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-synuclein in vitro
1 Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA
2 Department of Chemistry and Biochemistry, California Polytechnic State University, San Luis Obispo, California 93407, USA
3 Institute for Biological Instrumentation, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia
Reprint requests to: Anthony L. Fink, Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA; e-mail: enzyme{at}cats.ucsc.edu; fax: (831) 459-2935.
(RECEIVED August 30, 2002; FINAL REVISION January 16, 2003; ACCEPTED January 17, 2003)
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.0230903.
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Abstract |
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-synuclein has been implicated as a causative factor in Parkinson’s disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that -synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze -synuclein oligomerization. The formation of -synuclein–polycation complexes was not accompanied by significant structural changes in -synuclein. However, -synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating -synuclein aggregation.
Keywords: Parkinson’s disease; -synuclein; natively unfolded protein; polycation; electrostatic interactions; fibrillation; aggregation; oligomerization
Abbreviations: GAG, glycosaminoglycan • CD, circular dichroism • UV, ultraviolet • SAXS, small-angle X-ray scattering • PAGE, polyacrylamide gel-electrophoresis • PEI, polyethyleneimine • polyLys, poly-L-lysine • polyArg, poly-L-arginine • polyGlu, poly-L-glutamate
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Introduction |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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-Synuclein is a small (14 kD), highly conserved presynaptic protein that is abundant in various regions of the brain (Maroteaux et al. 1988; Jakes et al. 1994; Iwai et al. 1995). Structurally, purified -synuclein belongs to the family of natively unfolded proteins (Weinreb et al. 1996; Plaxco and Gross 1997; Wright and Dyson 1999; Dunker et al. 2001, 2002a, Dunker et al. b; Dunker and Obradovic 2001; Eliezer et al. 2001; Uversky et al. 2001a; Dyson and Wright 2002; Uversky 2002a,b), which are characterized by a unique combination of low overall hydrophobicity and large net charge (Uversky et al. 2000). Deposition of aggregated forms of -synuclein in neuronal or glial cytoplasm is a pathologic hallmark of several neurodegenerative diseases, including Parkinson’s disease, dementia with Lewy bodies, Lewy body variant of Alzheimer’s disease, and multiple system atrophy (Trojanowski et al. 1998; Lucking and Brice 2000; Goedert 2001). In vitro, the protein forms fibrils with morphologies and staining characteristics similar to those extracted from disease-affected brain (Spillantini et al. 1997; Conway et al. 1998, 2000; Crowther et al. 1998, 2000; Giasson et al. 1999; Narhi et al. 1999; Li et al. 2001). The kinetics of fibrillation are consistent with a nucleation-dependent mechanism (Conway et al. 1998; Wood et al. 1999), in which the critical early stage of the structural transformation involves a partially folded intermediate (Uversky et al. 2001a). The rate of -synuclein fibrillation has been shown to be significantly accelerated by various environmental factors, including low pH and high temperature (Uversky et al. 2001a), heparin and other GAGs (Cohlberg et al. 2002), metal cations (versky et al. 2001b) and pesticides (Uversky et al. 2001c; Manning-Bog et al. 2002).
A model has been suggested in which both the cations and pesticides interact with -synuclein to bring about a conformational change to a partially folded state with a high propensity to aggregate (Uversky et al. 2001b,c, 2002a). The mechanisms of partially folded intermediate formation are different for metal cations and pesticides. As the C-terminal region of -synuclein (about 40 amino acids) is very rich in acidic residues (Scheme 1) and thus highly negatively charged at neutral pH (the pI is 4.7), the resulting repulsive interactions are a major factor leading to the natively unfolded conformation of this protein. It has been suggested that the dominant effect of the metal ions on -synuclein conformational change and fibrillation is due to masking of the Coulombic charge–charge repulsion (Uversky et al. 2001b, 2002a). Structure-forming and fibrillation-accelerating effects of hydrophobic pesticides are consistent with the idea that the pesticide binds preferentially to the partially folded intermediate conformation, which is in equilibrium with the natively unfolded state, thereby increasing the population of the intermediate. The driving force for this is the existence of contiguous patches of hydrophobic surface in the intermediate and which are absent in the natively unfolded conformation (Uversky et al. 2001c; 2002a).
The acceleration seen for -synuclein fibrillation in the presence of anionic GAGs may be explained by binding of GAGs to a transient form of -synuclein. Binding sites on a variety of proteins for heparin invariably contain clusters of basic amino acid residues capable of binding to the negatively charged heparin polymer (Cardin and Weintraub 1989). The N-terminal region of -synuclein contains clusters of positive charge from its five repeats of the consensus sequence KTKEGV. It is likely that the transition of -synuclein from an unstructured state to one containing substantial ß-sheet creates a favorable conformation for GAG binding resulting in a high localized -synuclein concentration that favors fibrillation (Cohlberg et al. 2002).
In this study we show that -synuclein binds specifically to several unstructured polycations including polyethylenimine, poly-L-lysine, and poly-L-arginine. This interaction leads to the oligomerization of -synuclein, but is not accompanied by significant structural changes in this natively unfolded protein. However, the rate of -synuclein fibrillation is significantly accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the chemical nature of the polycation, its length, and concentration. We suggest that polycations accelerate -synuclein fibrillation by minimizing the electrostatic repulsion between negatively charged -synuclein molecules, resulting in a high localized concentration of -synuclein favoring fibrillation. Given the presence of naturally occurring polycations in the cytosol, these findings have implications with respect to the regulatory control of -synuclein aggregation in vivo and the etiology of Parkinson’s disease.
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Results |
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-Synuclein binds to polycations leading to association-synuclein to form soluble complexes with different polycations was evaluated by native polyacrylamide gel electrophoresis (nPAGE). When -synuclein was mixed with either polyethylenimine (PEI), poly-L-lysine (polyLys), or poly-L-arginine (polyArg), new bands corresponding to a -synuclein/polycation complex were observed (Fig. 1A
). Consequently, -synuclein binds to different polycations forming stable complexes. In contrast, no complex formation was observed between the polyanion poly-L-glutamate and -synuclein (Fig. 1B), indicating the requirement for a positively charged polymer for complex formation. Further, the structural requirements for complex formation of the polycation were rather general, as both polypeptides and polyethylenimines formed complexes.
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-synuclein/polycation complexes was accomplished by adding increasing amounts of PEI to -synuclein and monitoring the disappearance of free -synuclein by native PAGE (Fig. 1B
). When approximately 0.1–0.2 moles of PEI were added to 1 mole of -synuclein, all protein was complexed. This corresponds to approximately 10 molecules of -synuclein bound to one molecule of PEI of molecular weight 25,000 Daltons at saturation. An apparent association constant of 
1 µM may be estimated based on the minimal concentration of 0.35 µM PEI required to form the complex. Similar stoichiometries and affinities were observed for both polyLys and polyArg (data not shown).
Effect of polycations on -synuclein fibrillation
Fibrillation of -synuclein upon agitation in vitro is a hallmark of this protein, and is dependent on numerous experimental conditions (Uversky et al. 2001c). Figure 2A shows that -synuclein fibrillation was accelerated approximately 4–10-fold in the presence of a 0.002–0.004 molar ratio of the polycations polyLys, polyArg, and PEI, but not with the polyanion polyGlu. Interestingly, similar results have been recently reported for the complexes of human -synuclein with natural polycations, histones (J. Goers, A.B. Manning-Bog, A.L. McCormack, I.S. Millett, S. Doniach, D.A. Di Monte, V.N. Uversky, and A.L. Fink, in prep.); however, histones are more effective than the polycations studied here, bringing about accelerated fibrillation at lower concentrations. With histones -synuclein forms a tight 2:1 complex.
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-synuclein fibrillation was dependent on both polycation concentration and polymer size. For example, adding increasing amounts of PEI from 0 to 10 µg/mL (an approximate 0.002 mole equivalent compared -synuclein) led to a 10-fold decrease in lag time of fibril formation and to a considerable increase in the fibril elongation rate (Fig. 2B). Similar effects were observed with polyLys and polyArg (data not shown). The accelerating effect of the polycations also depended on their length, with longer polymers resulting in faster fibrillation (Fig. 2C). However, even short naturally occurring polyamines, such as spermine and spermidine, caused significant acceleration in -synuclein fibrillation: For instance, spermine was more effective than an equivalent short PEI, decreasing the lag time for fibrillation by 50% at 17 µg/mL (data not shown).
Electron microscopy showed that -synuclein fibrils formed in the presence of all the polycations studied were similar in their morphology to those grown in the absence of polycation (Fig. 3). Thus, incubation of -synuclein with different polycations resulted in substantial acceleration in the kinetics of -synuclein fibrillation, with the acceleration effect dependent on both polymer length and concentration.
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-synuclein binding to polycations-synuclein was investigated by far-UV circular dichroism. In the absence of polycations -synuclein possessed a far-UV CD spectrum typical of an essentially unfolded polypeptide chain (a major minimum in the vicinity of 198 nm with the absence of characteristic - or ß-bands in the 210–230-nm region). In the presence of polycations minimal changes were observed in the shape of this spectrum, indicating that complex formation with polycations did not cause significant change in -synuclein’s secondary structure (Fig. 4).
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Discussion |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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-synuclein specifically binds to a variety of polycations of different chemical nature, including polypeptides (polyLys and polyArg), synthetic polymers, such as polyethylenimine (PEI), and naturally occurring polyamines. The interaction of -synuclein with polycations is accompanied by oligomerization of the protein. Interestingly, none of the polycations studied had significant effect on the shape of the -synuclein far-UV CD spectrum, indicating minimal induction of ordered secondary structure (Fig. 4).
Intriguingly, polycations not only induced oligomerization but also significantly accelerated fibrillation of human -synuclein, with the magnitude of the acceleration being dependent on the nature of the polycation, its length, and concentration (Figs. 2, 3). We assume that the major driving force for the formation of these -synuclein-polycation complexes is electrostatic interaction between the negatively charged C-terminal domain of -synuclein and the positively charged side chains of the polycation. Such interactions may have two important consequences: (1) the partial masking of the Coulombic charge–charge repulsion both within the polypeptide chain and between the neighboring -synuclein molecules; (2) a considerable increase in the local concentration of -synuclein, which is known to be a factor favoring fast fibrillation (Uversky et al. 2001c). This underscores an important potential role of electrostatic interactions in protein aggregation. The lack of conformational change in the polycation complexes with -synuclein suggests that there is little secondary structure induced by the interaction, and thus that the interaction does not directly lead to formation of the partially folded intermediate of -synuclein. Consequently, the mechanism of polyamine-accelerated fibrillation of -synuclein is probably different from that of metals and protons.
It is worth noting that the ratio of -synuclein to PEI (10:1) is consistent with one -synuclein per 160 Å of polymer, assuming an extended unbranched conformation. This is also the length of the negatively charged C-terminal region of -synuclein in an extended conformation, consistent with the notion of very direct electrostatic interactions between the protein and polymer. The large molar excess of -synuclein over PEI could also be indicative of the binding of the polycation to an oligomeric, rather than monomeric, form of -synuclein, but the lack of conformation change argues against this. The lack of accelerated fibrillation with poly-Glu is not surprising, in view of the potential charge–charge repulsion. We ascribe the previously observed acceleration by negatively charged heparin due to specific heparin binding sites in the N-terminal region.
Significant amounts of naturally occurring polyamines (putrescine, spermidine, and spermine) are found in most cells, and those of the substantia nigra are no exception (Vivo et al. 2001). In fact, elevated levels of these polyamines have been reported in PD patients (Gomes-Trolin et al. 2002). Because polyamines are free radical scavengers and contribute to protecting cells from oxidative damage, the present findings are paradoxical in the context of a physiological role of polyamines as protective species in PD. Because our results unambiguously demonstrate that polyamines accelerate the fibrillation of -synuclein, there must be other protective mechanisms present in the cell that overcome the potential adverse effects of the polyamines. In addition, elevated neuronal levels of naturally occurring polyamines could be a contributing factor to Parkinson’s disease, and thus suggest new avenues for investigation, such as the levels of spermidine and spermine synthases.
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Materials and methods |
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TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
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-synuclein-synuclein were performed as previously described (Uversky et al. 2002b).
Supplies and chemicals
Thioflavin T (ThT), polyLys, polyArg, and PEI were obtained from Sigma. All other chemicals were of analytical grade from Fisher Chemicals.
Native and denaturing polyacrylamide gel electrophoresis (PAGE)
Denaturing sodium dodecyl sulfate PAGE (SDS-PAGE) was performed with PhastSystem 8–25% gradient gels (Amersham Biosciences). Samples of -synuclein–polycation fibrils were separated from soluble proteins by microcentrifugation at 18,000 x g for 25 min, washed once with buffer, and boiled for 10 min in 2% SDS prior to SDS-PAGE. Native electrophoresis was performed in 7.5% homogeneous gels for 30 min at 60 V in 0.088 M L-alanine, 0.025 M Tris-HCl at pH 8.8. All gels were stained with Coomassie blue.
Circular dichroism (CD) measurements
CD spectra were obtained on an AVIV 60DS spectrophotometer using -synuclein concentration of ~1.0 mg/mL at 23°C. Spectra were recorded in 0.01 cm cells from 250–190 nm with a step size of 1.0 nm, with a bandwidth of 1.5 nm, and an averaging time of 10 sec. For all spectra, an average of five scans was obtained. CD spectra of the appropriate buffers were recorded and subtracted from the protein spectra.
Fluorescence measurements
Fluorescence measurements were performed in semimicro quartz cuvettes (Hellma) with a 1-cm excitation light path using a FluoroMax-2 spectrofluorometer from Instruments S.A., Inc. All data were processed using DataMax/GRAMS software.
Fibril formation
Fibril formation of -synuclein in the presence or absence of polycations and polyanions was monitored in a fluorescence plate reader (Fluoroskan Ascent) as previously described (Uversky et al. 2002b).
Data evaluation of fibrillation kinetics
ThT fluorescence intensities were plotted as a function of time and fitted to a sigmoidal curve as previously described (Nielsen et al. 2001).
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Acknowledgments |
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The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
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References |
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