Methionine oxidation, α-synuclein and Parkinson's disease

Biochimica et Biophysica Acta 1703 (2005) 157 – 169 http://www.elsevier.com/locate/bba

Review

Methionine oxidation, a-synuclein and Parkinson’s disease Charles B. Glasera, Ghiam Yaminb, Vladimir N. Uverskyb, Anthony L. Finkb,* a

b

307 Greene Street, Mill Valley, CA 94941, United States Department of Chemistry and Biochemistry, University of California, 1156 High Street, Santa Cruz, CA 95064, USA Received 19 July 2004; received in revised form 18 October 2004; accepted 18 October 2004 Available online 2 November 2004

Abstract The aggregation of normally soluble a-synuclein in the dopaminergic neurons of the substantia nigra is a crucial step in the pathogenesis of Parkinson’s disease. Oxidative stress is believed to be a contributing factor in this disorder. Because it lacks Trp and Cys residues, mild oxidation of a-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides. Both oxidized and non-oxidized a-synucleins have similar unfolded conformations; however, the fibrillation of a-synuclein at physiological pH is completely inhibited by methionine oxidation. The inhibition results from stabilization of soluble oligomers of Met-oxidized asynuclein. Furthermore, the Met-oxidized protein also inhibits fibrillation of unmodified a-synuclein. The degree of inhibition of fibrillation by Met-oxidized a-synuclein is proportional to the number of oxidized methionines. However, the presence of metals can completely overcome the inhibition of fibrillation of the Met-oxidized a-synuclein. Since oligomers of aggregated a-synuclein may be cytotoxic, these findings indicate that both oxidative stress and environmental metal pollution could play an important role in the aggregation of a-synuclein, and hence possibly Parkinson’s disease. In addition, if the level of Met-oxidized a-synuclein was under the control of methionine sulfoxide reductase (Msr), then this could also be factor in the disease. D 2004 Elsevier B.V. All rights reserved. Keywords: Methionine oxidation; a-Synuclein; Parkinson’s disease

1. Introduction Many studies have indicated that oxidative stress is a risk factor for dopamine cell degeneration in Parkinson’s disease (PD) [1,13,38]. The healthy brain continuously generates high levels of reactive oxygen and nitrogen species. Paradoxically, it is least able to handle the high levels of reactive oxygen species (ROS) because of low levels of both antioxidant enzymes and cellular antioxidants [1,45]. The brain uses about 25% of respired oxygen even though it constitutes only 5% of body weight. Diseases involving oxidative stress can result from ineffective scavenger systems, insufficient concentrations of antioxidants, overproduction of free radicals or other oxidants, or a combination of all of these. Also, neurons have a very high

* Corresponding author. Tel.: +1 831 459 2744; fax: +1 831 459 2935. E-mail address: [email protected] (A.L. Fink). 1570-9639/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2004.10.008

membrane/volume ratio due to their unique shape, i.e., long thin extensions of axons and dendrites, and small cell bodies. Brain membranes have high levels of polyunsaturated fatty acids and thus are prime targets for oxygen and free radical damage. During normal respiration, the mitochondria generate water from oxygen, but also produce superoxide anion, hydrogen peroxide and hydroxyl free radical. In addition, the dopaminergic neurons in the substantia nigra, the region of the brain that is most affected in Parkinson’s disease, have a special sensitivity to free radicals, due to their high levels of dopamine [9] and Fe2+ [43,65]. The deposition of protein fibrils (amyloid) is a prominent feature of a number of protein conformational diseases or protein deposition diseases, including Alzheimer’s disease (AD), Parkinson’s disease, motor neuron disease (ALS), the prion diseases and many others (reviewed in Refs. [2,15,46,80,103]). Under normal cellular conditions, misfolded proteins are rapidly sequestered

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or eliminated from the intracellular environment by two different pathways, molecular chaperones or the ubiquitin proteasomal system. However, under particular pathological conditions, misfolded proteins may aggregate, initiating a cascade of events that leads eventually to cell death [3,10,17,25,29,40,41,48,49,76]. The aggregation process may be a consequence of increased levels of protein misfolding, abnormalities in the ubiquitin–proteasome system for removing damaged proteins, or problems with the chaperone systems, or a combination of all three. Parkinson’s disease, the second most common neurodegenerative disease, results from death of dopaminergic neurons in the substantia nigra. Some surviving nigral dopaminergic neurons contain cytosolic filamentous inclusions known as Lewy bodies (LBs) and Lewy neurites (LNs) [21]. Besides the substantia nigra, LBs and LNs also are found in other brain regions, such as the dorsal motor nucleus of the vagus, the nucleus basalis of Meynert, and the locus coeruleus [21]. In addition, abundant LBs and LNs in the cerebral cortex are neuropathological hallmarks of dementia with LBs, a common late-life dementia that is clinically similar to Alzheimer’s disease [75], in LB variant of Alzheimer’s disease [100], diffuse LB disease [98], multiple system atrophy [60,100], and several other neurodegenerative disorders, collectively known as synucleinopathies [28,101]. The major fibrillar material of LBs and LNs is a-synuclein [90]. The discovery that a missense mutation in the asynuclein gene resulted in autosomal dominantly inherited PD, and that this was accompanied by the accumulation of a-synuclein aggregates, sparked specific interest in the role of a-synuclein in this disease. Three different missense mutations in the a-synuclein gene, corresponding to A53T, A30P and E46K substitutions in a-synuclein, have now been identified in autosomal-dominantly inherited, earlyonset Parkinson’s disease [52,77,116]. The recent finding that triplication of the a-synuclein gene locus causes autosomal dominant PD with an average age of onset of 34 years [87] confirmed the critical role of a-synuclein aggregation in the etiology of PD [101]. The production of wild-type (WT) human a-synuclein in transgenic mice [62] or of WT, A30P, and A53T human a-synuclein in transgenic flies [20] leads to motor deficits and neuronal inclusions reminiscent of PD. All three proteins, as well as several Cterminal-truncated forms of recombinant a-synuclein, are able to assemble readily into filaments in vitro, with morphologies and staining characteristics similar to those extracted from disease-affected brain [11,84,90,112]. Furthermore, fibrils formed in vitro from a-synuclein and the familial mutant forms linked to Parkinson’s disease are typical amyloid fibrils, i.e., structurally and morphologically they resemble fibrils formed by other amyloidogenic proteins. Interestingly, the peptide derived from the central hydrophobic region of a-synuclein represents a constituent of Alzheimer’s plaques. This 35-amino-acid peptide, known as NAC (Non-Ah Component of Alzheimer’s disease

amyloid), was shown to amount to about 10% of the amyloid plaque [102]. Recently, synergistic interactions in the fibrillation of tau (involved in Alzheimer’s disease) and a-synuclein have been reported [26]. These observations indicate that a-synuclein is a key player in the pathogenesis of several neurodegenerative disorders. a-Synuclein is a small, acidic, natively unfolded (intrinsically disordered) and soluble protein that is found both in the cytosol and associated with presynaptic vesicles [106,111]. It contains 140 amino acid residues, which can be grouped in three regions [61]: The N-terminal region consisting of 60 residues containing four 11-amino-acid imperfect repeats with a hexameric consensus motif (KTKEGV), a central region comprising the amyloidogenic NAC sequence (residues 61–95) with two additional repeats, and the C-terminal region (residues 96–140), rich in acidic residues and prolines, suggesting a disordered conformation and containing three conserved tyrosine residues. The function of a-synuclein is unknown but its presence in presynaptic nerve terminals and its interaction with lipids and proteins suggest multiple functions, including lipid vesicle trafficking. Recent investigations of several diseasecausing amyloidogenic proteins, including a-synuclein, suggest that the cytotoxic species is probably not the actual fibrils, but rather, some earlier oligomeric species [7,8,19,36,44,47,54,55,64,78,117]. It is likely that such oligomers lead to membrane permeability and hence cell death. Numerous observations suggest that oxidative stress may be associated with Parkinson’s disease; for example, inhibitors of mitochondrial function (such as MPTP), which lead to release of reactive oxygen species (ROS), result in neuronal degeneration and loss of dopaminergic neurons. All amino acids are susceptible to oxidation, although their reactivities vary greatly [92,93]. Methionine and cysteine are the most readily oxidized amino acids and are unique in that the products of oxidation can, under some circumstances, be reduced back to the native amino acid residues. For example, methionine is easily oxidized to methionine sulfoxide (MetO) by H2O2, hypochlorite, chloramines, and peroxynitrite; all these oxidants are produced in biological systems [108]. However, this modification can be repaired by methionine sulfoxide reductase (Msr), which catalyzes the thioredoxin-dependent reduction of MetO back to methionine, both in vitro [70,97] and in vivo [66,70]. The details of Msr action are described below in the section on Msr. The nature of the relationship between protein oxidation, protein aggregation, and neurodegeneration in Parkinson’s disease are still unclear. There is very good data to support the premise that the aggregation of a-synuclein is a critical component of the etiology of PD, and substantial evidence suggests that oxidative stress is associated with PD; however, there are as yet no unambiguous data regarding the connection between a-synuclein oxidation and its aggregation and cytotoxicity. In fact, there are many

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contradictory results and conclusions regarding the effect of oxidative stress, including nitration, on a-synuclein. Because it lacks Trp and Cys residues, there are only limited options for the oxidation of a-synuclein under physiological conditions. Under relatively mild oxidation conditions, the only effect will be conversion of Met residues to the sulfoxides. Stronger oxidizing conditions, with the formation of peroxynitrite and its products including nitronium ion and hydroxyl free radical, will result in Tyr nitration and Tyr cross-linking and radicalinduced random cross-linking, which, depending on the site and nature of the cross-link, may or may not lead to enhanced aggregation. Although there have been numerous attempts to form a specific antibody to the methionine sulfoxide moiety, thus far none has been successful. This has hampered efforts to quantitate levels of oxidized methionine in biological fluids and to identify these derivatives in cellular aggregates. In this article, we review the structural properties and fibrillation characteristics of methionine oxidized a-synuclein and its Met-depleted mutants, and consider the effect of metals on the properties of the oxidized a-synuclein. In addition, the reversibility of this reaction catalyzed by Msr is considered. Finally, the implications of a-synuclein oxidation and Parkinson’s disease are discussed.

2. Effect of methionine oxidation on structural properties of human A-synuclein Human a-synuclein has four methionines, Met1, Met5, Met116, and Met127, all located outside the repeatcontaining region. Oxidation of a-synuclein was accomplished by incubation of the protein in the presence of 4% H2O2 for 20 min, resulting in the oxidation of all four methionines to the sulfoxide, and no other modifications, as confirmed by mass spectrometry [107].

Fig. 1. Conformational effects of methionine oxidation on a-synuclein. The effect of Met oxidation on the far-UV CD spectra of non-oxidized (solid line), and oxidized a-synuclein (dashed line). CD spectra were measured at pH 7.5 at 23 8C in 20 mM Tris–HCl buffer, pH 7.5. Protein concentration was 0.5 mg/ml.

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Fig. 2. Kinetics of fibrillation of non-oxidized (circles) and oxidized (squares) a-synuclein monitored by the enhancement of Thioflavin T fluorescence intensity. Measurements were performed at 37 8C, 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.

Structural properties of non-oxidized and oxidized forms of human recombinant a-synuclein have been compared using several biophysical approaches [107]. For example, the FTIR spectra of the amide I region (which corresponds to contributions from the carbonyl stretch, and is a measure of secondary structure) for both non-oxidized and oxidized a-synuclein are typical of a substantially unfolded polypeptide chain. However, the results of the deconvolution of the FTIR spectra showed that Met-oxidized a-synuclein contains somewhat less extended structure and slightly more disordered structure [107]. In agreement with the FTIR data, the far UV-CD spectrum of Met-oxidized a-synuclein (Fig. 1) is characterized by a slightly increased contribution of disordered structure in comparison with the non-oxidized protein (as manifested by a small increase in negative ellipticity in the vicinity of 196 nm and somewhat lower intensity in the vicinity of 222 nm). The increased degree of unfolding of the oxidized protein has been attributed to the decreased hydrophobicity of oxidized methionine [108] leading to the decrease in the overall hydrophobicity of the protein [107]. Another member of the synuclein family, hsynuclein, which lacks 11 residues of the central hydrophobic region of a-synuclein, has also been reported to have the properties expected of a random coil [106]. Thus, a decrease in the overall hydrophobicity in both h-synuclein and Met-oxidized a-synuclein leads to a more disordered conformation, compared to a-synuclein. Interestingly, both non-oxidized and oxidized forms of asynuclein possess almost identical far-UV CD spectra at acidic pH, which are consistent with pH-induced partial folding of the protein [107]. It has been shown that the pHinduced increase in ordered structure in natively unfolded members of the synuclein family represents an intramolecular process involving the formation of an amyloidogenic partially folded intermediate, and not self-association

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[104,106], which also appears to be the situation for the Met-oxidized form of human a-synuclein [107]. 3. Fibrillation of methionine oxidized A-synuclein Fibrillation kinetics of non-oxidized and oxidized asynuclein have been compared using the characteristic changes in the thioflavin T (ThT) fluorescence assay [107]. ThT is a fluorescent dye that interacts specifically with amyloid fibrils leading to a large increase in fluorescence intensity in the vicinity of 480 nm [72,73]. Fibril formation of non-oxidized a-synuclein at neutral pH is characterized by a typical sigmoidal curve, corresponding to a nucleated polymerization process (Fig. 2). In marked contrast, methionine-oxidized a-synuclein shows no evidence of fibril formation when incubated under standard conditions (pH 7.4, 378C, 100 mM NaCl) [107]. Rather surprisingly, oxidized a-synuclein was shown to form fibrils under conditions of low pH: the most likely explanation is that low pH favors formation of the critical amyloidogenic partially folded conformation of a-synuclein, and that this forms at low pH in the presence of Met sulfoxide, but not at neutral pH [107].

4. Oxidized a-synuclein inhibits fibrillation of the non-oxidized protein

Fig. 4. Size exclusion HPLC analysis of the association state of Metoxidized a-synuclein. The unoxidized monomer is shown for comparison (dotted line). The Met-oxidized a-synuclein elutes as an oligomer (solid line). The elution was monitored by the absorbance at 280 nm.

concentration of the oxidized protein led to almost complete inhibition of the non-oxidized protein fibrillation (Fig. 3) [107]. These results suggested that an interaction between oxidized and non-oxidized forms of human a-synuclein occurs, which inhibits the formation of nuclei, rather than growth of nascent fibrils. In fact, the inhibition of fibril formation by non-oxidized a-synuclein in the presence of oxidized protein originates from the formation of soluble hetero-oligomers, which are located off the fibrillation pathway (Fig. 4).

Intriguingly, the addition of methionine-oxidized asynuclein in a twofold molar excess to a 70 AM solution of non-oxidized a-synuclein substantially increased the duration of the lag-time, but had little effect on the rate constant for fibril growth. Further increase in the relative

5. Models of the non-oxidized and oxidized A-synuclein fibrillation

Fig. 3. Inhibition of fibrillation of non-oxidized a-synuclein in the presence of oxidized a-synuclein. The fibrillation kinetics were monitored with ThT in 20 mM Na-phosphate buffer, 100 mM NaCl, pH 7.5, using 70 AM nonoxidized a-synuclein solution in the absence (circles) or presence of Metoxidized protein in 1:2 (triangles), and 1:4 molar ratios (inverted triangles). The data for the oxidized protein alone is shown for comparison (squares).

Models for the aggregation of non-oxidized (A) and oxidized (B) a-synuclein at neutral pH are shown in Fig. 5 [107]. In the model, O and F correspond to soluble oligomers and fibrils, respectively; UN and UN* are the natively unfolded states of non-oxidized and oxidized asynuclein, respectively; and I is a partially folded intermediate. The Roman numerals indicate the major stages of the aggregation process. The model is based on the following assumptions, derived from experimental observations. Methionine oxidation leads to a decrease in a-synuclein residual structure (UN* vs. UN), due to the oxidation-induced increase in the overall polarity of the protein. This in turn leads to a shift in the equilibrium in stage I for the oxidized protein, UN*X I, in favor of the unfolded conformation UN*. Finally, stages II and III are arrested (or at least strongly inhibited) by the oxidation at neutral pH, but formation of soluble oligomers still occurs, stage IV (see Fig. 5B). Fig. 5C represents a model for the inhibitory effect of oxidized a-synuclein on fibrillation of the non-oxidized protein. In a mixture of non-oxidized and oxidized proteins, the conformational

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Fig. 5. Kinetic models for non-oxidized and oxidized a-synuclein aggregation. (A) Aggregation of the non-oxidized protein at neutral pH. (B) Aggregation of the oxidized protein at neutral pH. (C) Aggregation of a mixture of non-oxidized and oxidized proteins. In the model UN and UN* are the natively unfolded states of non-oxidized and oxidized a-synuclein, respectively; O and F correspond to soluble oligomers and fibrils, respectively; O* represents a hetero-oligomer containing both Met-oxidized and unoxidized a-synuclein; and I is a partially folded intermediate. The Roman numerals indicate the major stages of the aggregation process.

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of all methionines present in a given protein, as evidenced by mass spectral analysis [35]. Far-UV CD spectral analysis revealed that the substitution of one or two leucines for methionines did not affect the structure of natively unfolded a-synuclein. On the other hand, substitution of three Met residues by Leu residues leads to small but reproducible changes in the spectra, reflecting mutation-induced formation of small amount of ordered secondary structure [35]. The non-oxidized proteins possess slightly more ordered structure, most likely in a form of h-structure. This means that the oxidation of asynucleins led to the depletion in the amount of the residual secondary structure. Interestingly, the degree of the increase in disorder was more prominent for the WT protein and its single mutants, than for the double mutants, whereas the structure of triple mutants was almost unaffected by oxidation. This clearly demonstrates a role of Met oxidation in the enhancement of disordered structure [35]. The oxidation of methionines was shown to dramatically affect the fibrillation of the MetYLeu mutants. The degree of inhibition of fibrillation by MetO a-synuclein is proportional to the number of oxidized methionines. The results showed that with one oxidized Met, the kinetics of fibrillation were comparable to those for the control (nonoxidized), and with increasing numbers of methionine sulfoxides the kinetics of fibrillation became progressively slower (Fig. 6) [35]. Overall, the data support the hypothesis that the inhibitory effect on fibrillation due to methionine sulfoxide formation is uniformly distributed between the four methionines. Thus, comparison of the oxidationinduced decrease in the fibrillation rates of human asynuclein bearing different MetYLeu substitutions clearly showed that the oxidation of individual methionines has a cumulative effect on the fibrillation.

equilibrium is shifted toward the formation of soluble hetero-oligomers, O* [107].

6. Understanding the role of individual methionines in the fibrillation of Met-oxidized A-synuclein In order to determine the contribution of individual methionines to the conformational and aggregation behavior of human a-synuclein, a series of mutants containing methionine to leucine substitutions was created. This series includes three single mutants, M5L, M116L, M127L, each containing three Met residues; two double mutants, M5L/ M127L and M116/M127, each containing two Mets; a triple mutant, M5L/M116L/M127, which contains a single Met, and a triple mutant (Met1-)M116L/M127L, in which Met1 was removed by aminopeptidase, also containing a single Met; and a quadruple mutant, (Met1 )M5L/M116L/M127 in which Met1 was removed by aminopeptidase and which contained no Met residues [35]. The conditions used for the in vitro oxidation of the a-synucleins resulted in oxidation

Fig. 6. Comparison of the kinetics of fibrillation of Met-oxidized mutants of a-synuclein, showing that the inhibition increases with increasing numbers of oxidized methionine residues. Monitored by Thioflavin T fluorescence. Measurements were performed at 37 8C and pH 7.5 in 20 mM Naphosphate buffer containing 100 mM NaCl. Four oxidized Mets (circles); three oxidized Mets (black dots); two oxidized Mets (triangles); one oxidized Met (squares); and no oxidized Mets (inverted triangles).

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7. Metals can trigger fibrillation of methionine-oxidized A-synuclein The interaction of metal cations with a-synuclein induces a partially folded conformation, attributed to the counter ion-induced neutralization of the Coulombic charge–charge repulsion within the very negatively charged protein at neutral pH [105]. Furthermore, the interaction with metals induced formation of a comparable amount of ordered secondary structure in both non-oxidized and Met-oxidized forms of a-synuclein, most probably reflecting the stabilization of identical partially folded conformations [113]. The effect of metals on the fibrillation of Met-oxidized asynuclein was determined. For non-oxidized a-synuclein, the fibrillation rate increases dramatically in the presence of all metal cations investigated, including Li+, K+, Na+, Cs+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, Mg2+, Mn2+, Pb2+, Zn2+, Al3+, Co3+, Fe3+, and Ti3+[105,113]. Of particular note, heavy metals such as Hg2+ and Pb2+ (which are interesting because of their potential relevance to environment-induced Parkinsonism) were shown to be among the best accelerators of a-synuclein fibrillation, underlining a potential link between heavy metal exposure, enhanced level of a-synuclein fibrillation and Parkinson’s disease [113]. Methionine-oxidized a-synuclein readily formed fibrils in the presence of certain metal ions, such as Ti3+, Al3+, Zn2+, and Pb2+. However, not all metals were able to accelerate the fibrillation of the oxidatively modified asynuclein: for example, Hg2+, Cu2+, and Ca2+, although able to induce the partially folded conformation in the oxidized protein, did not induce its fibril formation. Furthermore, in the presence of Zn2+ and Pb2+, fibrillation of Met-oxidized a-synuclein (Fig. 7) was as accelerated as that of the nonoxidized protein, whereas Al3+ and Ti3+ showed a much less pronounced effect [113]. These effects were attributed to the

Fig. 7. Metals can overcome the inhibition of Met-oxidized a-synuclein: kinetics of fibrillation monitored by Thioflavin T fluorescence. Measurements were performed at 37 8C, pH 7.5, in the absence of metals (circles) or in the presence of Zn2+ (squares), Pb2+ (triangles), Ca2+ (diamonds). Solutions contained 5 mM metal and the protein concentration was 0.5 mg/ml.

relatively strong coordination of the zinc (and other metal) ion between (at least) two methionine sulfoxides. This bridging is assumed to aid the protein in adopting a necessary conformation for fibrillation, or possibly intermolecular cross-bridging, which could facilitate association and subsequent fibrillation [105]. To further investigate the effect of zinc on the fibrillation of Met-oxidized a-synuclein, the kinetics of fibrillation of various single, double, and triple MetYLeu substitutions of a-synuclein in the presence of zinc was analyzed [35]. The presence of Zn2+ not only induced dramatic acceleration of fibril formation for all the Met-oxidized mutants but their rates of fibrillation were faster than that of Met-oxidized WT protein. The efficiency of mutation-induced acceleration of fibrillation was proportional to the number of substituted methionines [35]. These observations suggest that Met sulfoxides of oxidized a-synuclein are not directly involved in the coordination of Zn2+, as had been assumed. This follows from the fact that a mutant with triple MetYLeu substitutions fibrillates faster than WT but it should fibrillate slower or not at all, if the oxidized Mets were involved in strong metal binding protein. Another possibility is that the Zn binding is occurring with His residue 50. In a study of metal binding and oxidation of amyloid-beta within isolated senile plaque cores by Raman microscopy, the authors found evidence for both methionine oxidation to the sulfoxide as well as Zn binding to histidine residues [16]. This method is a novel approach to studying Met oxidation and has considerable potential.

8. The Msr system The oxidation of methionine creates another level of complexity, in that a new asymmetric center is formed at the sulfur atom, giving two enantiomers, designated Met-R(O) and Met-S(O). These sulfoxides can be enzymatically reduced back to methionine by Msrs, which are widely dispersed in nature (e.g., in microbes, plants, and mammals), but may not be present in extracellular fluids [27]. MsrA enzymes are specific for the Met-S(O) enantiomer, while MsrB enzymes are specific for the Met-R(O) enantiomer. The nearly ubiquitous presence of the Msr system may be of special interest, since in the presence of the complementary enzymes, native Met is regenerated from its sulfoxide, thereby allowing for repair/control mechanisms for oxidized methionine residues [31,85,86]. This recycling process of oxidation and reduction, depending on its efficiency in any given cell or condition, may function in a number of biological processes including the scavenging of oxidants, cell signaling, the regulation of hormonal or enzymatic activities, or the reactivation of a critically important protein [57,94]. Some molecular chaperones/stress proteins are Met-rich, and are produced in significant concentrations under acute inflammatory and

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other stress conditions. It remains to be seen if they play a role in redox control through their Met residues. The study of heat shock (stress) proteins in plants will be discussed in detail in the chapter by Cecilia Sundby Emanuelsson. Reductive repair of oxidized methionines in a protein requires the presence of at least moderate amounts of two complementary reductases in active form unless the oxidation occurred in a highly stereospecific fashion [86]. In an oxidation study of the Met residues in calmodulin, there were indications of some selectivity (ratios of up to 2:1) in the formation of the two enantiomers at various methionine sites, dependent on the nature of the oxidizing species, and to a lesser extent on the location of the Met residue in the protein. It would be expected that the oxidation by an enzymatic mechanism would be more stereospecific and this has been reported for the oxidation of free Met with a flavin-containing monoxygenase system [50] with preferred formation of l-Met-d-SO. This enzyme might recognize highly specific Met-containing peptide sequences, but this has yet to be demonstrated (see chapter by Adnan Elfarra). The survival strategy of certain invasive microorganisms, which have both forms of the Msr enzyme as fused components of the same gene, may be very dependent on the cyclic oxidation and reduction of protein methionine residues to overcome host defenses [88]. It is important to note that in general, the oxidation of a methionine residue will give rise to a mixture of the two enantiomers at that site (although either catalytic oxidation or possible steric hindrance in the local environment of the Met in the protein could lead to a preponderance of one isomer). If both forms of Msr are present in active form in sufficient concentration, then the damaged proteins can be efficiently repaired, and the Met residues can act as an oxidant sink. If one enzyme predominates, however, then some or all of the products will be oxidized in the orientation, which is more stable to attack because of the lowered levels of the enzyme for that orientation. While some microorganisms have MsrA and MsrB co-linked to each other, in mammalian cells there is a distinct tissue distribution between MsrA and MsrB enzymes. In addition, there are at least three MsrB genes that also have their own distinctive distribution pattern. One of these MsrB enzymes is a selenoenzyme and its activity may be compromised due to dietary restrictions or oxidative inactivation. Another important but still unanswered question is as follows: If the Msr system is a factor in redox control in the brain, is there any reason to surmise that an Alzheimer’s or Parkinson’s patient may respond differently? It has been shown that an imbalance of redox control within the mitochondria can be a substantial contributor to cell death in PD. Toxicity is strongly associated with reduced levels of glutathione and increased levels of oxidized glutathione as well as oxidized cytochrome c. This can result in mitochondrial membrane breakdown and cell death by apoptosis [6,34,39,63]. The localization of Msr in the mitochondrial matrix was found using fusions with green

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fluorescence protein [33]. In addition, studies on rat liver cells demonstrated evidence for mitochondrial and cytosolic isoforms of Msr [110]. Of potential relevance in this regard is a report indicating a decline in the activity levels of Msr in various regions of the brains of Alzheimer’s patients [23], which has been attributed to post-translational modification of the reductase and/or a defect in translation resulting in inferior processing of the Msr RNA. This observation may be related to the mechanistic finding that the Msr action proceeds through a disulfide exchange [59], which also involves a sulfenic acid mechanism [5]. The efficiency of this pathway could be compromised under conditions of increased levels of nitric oxide, which may compete for the thiol sites to form the reversible S-nitroso derivatives with unpredictable consequences to the overall efficiency of the Msr system. Excessive production of nitric oxide has in fact been reported in both Parkinson’s and Alzheimer’s diseases [12,14,18,53,56]. Since the glutathione molecule can also be nitrosated, this offers additional opportunities for complexity [22]. It is interesting to note that the levels of thioredoxin and thioredoxin reductase are also reportedly modified in Alzheimer’s disease [58]. Thioredoxin is a potent inducer of protein disulfides and participates with Msr in the reduction of free or protein-bound methionine sulfoxide. Although there were increased levels of thioredoxin reductase, decreased levels of the substrate thioredoxin were found, suggesting that the enzyme will offer only limited protection against oxidative stress. Thioredoxin can also be nitrosated [96].

9. a-Synuclein oxidation and Parkinson’s disease The early stages of cellular protein oxidation provide a major challenge for investigation due to their complex nature, involving multiple selective and nonspecific chemical modifications, and in some cases the potential for reversibility. While the cysteine system has been extensively explored, due in part to the relative ease of analysis, the methionine conversion to sulfoxide, which can initiate, modify or inhibit biological processes, has received much less attention due to the difficulties in analysis. The most favored protein oxidation assay used today is the carbonyl assay. Carbonyls can form by a number of different pathways including direct oxidation of amino side chains, peptide bond cleavage reactions, modification of side chains with lipid peroxidation products, or with reducing sugars. This detection method generally uses 2,4-dinitrophenylhydrazine and is quite sensitive. However, although the carbonyl assay is valuable in estimating the total oxidative damage to a protein, it is limited in that none of the most susceptible amino acids to milder oxidative damage (i.e., methionine, histidine, tryptophan, cysteine, phenylalanine, and tyrosine) form carbonyls and therefore will not give a signal in this assay.

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It is known that hydrogen peroxide by itself will oxidize only the amino acids Met, Cys, and Trp, but the latter two amino acids are absent in a-synuclein, and as each of the four Met residues in a-synuclein is solvent exposed, all are expected to form sulfoxides, as observed. Interestingly, in vitro, the presence of low levels of hydrogen peroxide during incubations leads to inhibition of a-synuclein fibrillation, presumably due to oxidation of the Met residues. Since a number of conditions that lead to Met oxidation also lead to Tyr oxidation, it is important to also consider Tyr oxidation in the context of Parkinson’s disease. What should be kept in mind is that nitration of Tyr residues is a relatively common event, due to the prevalence of reactive products of NO, and nitrated long-lived proteins will accumulate sufficient levels of nitration to be detected [30,79,93,95,99]. The nitration is mediated by several active compounds, including peroxynitrite anion and nitrogen dioxide. Nitrotyrosine can also be formed by peroxidase oxidation of nitrite, a byproduct of nitric oxide metabolism [30]. These activities represent a shift from the signal transducing actions of nitric oxide to oxidative and potentially pathogenic pathways. Although tyrosine nitration is a clear marker of the contribution of nitric oxide to oxidative damage, one must keep in mind, however, that while this Tyr nitrated protein might be: ! ! ! ! !

necessary and sufficient to cause precipitation (or other effects), it also may be necessary but not sufficient, sufficient but not necessary (e.g., it may prove to be a pathway of minor importance), neither necessary nor sufficient, but a significant contributing factor to the disease process, an associated process that is not causative, but rather is concomitant with, or a result of, the disease.

A strong association between 3-nitrotyrosine levels and the accumulation of precipitated a-synuclein in the lesions associated with Parkinson’s disease has been reported; however, it is not clear whether the Tyr nitration was preor post-fibrillation [25]. Since the detection and analysis of Tyr nitration was based on immunological staining technology, the degree of conversion of Tyr to its nitrated forms was not measured, but could be low, which would support the idea that Tyr nitration is not a causal element of the disease. In fact, protein tyrosine nitration is generally a low yield process in vivo, although site-specific nitration focused on a particular protein tyrosine may result in modification of function and promotion of a biological effect [79]. To further assess these issues, a series of a-synuclein mutants, wherein one or more of the Tyr residues were replaced by Phe, were constructed, and both the mutant proteins and the wild-type protein were treated with either oxidative or nitrosative reagents [74]. With the nitrating agent peroxynitrite, Tyr residues were found to be necessary

for the formation of covalently cross-linked a-synuclein dimers and oligomers, since the a-synuclein mutant [4(Tyr/ Phe)] did not show the accumulation of covalently crosslinked oligomers. Tyrosine cross-linking of a-synuclein can occur under oxidizing conditions, and this has been proposed as the rate limiting step in a-synuclein fibril formation [51]. Oxidative treatment of a-synuclein with H2O2 and Cu2+ resulted in covalently cross-linked oligomers and fibrils, even in the absence of Tyr, since both wild-type and mutated proteins (including the 4(Tyr/Phe) mutant protein) gave similar results [74]. This observation indicates the presence of hydroxyl radicals, which would lead to nonspecific covalent crosslinking, i.e., not involving Tyr. The use of Cu2+ and H2O2 involves the well-known Fenton chemistry, which will result in the production of the potent hydroxyl free radical. This oxyradical can react with a wide variety of amino acids including Met, Lys, Arg, Pro, Thr, Glu, Trp, and His, and leads to nonspecific covalent cross-linking of proteins, not only through side chains, but also through the peptide backbone [32,71]. Oxidation of His residues was obligatory for the protein cross-linking in the study of hydroxyl radical mediated damage to crystallins, and could be the explanation for the cross-linking of a-synuclein lacking Tyr residues [32]. Strong support for the hypothesis that Tyr nitration per se does not induce fibrillation of a-synuclein comes from in vitro studies showing that nitrated a-synuclein did not fibrillate [114]. In this study, the nitration of synuclein was carried out with tetranitromethane, a tyrosine specific reagent, and the only modification to the protein used in the fibrillation experiments was the nitration of the Tyr residues (e.g., there was no di-Tyr cross-linking). The mechanism of this inhibition involves formation of stable soluble oligomers, which are located off the fibrillation pathway. Perhaps more importantly, nitrated a-synuclein was shown to inhibit fibrillation of nonmodified asynuclein, even at substoichiometric concentrations. In fact, effective inhibition of the fibrillation of nonmodified protein occurred even when the nitrated a-synuclein accounted for only 10% of total protein [114]. Thus, the results of in vivo investigations indicating that nitration is harmful suggest that nitration itself is not responsible for the deposition of asynuclein found in LBs, but more likely that the oligomers formed by nitrated a-synuclein are cytotoxic. In addition, other expected modifications that could have an impact on the results were not measured in the in vivo experiments. In particular, the possibility of covalent crosslinking through hydroxyl radical reactions was not investigated, and the oxidation state of the four Met residues in asynuclein in LBs needs to be evaluated. It is noteworthy that in the presence of physiological levels of carbon dioxide, peroxynitrite preferentially stimulates the nitration of Tyr over the oxidation of methionine [4]. However, this does not take into account numerous other oxidant species which may be present in the cellular environment and can oxidize Met to the sulfoxide. The reaction of superoxide with SOD

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produces H2O2, and superoxide is formed in large concentrations during normal respiration and is also formed from a variety of biochemical processes in the brain including NADPH oxidase, which is expressed in microglia, astrocytes, and neurons. As noted, H2O2 reacts with transition metal ions to generate hydroxyl radicals, a prime mediator of cell damage. Iron levels in the substantia nigra, the region of the brain that degenerates in Parkinson’s disease, are higher in patients with this disease, and genetic or pharmacological iron chelation prevents MPTP induced neurotoxicity bin vivoQ [43]. The reactivity of H2O2 in oxidative inflammatory cells can be enhanced by peroxidases, which produce hypochlorous acid (HOCl) and other hypohalous acids [38]. In the presence of high concentrations of free radical oxidants, Met sulfoxide can be oxidized further and irreversibly to the sulfone or to a number of free radical breakdown products [82]. These products might contribute to the final state of the aggregation but have not been investigated as yet. A further discussion of methionine oxidation product formation can be found in the chapters by Christian Schoeneich and Michael Davies. Protein aggregates can be pathogenic by several different routes, for example, by interfering with cell trafficking in neurons, by disrupting membrane structure or by sequestering proteins needed for cell survival; however, there is no direct correlation between inclusion formation and neurodegeneration, and other suggestions have been offered including the proposal that cytoplasmic protein inclusions may be an active, protective process that sequesters misfolded proteins [48]. In fact, increasing evidence suggest that oligomeric intermediates in aggregation may be the cytotoxic species in amyloidogenic diseases [7,19,36,44,54, 83,109]. Although the function of a-synuclein is unknown, it appears to interact with a large number of different partners, including synphilin-1, parkin, tyrosine hydroxylase, dopamine transporter, polyamines, phospholipase D, chaperones, and h-synuclein [37]. Under the rather mild oxidative conditions of low concentrations of hydrogen peroxide, only methionine residues are affected din vitroT and there is no cross-linking, but this is clearly sufficient to modify asynuclein, both structurally and in its capacity to fibrillate. Although it has not yet been investigated, the likelihood is that even this mild oxidation may alter the binding characteristics of a-synuclein for some of these associated proteins, and thus have major impact on its function.

10. Methionine oxidation in normal aging and in Parkinson’s disease There are several cellular components whose levels are known to change with aging and that could be important in the etiology of PD. For example, the levels of molecular chaperones are known to decrease with increasing age, and

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this is likely to have an important impact on the aggregation of a-synuclein. Similarly, increased levels of oxidized products are observed with increasing age; this includes membrane oxidation damage, and given the potential importance of membranes in the function and aggregation of a-synuclein, this could have important implications. The growing list of examples where the Met sulfoxide reductase system appears to play a distinctive role in slowing down the aging process raises the question as to whether it may also be a contributing factor in PD. Several reports indicate that overexpression of Msr leads to increased resistance to oxidative stress and increased cell survival rates [68,81,115]. Similarly, cells containing reduced activity of Msr are more sensitive to oxidative stress [66,67,69]. Msr activity exists in the lens of the eye [89] and protects the lens from oxidative damage [42]. The selective oxidation of the calcium regulatory proteins calmodulin (CAM) and Ca-ATPase occurs in vivo during aging. There is a progressive decline in the ability of CAM isolated from aged rat brain to activate the plasma membrane Ca-ATPase and the only residues modified are methionine residues [24]. In addition, there may be an adaptive response to oxidative stress that results in downregulating energy metabolism and the consequent generation of reactive oxygen species [91]. If Met-oxidized a-synuclein accumulates, it is anticipated to produce significant quantities of soluble oligomers, containing both oxidized and unoxidized a-synuclein. The cellular impact of this will depend on whether such oligomers have toxic effects or not. If they do, then clearly the accumulation of Met-oxidized a-synuclein could be a significant contributing factor to PD. There are at least two major factors that are required for a build-up of Metoxidized a-synuclein, namely mild oxidative stress, especially H2O2, and minimal reduction of oxidized Met by Msr, assuming that the oxidized Met residues in the oligomers are accessible to the Msr enzyme. An additional aspect of the role of Msr and a-synuclein in oxidative stress is the fact that a-synuclein is a major brain protein and thus a significant potential sink for oxidation through the oxidation of its methionines. This could be more significant in the dopaminergic neurons of the substantia nigra due to the oxidative instability of dopamine. The impact of this on the cell will be determined, in part, by the effectiveness of Msr to reduce the oxidized Met residues back to the normal form of a-synuclein, and the competition with oligomer formation of the Met-oxidized form. A further possible contributing factor relevant to PD is whether the Met-oxidized a-synuclein (monomeric or oligomeric) is recognized as a misfolded protein by the proteasome or other systems. Also, if cellular conditions result in high levels of protein turnover by the proteasome, the increased burden on cellular metabolism will result in increased respiration, leading to higher levels of reactive oxygen species. An interesting situation would arise if the catalytic activity one of the stereospecific Met sulfoxide

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reductases was present at a much higher level: This would result in conversion of a-synuclein to the Met-oxidized state, while still simultaneously permitting Met recycling to contribute to redox control, albeit at a less efficient level. An unknown feature at this point is whether the two isoforms of oxidized Met in proteins lead to differences in the function, structural stability, susceptibility to ubiquitination, and proteasome degradation or other proteolytic processing of the oxidized proteins. Thus, it is clear that methionine oxidation and Msr could play very important roles in the fate of a-synuclein, both under normal and pathological conditions.

11. Summary The aggregation of a-synuclein is a critical step in the etiology of Parkinson’s disease, and oxidative stress is believed to be a contributing factor in the disease. Mild oxidation of a-synuclein in vitro with hydrogen peroxide selectively converts all four methionine residues to the corresponding sulfoxides, leading to prevention of fibril formation, and build-up of a stable oligomer. Of potential great significance to the pathology, the presence of the Metoxidized protein also inhibits fibrillation of unmodified asynuclein. The degree of inhibition of fibrillation by Metoxidized a-synuclein is proportional to the number of oxidized methionines. Again, an important feature with respect to the disease state is the observation that the presence of metals can completely overcome the inhibition of fibrillation of Met-oxidized a-synuclein. Since the oligomers of Met-oxidized a-synuclein may be cytotoxic, their presence could be an important link to Parkinson’s disease. In addition, if the level of Met-oxidized a-synuclein was under the control of Msr, then this could also be factor in the disease. It is very likely that at least a significant fraction of the Met residues in a-synuclein will be oxidized in the dopaminergic neurons in the substantia nigra, perhaps at the same time as other events (e.g., Tyr nitration and/or cross-linking, interaction of the modified protein with various metals) are taking place. The overall effects of oxidation of a-synuclein in vivo, especially on protein cross-linking, aggregation and precipitation, on degradation, on redox control and on lipid interaction, have yet to be addressed.

References [1] M.F. Beal, Mitochondria, oxidative damage, and inflammation in Parkinson’s disease, Ann. N. Y Acad. Sci. 991 (2003) 120. [2] V. Bellotti, P. Mangione, M. Stoppini, Biological activity and pathological implications of misfolded proteins, Cell. Mol. Life Sci. 55 (1999) 977. [3] N.F. Bence, R.M. Sampat, R.R. Kopito, Impairment of the ubiquitin– proteasome system by protein aggregation, Science 292 (2001) 1552.

[4] B.S. Berlett, R.L. Levine, E.R. Stadtman, Carbon dioxide stimulates peroxynitrite-mediated nitration of tyrosine residues and inhibits oxidation of methionine residues of glutamine synthetase: both modifications mimic effects of adenylylation, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 2784. [5] S. Boschi-Muller, S. Azza, G. Branlant, E. coli methionine sulfoxide reductase with a truncated N terminus or C terminus, or both, retains the ability to reduce methionine sulfoxide, Protein Sci. 10 (2001) 2272. [6] E. Cadenas, K.J. Davies, Mitochondrial free radical generation, oxidative stress, and aging, Free Radic. Biol Med. 29 (2000) 222. [7] B. Caughey, P.T. Lansbury, Protofibrils, pores, fibrils, and neurodegeneration: separating the responsible protein aggregates from the innocent bystanders, Annu. Rev. Neurosci. 26 (2003) 267. [8] B.A. Chromy, R.J. Nowak, M.P. Lambert, K.L. Viola, L. Chang, P.T. Velasco, B.W. Jones, S.J. Fernandez, P.N. Lacor, P. Horowitz, C.E. Finch, G.A. Krafft, W.L. Klein, Self-assembly of Abeta(1–42) into globular neurotoxins, Biochemistry 42 (2003) 12749. [9] G. Cohen, Oxidative stress, mitochondrial respiration, and Parkinson’s disease, Ann. N. Y. Acad. Sci. 899 (2000) 112. [10] K.A. Conway, J.C. Rochet, R.M. Bieganski, P.T. Lansbury Jr., Kinetic stabilization of the alpha-synuclein protofibril by a dopamine-alpha-synuclein adduct, Science 294 (2001) 1346. [11] R.A. Crowther, R. Jakes, M.G. Spillantini, M. Goedert, Synthetic filaments assembled from C-terminally truncated alpha-synuclein, FEBS Lett. 436 (1998) 309. [12] M. Dahiyat, A. Cumming, C. Harrington, C. Wischik, J. Xuereb, F. Corrigan, G. Breen, D. Shaw, D. St. Clair, Association between Alzheimer’s disease and the NOS3 gene, Ann. Neurol. 46 (1999) 664. [13] T.M. Dawson, V.L. Dawson, Molecular pathways of neurodegeneration in Parkinson’s disease, Science 302 (2003) 819. [14] V.L. Dawson, T.M. Dawson, Nitric oxide neurotoxicity, J. Chem. Neuroanat. 10 (1996) 179. [15] C.M. Dobson, Protein misfolding, evolution and disease, Trends Biochem. Sci. 24 (1999) 329. [16] J. Dong, C.S. Atwood, V.E. Anderson, S.L. Siedlak, M.A. Smith, G. Perry, P.R. Carey, Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence, Biochemistry 42 (2003) 2768. [17] J.E. Duda, B.I. Giasson, Q. Chen, T.L. Gur, H.I. Hurtig, M.B. Stern, S.M. Gollomp, H. Ischiropoulos, V.M. Lee, J.Q. Trojanowski, Widespread nitration of pathological inclusions in neurodegenerative synucleinopathies, Am. J. Pathol. 157 (2000) 1439. [18] M. Ebadi, S.K. Sharma, Peroxynitrite and mitochondrial dysfunction in the pathogenesis of Parkinson’s disease, Antioxid. Redox Signal. 5 (2003) 319. [19] O.M. El Agnaf, G.B. Irvine, Aggregation and neurotoxicity of alphasynuclein and related peptides, Biochem. Soc. Trans. 30 (2002) 559. [20] M.B. Feany, W.W. Bender, A Drosophila model of Parkinson’s disease, Nature 404 (2000) 394. [21] L.S. Forno, Neuropathology of Parkinson’s disease, J. Neuropathol. Exp. Neurol. 55 (1996) 259. [22] M.W. Foster, J.S. Stamler, New insights into protein S-nitrosylation. Mitochondria as a model system, J. Biol. Chem. 279 (2004) 25891. [23] S.P. Gabbita, M.Y. Aksenov, M.A. Lovell, W.R. Markesbery, Decrease in peptide methionine sulfoxide reductase in Alzheimer’s disease brain, J. Neurochem. 73 (1999) 1660. [24] J. Gao, D. Yin, Y. Yao, T.D. Williams, T.C. Squier, Progressive decline in the ability of calmodulin isolated from aged brain to activate the plasma membrane Ca-ATPase, Biochemistry 37 (1998) 9536. [25] B.I. Giasson, J.E. Duda, I.V. Murray, Q. Chen, J.M. Souza, H.I. Hurtig, H. Ischiropoulos, J.Q. Trojanowski, V.M. Lee, Oxidative damage linked to neurodegeneration by selective alpha-synuclein nitration in synucleinopathy lesions, Science 290 (2000) 985.

C.B. Glaser et al. / Biochimica et Biophysica Acta 1703 (2005) 157–169 [26] B.I. Giasson, M.S. Forman, M. Higuchi, L.I. Golbe, C.L. Graves, P.T. Kotzbauer, J.Q. Trojanowski, V.M. Lee, Initiation and synergistic fibrillization of tau and alpha-synuclein, Science 300 (2003) 636. [27] C.B. Glaser, L. Karic, S. Parmelee, B.R. Premachandra, D. Hinkston, W.R. Abrams, Studies on the turnover of methionine oxidized alpha1-protease inhibitor in rats, Am. Rev. Respir. Dis. 136 (1987) 857. [28] M. Goedert, Parkinson’s disease and other alpha-synucleinopathies, Clin. Chem. Lab Med. 39 (2001) 308. [29] P.F. Good, A. Hsu, P. Werner, D.P. Perl, C.W. Olanow, Protein nitration in Parkinson’s disease, J. Neuropathol. Exp. Neurol. 57 (1998) 338. [30] S.A. Greenacre, H. Ischiropoulos, Tyrosine nitration: localisation, quantification, consequences for protein function and signal transduction, Free Radic. Res. 34 (2001) 541. [31] R. Grimaud, B. Ezraty, J.K. Mitchell, D. Lafitte, C. Briand, P.J. Derrick, F. Barras, Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase, J. Biol. Chem. 276 (2001) 48915. [32] P. Guptasarma, D. Balasubramanian, S. Matsugo, I. Saito, Hydroxyl radical mediated damage to proteins, with special reference to the crystallins, Biochemistry 31 (1992) 4296. [33] A. Hansel, L. Kuschel, S. Hehl, C. Lemke, H.J. Agricola, T. Hoshi, S.H. Heinemann, Mitochondrial targeting of the human peptide methionine sulfoxide reductase (MSRA), an enzyme involved in the repair of oxidized proteins, FASEB J. 16 (2002) 911. [34] M. Hashimoto, E. Rockenstein, L. Crews, E. Masliah, Role of protein aggregation in mitochondrial dysfunction and neurodegeneration in Alzheimer’s and Parkinson’s diseases, Neuromol. Med. 4 (2003) 21. [35] M.J. Hokenson, V.N. Uversky, J. Goers, G. Yamin, L.A. Munishkina, A.L. Fink, Role of individual methionines in the fibrillation of methionine-oxidized alpha-synuclein, Biochemistry 43 (2004) 4621. [36] M. Hoshi, M. Sato, S. Matsumoto, A. Noguchi, K. Yasutake, N. Yoshida, K. Sato, Spherical aggregates of beta-amyloid (amylospheroid) show high neurotoxicity and activate tau protein kinase I/ glycogen synthase kinase-3beta, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 6370. [37] H. Ischiropoulos, Oxidative modifications of alpha-synuclein, Ann. N. Y. Acad. Sci. 991 (2003) 93. [38] H. Ischiropoulos, J.S. Beckman, Oxidative stress and nitration in neurodegeneration: cause, effect, or association? J. Clin. Invest. 111 (2003) 163. [39] J.S. Isenberg, J.E. Klaunig, Role of the mitochondrial membrane permeability transition (MPT) in rotenone-induced apoptosis in liver cells, Toxicol. Sci. 53 (2000) 340. [40] P. Jenner, D.T. Dexter, J. Sian, A.H. Schapira, C.D. Marsden, Oxidative stress as a cause of nigral cell death in Parkinson’s disease and incidental Lewy body disease. The royal Kings and Queens Parkinson’s disease research group, Ann. Neurol. 32 (1992) S82 – S87 (Suppl.). [41] J.A. Johnston, C.L. Ward, R.R. Kopito, Aggresomes: a cellular response to misfolded proteins, J. Cell Biol. 143 (1998) 1883. [42] M. Kantorow, J.R. Hawse, T.L. Cowell, S. Benhamed, G.O. Pizarro, V.N. Reddy, J.F. Hejtmancik, Methionine sulfoxide reductase A is important for lens cell viability and resistance to oxidative stress, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 9654. [43] D. Kaur, F. Yantiri, S. Rajagopalan, J. Kumar, J.Q. Mo, R. Boonplueang, V. Viswanath, R. Jacobs, L. Yang, M.F. Beal, D. DiMonte, I. Volitaskis, L. Ellerby, R.A. Cherny, A.I. Bush, J.K. Andersen, Genetic or pharmacological iron chelation prevents MPTP-induced neurotoxicity in vivo: a novel therapy for Parkinson’s disease, Neuron 37 (2003) 899. [44] R. Kayed, E. Head, J.L. Thompson, T.M. McIntire, S.C. Milton, C.W. Cotman, C.G. Glabe, Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis, Science 300 (2003) 486. [45] N.P. Kedar, Can we prevent Parkinson’s and Alzheimer’s disease? J. Postgrad. Med. 49 (2003) 236.

167

[46] J.W. Kelly, The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways, Curr. Opin. Struck. Biol. 8 (1998) 101. [47] W.L. Klein, Abeta toxicity in Alzheimer’s disease: globular oligomers (ADDLs) as new vaccine and drug targets, Neurochem. Int. 41 (2002) 345. [48] R.R. Kopito, Aggresomes, inclusion bodies and protein aggregation, Trends Cell Biol. 10 (2000) 524. [49] R.R. Kopito, D. Ron, Conformational disease, Nat. Cell Biol. 2 (2000) E207 – E209. [50] R.J. Krause, S.L. Ripp, P.J. Sausen, L.H. Overby, R.M. Philpot, A.A. Elfarra, Characterization of the methionine S-oxidase activity of rat liver and kidney microsomes: immunochemical and kinetic evidence for FMO3 being the major catalyst, Arch. Biochem. Biophys. 333 (1996) 109. [51] S. Krishnan, E.Y. Chi, S.J. Wood, B.S. Kendrick, C. Li, W. GarzonRodriguez, J. Wypych, T.W. Randolph, L.O. Narhi, A.L. Biere, M. Citron, J.F. Carpenter, Oxidative dimer formation is the critical ratelimiting step for Parkinson’s disease alpha-synuclein fibrillogenesis, Biochemistry 42 (2003) 829. [52] R. Kruger, W. Kuhn, T. Muller, D. Woitalla, M. Graeber, S. Kosel, H. Przuntek, J.T. Epplen, L. Schols, O. Riess, Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease, Nat. Genet. 18 (1998) 106. [53] D.K. Lahiri, D. Chen, Y.W. Ge, M. Farlow, G. Kotwal, A. Kanthasamy, D.K. Ingram, N.H. Greig, Does nitric oxide synthase contribute to the pathogenesis of Alzheimer’s disease?: effects of beta-amyloid deposition on NOS in transgenic mouse brain with AD pathology, Ann. N. Y. Acad. Sci. 1010 (2003) 639. [54] H.A. Lashuel, D. Hartley, B.M. Petre, T. Walz, P.T. Lansbury Jr., Neurodegenerative disease: amyloid pores from pathogenic mutations, Nature 418 (2002) 291. [55] H.A. Lashuel, B.M. Petre, J. Wall, M. Simon, R.J. Nowak, T. Walz, P.T. Lansbury Jr., Alpha-synuclein, especially the Parkinson’s disease-associated mutants, forms pore-like annular and tubular protofibrils, J. Mol. Biol. 322 (2002) 1089. [56] C. Levecque, A. Elbaz, J. Clavel, F. Richard, J.S. Vidal, P. Amouyel, C. Tzourio, A. Alperovitch, M.C. Chartier-Harlin, Association between Parkinson’s disease and polymorphisms in the nNOS and iNOS genes in a community-based case-control study, Hum. Mol. Genet. 12 (2003) 79. [57] R.L. Levine, L. Mosoni, B.S. Berlett, E.R. Stadtman, Methionine residues as endogenous antioxidants in proteins, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 15036. [58] M.A. Lovell, C. Xie, S.P. Gabbita, W.R. Markesbery, Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer’s disease brain, Free Radic. Biol. Med. 28 (2000) 418. [59] W.T. Lowther, N. Brot, H. Weissbach, J.F. Honek, B.W. Matthews, Thiol-disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 6463. [60] C.B. Lucking, A. Brice, Alpha-synuclein and Parkinson’s disease, Cell. Mol. Life Sci. 57 (2000) 1894. [61] L. Maroteaux, J.T. Campanelli, R.H. Scheller, Synuclein: a neuronspecific protein localized to the nucleus and presynaptic nerve terminal., J. Neurosci. 8 (1988) 2804. [62] E. Masliah, E. Rockenstein, I. Veinbergs, M. Mallory, M. Hashimoto, A. Takeda, Y. Sagara, A. Sisk, L. Mucke, Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neurodegenerative disorders, Science 287 (2000) 1265. [63] M.P. Mattson, W. Duan, bApoptoticQ biochemical cascades in synaptic compartments: roles in adaptive plasticity and neurodegenerative disorders, J. Neurosci. Res. 58 (1999) 152. [64] T.A. Mirzabekov, M.C. Lin, B.L. Kagan, Pore formation by the cytotoxic islet amyloid peptide amylin, J. Biol. Chem. 271 (1996) 1988.

168

C.B. Glaser et al. / Biochimica et Biophysica Acta 1703 (2005) 157–169

[65] T. Moos, E.H. Morgan, The metabolism of neuronal iron and its pathogenic role in neurological disease: review, Ann. N. Y. Acad. Sci. 1012 (2004) 14. [66] J. Moskovitz, S. Bar-Noy, W.M. Williams, J. Requena, B.S. Berlett, E.R. Stadtman, Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals, Proc. Natl. Acad. Sci. U. S. A. 98 (2001) 12920. [67] J. Moskovitz, B.S. Berlett, J.M. Poston, E.R. Stadtman, The yeast peptide-methionine sulfoxide reductase functions as an antioxidant in vivo, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 9585. [68] J. Moskovitz, E. Flescher, B.S. Berlett, J. Azare, J.M. Poston, E.R. Stadtman, Overexpression of peptide-methionine sulfoxide reductase in Saccharomyces cerevisiae and human T cells provides them with high resistance to oxidative stress, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14071. [69] J. Moskovitz, E.R. Stadtman, Selenium-deficient diet enhances protein oxidation and affects methionine sulfoxide reductase (MsrB) protein level in certain mouse tissues, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 7486. [70] J. Moskovitz, H. Weissbach, N. Brot, Cloning the expression of a mammalian gene involved in the reduction of methionine sulfoxide residues in proteins, Proc. Natl. Acad. Sci. U. S. A. 93 (1996) 2095. [71] J. Moskovitz, M.B. Yim, P.B. Chock, Free radicals and disease, Arch. Biochem. Biophys. 397 (2002) 354. [72] H. Naiki, K. Higuchi, M. Hosokawa, T. Takeda, Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T, Anal. Biochem. 177 (1989) 244. [73] H. Naiki, K. Higuchi, K. Matsushima, A. Shimada, W.H. Chen, M. Hosokawa, T. Takeda, Fluorometric examination of tissue amyloid fibrils in murine senile amyloidosis: use of the fluorescent indicator, thioflavine T, Lab. Invest. 62 (1990) 768. [74] E.H. Norris, B.I. Giasson, H. Ischiropoulos, V.M. Lee, Effects of oxidative and nitrative challenges on alpha-synuclein fibrillogenesis involve distinct mechanisms of protein modifications, J. Biol. Chem. 278 (2003) 27230. [75] H. Okazaki, L.E. Lipkin, S.M. Aronson, Diffuse intracytoplasmic ganglionic inclusions (Lewy type) associated with progressive dementia and quadriparesis in flexion, J. Neuropathol. Exp. Neurol. 20 (1961) 237. [76] S.R. Paik, H.J. Shin, J.H. Lee, Metal-catalyzed oxidation of alphasynuclein in the presence of Copper(II) and hydrogen peroxide, Arch. Biochem. Biophys. 378 (2000) 269. [77] M.H. Polymeropoulos, C. Lavedan, E. Leroy, S.E. Ide, A. Dehejia, A. Dutra, B. Pike, H. Root, J. Rubenstein, R. Boyer, E.S. Stenroos, S. Chandrasekharappa, A. Athanassiadou, T. Papapetropoulos, W.G. Johnson, A.M. Lazzarini, R.C. Duvoisin, G. Di Iorio, L.I. Golbe, R.L. Nussbaum, Mutation in the alpha-synuclein gene identified in families with Parkinson’s disease, Science 276 (1997) 2045. [78] I. Qahwash, K.L. Weiland, Y. Lu, R.W. Sarver, R.F. Kletzien, R. Yan, Identification of a mutant amyloid peptide that predominantly forms neurotoxic protofibrillar aggregates, J. Biol. Chem. 278 (2003) 23187. [79] R. Radi, Nitric oxide, oxidants, and protein tyrosine nitration, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 4003. [80] J.C. Rochet, P.T. Lansbury Jr., Amyloid fibrillogenesis: themes and variations, Curr. Opin. Struct. Biol. 10 (2000) 60. [81] H. Ruan, X.D. Tang, M.L. Chen, M.L. Joiner, G. Sun, N. Brot, H. Weissbach, S.H. Heinemann, L. Iverson, C.F. Wu, T. Hoshi, M.L. Chen, M.A. Joiner, S.H. Heinemann, High-quality life extension by the enzyme peptide methionine sulfoxide reductase, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 2748. [82] C. Schoneich, D. Pogocki, G.L. Hug, K. Bobrowski, Free radical reactions of methionine in peptides: mechanisms relevant to betaamyloid oxidation and Alzheimer’s disease, J. Am. Chem. Soc. 125 (2003) 13700. [83] B.R. Sellman, B.L. Kagan, R.K. Tweten, Generation of a membranebound, oligomerized pre-pore complex is necessary for pore

[84]

[85]

[86]

[87]

[88]

[89]

[90]

[91] [92]

[93] [94]

[95]

[96]

[97]

[98]

[99]

[100]

[101]

formation by Clostridium septicum alpha toxin, Mol. Microbiol. 23 (1997) 551. L.C. Serpell, J. Berriman, R. Jakes, M. Goedert, R.A. Crowther, Fiber diffraction of synthetic alpha-synuclein filaments shows amyloid-like cross-beta conformation, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 4897. V.S. Sharov, D.A. Ferrington, T.C. Squier, C. Schoneich, Diastereoselective reduction of protein-bound methionine sulfoxide by methionine sulfoxide reductase, FEBS Lett. 455 (1999) 247. V.S. Sharov, C. Schoneich, Diastereoselective protein methionine oxidation by reactive oxygen species and diastereoselective repair by methionine sulfoxide reductase, Free Radic. Biol. Med. 29 (2000) 986. A.B. Singleton, M. Farrer, J. Johnson, A. Singleton, S. Hague, J. Kachergus, M. Hulihan, T. Peuralinna, A. Dutra, R. Nussbaum, S. Lincoln, A. Crawley, M. Hanson, D. Maraganore, C. Adler, M.R. Cookson, M. Muenter, M. Baptista, D. Miller, J. Blancato, J. Hardy, K. Gwinn-Hardy, Alpha-synuclein locus triplication causes Parkinson’s disease, Science 302 (2003) 841. E.P. Skaar, D.M. Tobiason, J. Quick, R.C. Judd, H. Weissbach, F. Etienne, N. Brot, H.S. Seifert, The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10108. A. Spector, R. Scotto, H. Weissbach, N. Brot, Lens methionine sulfoxide reductase, Biochem. Biophys. Res. Commun. 108 (1982) 429. M.G. Spillantini, R.A. Crowther, R. Jakes, M. Hasegawa, M. Goedert, a-Synuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies, Proc Natl Acad Sci. U. S. A. 95 (1998) 6469. T.C. Squier, Oxidative stress and protein aggregation during biological aging, Exp. Gerontol. 36 (2001) 1539. E.R. Stadtman, Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions, Annu. Rev. Biochem. 62 (1993) 797. E.R. Stadtman, Protein oxidation in aging and age-related diseases, Ann. N. Y. Acad. Sci. 928 (2001) 22. E.R. Stadtman, J. Moskovitz, B.S. Berlett, R.L. Levine, Cyclic oxidation and reduction of protein methionine residues is an important antioxidant mechanism, Mol. Cell. Biochem. 234–235 (2002) 3. E.R. Stadtman, C.N. Oliver, P.E. Starke-Reed, S.G. Rhee, Agerelated oxidation reaction in proteins, Toxicol. Ind. Health 9 (1993) 187. V.V. Sumbayev, S-nitrosylation of thioredoxin mediates activation of apoptosis signal-regulating kinase 1, Arch. Biochem. Biophys. 415 (2003) 133. H. Sun, J. Gao, D.A. Ferrington, H. Biesiada, T.D. Williams, T.C. Squier, Repair of oxidized calmodulin by methionine sulfoxide reductase restores ability to activate the plasma membrane CaATPase, Biochemistry 38 (1999) 105. A. Takeda, M. Mallory, M. Sundsmo, W. Honer, L. Hansen, E. Masliah, Abnormal accumulation of NACP/a-Synuclein in neurodegenerative disorders, Am. J. Pathol. 152 (1998) 367. M. Tien, B.S. Berlett, R.L. Levine, P.B. Chock, E.R. Stadtman, Peroxynitrite-mediated modification of proteins at physiological carbon dioxide concentration: pH dependence of carbonyl formation, tyrosine nitration, and methionine oxidation, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 7809. J.Q. Trojanowski, M. Goedert, T. Iwatsubo, V.M. Lee, Fatal attractions: abnormal protein aggregation and neuron death in Parkinson’s disease and Lewy body dementia, Cell Death Differ. 5 (1998) 832. J.Q. Trojanowski, V.M. Lee, Parkinson’s disease and related alphasynucleinopathies are brain amyloidoses, Ann. N. Y. Acad. Sci. 991 (2003) 107.

C.B. Glaser et al. / Biochimica et Biophysica Acta 1703 (2005) 157–169 [102] K. Ueda, H. Fukushima, E. Masliah, Y. Xia, A. Iwai, M. Yoshimoto, D.A. Otero, J. Kondo, Y. Ihara, T. Saitoh, Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease, Proc. Natl. Acad. Sci. U. S. A. 90 (1993) 11282. [103] V.N. Uversky, J.R. Gillespie, A. Talapatra, A.L. Fink, Protein deposits as the molecular basis of amyloidosis: Part I. Systemic amyloidosis, Med. Sci. Monit. 5 (1999) 1001. [104] V.N. Uversky, J. Li, A.L. Fink, Evidence for a partially folded intermediate in alpha-synuclein fibril formation, J. Biol. Chem. 276 (2001) 10737. [105] V.N. Uversky, J. Li, A.L. Fink, Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular link between Parkinson’s disease and heavy metal exposure, J. Biol. Chem. 276 (2001) 44284. [106] V.N. Uversky, J. Li, P.O. Souillac, I.S. Millett, S. Doniach, R. Jakes, M. Goedert, A.L. Fink, Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins, J. Biol. Chem. 277 (2002) 11970. [107] V.N. Uversky, G. Yamin, P.O. Souillac, J. Goers, C.B. Glaser, A.L. Fink, Methionine oxidation inhibits fibrillation of human alphasynuclein in vitro, FEBS Lett. 517 (2002) 239. [108] W. Vogt, Oxidation of methionyl residues in proteins: tools, targets, and reversal, Free Radic. Biol. Med. 18 (1995) 93. [109] M.J. Volles, P.T. Lansbury Jr., Vesicle permeabilization by protofibrillar alpha-synuclein is sensitive to Parkinson’s disease-linked mutations and occurs by a pore-like mechanism, Biochemistry 41 (2002) 4595.

169

[110] S. Vougier, J. Mary, B. Friguet, Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells, Biochem. J. 373 (2003) 531. [111] P.H. Weinreb, W. Zhen, A.W. Poon, K.A. Conway, P.T. Lansbury Jr., NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded, Biochemistry 35 (1996) 13709. [112] S.J. Wood, J. Wypych, S. Steavenson, J.C. Louis, M. Citron, A.L. Biere, Alpha-synuclein fibrillogenesis is nucleation-dependent. Implications for the pathogenesis of Parkinson’s disease, J. Biol. Chem. 274 (1999) 19509. [113] G. Yamin, C.B. Glaser, V.N. Uversky, A.L. Fink, Certain metals trigger fibrillation of methionine-oxidized {alpha}-synuclein, J. Biol. Chem. 278 (2003) 27630. [114] G. Yamin, V.N. Uversky, A.L. Fink, Nitration inhibits fibrillation of human alpha-synuclein in vitro by formation of soluble oligomers, FEBS Lett. 542 (2003) 147. [115] O. Yermolaieva, R. Xu, C. Schinstock, N. Brot, H. Weissbach, S.H. Heinemann, T. Hoshi, Methionine sulfoxide reductase A protects neuronal cells against brief hypoxia/reoxygenation, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 1159. [116] J.J. Zarranz, J. Alegre, J.C. Gomez-Esteban, E. Lezcano, R. Ros, I. Ampuero, L. Vidal, J. Hoenicka, O. Rodriguez, B. Atares, V. Llorens, T.E. Gomez, T. del Ser, D.G. Munoz, J.G. de Yebenes, The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia, Ann. Neurol. 55 (2004) 164. [117] M. Zhu, J. Li, A.L. Fink, The association of alpha-synuclein with membranes affects bilayer structure, stability, and fibril formation, J. Biol. Chem. 278 (2003) 40186.