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α-Synuclein-mediated defense against oxidative stress via modulation of glutathione peroxidase
Biochimica et Biophysica Acta 1834 (2013) 972–976
Contents lists available at SciVerse ScienceDirect
Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap
α-Synuclein-mediated defense against oxidative stress via modulation of glutathione peroxidase Hee Jung Koo 1, Jee Eun Yang 1, Jae Hyung Park, Daekyun Lee, Seung R. Paik ⁎ School of Chemical and Biological Engineering, Institute of Chemical Processes, College of Engineering, Seoul National University, Seoul 151-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 November 2012 Received in revised form 28 February 2013 Accepted 7 March 2013 Available online 16 March 2013 Keywords: α-Synuclein Glutathione peroxidase Oxidative stress Amyloidogenesis Parkinson's disease
a b s t r a c t In this report, mutual effect of α-synuclein and GPX-1 is investigated to unveil their involvement in the PD pathogenesis in terms of cellular defense mechanism against oxidative stress. Biochemical and immunocytochemical studies showed that α-synuclein enhanced the GPX-1 activity with Kd of 17.3 nM and the enzyme in turn markedly enhanced in vitro fibrillation of α-synuclein. Transmission electron microscopy revealed the fibrillar meshwork of α-synuclein containing GPX-1 located in locally concentrated islets. The entrapped enzyme was demonstrated to be protected in a latent form and its activity was fully recovered as released from the matrix. Therefore, novel defensive roles of α-synuclein and its amyloid fibrils against oxidative stress are suggested as the GPX-1 stimulator and the active depot for the enzyme, respectively. © 2013 Elsevier B.V. All rights reserved.
1. Introduction α-Synuclein is a natively unstructured protein that is abundant in presynaptic nerve terminals. This protein has significant implication in the pathogenesis of Parkinson's disease (PD) by participating in the formation of radiating amyloid fibrils found on Lewy bodies (LBs), a pathological signature of the disease [1,2]. Mechanism of how this disordered protein is polymerized into the highly ordered suprastructure is yet to be fully understood, and pathological roles of the fibrillar aggregates are still controversial. Oxidative stress has been suggested to be a part of PD pathogenesis by influencing the α-synuclein amyloidogenesis. Reactive oxygen species (ROS) resulting from mitochondrial dysfunction or dopamine hydrolysis lead to oxidative modification of α-synuclein, affecting its fibrillation process [3–5]. Tyrosine nitration and dityrosine cross-link of α-synuclein result in accumulation of the modified aggregative species and subsequent accelerated aggregation under the condition of impaired proteasomal degradation in PD [6–8]. It becomes apparent that cellular oxidative system is crucial for the pathogenesis of PD by processing ROS.
Abbreviations: GPX-1, glutathione peroxidase-1; PD, Parkinson's disease; LBs, Lewy bodies; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, glutathione disulfide ⁎ Corresponding author at: School of Chemical and Biological Engineering, Seoul National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151-744, Republic of Korea. Tel.: +82 2 880 7402; fax: +82 2 888 1604. E-mail address: [email protected] (S.R. Paik). 1 These authors contributed equally to this study. 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.03.008
However, a direct connection of α-synuclein and its fibrillation with cellular anti-oxidative system has been rarely investigated. Selenocysteine-containing glutathione peroxidases (GPXs) are important antioxidant enzymes protecting tissues from oxidative damages by removing hydrogen peroxide and a range of organic peroxides. Among several isoforms of GPXs with diverse tissue and substrate specificities, the homotetrameric GPX-1 is the most abundant cytoplasmic enzyme [9]. GPX-1 primarily catalyzes the reaction of converting hydrogen peroxide to water by oxidizing reduced glutathione (GSH) to glutathione disulfide (GSSG) in cytoplasm [10]. GPX-1 has only a few interactive proteins reported. Non-receptor tyrosine kinases such as c-Abl and Arg were found to modulate GPX-1 activity by the kinase-mediated phosphorylation [11]. Selenium binding protein-1 bound to GPX-1 and repressed its antioxidant activity [12]. Because of its crucial role in coping with oxidative stress, GPX-1 has been implicated in development of many diseases and their prevention, which includes cancer, neurodegenerative diseases, and cardiovascular disease [13,14]. Recent finding revealed that GPX-1 was co-localized with α-synuclein in LBs found in the brain tissues of PD patients, suggesting a neuroprotective role of GPX-1 toward the disease [15]. Since molecular-level inter-relationship between the two co-localized proteins is basically unknown, their mutual effects have been investigated in this study by assessing the accelerated fibrillation of α-synuclein by GPX-1 and the stimulated GPX-1 activity by α-synuclein. The results have led us to suggest an anti-oxidative role of α-synuclein and its fibrillation by modulating GPX-1, which may provide us a novel insight into pathophysiological role(s) of
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α-synuclein and GPX-1 in terms of controlling both amyloidogenesis and oxidative stress.
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3. Results and discussion 3.1. α-Synuclein enhances antioxidant activity of glutathione peroxidase
2. Materials and methods 2.1. Materials Human recombinant α-synuclein was prepared according to the procedure previously described [16]. GPX-1 (bovine erythrocytes), glutathione reductase, GSH, cumen hydroperoxide, and NADPH were from Sigma. Mouse anti-α-synuclein and goat anti-GPX-1 antibodies were obtained from Santa Cruz, and goat anti-mouse Alexa Fluor 488 and rabbit anti-goat Alexa Fluor 633 were from Invitrogen. Gold (10 nm)-conjugated anti-goat antibody was provided by Abcam. Thioflavin-T was purchased from Sigma.
To investigate mutual interactions between α-synuclein and GPX-1, we first examined an effect of α-synuclein on the antioxidant activity of GPX-1. The GPX-1 activity was monitored with a coupling system employing cumene hydroxide and GSH in the presence of
2.2. GPX-1 assay GPX-1 activity was measured by a coupling system employing cumen hydroperoxide and GSH in the presence of glutathione reductase and NADPH. Oxidized glutathione formed by GPX-1 reaction (H2O2 + 2GSH → GSSG + 2H2O) was continuously reduced by glutathione reductase activity (GSSG + NADPH + H+ → 2GSH + NADP+). GPX-1 activity was calculated from the decrease in NADPH absorbance at 340 nm. The assay mixture included 0.1 mM each of cumen hydroperoxide, GSH, and NADPH and 0.1 unit glutathione reductase in 50 mM Tris/ Cl (pH 7.6) containing 5 mM EDTA. Protein samples were lastly added into the assay mixture and the measurement was performed immediately. 2.3. Amyloid fibril formation 20 μM α-synuclein was incubated in the absence and presence of various amounts of GPX-1 at 37 °C with vigorous shaking in 20 mM MES, pH 6.5. Amyloid fibrillation was monitored by thioflavin-T binding fluorescence at 482 nm with an excitation at 450 nm. An aliquot (20 μl) of the reaction mixture was combined with thioflavin-T (5 μM) in 50 mM glycine (pH 8.5) to a final volume of 200 μl, and then the fluorescence intensity was measured with LS-55 luminescence spectrometer (Perkin-Elmer). Morphological analyses of amyloid fibrils were carried out with JEM1010 transmission electron microscope (JEOL). An aliquot (20 μl) of the sample was adsorbed onto a 200 mesh carbon-coated copper grid (Ted Pella Inc.) for 3 min, and the excess liquid was removed with a filter paper. The adsorbed sample was then negatively stained with 2% uranyl acetate for 1 min in the dark. 2.4. Immunochemical analyses For immunocytochemistry, neuroblastoma SH-SY5Y cells cultured on the coverslips were fixed in ice-cold methanol/acetone solution (1:1) at −20 °C for 10 min, washed and blocked with a blocking buffer (1% bovine serum albumin in phosphate buffered saline) for 30 min at 37 °C. The fixed cells were incubated in the blocking buffer containing mouse anti-α-synuclein antibody and goat anti-GPX-1 antibody for 2 h, and then incubated with the fluorescent secondary antibodies of anti-mouse Alexa Fluor 488 and rabbit anti-goat Alexa Fluor 633 for 1 h in the dark. After washing with the blocking buffer, the cells were mounted with a mounting medium and examined using a LSM510 confocal microscope (Carl Zeiss). For immunogold labeling of the entrapped GPX-1 within amyloid fibrils, the sample was adsorbed onto a carbon-coated copper grid for 15 min at room temperature. After removing excess liquid with a filter paper, the adsorbed sample was incubated successively with goat anti-GPX-1 antibody and gold-conjugated anti-goat antibody for 1 h each. After washing with 20 mM MES (pH 6.5), the adsorbed sample was negatively stained with 2% uranyl acetate and analyzed using a transmission electron microscope.
Fig. 1. α-Synuclein-mediated enhancement of GPX-1 activity. (A) GPX-1 activities with increased α-synuclein concentrations. GPX-1 (5 nM) was pre-incubated with 0 (black), 56 (red), 112 (blue), 224 (cyan), 280 (green), and 560 nM (pink) of α-synuclein at 4 °C for 20 min, and the enzyme activity was monitored by observing the decrease in NADPH absorbance at 340 nm. (B) Initial activities of GPX-1 are plotted as a function of α-synuclein in molarity. Double reciprocal plot was performed to obtain the dissociation constant between GPX-1 and α-synuclein (inset). (C) Immunocytochemical analysis of SH-SY5Y neuroblastoma cell-line. Co-localization of GPX-1 and α-synuclein was visualized by staining cells with anti-α-synuclein and anti-GPX-1 antibodies. Scale bar, 50 μm.
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glutathione reductase/NADPH [17]. The enzyme was pre-incubated with α-synuclein at 4 °C for 20 min before assessed by observing the decrease in NADPH absorbance at 340 nm due to the reduction of GSSG back to GSH by glutathione reductase. Significant enhancement of GPX-1 activity was observed with α-synuclein (Fig. 1). As α-synuclein level increased, GPX-1 activity became continuously increased as evidenced by the increased negative slopes reflecting enhanced NADPH consumption (Fig. 1A). Neither glutathione reductase activity nor oxidation of NADPH was affected by α-synuclein. Direct molecular interaction between α-synuclein and GPX-1 was evaluated by plotting the initial activities of GPX-1 obtained as changes of moles of NADPH per min per mg protein as a function of α-synuclein in molarity (Fig. 1B). The enhanced GPX-1 activities exhibited a hyperbolic relationship with α-synuclein, indicating a possible specific binding between the two proteins. By performing a double reciprocal plot, their dissociation constant was estimated as 17.3 nM (Fig. 1B, inset). The α-synuclein-mediated enhancement of GPX-1 activity and the exceptionally high molecular affinity would suggest a novel physiological role of α-synuclein in cellular anti-oxidative processes. In fact, a direct and tight interaction between the two proteins under physiological condition was demonstrated with immunocytochemcial analysis of neuroblastoma cell-line, SH-SY5Y. Confocal microscopic images of immunofluorescence-stained cells with anti-α-synuclein and anti-GPX-1 antibodies showed that both intrinsic proteins were found to be co-localized in SH-SY5Y cells (Fig. 1C). Taken together, these results allow us to suggest a physiological function of α-synuclein as a GPX's natural partner in cellular environment working as a GPX-1 activator and thus acting as an anti-oxidative agent. 3.2. Glutathione peroxidase accelerates amyloid fibrillation of α-synuclein Since GPX-1 was found to exist in α-synuclein-enriched Lewy bodies [15], GPX-1 effect on the amyloidogenesis of α-synuclein has been examined. Typical kinetic study using amyloid-specific Thioflavin-T revealed that in vitro fibrillation of α-synuclein was remarkably accelerated by GPX-1 in a dose-dependent manner (Fig. 2A). Substoichiometric amount of GPX-1 at 0.05 μM was sufficient to show the facilitated fibrillation of α-synuclein at 20 μM by shortening the lag period and increasing the final Thioflavin-T binding fluorescence. As the GPX-1 level increased twice, the lag further decreased to 20 h from 40 h obtained at its absence. Moreover, the final Thioflavin-T binding fluorescence also increased by almost two-fold from that of the kinetics obtained with α-synuclein alone. Based on the high affinity with Kd at nM range, the tight α-synuclein-GPX-1 complex would provide a nucleation center for the accelerated and enhanced amyloidogenesis although it cannot be completely ruled out that GPX-1 itself could act as a template at which α-synuclein continuously turns into an aggregative form. To demonstrate whether the activity of GPX-1 is crucial for the GPX-1-mediated acceleration of α-synuclein fibrillation, the fibrillation kinetics was carried out with GPX-1 in varying activities obtained via pre-incubating the enzyme at 37 °C by itself (Supplementary Fig. S1). As the enzyme with decreasing activities from 100%, 50%, to 25% was added to the fibrillation mixture at a molar ratio of 1:200 between GPX-1 and α-synuclein, the shortened lag period was extended from 13.1 h to 15.6 and 25.5 h, respectively, whereas it required about 40 h of the lag for the protein to be fibrillated in the absence of the enzyme (Fig. 2B). In addition, the augmented final fluorescence obtained with the enzyme at its full activity proportionally decreased as the enzymatic activity was inactivated (Fig. 2B). These findings clearly indicate that the biochemically active GPX-1 is required for the enzyme-mediated acceleration of α-synuclein fibrillation by both shortening the lag phase and increasing the final fibrillation level. Addition of
Fig. 2. GPX-1-mediated acceleration of the amyloid fibrillation of α-synuclein. (A) Aggregation kinetics of α-synuclein in the presence of GPX-1. α-Synuclein (20 μM) was incubated with 0.05 (▼) and 0.1 μM GPX-1 (△) at 37 °C with vigorous shaking. Amyloid fibrillation was monitored by thioflavin-T binding fluorescence at 482 nm (emission at 450 nm) during incubation. α-Synuclein (●) and GPX-1 (○) were separately incubated as controls. (B) GPX-1 activity-dependency of the amyloid fibrillation of α-synuclein. Partially inactivated GPX-1 was obtained at various time points during pre-incubation of the enzyme alone at 37 °C. α-Synuclein (20 μM) was fibrillated with 0.1 μM GPX-1 with decreasing activities from 100% (○), 50% (▼), and 25% (△).
completely inactivated GPX-1 obtained via heating at 100 °C resulted in a fibrillation kinetics indistinguishable from that of α-synuclein alone (Supplementary Fig. S2). 3.3. Entangled fibrillar aggregates of α-synuclein acting as a depot for active GPX-1 Morphological comparison of the final fibrillar aggregates of α-synuclein obtained in the absence and presence of GPX-1 revealed that the fibrils resulted from the accelerated fibrillation with the enzyme were densely entangled (Fig. 3A). Immunogold labeling was performed with anti-GPX-1 antibody in order to evaluate the GPX-1 distribution within the entanglement. The enzymes were found to be locally clustered into the GPX-1-islets within the fibrillar meshwork (Fig. 3B). This entrapment of GPX-1 into the α-synuclein fibrils which has occurred during the in vitro fibrillation process is compatible with the previous observation of their co-existence in the Lewy bodies of PD patient's brain [15]. Since the enhanced fibrillation of α-synuclein has required the active GPX-1, the enzymatic activity of the entrapped GPX-1 was examined by resuspending the GPX-1-containing fibrils in the assay mixture, and monitored for the decrease of NADPH absorbance at 340 nm. Impressively, the entrapped GPX-1 retained most of their activities during the prolonged incubation of the fibrillation for 70 h while the activity was almost completely vanished following only
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Fig. 3. Localization of GPX-1 within entangled α-synuclein fibrils. (A) Morphologies of GPX-1-induced α-synuclein fibrils. Scale bar, 0.5 μm. (B) Distribution of GPX-1 within the entangled fibrils. Immunogold labeling was performed with anti-GPX-1 and gold-conjugated secondary antibodies. Dashed circles indicate GPX-1-islets. All samples for TEM analysis were negatively stained with 2% uranyl acetate on carbon-coated copper grids. Scale bar, 200 nm.
2 h of incubation at 37 °C in the absence of the fibrils (Fig. 4A and Supplementary Fig. S1). The time course of the entrapped GPX-1 activity exhibited hysteresis, indicating that the NADPH consumption gradually increased as a function of time (Fig. 4B). By plotting slopes of the tangent lines of the activity trace at various time points, the GPX-1 activity previously entrapped in the matrix was shown to increase and then the activity remained unaffected when the substrate was completely consumed (Fig. 4B inset). To account for the hysteresis in activity, the enzyme-entrapped fibrillar matrix was successively extracted with a fixed amount (100 μl) of 20 mM MES, pH 6.5, and the GPX-1 activity in the extracts was measured. Interestingly, the extracted activity also exhibited the hysteresis when compared with a control of GPX-1 (Fig. 4C). As the extraction proceeded, the GPX-1 was continuously released in solution with subsequently diminished activities. In addition, the hysteresis became less apparent as the extracted GPX-1 activity decreased (Fig. 4C inset). This result indicates that the GPX-1 would be entrapped in a possible latent form within the fibrillar matrix, which can be slowly activated upon its release. Moreover, the gradual increase in the GPX-1 activity observed with the GPX-1 containing matrix (Fig. 4B) would be also due to the slow release of the enzyme from the matrix. Based on our findings, antioxidative function of α-synuclein has been suggested in terms of mutual activation between GPX-1 and
Fig. 4. Enzymatic activity of the entrapped GPX-1. (A) Protection of GPX-1 activity within the fibrillar matrix. GPX-1 activity was monitored after the α-synuclein fibrillation was carried out in the presence of GPX-1 for 3 days at 37 °C. Initial and 2 h-aged activities of GPX-1 at 37 °C were shown as controls. (B) Time course of the entrapped GPX-1 activity. The GPX-1-containing fibrils produced after 70 h fibrillation were obtained by centrifugation at 16,100 ×g for 30 min, and resuspended in the GPX-1 assay mixture. The enzyme activity was monitored by observing the decrease in NADPH absorbance at 340 nm. The inset is the result of a polynomial fitting and its first derivative plotting of the pink line. (C) Reactivation of GPX-1 released from fibrils. Successive extraction of the GPX-1-entrapped fibrillar matrix was carried out with 100 μl of 20 mM MES (pH 6.5) and each extract was subjected to the activity measurement. Black-dashed lines indicate initial and final activities of the released enzyme in the first extract. GPX-1 without the prior entrapment was used as a control (red). The enzyme activities in the successive extracts were shown (inset); number indicates the extraction order.
amyloidogenesis of α-synuclein (Scheme 1). The H2O2-removing process of GPX-1 is significantly activated by interacting with α-synuclein, and the α-synuclein-GPX-1 complex provides a possible nucleation center for the accelerated amyloid fibril formation of
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funded by the Ministry of Education, Science, and Technology (2012-0002064 and 2012R1A2A2A01013582). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2013.03.008. References
Scheme 1. Mutual relationship between GPX-1 and the amyloidogenesis of α-synuclein.
α-synuclein, which results in the entangled filamentous aggregates with the GPX-1 deposited in a locally concentrated fashion. In addition, we have previously reported that oxidized GSH (GSSG) also enhanced the amyloidogenesis of α-synuclein [18]. Taken together, the physiological function of α-synuclein and its amyloidogenesis can be understood with respect to the anti-oxidative GPX-1 system including glutathione turnover. Therefore, it can be considered that the mutual effects between α-synuclein and GPX-1 would contribute to strengthen the cellular defense against toxic insults; as an activator of cellular GPX-1, α-synuclein improves the enzyme's antioxidant activity by removing toxic H2O2 with concomitant formation of GSSG, and the resulting α-synuclein-GPX-1 complex along with GSSG eradicates the toxic oligomers of α-synuclein by facilitating the innocuous amyloid fibril formation [1]. Under pathological conditions, α-synuclein has been noted to be involved in the oxidative stress via causing mitochondrial dysfunction or direct generation of ROS from its aggregates [19–21]. In this report, however, we have suggested that α-synuclein might be a novel physiological partner of GPX-1, which improves the enzyme's capability of preventing oxidative damages. Especially, brain tissues have been vulnerable to oxidative insults because of high oxygen consumption to fulfill high energy demand and relatively low activity of GPX-1 compared to other tissues, which may become exacerbated in PD due to the substantial decrease of GSH by 40% in the affected region of substantia nigra [22–24]. Therefore, it is hypothesized that abundant pre-synaptic α-synuclein may complement the withered antioxidant defense of nerve tissues by acting as an activator of cellular GPX-1. More surprisingly, we also found that the GPX-1 was entrapped within the entangled fibrils by forming the GPX-1 islets during the accelerated α-synuclein fibrillation, which could be slowly released and also partly activated in the surroundings after an extended period of protection of its activity from the facile deactivation of GPX-1 in solution. These facts would suggest that α-synuclein fibrils can act as a biologically meaningful depot to retain GPX-1 in a latent form. Consequently, α-synuclein and its fibrils could be considered to be involved in antioxidative processes within neuronal cells particularly susceptible to oxidative stresses. Although the issue of toxic effects of amyloid fibrils is still controversial, these well-promoted pathological structures now could be conceptually transformed into a physiologically active component of cellular biogenesis. Acknowledgements The first two authors, KHJ and EJY, contributed equally to the study. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF)
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α-Synuclein-mediated defense against oxidative stress via modulation of glutathione peroxidase Hee Jung Koo 1, Jee Eun Yang 1, Jae Hyung Park, Daekyun Lee, Seung R. Paik ⁎ School of Chemical and Biological Engineering, Institute of Chemical Processes, College of Engineering, Seoul National University, Seoul 151-744, Republic of Korea
a r t i c l e
i n f o
Article history: Received 6 November 2012 Received in revised form 28 February 2013 Accepted 7 March 2013 Available online 16 March 2013 Keywords: α-Synuclein Glutathione peroxidase Oxidative stress Amyloidogenesis Parkinson's disease
a b s t r a c t In this report, mutual effect of α-synuclein and GPX-1 is investigated to unveil their involvement in the PD pathogenesis in terms of cellular defense mechanism against oxidative stress. Biochemical and immunocytochemical studies showed that α-synuclein enhanced the GPX-1 activity with Kd of 17.3 nM and the enzyme in turn markedly enhanced in vitro fibrillation of α-synuclein. Transmission electron microscopy revealed the fibrillar meshwork of α-synuclein containing GPX-1 located in locally concentrated islets. The entrapped enzyme was demonstrated to be protected in a latent form and its activity was fully recovered as released from the matrix. Therefore, novel defensive roles of α-synuclein and its amyloid fibrils against oxidative stress are suggested as the GPX-1 stimulator and the active depot for the enzyme, respectively. © 2013 Elsevier B.V. All rights reserved.
1. Introduction α-Synuclein is a natively unstructured protein that is abundant in presynaptic nerve terminals. This protein has significant implication in the pathogenesis of Parkinson's disease (PD) by participating in the formation of radiating amyloid fibrils found on Lewy bodies (LBs), a pathological signature of the disease [1,2]. Mechanism of how this disordered protein is polymerized into the highly ordered suprastructure is yet to be fully understood, and pathological roles of the fibrillar aggregates are still controversial. Oxidative stress has been suggested to be a part of PD pathogenesis by influencing the α-synuclein amyloidogenesis. Reactive oxygen species (ROS) resulting from mitochondrial dysfunction or dopamine hydrolysis lead to oxidative modification of α-synuclein, affecting its fibrillation process [3–5]. Tyrosine nitration and dityrosine cross-link of α-synuclein result in accumulation of the modified aggregative species and subsequent accelerated aggregation under the condition of impaired proteasomal degradation in PD [6–8]. It becomes apparent that cellular oxidative system is crucial for the pathogenesis of PD by processing ROS.
Abbreviations: GPX-1, glutathione peroxidase-1; PD, Parkinson's disease; LBs, Lewy bodies; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, glutathione disulfide ⁎ Corresponding author at: School of Chemical and Biological Engineering, Seoul National University, 599 Gwanak-Ro, Gwanak-Gu, Seoul 151-744, Republic of Korea. Tel.: +82 2 880 7402; fax: +82 2 888 1604. E-mail address: [email protected] (S.R. Paik). 1 These authors contributed equally to this study. 1570-9639/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbapap.2013.03.008
However, a direct connection of α-synuclein and its fibrillation with cellular anti-oxidative system has been rarely investigated. Selenocysteine-containing glutathione peroxidases (GPXs) are important antioxidant enzymes protecting tissues from oxidative damages by removing hydrogen peroxide and a range of organic peroxides. Among several isoforms of GPXs with diverse tissue and substrate specificities, the homotetrameric GPX-1 is the most abundant cytoplasmic enzyme [9]. GPX-1 primarily catalyzes the reaction of converting hydrogen peroxide to water by oxidizing reduced glutathione (GSH) to glutathione disulfide (GSSG) in cytoplasm [10]. GPX-1 has only a few interactive proteins reported. Non-receptor tyrosine kinases such as c-Abl and Arg were found to modulate GPX-1 activity by the kinase-mediated phosphorylation [11]. Selenium binding protein-1 bound to GPX-1 and repressed its antioxidant activity [12]. Because of its crucial role in coping with oxidative stress, GPX-1 has been implicated in development of many diseases and their prevention, which includes cancer, neurodegenerative diseases, and cardiovascular disease [13,14]. Recent finding revealed that GPX-1 was co-localized with α-synuclein in LBs found in the brain tissues of PD patients, suggesting a neuroprotective role of GPX-1 toward the disease [15]. Since molecular-level inter-relationship between the two co-localized proteins is basically unknown, their mutual effects have been investigated in this study by assessing the accelerated fibrillation of α-synuclein by GPX-1 and the stimulated GPX-1 activity by α-synuclein. The results have led us to suggest an anti-oxidative role of α-synuclein and its fibrillation by modulating GPX-1, which may provide us a novel insight into pathophysiological role(s) of
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α-synuclein and GPX-1 in terms of controlling both amyloidogenesis and oxidative stress.
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3. Results and discussion 3.1. α-Synuclein enhances antioxidant activity of glutathione peroxidase
2. Materials and methods 2.1. Materials Human recombinant α-synuclein was prepared according to the procedure previously described [16]. GPX-1 (bovine erythrocytes), glutathione reductase, GSH, cumen hydroperoxide, and NADPH were from Sigma. Mouse anti-α-synuclein and goat anti-GPX-1 antibodies were obtained from Santa Cruz, and goat anti-mouse Alexa Fluor 488 and rabbit anti-goat Alexa Fluor 633 were from Invitrogen. Gold (10 nm)-conjugated anti-goat antibody was provided by Abcam. Thioflavin-T was purchased from Sigma.
To investigate mutual interactions between α-synuclein and GPX-1, we first examined an effect of α-synuclein on the antioxidant activity of GPX-1. The GPX-1 activity was monitored with a coupling system employing cumene hydroxide and GSH in the presence of
2.2. GPX-1 assay GPX-1 activity was measured by a coupling system employing cumen hydroperoxide and GSH in the presence of glutathione reductase and NADPH. Oxidized glutathione formed by GPX-1 reaction (H2O2 + 2GSH → GSSG + 2H2O) was continuously reduced by glutathione reductase activity (GSSG + NADPH + H+ → 2GSH + NADP+). GPX-1 activity was calculated from the decrease in NADPH absorbance at 340 nm. The assay mixture included 0.1 mM each of cumen hydroperoxide, GSH, and NADPH and 0.1 unit glutathione reductase in 50 mM Tris/ Cl (pH 7.6) containing 5 mM EDTA. Protein samples were lastly added into the assay mixture and the measurement was performed immediately. 2.3. Amyloid fibril formation 20 μM α-synuclein was incubated in the absence and presence of various amounts of GPX-1 at 37 °C with vigorous shaking in 20 mM MES, pH 6.5. Amyloid fibrillation was monitored by thioflavin-T binding fluorescence at 482 nm with an excitation at 450 nm. An aliquot (20 μl) of the reaction mixture was combined with thioflavin-T (5 μM) in 50 mM glycine (pH 8.5) to a final volume of 200 μl, and then the fluorescence intensity was measured with LS-55 luminescence spectrometer (Perkin-Elmer). Morphological analyses of amyloid fibrils were carried out with JEM1010 transmission electron microscope (JEOL). An aliquot (20 μl) of the sample was adsorbed onto a 200 mesh carbon-coated copper grid (Ted Pella Inc.) for 3 min, and the excess liquid was removed with a filter paper. The adsorbed sample was then negatively stained with 2% uranyl acetate for 1 min in the dark. 2.4. Immunochemical analyses For immunocytochemistry, neuroblastoma SH-SY5Y cells cultured on the coverslips were fixed in ice-cold methanol/acetone solution (1:1) at −20 °C for 10 min, washed and blocked with a blocking buffer (1% bovine serum albumin in phosphate buffered saline) for 30 min at 37 °C. The fixed cells were incubated in the blocking buffer containing mouse anti-α-synuclein antibody and goat anti-GPX-1 antibody for 2 h, and then incubated with the fluorescent secondary antibodies of anti-mouse Alexa Fluor 488 and rabbit anti-goat Alexa Fluor 633 for 1 h in the dark. After washing with the blocking buffer, the cells were mounted with a mounting medium and examined using a LSM510 confocal microscope (Carl Zeiss). For immunogold labeling of the entrapped GPX-1 within amyloid fibrils, the sample was adsorbed onto a carbon-coated copper grid for 15 min at room temperature. After removing excess liquid with a filter paper, the adsorbed sample was incubated successively with goat anti-GPX-1 antibody and gold-conjugated anti-goat antibody for 1 h each. After washing with 20 mM MES (pH 6.5), the adsorbed sample was negatively stained with 2% uranyl acetate and analyzed using a transmission electron microscope.
Fig. 1. α-Synuclein-mediated enhancement of GPX-1 activity. (A) GPX-1 activities with increased α-synuclein concentrations. GPX-1 (5 nM) was pre-incubated with 0 (black), 56 (red), 112 (blue), 224 (cyan), 280 (green), and 560 nM (pink) of α-synuclein at 4 °C for 20 min, and the enzyme activity was monitored by observing the decrease in NADPH absorbance at 340 nm. (B) Initial activities of GPX-1 are plotted as a function of α-synuclein in molarity. Double reciprocal plot was performed to obtain the dissociation constant between GPX-1 and α-synuclein (inset). (C) Immunocytochemical analysis of SH-SY5Y neuroblastoma cell-line. Co-localization of GPX-1 and α-synuclein was visualized by staining cells with anti-α-synuclein and anti-GPX-1 antibodies. Scale bar, 50 μm.
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glutathione reductase/NADPH [17]. The enzyme was pre-incubated with α-synuclein at 4 °C for 20 min before assessed by observing the decrease in NADPH absorbance at 340 nm due to the reduction of GSSG back to GSH by glutathione reductase. Significant enhancement of GPX-1 activity was observed with α-synuclein (Fig. 1). As α-synuclein level increased, GPX-1 activity became continuously increased as evidenced by the increased negative slopes reflecting enhanced NADPH consumption (Fig. 1A). Neither glutathione reductase activity nor oxidation of NADPH was affected by α-synuclein. Direct molecular interaction between α-synuclein and GPX-1 was evaluated by plotting the initial activities of GPX-1 obtained as changes of moles of NADPH per min per mg protein as a function of α-synuclein in molarity (Fig. 1B). The enhanced GPX-1 activities exhibited a hyperbolic relationship with α-synuclein, indicating a possible specific binding between the two proteins. By performing a double reciprocal plot, their dissociation constant was estimated as 17.3 nM (Fig. 1B, inset). The α-synuclein-mediated enhancement of GPX-1 activity and the exceptionally high molecular affinity would suggest a novel physiological role of α-synuclein in cellular anti-oxidative processes. In fact, a direct and tight interaction between the two proteins under physiological condition was demonstrated with immunocytochemcial analysis of neuroblastoma cell-line, SH-SY5Y. Confocal microscopic images of immunofluorescence-stained cells with anti-α-synuclein and anti-GPX-1 antibodies showed that both intrinsic proteins were found to be co-localized in SH-SY5Y cells (Fig. 1C). Taken together, these results allow us to suggest a physiological function of α-synuclein as a GPX's natural partner in cellular environment working as a GPX-1 activator and thus acting as an anti-oxidative agent. 3.2. Glutathione peroxidase accelerates amyloid fibrillation of α-synuclein Since GPX-1 was found to exist in α-synuclein-enriched Lewy bodies [15], GPX-1 effect on the amyloidogenesis of α-synuclein has been examined. Typical kinetic study using amyloid-specific Thioflavin-T revealed that in vitro fibrillation of α-synuclein was remarkably accelerated by GPX-1 in a dose-dependent manner (Fig. 2A). Substoichiometric amount of GPX-1 at 0.05 μM was sufficient to show the facilitated fibrillation of α-synuclein at 20 μM by shortening the lag period and increasing the final Thioflavin-T binding fluorescence. As the GPX-1 level increased twice, the lag further decreased to 20 h from 40 h obtained at its absence. Moreover, the final Thioflavin-T binding fluorescence also increased by almost two-fold from that of the kinetics obtained with α-synuclein alone. Based on the high affinity with Kd at nM range, the tight α-synuclein-GPX-1 complex would provide a nucleation center for the accelerated and enhanced amyloidogenesis although it cannot be completely ruled out that GPX-1 itself could act as a template at which α-synuclein continuously turns into an aggregative form. To demonstrate whether the activity of GPX-1 is crucial for the GPX-1-mediated acceleration of α-synuclein fibrillation, the fibrillation kinetics was carried out with GPX-1 in varying activities obtained via pre-incubating the enzyme at 37 °C by itself (Supplementary Fig. S1). As the enzyme with decreasing activities from 100%, 50%, to 25% was added to the fibrillation mixture at a molar ratio of 1:200 between GPX-1 and α-synuclein, the shortened lag period was extended from 13.1 h to 15.6 and 25.5 h, respectively, whereas it required about 40 h of the lag for the protein to be fibrillated in the absence of the enzyme (Fig. 2B). In addition, the augmented final fluorescence obtained with the enzyme at its full activity proportionally decreased as the enzymatic activity was inactivated (Fig. 2B). These findings clearly indicate that the biochemically active GPX-1 is required for the enzyme-mediated acceleration of α-synuclein fibrillation by both shortening the lag phase and increasing the final fibrillation level. Addition of
Fig. 2. GPX-1-mediated acceleration of the amyloid fibrillation of α-synuclein. (A) Aggregation kinetics of α-synuclein in the presence of GPX-1. α-Synuclein (20 μM) was incubated with 0.05 (▼) and 0.1 μM GPX-1 (△) at 37 °C with vigorous shaking. Amyloid fibrillation was monitored by thioflavin-T binding fluorescence at 482 nm (emission at 450 nm) during incubation. α-Synuclein (●) and GPX-1 (○) were separately incubated as controls. (B) GPX-1 activity-dependency of the amyloid fibrillation of α-synuclein. Partially inactivated GPX-1 was obtained at various time points during pre-incubation of the enzyme alone at 37 °C. α-Synuclein (20 μM) was fibrillated with 0.1 μM GPX-1 with decreasing activities from 100% (○), 50% (▼), and 25% (△).
completely inactivated GPX-1 obtained via heating at 100 °C resulted in a fibrillation kinetics indistinguishable from that of α-synuclein alone (Supplementary Fig. S2). 3.3. Entangled fibrillar aggregates of α-synuclein acting as a depot for active GPX-1 Morphological comparison of the final fibrillar aggregates of α-synuclein obtained in the absence and presence of GPX-1 revealed that the fibrils resulted from the accelerated fibrillation with the enzyme were densely entangled (Fig. 3A). Immunogold labeling was performed with anti-GPX-1 antibody in order to evaluate the GPX-1 distribution within the entanglement. The enzymes were found to be locally clustered into the GPX-1-islets within the fibrillar meshwork (Fig. 3B). This entrapment of GPX-1 into the α-synuclein fibrils which has occurred during the in vitro fibrillation process is compatible with the previous observation of their co-existence in the Lewy bodies of PD patient's brain [15]. Since the enhanced fibrillation of α-synuclein has required the active GPX-1, the enzymatic activity of the entrapped GPX-1 was examined by resuspending the GPX-1-containing fibrils in the assay mixture, and monitored for the decrease of NADPH absorbance at 340 nm. Impressively, the entrapped GPX-1 retained most of their activities during the prolonged incubation of the fibrillation for 70 h while the activity was almost completely vanished following only
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Fig. 3. Localization of GPX-1 within entangled α-synuclein fibrils. (A) Morphologies of GPX-1-induced α-synuclein fibrils. Scale bar, 0.5 μm. (B) Distribution of GPX-1 within the entangled fibrils. Immunogold labeling was performed with anti-GPX-1 and gold-conjugated secondary antibodies. Dashed circles indicate GPX-1-islets. All samples for TEM analysis were negatively stained with 2% uranyl acetate on carbon-coated copper grids. Scale bar, 200 nm.
2 h of incubation at 37 °C in the absence of the fibrils (Fig. 4A and Supplementary Fig. S1). The time course of the entrapped GPX-1 activity exhibited hysteresis, indicating that the NADPH consumption gradually increased as a function of time (Fig. 4B). By plotting slopes of the tangent lines of the activity trace at various time points, the GPX-1 activity previously entrapped in the matrix was shown to increase and then the activity remained unaffected when the substrate was completely consumed (Fig. 4B inset). To account for the hysteresis in activity, the enzyme-entrapped fibrillar matrix was successively extracted with a fixed amount (100 μl) of 20 mM MES, pH 6.5, and the GPX-1 activity in the extracts was measured. Interestingly, the extracted activity also exhibited the hysteresis when compared with a control of GPX-1 (Fig. 4C). As the extraction proceeded, the GPX-1 was continuously released in solution with subsequently diminished activities. In addition, the hysteresis became less apparent as the extracted GPX-1 activity decreased (Fig. 4C inset). This result indicates that the GPX-1 would be entrapped in a possible latent form within the fibrillar matrix, which can be slowly activated upon its release. Moreover, the gradual increase in the GPX-1 activity observed with the GPX-1 containing matrix (Fig. 4B) would be also due to the slow release of the enzyme from the matrix. Based on our findings, antioxidative function of α-synuclein has been suggested in terms of mutual activation between GPX-1 and
Fig. 4. Enzymatic activity of the entrapped GPX-1. (A) Protection of GPX-1 activity within the fibrillar matrix. GPX-1 activity was monitored after the α-synuclein fibrillation was carried out in the presence of GPX-1 for 3 days at 37 °C. Initial and 2 h-aged activities of GPX-1 at 37 °C were shown as controls. (B) Time course of the entrapped GPX-1 activity. The GPX-1-containing fibrils produced after 70 h fibrillation were obtained by centrifugation at 16,100 ×g for 30 min, and resuspended in the GPX-1 assay mixture. The enzyme activity was monitored by observing the decrease in NADPH absorbance at 340 nm. The inset is the result of a polynomial fitting and its first derivative plotting of the pink line. (C) Reactivation of GPX-1 released from fibrils. Successive extraction of the GPX-1-entrapped fibrillar matrix was carried out with 100 μl of 20 mM MES (pH 6.5) and each extract was subjected to the activity measurement. Black-dashed lines indicate initial and final activities of the released enzyme in the first extract. GPX-1 without the prior entrapment was used as a control (red). The enzyme activities in the successive extracts were shown (inset); number indicates the extraction order.
amyloidogenesis of α-synuclein (Scheme 1). The H2O2-removing process of GPX-1 is significantly activated by interacting with α-synuclein, and the α-synuclein-GPX-1 complex provides a possible nucleation center for the accelerated amyloid fibril formation of
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funded by the Ministry of Education, Science, and Technology (2012-0002064 and 2012R1A2A2A01013582). Appendix A. Supplementary data Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.bbapap.2013.03.008. References
Scheme 1. Mutual relationship between GPX-1 and the amyloidogenesis of α-synuclein.
α-synuclein, which results in the entangled filamentous aggregates with the GPX-1 deposited in a locally concentrated fashion. In addition, we have previously reported that oxidized GSH (GSSG) also enhanced the amyloidogenesis of α-synuclein [18]. Taken together, the physiological function of α-synuclein and its amyloidogenesis can be understood with respect to the anti-oxidative GPX-1 system including glutathione turnover. Therefore, it can be considered that the mutual effects between α-synuclein and GPX-1 would contribute to strengthen the cellular defense against toxic insults; as an activator of cellular GPX-1, α-synuclein improves the enzyme's antioxidant activity by removing toxic H2O2 with concomitant formation of GSSG, and the resulting α-synuclein-GPX-1 complex along with GSSG eradicates the toxic oligomers of α-synuclein by facilitating the innocuous amyloid fibril formation [1]. Under pathological conditions, α-synuclein has been noted to be involved in the oxidative stress via causing mitochondrial dysfunction or direct generation of ROS from its aggregates [19–21]. In this report, however, we have suggested that α-synuclein might be a novel physiological partner of GPX-1, which improves the enzyme's capability of preventing oxidative damages. Especially, brain tissues have been vulnerable to oxidative insults because of high oxygen consumption to fulfill high energy demand and relatively low activity of GPX-1 compared to other tissues, which may become exacerbated in PD due to the substantial decrease of GSH by 40% in the affected region of substantia nigra [22–24]. Therefore, it is hypothesized that abundant pre-synaptic α-synuclein may complement the withered antioxidant defense of nerve tissues by acting as an activator of cellular GPX-1. More surprisingly, we also found that the GPX-1 was entrapped within the entangled fibrils by forming the GPX-1 islets during the accelerated α-synuclein fibrillation, which could be slowly released and also partly activated in the surroundings after an extended period of protection of its activity from the facile deactivation of GPX-1 in solution. These facts would suggest that α-synuclein fibrils can act as a biologically meaningful depot to retain GPX-1 in a latent form. Consequently, α-synuclein and its fibrils could be considered to be involved in antioxidative processes within neuronal cells particularly susceptible to oxidative stresses. Although the issue of toxic effects of amyloid fibrils is still controversial, these well-promoted pathological structures now could be conceptually transformed into a physiologically active component of cellular biogenesis. Acknowledgements The first two authors, KHJ and EJY, contributed equally to the study. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF)
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