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Curcumin Prevents Aggregation in α-Synuclein by Increasing Reconfiguration Rate*

  • Basir Ahmad
    Affiliations
    Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48824
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  • Lisa J. Lapidus
    Correspondence
    To whom correspondence should be addressed: Dept. of Physics and Astronomy, Michigan State University, 4223 Biomedical Physical Sciences, East Lansing, MI 48824. Tel.: 517-884-5656; Fax: 517-353-4500
    Affiliations
    Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan 48824
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  • Author Footnotes
    * This work was supported by National Science Foundation Grant MCB-0825001.
    This article contains supplemental “Results,” Figs. S1–S4, Tables S1 and S2, Equations S1 and S2, and additional references.
Open AccessPublished:January 20, 2012DOI:https://doi.org/10.1074/jbc.M111.325548
      α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-β component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

      Introduction

      α-Synuclein aggregation is involved in, and likely the cause of, Parkinson disease (
      • Goedert M.
      α-Synuclein and neurodegenerative diseases.
      ). Although α-synuclein is commonly thought of as intrinsically disordered, a recent report demonstrated that, in human cells, it exists in a helical tetramer that does not easily aggregate (
      • Bartels T.
      • Choi J.G.
      • Selkoe D.J.
      α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation.
      ). This suggests that the physiological pathway for aggregation is first unfolding of the tetramer to kinetically trapped monomers and then reassociation to a disordered aggregate and eventually fibrillar Lewy bodies. Therefore, preventing reassociation of the monomers is a useful therapeutic strategy. Many researchers in the past several years have investigated the interaction of potential aggregation inhibitors with oligomers of various sizes and fibrils, but there have been few observations of inhibitors with monomers, primarily because spectroscopic detection is difficult (
      • Amer D.A.
      • Irvine G.B.
      • El-Agnaf O.M.
      Inhibitors of α-synuclein oligomerization and toxicity: a future therapeutic strategy for Parkinson disease and related disorders.
      ,
      • Caruana M.
      • Högen T.
      • Levin J.
      • Hillmer A.
      • Giese A.
      • Vassallo N.
      Inhibition and disaggregation of α-synuclein oligomers by natural polyphenolic compounds.
      ,
      • Ehrnhoefer D.E.
      • Bieschke J.
      • Boeddrich A.
      • Herbst M.
      • Masino L.
      • Lurz R.
      • Engemann S.
      • Pastore A.
      • Wanker E.E.
      EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers.
      ,
      • Lamberto G.R.
      • Binolfi A.
      • Orcellet M.L.
      • Bertoncini C.W.
      • Zweckstetter M.
      • Griesinger C.
      • Fernández C.O.
      Structural and mechanistic basis behind the inhibitory interaction of PcTS on α-synuclein amyloid fibril formation.
      ,
      • Zhu M.
      • Rajamani S.
      • Kaylor J.
      • Han S.
      • Zhou F.
      • Fink A.L.
      The flavonoid baicalein inhibits fibrillation of α-synuclein and disaggregates existing fibrils.
      ).
      We have recently investigated the chain dynamics of disordered monomeric α-synuclein under a variety of aggregation conditions and found that the internal reconfiguration rate (or the rate of intramolecular diffusion) is fast under conditions in which aggregation is inhibited and slows when aggregation is more likely (
      • Ahmad B.
      • Chen Y.
      • Lapidus L.J.
      ). We interpreted these observations with a model in which the first step of aggregation is kinetically controlled by the reconfiguration rate of the disordered monomer. When intramolecular diffusion is fast compared with bimolecular association, aggregation is unlikely because exposed hydrophobes quickly reconfigure, but if intramolecular diffusion slows to the same rate as bimolecular association, aggregation becomes more likely. A logical extension of this model is that aggregation inhibitors prevent bimolecular association by raising the reconfiguration (or the rate of intramolecular diffusion) of the disordered protein.
      Intramolecular diffusion is the random motion of one part of the protein chain relative to another. To measure intramolecular diffusion, we used the Trp-Cys contact quenching method by which tryptophan is excited to a long-lived triplet state that is quenched on contact with cysteine within the same protein chain. Measurement of this rate of quenching at various temperatures and viscosities allows the extraction of the rate of diffusion between these two points in the chain.
      In this work, we investigated the effect of the small molecule curcumin on the intramolecular diffusion of α-synuclein. Curcumin, a compound found in the spice turmeric, has been shown to have many medicinal properties and inhibits aggregation of the Alzheimer amyloid-β peptide (
      • Yang F.
      • Lim G.P.
      • Begum A.N.
      • Ubeda O.J.
      • Simmons M.R.
      • Ambegaokar S.S.
      • Chen P.P.
      • Kayed R.
      • Glabe C.G.
      • Frautschy S.A.
      • Cole G.M.
      Curcumin inhibits formation of amyloid β-oligomers and fibrils, binds plaques, and reduces amyloid in vivo.
      ). Ιn α-synuclein, curcumin has been shown to inhibit fibril formation and increase solubility, but the physical basis of the aggregation inhibition is not known (
      • Pandey N.
      • Strider J.
      • Nolan W.C.
      • Yan S.X.
      • Galvin J.E.
      Curcumin inhibits aggregation of α-synuclein.
      ). We found that curcumin strongly bound to the monomer and completely inhibited aggregation, and with curcumin, intramolecular diffusion of α-synuclein was increased by >10-fold at 40 °C compared with the protein alone.

      EXPERIMENTAL PROCEDURES

      α-Synuclein Mutation, Expression, and Purification

      The α-synuclein plasmid was a kind gift from Gary Pielak (University of North Carolina, Chapel Hill, NC). Mutants Y39W/A69C and A69C/F94W of α-synuclein were created using the QuikChange site-directed mutagenesis kit (Stratagene). The mutations were confirmed by DNA sequencing. The wild-type and mutant proteins were expressed in Escherichia coli BL21(DE3) cells transformed with the T7-7 plasmid and purified as described previously (
      • Uversky V.N.
      • Li J.
      • Fink A.L.
      Evidence for a partially folded intermediate in α-synuclein fibril formation.
      ). The purity of the mutants was confirmed by SDS-PAGE to be >95%. The protein concentration was determined from the absorbance at 280 nm using an extinction coefficient of 11460 m−1 cm−1. Stock solutions of ∼200 μm were stored at −80 °C in 25 mm sodium phosphate buffer (pH 7.4) with 1 mm tris(2-carboxyethyl)phosphine (TCEP).
      The abbreviations used are:
      TCEP
      tris(2-carboxyethyl)phosphine
      TFE
      trifluoroethanol
      ThT
      thioflavin T.
      An aliquot was thawed and filtered shortly before each experiment.

      Aggregation Inhibition Studies

      The effect of curcumin on the inhibition of α-synuclein aggregation was measured under two aggregation conditions. First, fibril formation in the absence and presence of curcumin (curcumin/protein molar ratio of 1.5) was initiated by stirring the protein, at a concentration of 48 μm, in 25 mm phosphate buffer (pH 7.4), 150 mm NaCl, and 1 mm TCEP at 37 °C (
      • Uversky V.N.
      • Li J.
      • Fink A.L.
      Evidence for a partially folded intermediate in α-synuclein fibril formation.
      ). Second, soluble oligomer formation in the absence and presence of curcumin (curcumin/protein molar ratio of 1.5) was started by incubating 5 μm α-synuclein in 10% (v/v) trifluoroethanol (TFE), 25 mm phosphate buffer (pH 7.4), and 1 mm TCEP at 37 °C (
      • Anderson V.L.
      • Ramlall T.F.
      • Rospigliosi C.C.
      • Webb W.W.
      • Eliezer D.
      Identification of a helical intermediate in trifluoroethanol-induced α-synuclein aggregation.
      ).
      At regular time intervals, individual aliquots of 60 (in one experiment, 10) μl of each sample preincubated without or with curcumin (curcumin/α-synuclein ratio of 1.5:1) were mixed with 440 (in one experiment, 490) μl of 25 μm thioflavin T (ThT) solution and 25 mm phosphate buffer (pH 7.4), and the aggregation kinetics were followed by measurements of ThT fluorescence at 480 nm and far-UV CD at 217 nm, respectively. The ThT fluorescence was measured using a Jobin Yvon SPEX FluoroLog-3 spectrofluorometer equipped with a temperature-controlled cell holder. The excitation and emission wavelengths were 440 and 480 nm, respectively. A 10-mm path length quartz cell and an excitation and emission slit width of 5 nm were used. Far-UV CD data were obtained with an Applied Photophysics Chirascan spectropolarimeter equipped with a temperature-controlled cell holder.

      Conformational Studies

      Intrinsic Fluorescence

      Tryptophan fluorescence measurements were carried out on a Jobin Yvon SPEX FluoroLog-3 spectrofluorometer equipped with a temperature-controlled cell holder. The fluorescence spectra were measured at 25 °C with a 1-cm path length cell, exciting at 295 nm. Both excitation and emission slits were set at 5 nm.

      Circular Dichroism

      CD measurements were carried out with an Applied Photophysics Chirascan spectropolarimeter equipped with a temperature-controlled cell holder. Spectra were recorded with a 0.5–4-s adaptive integration time and a 1-nm bandwidth. Each spectrum was the average of four scans. Far- and near-UV CD spectra were taken at protein concentrations of 5 and 25 μm with 0.1- and 1.0-cm path length cells, respectively.

      Trp-Cys Contact Quenching Studies

      Shortly before the experiment, a 300-μl aliquot of the protein with or without the desired concentration of curcumin was diluted 10:1 in 25 mm sodium phosphate buffer (pH 7.4), 1 mm TCEP, and various sucrose concentrations that had been bubbled with N2O to eliminate oxygen and scavenge solvated electrons created in the UV laser pulse. Triplet lifetime decay kinetics were measured with an instrument similar to one described previously (
      • Singh V.R.
      • Kopka M.
      • Chen Y.
      • Wedemeyer W.J.
      • Lapidus L.J.
      Dynamic similarity of the unfolded states of proteins L and G.
      ). Briefly, the tryptophan triplet was excited by a 10-ns laser pulse at 289 nm created from the fourth harmonic of an Nd:YAG laser (Continuum) and a 1-meter Raman cell filled with 450 p.s.i. of D2 gas. The triplet population was probed at 441 nm by a HeCd laser (Kimmon). The probe and a reference beam were measured with silicon detectors and combined in a differential amplifier (DA 1853A, LeCroy) with an additional stage of a 350-MHz preamplifier (SR445A, Stanford Research Systems). The total gain was 50-fold. The temperature and viscosity were controlled as described previously (
      • Ahmad B.
      • Chen Y.
      • Lapidus L.J.
      ). The variation of solution viscosity was achieved with the addition of different concentrations of sucrose. Measurement of each sample at five temperatures took ∼20 min, so aggregation during this time was minimal. The viscosity of each solvent at each temperature was measured independently using a cone-cup viscometer (Brookfield Engineering).

      DISCUSSION

      We have previously shown that α-synuclein, unique among disordered sequences, compacts and diffuses more slowly as temperature is increased (
      • Ahmad B.
      • Chen Y.
      • Lapidus L.J.
      ). Examining other conditions under which aggregation is enhanced (low pH or the familial mutation A30P), we found a good correlation between the rate of intramolecular diffusion and the rate of aggregation. When diffusion is fast (D ∼ 10−6 cm2 s−1), such as is observed for most intrinsically disordered sequences, the protein reconfigures too fast to make stable bimolecular interactions with another protein chain, but when the reconfiguration rate is about the same as the bimolecular encounter rate, stable interactions are more likely, and aggregation can proceed. This accounts for the dramatic increase in the aggregation rate of α-synuclein at 40 °C compared with 0 °C.
      In this work, we examined the effect of curcumin binding on the intramolecular diffusion of α-synuclein. There is little difference in D at T = 0 °C, but the difference widens with increasing temperature. At T = 40 °C and an equal molar ratio of curcumin to protein, D is 15 times higher than with no curcumin. This difference widens to 30 times at 1.5:1 curcumin/protein, the highest ratio measurable in our instrument, which suggests that multiple curcumin molecules bound to a single protein further increase D.
      The Trp fluorescence data suggest that one preferred binding site for curcumin is near position 94. Molecular mechanics simulations of Alzheimer peptides have shown that curcumin preferentially associates with alanine and other aliphatic residues (
      • Kumar P.
      • Pillay V.
      • Choonara Y.E.
      • Modi G.
      • Naidoo D.
      • du Toit L.C.
      In silico theoretical molecular modeling for Alzheimer disease: the nicotine-curcumin paradigm in neuroprotection and neurotherapy.
      ). Between residues 60 and 100, there are 15 aliphatic residues (alanine and valine plus Leu-100), and in particular, there are three alanines in a row at positions 89–91. We propose this as a possible binding site. Having made one or more bonds between the side chains and the curcumin, the aromatic rings of the molecule are then available to interact with any of the nearby hydrophobic residues, creating a hydrophobic cluster of residues close in sequence.
      Thus, it appears that one or more curcumin molecules bound to α-synuclein rescue the protein from the slow diffusion regime that promotes aggregation. Because the reaction-limited rates are correlated with temperature and the diffusion-limited rates are inversely correlated, by extension, the chain volume and diffusion coefficient are inversely correlated. We conclude that curcumin disrupts long-range interactions within the chain, allowing it to more quickly reconfigure. Fig. 6 shows a schematic of this behavior. Typically, α-synuclein is a fairly compact disordered protein with many long-range interactions within the chain (gray circles). This makes reconfiguration fairly slow (upper row) and allows exposed hydrophobes to associate with other chains, making oligomers, which eventually rearrange into larger fibrillar species. With the addition of curcumin (middle row), the chains become less compact, and intramolecular interactions are more short-range, allowing faster reconfiguration. Faster reconfiguration allows the chains to escape from bimolecular association (lower row) and prevents further aggregation steps.
      Figure thumbnail gr6
      FIGURE 6Schematic of the action of curcumin on α-synuclein in bimolecular association and subsequent aggregation steps.
      Future work should investigate whether this property is common in aggregation inhibitors. For example, as a control experiment, we measured intramolecular diffusion of the protein in the presence of N-acetylleucine, a hydrophobic amino acid, and found that the diffusion coefficient was unchanged (supplemental Fig. S4), suggesting that the ability of curcumin to affect reconfiguration is somewhat unique.
      This assay yields unique information about the mechanism of aggregation inhibition at the first step of the process. More common assays, such as ThT fluorescence, are not sensitive to monomer/monomer interactions, which are the preferred step for an inhibitor to act on. One potential danger with inhibiting a later step of the aggregation pathway is that accumulation of a toxic intermediate could make toxicity worse (
      • Ross C.A.
      • Poirier M.A.
      Protein aggregation and neurodegenerative disease.
      ). Therefore, this measurement should become a common assay in the development of new Parkinson drug candidates that prevent aggregation at the first step.

      Acknowledgments

      We thank Charles Hoogstraten for helpful discussions, Gary Pielak for the kind gift of the α-synuclein plasmid, and Terry Ball for mutation and expression of the protein. We acknowledge the support of the Michigan State University High Performance Computing Center and the Institute for Cyber Enabled Research.

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