Certain Metals Trigger Fibrillation of Methionine-oxidized
␣
-Synuclein*
Received for publication, March 31, 2003, and in revised form, May 13, 2002
Published, JBC Papers in Press, May 16, 2003, DOI 10.1074/jbc.M303302200
Ghiam Yamin, Charles B. Glaser‡, Vladimir N. Uversky, and Anthony L. Fink§
From the Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064
The aggregation and fibrillation of
␣
-synuclein has
been implicated as a key step in the etiology of Parkin-
son’s disease and several other neurodegenerative dis-
orders. In addition, oxidative stress and certain envi-
ronmental factors, including metals, are believed to
play an important role in Parkinson’s disease. Previ-
ously, we have shown that methionine-oxidized human
␣
-synuclein does not fibrillate and also inhibits fibrilla-
tion of unmodified
␣
-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 sta-
bilization of the monomeric form of Met-oxidized
␣
-synuclein. We have now examined the effect of several
metals on the structural properties of methionine-oxi-
dized human
␣
-synuclein and its propensity to fibrillate.
The presence of metals induced partial folding of both
oxidized and non-oxidized
␣
-synucleins, which are in-
trinsically unstructured under conditions of neutral pH.
Although the fibrillation of
␣
-synuclein was completely
inhibited by methionine oxidation, the presence of cer-
tain metals (Ti
3ⴙ
,Zn
2ⴙ
,Al
3ⴙ
, and Pb
2ⴙ
) overcame this
inhibition. These findings indicate that a combination of
oxidative stress and environmental metal pollution
could play an important role in triggering the fibrilla-
tion of
␣
-synuclein and thus possibly Parkinson’s
disease.
Parkinson’s disease (PD)
1
is the second most common neu-
rodegenerative disorder after Alzheimer’s disease. Clinical
symptoms of PD (tremor, rigidity, and bradykinesia) are attrib-
uted 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
␣
-synuclein (3). The
mutations A53T and A30P in
␣
-synuclein have been identified
in autosomal-dominantly inherited, early onset PD (4, 5). Fur-
thermore, the production of
␣
-synuclein in transgenic mice (6)
or in transgenic flies (7) leads to motor deficits and neuronal
inclusions reminiscent of PD. All this implicates
␣
-synuclein in
the pathogenesis of PD.
␣
-Synuclein is a small (14 kDa), highly conserved presynap-
tic protein that is abundant in various regions of the brain (8,
9). Structurally, purified
␣
-synuclein belongs to the rapidly
growing family of intrinsically unstructured or natively un-
folded proteins (10, 11), which have little or no ordered struc-
ture under physiological conditions due to a unique combina-
tion of low overall hydrophobicity and large net charge (12).
␣
-Synuclein readily assembles into amyloid-like fibrils in vitro
with morphologies and staining characteristics similar to those
extracted from disease-affected brain (11, 13–18). 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 sug-
gests 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 re-
ported that direct interaction of
␣
-synuclein with metal ions
(21) or pesticides leads to accelerated fibrillation (22–24).
Oxidative injury is also suspected as another causative agent
in the pathogenesis of PD (25, 26). The existence of nitrated
␣
-synuclein (i.e. protein containing the product of the tyrosine
oxidation, 3-nitrotyrosine) accumulation in Lewy bodies has
been demonstrated (27–29). Accumulation of another product
of tyrosine oxidation, dityrosine, has been detected in vitro
during experiments on the aggregation of
␣
-synuclein in the
presence of copper and H
2
O
2
(30) or catecholamines (31) and
leads to accelerated fibrillation of
␣
-synuclein (32). The methi-
onine side chain is the most readily oxidized amino acid in
␣
-synuclein, and the four methionines, Met-1, Met-5, Met-116,
and Met-127, are easily oxidized in vitro in the presence of
H
2
O
2
. Interestingly, however, oxidation of the methionine res-
idues of
␣
-synuclein to the sulfoxides, rather than accelerating
fibrillation, was found to prevent it (33). Furthermore, and
most importantly, the presence of the methionine-oxidized
␣
-synuclein was found to completely inhibit fibrillation of the
unmodified protein at ratios of ⱖ4:1 (33). Given the potential
role of metals in the pathological aggregation of
␣
-synuclein
and the known strong coordination of some metals to sulfox-
ides, we decided to investigate the structural and fibrillation
properties of Met-oxidized
␣
-synuclein in the presence of sev-
eral metals to shed more light on the combined effect of envi-
ronmental factors (metals) and oxidative damage (methionine
oxidation to the sulfoxide) on
␣
-synuclein.
MATERIALS AND METHODS
Expression and Purification of Human
␣
-Synuclein—Human recom-
binant
␣
-synuclein was expressed in the Escherichia coli BL21(DE3)
cell line transfected with pRK172/
␣
-synuclein wild-type plasmid (kind
gift of M. Goedert, MRC Cambridge) and purified as described previ-
ously (33). Purity of the
␣
-synuclein was determined by SDS-polyacryl-
* This work was supported by Grant NS39985 from The National
Institutes of Health. The costs of publication of this article were de-
frayed 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.
‡ Present address: 307 Greene St., Mill Valley, CA 94941.
§ To whom correspondence should be addressed. Tel.: 831-459-2744;
Fax: 831-459-2935; E-mail: enzyme@cats.ucsc.edu.
1
The abbreviations used are: PD, Parkinson’s disease; ThT,
thioflavin T.
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 30, Issue of July 25, pp. 27630–27635, 2003
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
This paper is available on line at http://www.jbc.org27630
