Certain Metals Trigger Fibrillation of Methionine-oxidized {alpha}-Synuclein*

Ghiam Yamin, Charles B. Glaser {ddagger}, Vladimir N. Uversky and Anthony L. Fink §

From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064

Received for publication, March 31, 2003 , and in revised form, May 13, 2002.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aggregation and fibrillation of {alpha}-synuclein has been implicated as a key step in the etiology of Parkinson's disease and several other neurodegenerative disorders. In addition, oxidative stress and certain environmental factors, including metals, are believed to play an important role in Parkinson's disease. Previously, we have shown that methionine-oxidized human {alpha}-synuclein does not fibrillate and also inhibits fibrillation of unmodified {alpha}-synuclein (Uversky, V. N., Yamin, G., Souillac, P. O., Goers, J., Glaser, C. B., and Fink, A. L. (2002) FEBS Lett. 517, 239–244). Using dynamic light scattering, we show that the inhibition results from stabilization of the monomeric form of Met-oxidized {alpha}-synuclein. We have now examined the effect of several metals on the structural properties of methionine-oxidized human {alpha}-synuclein and its propensity to fibrillate. The presence of metals induced partial folding of both oxidized and non-oxidized {alpha}-synucleins, which are intrinsically unstructured under conditions of neutral pH. Although the fibrillation of {alpha}-synuclein was completely inhibited by methionine oxidation, the presence of certain metals (Ti3+, Zn2+, Al3+, and Pb2+) overcame this inhibition. These findings indicate that a combination of oxidative stress and environmental metal pollution could play an important role in triggering the fibrillation of {alpha}-synuclein and thus possibly Parkinson's disease.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Parkinson's disease (PD)1 is the second most common neurodegenerative disorder after Alzheimer's disease. Clinical symptoms of PD (tremor, rigidity, and bradykinesia) are attributed to the progressive loss of dopaminergic neurons from the substantia nigra. Some surviving nigral dopaminergic neurons contain cytosolic filamentous inclusions known as Lewy bodies and Lewy neurites (1, 2), a major fibrillar component of which was shown to be the presynaptic protein {alpha}-synuclein (3). The mutations A53T and A30P in {alpha}-synuclein have been identified in autosomal-dominantly inherited, early onset PD (4, 5). Furthermore, the production of {alpha}-synuclein in transgenic mice (6) or in transgenic flies (7) leads to motor deficits and neuronal inclusions reminiscent of PD. All this implicates {alpha}-synuclein in the pathogenesis of PD.

{alpha}-Synuclein is a small (14 kDa), highly conserved presynaptic protein that is abundant in various regions of the brain (8, 9). Structurally, purified {alpha}-synuclein belongs to the rapidly growing family of intrinsically unstructured or natively unfolded proteins (10, 11), which have little or no ordered structure under physiological conditions due to a unique combination of low overall hydrophobicity and large net charge (12). {alpha}-Synuclein readily assembles into amyloid-like fibrils in vitro with morphologies and staining characteristics similar to those extracted from disease-affected brain (11, 1318). Fibrillation occurs via a nucleation-dependent polymerization mechanism (14, 17) with a critical initial structural transformation from the unfolded conformation to a partially folded intermediate (11).

The cause of PD is unknown, but considerable evidence suggests a multifactorial etiology involving genetic susceptibility and environmental factors. Recent work has shown that, except in extremely rare cases, there appears to be no direct genetic basis of PD (19). However, several studies have implicated environmental factors, especially pesticides and metals (20). In agreement with these observations, it has been recently reported that direct interaction of {alpha}-synuclein with metal ions (21) or pesticides leads to accelerated fibrillation (2224).

Oxidative injury is also suspected as another causative agent in the pathogenesis of PD (25, 26). The existence of nitrated {alpha}-synuclein (i.e. protein containing the product of the tyrosine oxidation, 3-nitrotyrosine) accumulation in Lewy bodies has been demonstrated (2729). Accumulation of another product of tyrosine oxidation, dityrosine, has been detected in vitro during experiments on the aggregation of {alpha}-synuclein in the presence of copper and H2O2 (30) or catecholamines (31) and leads to accelerated fibrillation of {alpha}-synuclein (32). The methionine side chain is the most readily oxidized amino acid in {alpha}-synuclein, and the four methionines, Met-1, Met-5, Met-116, and Met-127, are easily oxidized in vitro in the presence of H2O2. Interestingly, however, oxidation of the methionine residues of {alpha}-synuclein to the sulfoxides, rather than accelerating fibrillation, was found to prevent it (33). Furthermore, and most importantly, the presence of the methionine-oxidized {alpha}-synuclein was found to completely inhibit fibrillation of the unmodified protein at ratios of >=4:1 (33). Given the potential role of metals in the pathological aggregation of {alpha}-synuclein and the known strong coordination of some metals to sulfoxides, we decided to investigate the structural and fibrillation properties of Met-oxidized {alpha}-synuclein in the presence of several metals to shed more light on the combined effect of environmental factors (metals) and oxidative damage (methionine oxidation to the sulfoxide) on {alpha}-synuclein.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression and Purification of Human {alpha}-Synuclein—Human recombinant {alpha}-synuclein was expressed in the Escherichia coli BL21(DE3) cell line transfected with pRK172/{alpha}-synuclein wild-type plasmid (kind gift of M. Goedert, MRC Cambridge) and purified as described previously (33). Purity of the {alpha}-synuclein was determined by SDS-polyacrylamide gel electrophoresis, UV absorbance spectroscopy, and mass spectrometry.

Supplies and Chemicals—Thioflavin T (ThT) was obtained from Sigma. ZnSO4 and CaCl2 (analytical grade) were from Fisher. Analytical grade Ti2(SO4)3, CuCl2, and Hg(CH3CO2)2 were from Aldrich, whereas AlCl3 and PbO2 were from Mallinckrodt Chemical Works and Matheson Coleman & Bell, respectively. All other chemicals were of analytical grade from Fisher. All buffers and solutions were prepared with nanopure water and stored in plastic vials.

Oxidation of {alpha}-Synuclein by Hydrogen Peroxide—Oxidation of {alpha}-synuclein by H2O2 was performed as described previously (33).

Circular Dichroism (CD) Measurements—CD spectra were recorded on an AVIV 60DS spectrophotometer (Lakewood, NJ) using {alpha}-synuclein concentrations of 1.0 mg/ml and a 0.1-mm path length cell. Spectra were recorded 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 s. For all spectra, an average of five scans was obtained. CD spectra of the appropriate buffers were recorded and subtracted from the protein spectra.

Electron Microscopy—Transmission electron micrographs were collected using a JEOL JEM-100B microscope operating with an accelerating voltage of 80 kV. Typical nominal magnifications were x75,000. Samples were deposited on Formavar-coated 300-mesh copper grids and negatively stained with 1% aqueous uranyl acetate.

Fibril Formation Assay—Fibril formation of oxidized and non-oxidized {alpha}-synuclein in the presence of various metals was monitored using the ThT assay in a fluorescence plate reader (Fluoroskan Ascent) as described previously (33). Standard conditions were 35 µM {alpha}-synuclein, pH 7.5, 20 mM Tris-HCl buffer, 37 °C, with agitation. ThT fluorescence was excited at 450 nm, and the emission wavelength was 482 nm.

Estimation of Hydrodynamic Dimensions—Dynamic light scattering was used to determine the Stokes radii with a DynaPro Molecular Sizing Instrument (Protein Solutions, Lakewood, NJ) using a 1.5-mm path length 12-µl quartz cuvette. Prior to measurement, solutions were filtered with a 0.1-µm Whatman Anodisc-13 filter.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Effect of Methionine Oxidation and Metal Binding on {alpha}-Synuclein Conformation—We first examined the effect of methionine oxidation on the conformation of {alpha}-synuclein and then the effects of selected metals on the conformation of unmodified and Met-oxidized {alpha}-synuclein. Fig. 1 compares far-UV CD spectra measured for the non-oxidized (Fig. 1A) and Metoxidized (Fig. 1B) forms of human {alpha}-synuclein in the absence or presence of several polyvalent cations. The spectra show that oxidized {alpha}-synuclein is slightly more unfolded than non-oxidized {alpha}-synuclein in the absence of cations. This is manifested by a small increase in negative ellipticity in the vicinity of 198 nm and somewhat lower intensity in the vicinity of 222 nm. This increased degree of disorder has been attributed to the decreased hydrophobicity of the oxidized methionines, leading to a decrease in the overall hydrophobicity of the protein (33).



View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1.
Conformational effects of methionine oxidation and metals on {alpha}-synuclein. The effects of metal binding on the far-UV CD spectra of non-oxidized (A) and oxidized {alpha}-synuclein (B) are shown. CD spectra were measured at pH 7.5 in the absence (filled circles, solid lines) or presence of 5 mM of several metal cations: Al3+ (open circles, dotted lines); Zn2+ (filled squares, short dashed lines); Cu2+ (inverted open triangles, dotted-dashed lines) and Ca2+ (inverted filled triangles, long dashed lines). Measurements were carried out at 23 °C in 20 mM Tris-HCl buffer, pH 7.5. Protein concentration was 0.5 mg/ml.

 

Previously, we demonstrated that the interaction of metal cations with natively unfolded {alpha}-synuclein induced a partially folded conformation (21). This transition was attributed to the counter ion-induced neutralization of the coulombic charge-charge repulsion within the very negatively charged protein at neutral pH (21). In agreement with this observation, Fig. 1 shows that in the presence of metals, definite changes occur in the far-UV CD spectra of both non-oxidized and oxidized forms of {alpha}-synuclein. In particular, a decrease in the minimum at 196 nm was accompanied by an increase in negative intensity around 222 nm, reflecting metal binding-induced formation of secondary structure (Fig. 1). Significantly, Fig. 1 shows that binding of the metals induced comparable structural changes in both oxidized and unmodified proteins, most probably reflecting the stabilization of identical partially folded conformations. Thus, Met-oxidized {alpha}-synuclein is slightly more unfolded than non-oxidized protein, but in the presence of metal ions, it adopts a similar partially folded conformation. Our previous studies have shown that formation of such a partially folded conformation correlates with accelerated fibrillation, as is seen with the effect of metals on non-oxidized {alpha}-synuclein (21).

The Effect of Metal Binding on Fibrillation of Methionine-oxidized {alpha}-Synuclein—Next, we determined the effect of the metals on the fibrillation of Met-oxidized {alpha}-synuclein. ThT is a fluorescent dye that interacts with amyloid fibrils, leading to an increase in the fluorescence intensity in the vicinity of 480 nm (34). Fig. 2 compares fibrillation patterns of non-oxidized (Fig. 2A) and oxidized {alpha}-synuclein (Fig. 2B) in the absence and presence of several metal cations monitored by ThT fluorescence. Fibril formation for the non-oxidized {alpha}-synuclein at neutral pH was characterized by a typical sigmoidal curve. In agreement with earlier studies (24), the fibrillation rate increased dramatically in the presence of all metal cations investigated (Fig. 2A). The list of the previously analyzed cations (Li+, K+, Na+, Cs+, Ca2+, Co2+, Cd2+, Cu2+, Fe2+, Mg2+, Mn2+, Zn2+, Co3+, Al3+, and Fe3+) has been extended to consider the effect of Hg2+, Pb2+, and Ti3+. Interestingly, Hg2+ and Pb2+, which are of particular relevance to environment-induced Parkinsonism, are among the most effective accelerators of {alpha}-synuclein fibrillation. This underlines, once again, a potential link between heavy metal exposure, enhanced {alpha}-synuclein fibrillation, and Parkinson's disease.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 2.
Kinetics of fibrillation of non-oxidized (A) and oxidized (B) {alpha}-synuclein monitored by the enhancement of thioflavin T fluorescence intensity. Measurements were performed at 37 °C in the absence of metals (black circles) or in the presence of Ti3+ (open circles), Zn2+ (black triangles), Pb2+ (open triangles), Hg2+ (black squares), Cu2+ (open squares), Ca2+ (black diamonds), and Al3+ (open diamonds). Solutions contained 5 mM metal salts except for the Ti3+, where the concentration was decreased to 0.1 mM due to the low solubility. Measurements were carried out in 20 mM Tris-HCl buffer, pH 7.5. Protein concentration was 0.5 mg/ml. ThT fluorescence was excited at 450 nm, and the emission wavelength was 482 nm.

 

In contrast, there was no evidence of fibril formation by methionine-oxidized {alpha}-synuclein at neutral pH (Fig. 2B). Previously, we showed that the inhibitory effect of methionine oxidation on {alpha}-synuclein fibrillation can be eliminated under conditions of low pH, due to the formation of a partially folded intermediate reflecting protonation of the carboxylate groups (33). In view of this observation, and the observation that metal cations induce partial folding of oxidized {alpha}-synuclein (Fig. 1), one might expect that fibrillation of the methionine-oxidized protein would occur in the presence of metals. In accord with this hypothesis, methionine-oxidized {alpha}-synuclein readily formed fibrils in the presence of certain metal ions, such as Ti3+, Al3+, Zn2+, and Pb2+ (Fig. 2B and Table I). However, not all metals were able to accelerate the fibrillation of methionine-oxidized {alpha}-synuclein: for example, Hg2+, Cu2+, and Ca2+, although able to induce the partially folded conformation in the oxidized protein, did not induce its fibril formation (at least not within the time scale examined). Moreover, Fig. 2 and Table I show that in the presence of Zn2+ and Pb2+, fibrillation of the oxidized {alpha}-synuclein was as accelerated as for the non-oxidized protein, whereas Al3+ and Ti3+ showed a less pronounced effect. The morphology of the fibrillar material formed by the non-oxidized and oxidized {alpha}-synuclein in the presence of several metal cations was analyzed by transmission electron microscopy, and both forms of {alpha}-synuclein formed typical amyloid fibrils, as shown in Fig. 3.


View this table:
[in this window]
[in a new window]
 
TABLE I
The effect of metal cations on the fibrillation of methionine-oxidized {alpha}-synuclein

Kinetic parameters of non-oxidized and oxidized {alpha}-synuclein fibrillation in the presence of different metal cations, monitored by ThT fluorescence. Typical errors (S.D.) were 15% on the lag times and t1/2 and 20% on the rate constants. Conditions were 35 µM {alpha}-synuclein, pH 7.5, 20 mM Tris-HCl buffer, 37 °C, with agitation.

 


View larger version (155K):
[in this window]
[in a new window]
 
FIG. 3.
Negatively stained transmission electron micrographs of different {alpha}-synuclein fibrils induced in non-oxidized and oxidized protein by different metals. A, Met oxidized {alpha}-synuclein control. B, {alpha}-synuclein control. C, oxidized {alpha}-synuclein in the presence of calcium. D, oxidized {alpha}-synuclein in the presence of aluminum. E, oxidized {alpha}-synuclein in the presence of lead. F, oxidized {alpha}-synuclein in the presence of zinc.

 

Dynamic Light Scattering Experiments to Monitor Hydrodynamic Size—There are a number of possible mechanisms whereby methionine oxidation could inhibit {alpha}-synuclein fibrillation. One of these would be through stabilization of off-pathway oligomers, and another would be through the capping nascent fibrils. To investigate these possibilities, we monitored the association state of Met-oxidized {alpha}-synuclein during its incubation, in the absence and presence of metal ions, using dynamic light scattering (Fig. 4). Given the nature of the experimental measurements, populations of oligomers of less than 5–10% are not considered significant. Since the data shown in Fig. 4 are only for soluble protein, the total concentrations may be different in the different panels of the figure.



View larger version (49K):
[in this window]
[in a new window]
 
FIG. 4.
Population of oligomers during {alpha}-synuclein fibrillation determined from dynamic light scattering. Top row, unmodified {alpha}-synuclein: control (A), Ca2+ (B), Zn2+ (C). Bottom row, corresponding methionine-oxidized {alpha}-synuclein: control (D), Ca2+ (E), Zn2+ (F). The height of the bars represents the population of the species, and the position of the bars reflects the time of incubation and size of the species.

 

Met-oxidized {alpha}-synuclein remained monomeric for >100 h under standard incubation conditions (35 µM {alpha}-synuclein, pH 7.5, 37 °C, with agitation), as shown in Fig. 4D, indicating that neither oligomers nor fibrils were formed in statistically significant amounts. In contrast, unmodified {alpha}-synuclein remained predominantly monomeric for the first 20 h (corresponding to the lag time) but then showed dimers and higher oligomers at longer times (in addition to fibrils), as shown in Fig. 4A. Thus, the conversion of methionine to its sulfoxide must, in some way, prevent formation of the critical partially folded intermediate conformation and subsequent association into fibrils. In the presence of Zn2+, which leads to fibril formation from the Met-oxidized {alpha}-synuclein, the monomer is the only species initially present. However, at later times, in addition to fibrils, soluble oligomers were detected, amounting to as much as 30% of the total protein and having an Rs of ~40 nm, similar to the size of the oligomers observed with the unmodified protein. In contrast, in the presence of Ca2+, which does not lead to fibrils with Met-oxidized {alpha}-synuclein, only the monomer was detected during the incubation. Since these two metals reflect the observed behavior of the two types of metal ion-induced effects in the other properties investigated, their behavior is considered representative of other metal ions.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress is believed to be a factor in the etiology of Parkinson's disease, and the methionine residues of {alpha}-synuclein are the most easily oxidized side chains in the protein. Therefore, our previous discovery that methionine-oxidized {alpha}-synuclein, which is expected to represent one of the most common products of oxidative damage to {alpha}-synuclein, fails to form fibrils and inhibits fibrillation of unmodified {alpha}-synuclein was rather surprising, although oxidation of the single methionine residue in A{beta} has also been shown to attenuate fibrillation of A{beta} (35).

Previously, we have shown that formation of a partially folded intermediate is a critical initial step of the {alpha}-synuclein fibrillogenesis (11) and that {alpha}-synuclein fibrillation is accelerated under conditions that stabilize such an intermediate state, e.g. at acidic pH or high temperature (11, 36) or in the presence of metal cations (21). A contributing factor to the inhibition of methionine-oxidized {alpha}-synuclein fibrillation is believed to be the slightly increased stabilization of the natively unfolded conformation (33).

The data presented here are consistent with the conclusion that interaction of methionine-oxidized {alpha}-synuclein with certain metals modulates its conformational properties and propensity for fibrillation. Whereas all the metals studied are able to induce partial folding in this intrinsically unstructured (natively unfolded) protein, not all cations are equal in their abilities to eliminate the inhibitory effect of methionine oxidation on {alpha}-synuclein fibrillation. In particular, the fibrillation rates were very close for oxidized and non-oxidized {alpha}-synuclein in the presence of Zn2+ and Pb2+; however, fibrillation was still inhibited in the presence of Hg2+, Cu2+, and Ca2+. This observation indicates that factors other than electrostatic interactions must play an important role in overcoming the inhibition of {alpha}-synuclein fibrillation caused by methionine oxidation. One such factor is undoubtedly the known propensity for certain metals to strongly coordinate with sulfoxides, leading to very stable complexes (37). In particular, for some metal ions, bridging between two sulfoxides is favored. Such intermolecular or intramolecular coordination of two (or more) methionine sulfoxides could significantly affect the fibrillation. In particular, we propose that stable intermolecular bridging metal complexes would significantly promote fibrillation: thus, the presence of Zn2+ or Pb2+ leads to intermolecular cross-bridging, which facilitates the association of Met-oxidized {alpha}-synuclein and leads to its subsequent fibrillation. Metals such as Hg2+ and Cu2+, which may also form sulfoxide bridges, may be limited to intramolecular coordination due to different ligand bonding. The results show that in those conditions where fibrillation occurs, large soluble oligomers are present at the latter stages of the lag time and during the fibril growth stage of the aggregation process.

With regard to the biological relevance of these observations, it is becoming clear that many factors can affect the rate of {alpha}-synuclein fibrillation, suggesting that in dopaminergic neurons, there is a balance between factors that can accelerate fibrillation and those that inhibit or prevent it. It is likely that there are chaperones or chaperone-like species that are important in minimizing {alpha}-synuclein aggregation under normal conditions. In our earlier study, showing that the addition of Met-oxidized {alpha}-synuclein inhibited fibrillation of the non-oxidized form (33), we suggested that the methionine residues in {alpha}-synuclein may be used by the cells as a natural scavenger of reactive oxygen species, since (a) methionine can react with essentially all of the known oxidants found in normal and pathological tissues; (b) {alpha}-synuclein is a very abundant brain protein; (c) it has recently been shown that the concentration of {alpha}-synuclein could be increased significantly as a result of the neuronal response to toxic insult (23); and (d) methionine sulfoxide residues in proteins can be cycled back to their native methionines by methionine sulfoxide reductase (38), a process that might protect other functionally essential residues from oxidative damage (39). It should be noted, however, that the efficiency of this regeneration system must take into account the finding that methionine oxidation forms the sulfoxide in two diastereoisomer forms and that stereoselective oxidation can sometimes occur, dependent on both the structural restraints in the region of the methionine molecule and on the oxidant itself (40). Each methionine sulfoxide isomer can be reduced back to its original methionine state, provided that the corresponding complementary reductase is present and active (41).

The balance between the protective antioxidant role of the methionine residues that is enhanced by this recycling and the protective antifibrillation effect of oxidized methionine residues in {alpha}-synuclein may fail under conditions of environmental pollution due to exposure of a person to lead, aluminum, zinc, titanium, and other metals. We assume that in the presence of the enhanced concentrations of such industrial pollutants, toxic insult-induced up-regulation of {alpha}-synuclein may no longer play a protective role; rather, it may represent a risk factor, leading to metal-triggered fibrillation of the methionine-oxidized protein.


    FOOTNOTES
 
* This work was supported by Grant NS39985 from The National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Present address: 307 Greene St., Mill Valley, CA 94941. Back

§ To whom correspondence should be addressed. Tel.: 831-459-2744; Fax: 831-459-2935; E-mail: enzyme{at}cats.ucsc.edu.

1 The abbreviations used are: PD, Parkinson's disease; ThT, thioflavin T. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Lewy, F. H. (1912) in Handbuch der Neurologie (Lewandowski, M., ed), pp. 920–933, Springer, Berlin
  2. Forno, L. S. (1996) J. Neuropathol. Exp. Neurol. 55, 259–272[Medline] [Order article via Infotrieve]
  3. Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Nature 388, 839–840[CrossRef][Medline] [Order article via Infotrieve]
  4. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., and Nussbaum, R. L. (1997) Science 276, 2045–2047[Abstract/Free Full Text]
  5. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Nat. Genet. 18, 106–108[Medline] [Order article via Infotrieve]
  6. Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A., Sagara, Y., Sisk, A., and Mucke, L. (2000) Science 287, 1265–1269[Abstract/Free Full Text]
  7. Feany, M. B., and Bender, W. W. (2000) Nature 404, 394–398[CrossRef][Medline] [Order article via Infotrieve]
  8. Maroteaux, L., Campanelli, J. T., and Scheller, R. H. (1988) J. Neurosci. 8, 2804–2815[Abstract]
  9. Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., de Silva, H. A., Kittel, A., and Saitoh, T. (1995) Neuron 14, 467–475[Medline] [Order article via Infotrieve]
  10. Weinreb, P. H., Zhen, W. G., Poon, A. W., Conway, K. A., and Lansbury, P. T., Jr. (1996) Biochemistry 35, 13709–13715[CrossRef][Medline] [Order article via Infotrieve]
  11. Uversky, V. N., Li, J., and Fink, A. L. (2001) J. Biol. Chem. 276, 10737–10744[Abstract/Free Full Text]
  12. Uversky, V. N., Gillespie, J. R., and Fink, A. L. (2000) Proteins 41, 415–427[CrossRef][Medline] [Order article via Infotrieve]
  13. Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Nat. Med. 4, 1318–1320[CrossRef][Medline] [Order article via Infotrieve]
  14. Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 571–576[Abstract/Free Full Text]
  15. Crowther, R. A., Jakes, R., Spillantini, M. G., and Goedert, M. (1998) FEBS Lett. 436, 309–312[CrossRef][Medline] [Order article via Infotrieve]
  16. Giasson, B. I., Uryu, K., Trojanowski, J. Q., and Lee, V. M. Y. (1998) J. Biol. Chem. 274, 7619–7622[Abstract/Free Full Text]
  17. Wood, S. J., Wypych, J., Steavenson, S., Louis, J. C., Citron, M., and Biere, A. L. (1999) J. Biol. Chem. 274, 19509–19512[Abstract/Free Full Text]
  18. Serpell, L. C., Berriman, J., Jakes, R., Goedert, M., and Crowther, R. A. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4897–4902[Abstract/Free Full Text]
  19. Tanner, C. M., Ottman, R., Goldman, S. M., Ellenberg, J., Chan, P., Mayeux, R., and Langston, J. W. (1999) JAMA (J. Am. Med. Assoc.) 281, 341–346[Abstract/Free Full Text]
  20. Tanner, C. M. (1989) Trends Neurosci. 12, 49–54[CrossRef][Medline] [Order article via Infotrieve]
  21. Uversky, V. N., Li, J., and Fink, A. L. (2001) J. Biol. Chem. 276, 44284–44296[Abstract/Free Full Text]
  22. Uversky, V. N., Li, J., and Fink, A. L. (2001) FEBS Lett. 500, 105–108[CrossRef][Medline] [Order article via Infotrieve]
  23. Manning-Bog, A. B., McCormack, A. L., Li, J., Uversky, V. N., Fink, A. L., and Di Monte, D. A. (2002) J. Biol. Chem. 277, 1641–1644[Abstract/Free Full Text]
  24. Uversky, V. N., Li, J., Bower, K., and Fink, A. L. (2002) Neurotoxicology 23, 527–536[CrossRef][Medline] [Order article via Infotrieve]
  25. Jenner, P., Dexter, D. T., Sian, J., Schapira, A. H., and Marsden, C. D. (1992) Ann. Neurol. 32, (suppl.) S82–S87[Medline] [Order article via Infotrieve]
  26. Yoritaka, A., Hattori, N., Uchida, K., Tanaka, M., Stadtman, E. R, and Mizuno, Y. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 2696–2701[Abstract/Free Full Text]
  27. Good, P. F., Hsu, A., Werner, P., Perl, D. P., and Olanow, C. W. (1998) J. Neuropathol. Exp. Neurol. 57, 338–342[Medline] [Order article via Infotrieve]
  28. Duda, J. E., Giasson, B. I., Chen, Q., Gur, T. L., Hurtig, H. I., Stern, M. B., Gollomp, S. M., Ischiropoulos, H., Lee, V. M., and Trojanowski, J. Q. (2000) Am. J. Pathol. 157, 1439–1445[Abstract/Free Full Text]
  29. Giasson, B. I., Duda, J. E., Murray, I. V., Chen, Q., Souza, J. M., Hurtig, H. I., Ischiropoulos, H., Trojanowski, J. Q., and Lee, V. M. (2000) Science 290, 985–989[Abstract/Free Full Text]
  30. Paik, S. R., Shin, H. J., and Lee, J. H. (2000) Arch. Biochem. Biophys. 378, 269–277[CrossRef][Medline] [Order article via Infotrieve]
  31. Conway, K. A., Rochet, J. C., Bieganski, R. M., and Lansbury, P. T., Jr. (2001) Science 294, 1346–1349[Abstract/Free Full Text]
  32. Krishnan, S., Chi, E. Y., Wood, S. J., Kendrick, B. S., Li, C., Garzon-Rodriguez, W., Wypych, J., Randolph, T. W., Narhi, L. O., Biere, A. L., Citron, M., and Carpenter, J. F. (2003) Biochemistry 42, 829–837[CrossRef][Medline] [Order article via Infotrieve]
  33. Uversky, V. N., Yamin, G., Souillac, P. O., Goers, J., Glaser, C. B., and Fink, A. L. (2002) FEBS Lett. 517, 239–244[CrossRef][Medline] [Order article via Infotrieve]
  34. Naiki, H., Higuchi, K., Matsushima, K., Shimada, A., Chen, W. H., Hosokawa, M., and Takeda, T. (1990) Lab. Invest. 62, 768–773[Medline] [Order article via Infotrieve]
  35. Butterfield, D. A., and Kanski, J. (2002) Peptides (Elmsford) 23, 1299–1309[Medline] [Order article via Infotrieve]
  36. Uversky, V. N., Lee, H. J., Li, J., Fink, A. L., and Lee, S. J. (2001) J. Biol. Chem. 276, 43495–43498[Abstract/Free Full Text]
  37. Cotton, F. A., and Francis, R. (1960) J. Amer. Chem. Soc. 82, 2986–2991
  38. Levine, R. L., Mosoni, L., Berlett, B. S., and Stadtman, E. R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 15036–15040[Abstract/Free Full Text]
  39. Reddy, P. S., and Bhagyalakshmi, A. (1994) Ecotoxicol. Environ. Saf. 29, 255–264[Medline] [Order article via Infotrieve]
  40. Sharov, V. S., Ferrington, D. A., Squier, T. C., and Schoneich, C. (1999) FEBS Lett. 455, 247–250[CrossRef][Medline] [Order article via Infotrieve]
  41. Sharov, V. S., and Schoneich, C. (2000) Free Radic. Biol. Med. 29, 986–994[CrossRef][Medline] [Order article via Infotrieve]