Accepted 29 September 2008
Keywords:-synucleinIntrinsically disordered proteinSmoking
neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that
Biochimica et Biophysica Acta 1794 (2009) 282290
Contents lists available at ScienceDirect
Biochimica et Bi
j ourna l homepage: www.e lsdisorder after Alzheimer's disease. PD is a slowly progressiveneurodegenerative disease caused by the loss of nerve cells in a partof the mid-brain known as the substantia nigra. The cells in the sub-stantia nigra are responsible for dopamine production, a chemicalmessenger involved inmovement coordination. PD is developedwhenthese cells are damaged or destroyed causing different signs of PD:resting tremors, slowness of movement, stiffness of the limbs andbalance problems . The surviving nigral dopaminergic neuronscontain specic proteinaceous inclusions, Lewy bodies (LB) and Lewyneurites (LN), which are also found in several neurodegenerative
The precise molecular mechanism that leads to the death of thecells in the substantia nigra is still unknown. However, substantialevidence has suggested that the brillation of -synuclein is a criticalstep in the pathogenesis of PD (reviewed in ). -synuclein is amajor brillar component of Lewy bodies and Lewy neuritis . It is atypical natively unfolded protein, which possess little orderedstructure at physiological conditions [6,7,9,11,12]. synuclein con-tains 140 amino acid residues and lacks both cysteine and tryptophanresidues . The sequence of -synuclein is divided into threeregions (Scheme 1):diseases [2,3]. It is estimated that 1.5 millio
Corresponding author. Center for ComputationalDepartment of Biochemistry and Molecular Biology,Medicine, 410 W. 10th Street, HS 5009, Indianapolis, IN6448; fax: +1 317 278 9217.
E-mail address: email@example.com (V.N. Uversky).1 Prof. Anthony L. Fink has passed away on March 2,
1570-9639/$ see front matter 2008 Elsevier B.V. Aldoi:10.1016/j.bbapap.2008.09.026common ageing-relatedmon neurodegenerative
age 50, PD is generally considered as an aging-related disease, andapproximately one of every 100 persons over the age of 65 in the USsuffers from this disorder .Parkinson's disease (PD) is the mostmovement disorder and second most com1. IntroductionParkinson's diseaseNicotineHydroquinoneFibrillationMisfoldingare neuro-protective. The brillation of -synuclein was studied in relation to ve different compoundsfound in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioavin T assays,gel electrophoresis, size exclusion chromatographyhigh performance liquid chromatography (SECHPLC)and atomic force microscopy (AFM) were utilized to monitor the rate of -synuclein brillation and theinhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit -synuclein bril formation in a concentration-dependent manner, with nicotine being more effective. TheSECHPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of theoligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomerswith an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of thecigarette smoke components on the A53T mutant brillation. These results show that nicotine andhydroquinone inhibit -synuclein brillation and stabilize soluble oligomeric forms. This information can beused to understand the molecular mechanism of the nicotine and hydroquinone action to developtherapeutic solutions for PD.
2008 Elsevier B.V. All rights reserved.
by PD. Since only a small percentage of patients are diagnosed beforeAvailable online 25 October 2008
smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals thatArticle history:Received 24 July 2008
-synuclein is a small presyfunction is unknown, the aggregated form of -synuclein is a pathological hallmark of severalSmoking and Parkinson's disease: Does n
Dong-Pyo Hong a, Anthony L. Fink a,1, Vladimir N. Uva Department of Chemistry and Biochemistry, University of California at Santa Cruz, Santab Center for Computational Biology and Bioinformatics, Department of Biochemistry and MIndiana University School of Medicine, Indianapolis, IN 46202, USAc Institute for Biological Instrumentation, Russian Academy of Sciences, 142290 Pushchino,
a b s t r a c ta r t i c l e i n f o
Received in revised form 9 September 2008n Americans are affected
Biology and Bioinformatics,Indiana University School of46202, USA. Tel.: +1 317 278
l rights reserved.otine affect -synuclein brillation?
ky b,c,z, California 95064, USAcular Biology, Institute for Intrinsically Disordered Protein Research,
scow Region, Russia
tic protein (14,460 D) that is abundantly distributed in the brain. Although, its
evie r.com/ locate /bbapap(1) The N-terminal region, which represents residues 160,contains 11-amino acid imperfect repeats with a consensus motif(KTKEGV).
(2) The central region, which represents residues 6195, contains ahighly amyloidogenic NAC region and two additional repeats.
(3) The C-terminal region, which represents residues 96140, isrich in acidic residues and prolines, which are suggested to adopt adisordered conformation .
smokers which suggests that nicotine exposure prevents againstneuronal insults . Furthermore, nicotine administered orally bygum or transdermally by the patch has been shown to improvesymptoms of Parkinson's disease . Intriguingly, it has been
Scheme 1. -synuclein sequence. The three sites of early-onset PD-linked mutations (positions 30, 46 and 53) are highlighted.
283D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 282290The competing kinetic pathways for the aggregation of -synuclein begin with a natively unfolded -synuclein monomer,which partially folds into an aggregation-prone intermediate .Depending on the conditions, this intermediate can form threedifferent products: soluble oligomers, insoluble amorphous aggre-gates or insoluble brils [6,7,9] (Scheme 2). The state that -synucleinadopts depends on changes in the environmental conditions such asdecrease in pH, or temperature increase, evolving to the formation ofthe partially folded intermediate [6,7,9,12].
The involvement of -synuclein in PD was suggested by previousstudies of families with autosomal dominant early-onset of familial PD(FPD) in which three separate mutations in the -synuclein geneA53T, A30P and E46K were linked to the disease . All three FPDmutations alter the rate of -synuclein aggregation in vitro, but onlythe A53T mutation accelerates bril formation as compared to thewild type proteins, whereas A30P and E46K increase rates ofamorphous aggregation .
It is believed thatmany environmental and genetic factors affect-synuclein brillation. Factors that accelerate the -synuclein brilla-tion include certain pesticides, metals, lipids, membranes, polycations,glycosaminoglycans (GAGs), and macromolecular crowding [6,7,9].This acceleration is due to the conditions that increase theconcentration of the amyloidogenic intermediate. Oxidative modica-tion was suggested to play an important role in the PD pathogenesisand one of the major factors triggering -synuclein aggregation wasshown to be the formation of free radicals (e.g., see ).
On the contrary, inhibition of brillation occurs when themonomer or non-brillogenic oligomers are stabilized [6,7,9]. Somecompounds in cigarette smoke might cause such inhibition, asepidemiological studies revealed that the smoke compounds sig-nicantly decrease the risk of PD [22,23]. In cigarette smoke, out of themore than 3800 identied compounds, anabasine, cotinine, hydro-quinone, nornicotine, and especially nicotine, potentially representsuch inhibitory compounds. Nicotine is a good candidate forinvestigation because of several reasons. First, nicotine stimulatesstriatal dopamine neurons that are damaged in PD . Second,epidemiological studies have shown that PD is less prevalent inScheme 2. Multiple pathways for -synuclein aggregation. Nu represents the nativelyunfolded -synuclein monomer.reported that nicotine possesses both pro-oxidant and antioxidantproperties . It has been pointed out that because of the nicotine'sbenecial role in the treatment of certain neurodegenerative diseases,its involvement in free radical production or its ability to act as anantioxidant requires careful study to further evaluate its overalltherapeutic usefulness . In addition to nicotine, numerous agentsin tobacco products could modulate biological functions and thedevelopment of PD .
In this study, the effect of nicotine, and three structurally similarcompounds, anabasine, cotinine and nornicotine (Scheme 3), on -synuclein brillation were investigated. The goal was to understandhow these compounds affect the rate of brillation. The results werecompared to those obtained for hydroquinone, a known inhibitor of-synuclein brillation, which is structurally different from the foursmoke compounds (Scheme 3). Thioavin T (ThT) assays, gelelectrophoresis, SECHPLC and atomic force microscope (AFM) wereused to investigate the inhibitory effects. Our results revealed thatnicotine and hydroquinone show strong inhibitory effects on -synuclein brillation by stabilizing oligomers. These ndings mayshed light on novel therapeutic solutions for PD based on the structureof the compounds.
2. Materials and methods
The chemical compounds such as nicotine and cotinine wereobtained from Sigma, hydroquinone was purchased from AcrosOrganics, whereas nornicotine and ThT were from Fluka.Scheme 3. Chemical structures of nicotine, cotinine, anabasine, nornicotine, andhydroquinone.
284 D.-P. Hong et al. / Biochimica et Biophysica Acta 1794 (2009) 2822902.2. Expression and purication of human recombinant -synuclein
Human wild type -synuclein was expressed using E. coli BL21(DE3) cell line transfected with pRK172/-synuclein plasmid (gener-ously donated by M. Goedert, MRC Cambridge). Expression andpurication of human recombinant -synuclein and its mutant fromE. coli were performed as previously described . Culture askscontaining 1 l of LB media were inoculated with 6 ml of a night pre-culture each and subsequently incubated at 37 C at 250 rpm until themedia reached A500=0.91. The culture was then induced withisopropyl-b-D-thiogalactopyranoside (IPTG) and incubated foranother 5 h . The cells were collected by centrifugation at4000 rpm for 15 min at 4 C. The cell pellets were frozen and keptovernight at 20 C. On the next day, pellets were thawed at roomtemperature and redissolved in 40 ml of lysis buffer (50 mM NaCl,20 mM TrisHCl, 0.10% Triton- 100, 0.20 mM phenylmethylsulfonyuoride (PMSF) pH 7.5).
Sonication at 60% power for 3.5 min total with seven 30 secondsonication bursts and 40 s of rest in between each burst was used tolyse the cells. Subsequently, ammonium sulfate was slowly added toachieve 30% saturation with vigorous stirring on ice. 10 min of gentlestirring completed the precipitation. In order to precipitate the celldebris, the solution was centrifuged at 13,000 rpm for 15 min at 4 C.The supernatant was separated from the pellet and the pellet wasdiscarded. The supernatant was brought to 50% saturation withammonium sulfate. The solution was centrifuged at 13,000 rpm for15 min at 4 C. The supernatant was separated from the pellet andthen discarded. The pellet was saved, dissolved in 50 ml of 10 mMTrisHCl (pH 7.5) and dialyzed against 4 l of 50 mM NaCl, 20 mMTrisHCl (pH 7.5) for 2 h. The dialysis buffer was changed after 2 hand then changed three more times overnight. In order to purify theprotein, the sample from the dialysis was loaded onto a DEAE-Sepharose Fast Flow column previously equilibrated with 5-bedvolume of 50 mM NaCl, 20 mM TrisHCl, and pH 7.2. About 80fractions were collected, and the presence of -synuclein wasveried by using SDS-PAGE. In order to remove the salt from theprotein, the fractions that contained -synuclein were dialyzedagainst deionized water for at least 36 h at 4 C. After the dialysis, theprecipitate was removed by centrifugation. Protein purity evaluatedby mass-spectrometry, SECHPLC and SDS-PAGE was close to 98%.The protein concentration was then determined by A275. The proteinwas frozen in liquid nitrogen and then lyophilized.
2.3. Fibrillation of WT -synuclein and the ThT assay
The lyophilized -synuclein was dissolved in 1 ml of 0.02 MNaPO4 (Pi) buffer, 0.1 M NaCl and 0.01% NaN3, pH 7.5. In order toremove any insoluble material, the sample was centrifuged in thecold room for 30 min at 13.2 rpm. The supernatant was thenanalyzed by UV spectrophotometry to estimate protein concentra-tion and by mass-spectrometry, SECHPLC and SDS-PAGE toevaluate the presence of aggregated material. At the beginning ofthe brillation studies, protein samples were predominantly mono-meric and did not contain noticeable amounts of any oligomericforms. Assay solutions contained -synuclein at a concentration of1.0 mg/ml, 20 M ThT with various concentrations of smokecompounds as indicated. A volume of 150 l of the mixture waspipetted into a well of a 96-well plate (white plastic, clear bottom),and a 1/8th in diameter. Teon sphere (McMaster-Carr, Los Angeles)was added. Each sample was run in triplicate or quadruplicate. Theplates were sealed with Mylar plate sealers (Dynex). The plate wasloaded into a uorescence plate reader (Fluoroskan Ascent) andincubated at 37 C with shaking at 600 rpm with a shaking diameterof 2 mm. The uorescence was measured at 30 min intervals withexcitation at 450 nm and emission at 485 nm, with a sampling time
of 100 ms.The data were t to a sigmoidal curve described by the empiricalequation  using SigmaPlot software:
F = Fi +mit +Ff +mf t 1 + e
where F is the uorescence intensity and t50 is the time to 50% ofmaximal uorescence. The initial baseline during the lag time isdescribed by Fi +mit. The nal baseline after the growth phase hasended is described by Ff +mft. The apparent rate constant, kapp, for thegrowth of brils is given by 1/, the lag time is calculated as t50 2and the amplitude, amp, is given by Ff Fi. Although Eq. 1 gave verygood ts for the ThT kinetic proles, the expression is strictly a simpleempirical means of providing kinetic parameters for comparing ratesof brillation from different samples and does not directly reect theunderlying complex kinetic scheme.
Aliquots of the supernatant were airfuged for 30 min at 20 psi(75,000 rpm) at the end of the brillation. 3 l of the supernatant and1 l of staining gel were added into an Eppendorf tube, boiled for5 min and spun down at 14,000 rpm for 1 min. The amount of solubleprotein present in the supernatant was monitored using theCoomassie-blue staining of the sodium dodecyl sulfate-polyacryla-mide gel electrophoresi...