequence of PrP106–126, more hydrophilic solutes like hydroxyectoine and MG seem to interact with only polar head of this peptide, therefore amyloid formation can occur via interaction of remaining hydrophobic tail. On the other hand, the inhibitory action of ectoine and MGA can be explained by preferential exclusion mechanism. Since our results depict that the behavior of solute was highly dependent on its structure, it could be inferred that the preferential binding property of the compatible solute could play a key role in the prion inhibition mechanism. Thus, it is hypothesized that the preferential hydration of PrP106–126 helps the solutes to interfere with the hydrogen bonded monomer–monomer interactions of PrPsc thereby depriving the binding energy necessary for the growth of b-sheet and hence thermodynamically controls the prion fibril formation. In conclusion, our findings with ectoine and MGA evidenced that they have successfully inhibited PrP106–126 amyloid formation and reduced their cytotoxicity in human neuroblastoma cells. The findings presented here on these naturally occurring organic solutes represent their ability to be used as potential drug candidates against prion diseases.
Fig. 3. AFM images of PrP106–126 samples (400 lM) incubated with SSMs (1 M each) for 3 days at 37 C. (A) Control. (B) Ectoine. (C) MGA. The images were taken at saturation phase in tapping mode (5 lm · 5 lm).
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Fig. 4. Dose–response of SH-SY5Y death induced by PrP106–126 (2 lM) with and without either (A) ectoine or (B) MGA at different concentrations. The final concentrations of these solutes in the cell culture media were 2.5 lM, 0.1 mM, 1.0 mM, and 10 mM, respectively. As a positive control, cell viability in the presence of the solutes only (10 mM, final concentration) was plotted. The error bars were calculated with a 95% confidence level. Statistical analysis was performed by means of one-way analysis of variance (ANOVA). A P-value less than or equal to 0.5 was considered statistically significant.
Acknowledgments This work was supported by grants from BioGreen 21 Program (20070301034038), Rural Development Administration, Republic of Korea, and bitop AG (GB08220), Germany. References [1] A. Aguzzi, M. Polymenidou, Mammalian prion biology: one century of evolving concepts, Cell 116 (2004) 313–327. [2] S.B. Prusiner, Prion diseases and the BSE crisis, Science 278 (1997) 245–251. [3] S.B. Prusiner, Prions, Proc. Natl. Acad. Sci. USA 95 (1998) 13363– 13383. [4] C. Kuwahara, A.M. Takeuchi, T. Nishimura, K. Haraguchi, A. Kubosaki, Y. Matsumoto, K. Saeki, Y. Matsumoto, T. Yokoyama, S. Itohara, T. Onodera, Prions prevent neuronal cell-line death, Nature 400 (1999) 225–226. [5] D.A. Harris, H.L. True, New insights into prion structure and toxicity, Neuron 50 (2006) 353–357. [6] M.F. Jobling, L.R. Stewart, A.R. White, C. McLean, A. Friedhuber, F. Maher, K. Beyreuther, C.L. Masters, C.J. Barrow, S.J. Collins, R. Cappai, The hydrophobic core sequence modulates the neurotoxic and secondary structure properties of the prion peptide 106–126, J. Neurochem. 73 (1999) 1557–1565. [7] M. Salmona, P. Malesani, L. De Gioia, S. Gorla, M. Bruschi, A. Molinari, F. Della Vedova, B. Pedrotti, M.A. Marrari, T. Awan, O. Bugiani, G. Forloni, F. Tagliavini, Molecular determinants of the physicochemical properties of a critical prion protein region comprising residues 106–126, Biochem. J. 342 (1999) 207–214. [8] F. Tagliavini, F. Prelli, L. Verga, G. Giaccone, R. Sarma, P. Gorevic, B. Ghetti, F. Passerini, E. Ghibaudi, G. Forloni, M. Salmona, O.
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Biochemical and Biophysical Research Communications 365 (2008) 808–813 www.elsevier.com/locate/ybbrc
Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106–126 Mathumai Kanapathipillai a, Sook Hee Ku b, Koyeli Girigoswami b, Chan Beum Park b
a,b,*
a Department of Chemical and Materials Engineering, Arizona State University, P.O. Box 876006, Tempe, AZ 85287, USA Institute for the Bio-Century and Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
Received 6 November 2007 Available online 26 November 2007
Abstract In prion diseases, the posttranslational modification of host-encoded prion protein PrPc yields a high b-sheet content modified protein PrPsc, which further polymerizes into amyloid fibrils. PrP106–126 initiates the conformational changes leading to the conversion of PrPc to PrPsc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106–126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106–126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity. 2007 Elsevier Inc. All rights reserved. Keywords: Prion; Small stress molecules; Ectoine; Mannosylglyceramide; Cytotoxicity
Prions are infectious particles that cause transmissible spongiform encephalopathies in animals and humans [1]. The infectious prions are composed of PrPsc, a conformationally altered form of a host-encoded glycoprotein PrPc [2,3]. PrPc is modified posttranslationally to yield a high b-sheet content structure that further polymerizes into amyloid fibrils. Although the two isoforms are chemically identical, they possess very different physicochemical properties. The mechanism by which PrPc conAbbreviations: HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; SSM, small stress molecule; MG, mannosylglycerate; MGA, mannosylglyceramide; AFM, atomic force microscopy; PrPc, normal cellular prion protein; PrPsc, infectious isoform of PrPc; PB, phosphate buffer. * Corresponding author. Address: Institute for the Bio-Century and Department of Materials Science and Engineering, Korea Advanced Institute of Science and Technology, Guseong-dong, Daejeon 305-701, Republic of Korea. Fax: +82 42 869 3310. E-mail address: [email protected] (C.B. Park). 0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.11.074
tributes to prion-induced neurotoxicity still remains unclear. One hypothesis is that, PrPc normally possesses a neuroprotective function [4] that is abolished or subverted by interaction with PrPsc [5]. Due to the toxicity and handling difficulties of the whole prion protein, several PrP peptide fragments were used for in vitro studies. Among those, PrP106–126 is conserved among various species and is suggested to be one of the important regions of conformational change initiation, leading to the conversion of PrPc to PrPsc [6,7]. PrP106–126 also exhibited a combination of b-sheet and random coil structures under different experimental conditions [8,9]. It aggregates into protease-resistant amyloid fibrils thereby inducing neuronal cell death by apoptosis, and causing proliferation and hypertrophy of cultured glia [10,11]. Furthermore, the toxicity of PrP106–126 is similar to that of PrPsc as both require the expression of PrPc to cause cell death [12,13].
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Molecules, which can stabilize the structure of PrPc or convert PrPsc into the normal form, can be potential drug candidates for prion related diseases. Small stress molecules (SSMs) are formed in microorganisms during stressed conditions to prevent protein denaturation and hence to maintain protein stability and function [14,15]. Furthermore, these naturally occurring solutes are known to be non-toxic to cellular environment even at concentrations as high as 1 M, which makes them more promising for drug design [16,17]. Recently, we reported that several SSMs could successfully inhibit the amyloid formation of bovine insulin and Alzheimer’s Ab42 peptides, and thereby reduce the neurotoxicity of Ab42 aggregates [18,19]. Here, our study explores, initially, the role of four SSMs—ectoine, hydroxyectoine, mannosylglyceramide (MGA), and mannosylglycerate (MG) during PrP106– 126 aggregation. The ectoines (ectoine and hydroxyectoine) are highly water-soluble, zwitterionic low-molecular weight molecules. They are biologically inert, do not interfere with most enzymatic and binding reactions, and are also highly compatible with cell metabolism. Ectoine demonstrates its protective effect on proteins and enzymes through protection from thermal stress, proteolysis, and change of pH or the salt concentration. They also exert their stabilizing effect on different proteins, nucleic acids, membranes, and whole cells [14,20–24]. MG and MGA, also known as Firoin and Firoin-A, respectively, are the examples of carbohydrate extremolytes which play a role in the high temperature and osmotic adaptation of thermophilic and hyperthermophilic organisms [23,25]. The chemically reactive end of the sugar of this group of extremolytes forms a glycosidic bond with a hydroxyl group of glyceric acid or glyceramide. In microorganisms, the anionic MG is accumulated in response to heat stress, whereas uncharged MGA increases with elevated NaCl levels [26]. Here we report that the small stress molecules, especially MGA and ectoine, inhibit amyloid formation of PrP106–126 in vitro. In this study, the aggregation kinetics of the PrP106–126 fragment, which was thought to be responsible for its toxicity, was observed by turbidity assay. It was evinced that ectoine and MGA could inhibit aggregation of PrP106–126, whereas hydroxyectoine and MG hardly had any effect on the aggregation of this peptide fragment. The AFM analysis of the samples containing these two compounds also corroborated our findings of turbidity assay showing lower amount of aggregates. Cell viability assay was done to study whether these solutes have any protective effect on neuronal cells treated with the prion peptide aggregate. The results depicted that with the increase in concentration of ectoine and MGA, the cell viability also increased interpreting the protective role of these compounds. Thus, our findings can be useful for designing these naturally occurring compounds as inhibitors of prion amyloid formation, thereby yielding a protective tool against prion-induced cell death.
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Materials and methods In vitro PrP106–126 amyloid formation. Human PrP106–126 was purchased from Bachem (Athens, GA, USA) and ectoine, hydroxyectoine, MGA, and MG were provided by bitop AG (Witten, Germany). All other chemicals and reagents were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Stock solution of PrP106–126 was prepared as described earlier [19]. Briefly, PrP106–126 was dissolved in 100% 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), sonicated in water bath, and was then aliquoted in sterile eppendorf tubes. After initial drying at room temperature and further drying with nitrogen gas, it was stored at 20 C. PrP106–126 peptide was further dissolved in appropriate buffers at the desired concentrations as per experimental conditions. Turbidity assay. Samples with PrP106–126 (20 lM) in 200 mM phosphate buffer (PB), pH 5.5, at 37 C were co-incubated with or without any one of these solutes, namely—ectoine, hydroxyectoine, MG or MGA (each 100 mM), in a 1 ml cuvette. Aggregation was measured by turbidity at 600 nm vs. PB as a blank [7]. Samples were thoroughly mixed before each reading to suspend the fibrils and aggregates uniformly. Optical densities were measured after 2 and 5 days at ambient conditions. Aggregation kinetics study. Turbidity was measured for kinetics study of ectoine and MGA at different concentrations (25, 50, 75, and 100 mM). Samples containing PrP106–126 (20 lM) in 200 mM PB, pH 5.5 was coincubated with different concentrations of ectoine and MGA, at 37 C in a 1 ml cuvette. The absorbance measurements were carried out for 7 days, by recording the absorbance of the samples every 24 h, using UV–VIS spectrophotometer (Schimadzu Co., Kyoto, Japan). Atomic force microscopy (AFM). Samples were imaged using a multimode AFM equipped with a Nanoscope III controller (Digital Instruments Inc., CA, USA) according to [27]. For AFM analysis, PrP106–126 aggregates were formed in a 200 mM PB, pH 5.5 at a concentration of 400 lM at 37 C in 3 days with or without either ectoine (1 M) or MGA (1 M). Incubation was done in sealed glass vials to prevent any possible evaporation. Samples were prepared by placing 5 ll of the pre-incubated solution on freshly cleaved mica (Ted Pella Inc., Redding, CA) at room temperature for 60 s, washed twice with 50 ll of deionized water, and blown dried with nitrogen gas. Image data were obtained in tapping mode under ambient conditions at a scan frequency of 1–2 Hz with etched silicon TESP nanoprobes (Veeco Metrology, LLC, Santa Barbara, CA) operating at a resonant frequency of 306–444 kHz. A scan area of 5 · 5 lm was imaged for two different samples in each case. MTT assay. Human neuroblastoma cells (SH-SY5Y), provided by Dr. Michael Sierks at Arizona State University, were maintained in growth medium with 40% minimal essential medium (MEM), 40% Ham’s modified F-12, 18% fetal bovine serum (FBS), 1% L-glutamine (3.6 mM), and 1% penicillin/streptomycin antibiotics in 5% CO2 at 37 C. FBS, penicillin/ streptomycin antibiotics, and L-glutamine were purchased from Invitrogen (Carlsbad, CA, USA). MEM and Ham’s F-12 were purchased from Irvine Scientific (Santa Ana, CA, USA). The cells were cultured in flasks and were plated at a concentration of 104 cells/100 ll medium per well in a 96well polystyrene plate. The cells were incubated at 37 C for 24 h for attachment to the bottom of the wells. PrP106–126 (200 lM in 200 mM PB, pH 5.5) were 24 h pre-incubated with/without ectoine or MGA (at different concentrations) and further diluted 10 times in 200 mM PB, pH 7.4, yielding the concentrations as—PrP106–126, 20 lM; ectoine or MGA, 25 lM, 1 mM, 10 mM, and 100 mM. Upon addition to the cell cultures they were further diluted 10 times with final concentrations of PrP106–126 as 2 lM. The same volume of 200 mM PB, pH 7.4, was added to the control cultures. The final concentrations of the SSMs in the culture medium were 2.5 lM, 0.1 mM, 1.0 mM, and 10 mM, respectively. Then, the treated cells were incubated for an additional 48 h at 37 C. For the cell viability assay, 10 ll of 5 mg/ml MTT [(3-4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide] in 1· RPMI 1640 was added to each well, and cell survival was determined according to Shearman et al. [28]. The plates were centrifuged and the medium was aspirated after 3 h incubation at 37 C and the blue formazan crystals were dissolved with a solution of 2-propanol containing 0.1 N HCl (100 ll). The absorbance of
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each sample was measured at 560 nm using a multiwell Victor3 microplate reader (Perkin-Elmer Inc.). Four replicate wells were used for each sample and control. Each experiment was repeated twice, and the average value was taken. Cell viability was found by dividing the sample absorbance values by the control sample absorbance.
Results and discussion Molecules that stabilize the conformation of PrPc or destabilize the infected PrPsc conformation could be effective targets for therapeutics related to prion disorders. Increasing evidences reveal that there exist some stabilization effects of SSMs on proteins against heating as well as freeze-drying [14,20]. Therefore, it is conceivable that such SSMs can inhibit the amyloid formation of amyloidogenic proteins via stabilizing their monomers. In the present study we have selected four SSMs—ectoine, hydroxyectoine, MG, and MGA due to their unique properties. The chemical structures of these SSMs are shown in Fig. 1A. Due to the relation of PrP106–126 aggregation with toxicity, the aggregation kinetics of PrP106–126 was observed using turbidometry in this work. Absorbance was measured under ambient conditions at 48 h and at 120 h for samples containing PrP106–126 peptide in PB, co-incubated with or without ectoine, hydroxyectoine, MG, and MGA (Fig. 1B). The absorbance of samples of the prion peptide co-incubated with ectoine and MGA indicated a decrease in aggregation, whereas aggregation of the peptide fragment in the presence of hydroxyectoine and MG was similar in amount to that of control containing PrP106–126 only. According to initial turbidity data, ectoine and MGA were selected as possible inhibitors of prion aggregation and concentration effect on the aggre-
gate formation was further monitored for these compounds only (Fig. 2). The prion amyloid formation followed a typical sigmoidal curve with an initial lag phase, followed by elongation and saturation phase. The lag time for each sample increased with concentration of solutes except the sample containing PrP106–126 only. In amyloid aggregate formation process, the lag time indicated the formation of stable nuclei whereas the elongation phase implicated the growth of fibrils [29]. AFM analysis of the PrP106–126 samples co-incubated with or without either ectoine or MGA were done to visualize their morphology. PrP106–126 samples with or without either ectoine or MGA was incubated for 3 days and the representative images were taken for each sample (Fig. 3). It was clear from these images that in control samples higher amount of aggregates were found whereas the samples co-incubated with ectoine or MGA showed scarce amount of aggregation. Thus, from the turbidity assay (Fig. 2) and AFM analysis (Fig. 3) we can adumbrate that ectoine and MGA could be potential inhibitors of prion aggregation. As ectoine and MGA were found to exhibit the property of decreasing the cytotoxic aggregates of prion peptide, it becomes necessary to excavate whether they can be used safely for the treatment of prion disorders. As revealed from the earlier studies that these solutes were non-toxic at even high concentrations [16,17], we wanted to check whether they had any protective role for the cells treated with prion peptide. For this purpose, the viabilities of human neuroblastoma cells (SH-SY5Y) were found using MTT assay with PrP106–126 (2 lM) alone and samples co-incubated with ectoine or MGA at different sets of concentrations. To see the effect of only solutes, cells were also incubated with ectoine or MGA only in absence of
Fig. 1. (A) Chemical structures of SSMs—ectoine, hydroxyectoine, MG, and MGA. (B) Effect of small stress molecules (SSMs)—ectoine, hydroxyectoine, MG, and MGA on prion amyloid formation. Bar graph depicting turbidity of PrP106–126 (20 lM) amyloid formation when co-incubated with the SSMs (100 mM each) at 37 C.
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Fig. 2. Effect of concentration of (A) ectoine and (B) MGA on prion amyloid formation. Data shown are turbidity time profile for PrP106–126 (20 lM) co-incubated with these two solutes at concentrations of 25, 50, 75, and 100 mM, respectively.
PrP106–126. Cells co-incubated with ectoine or MGA together with PrP106–126 showed high survival rate compared to control samples (Fig. 4A and B). The sample con-
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taining 2 lM PrP106–126 showed about a 34% decrease in cell viability compared to the control cells, while the samples co-incubated with ectoine or MGA showed decrease in the viability ranging from 8% to 26% and 12% to 30% depending on the concentration of solute, respectively. These results illustrate that ectoine and MGA could be a potential inhibitor for prion-induced toxicity. The focus of our study was to find out the efficacy of small stress solutes as anti-amyloidogenic therapeutics for prion disorders. Our results indicated that ectoine and MGA partially inhibited the aggregation and neurotoxicity of PrP106–126, but hydroxyectoine and MG did not. Hydroxyectoine is more hydrophilic than ectoine because of hydroxyl group, and MG has negative charge on its carboxyl group. PrP106–126 consists of N-terminal polar head (KTNMKHM) and long hydrophobic tail (AGAAAAGAVVGGLG) and the latter is believed to be responsible for amyloid formation [6,8,30]. Considering the sequence of PrP106–126, more hydrophilic solutes like hydroxyectoine and MG seem to interact with only polar head of this peptide, therefore amyloid formation can occur via interaction of remaining hydrophobic tail. On the other hand, the inhibitory action of ectoine and MGA can be explained by preferential exclusion mechanism. Since our results depict that the behavior of solute was highly dependent on its structure, it could be inferred that the preferential binding property of the compatible solute could play a key role in the prion inhibition mechanism. Thus, it is hypothesized that the preferential hydration of PrP106–126 helps the solutes to interfere with the hydrogen bonded monomer–monomer interactions of PrPsc thereby depriving the binding energy necessary for the growth of b-sheet and hence thermodynamically controls the prion fibril formation. In conclusion, our findings with ectoine and MGA evidenced that they have successfully inhibited PrP106–126 amyloid formation and reduced their cytotoxicity in human neuroblastoma cells. The findings presented here on these naturally occurring organic solutes represent their ability to be used as potential drug candidates against prion diseases.
Fig. 3. AFM images of PrP106–126 samples (400 lM) incubated with SSMs (1 M each) for 3 days at 37 C. (A) Control. (B) Ectoine. (C) MGA. The images were taken at saturation phase in tapping mode (5 lm · 5 lm).
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Fig. 4. Dose–response of SH-SY5Y death induced by PrP106–126 (2 lM) with and without either (A) ectoine or (B) MGA at different concentrations. The final concentrations of these solutes in the cell culture media were 2.5 lM, 0.1 mM, 1.0 mM, and 10 mM, respectively. As a positive control, cell viability in the presence of the solutes only (10 mM, final concentration) was plotted. The error bars were calculated with a 95% confidence level. Statistical analysis was performed by means of one-way analysis of variance (ANOVA). A P-value less than or equal to 0.5 was considered statistically significant.
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