doi:10.1016/j.jmb.2009.09.008 J. Mol. Biol. (2009) 394, 94–107
Available online at www.sciencedirect.com
Molecular Insights into the Interaction betweenα-Synuclein and Docosahexaenoic Acid
Giorgia De Franceschi1, Erica Frare1, Luigi Bubacco2, Stefano Mammi3,Angelo Fontana1 and Patrizia Polverino de Laureto1⁎
1CRIBI Biotechnology Centre,University of Padova, Viale G.Colombo 3, 35121 Padova, Italy2Department of Biology,University of Padova, Viale G.Colombo 3, 35121 Padova, Italy3Department of ChemicalSciences, University of Padova,Via Marzolo 1, 35131 Padova,Italy
Received 5 June 2009;received in revised form3 September 2009;accepted 3 September 2009Available online8 September 2009
α-Synuclein (α-syn) is a 140-residue protein of unknown function, involvedin several neurodegenerative disorders, such as Parkinson's disease.Recently, the possible interaction between α-syn and polyunsaturatedfatty acids has attracted a strong interest. Indeed, lipids are able to triggerthe multimerization of the protein in vitro and in cultured cells.Docosahexaenoic acid (DHA) is one of the main fatty acids (FAs) incerebral gray matter and is dynamically released following phospholipidhydrolysis. Moreover, it has been found in high levels in brain areascontaining α-syn inclusions in patients affected by Parkinson's disease.Debated and unsolved questions regard the nature of the molecularinteraction between α-syn and DHA and the effect exerted by the protein onthe aggregated state of the FA. Here, we show that α-syn is able to stronglyinteract with DHA and that a mutual effect on the structure of the proteinand on the physical state of the lipid derives from this interaction. α-Synacquires an α-helical conformation in a simple two-state transition. Thebinding of the protein to the FA leads to a reduction of the size of thespontaneously formed aggregated species of DHA as well as of the criticalaggregate concentration of the lipid. Specifically, biophysical methods andelectron microscopy observations indicated that the FA forms oil droplets inthe presence of α-syn. Limited proteolysis experiments showed that, whenthe protein is bound to the FA oil droplets, it is initially cleaved in the 89–102region, suggesting that this chain segment is sufficiently flexible or unfoldedto be protease-sensitive. Subsequent proteolytic events produce fragmentscorresponding to the first 70–80 residues that remain structured and showhigh affinity for the lipid. The fact that a region of the polypeptide chainremains accessible to proteases, when interacting with the lipid, suggeststhat this region could be involved in other interactions, justifying theambivalent propensity of α-syn towards folding or aggregation in thepresence of FAs.
α-Synuclein (α-syn) is a 140-amino-acid, nativelyunfolded protein of still unknown function, charac-terized by seven repeats (KTKEGV) in the N-terminal region; by a hydrophobic central region,formed by residues 61–95 (non-amyloid-β com-ponent); and by acidic stretches in the C-terminalpart. It is highly expressed in the central nervoussystem, particularly at the presynaptic nerve term-inals. It is the major component of Lewy bodies, thecytoplasmic proteinaceous aggregates pathogno-monic for Parkinson's disease (PD).1 These filamen-
tous aggregates have a fibrillar structure with across-β-sheet core typical of amyloids.1,2 Mutationsor overexpression of the human α-syn gene has beenlinked to early-onset autosomal dominant PD.3–6
Despite the evidence for a key function of α-syn inthe onset of PD, there is little information about itsphysiological function in the brain. α-Syn wassuggested to be involved in synaptic plasticity7 andin regulation of dopamine neurotransmission8 andto act as a chaperone.9 The physiological function ofα-syn is also correlated with lipids and membranes,since this protein seems to modulate presynapticvesicle pool size and vesicle recycling.10,11 α-Syn isprincipally a soluble cytosolic protein, but it was alsofound reversibly associated with membranes.12,13 Invitro studies suggest that, although unfolded insolution, α-syn acquires an α-helical structure in thepresence of SDS micelles14 or membrane mimeticsystems rich in acidic phospholipids, such asphosphatidylserine and phosphatidic acid.12 Theinteraction with membranes appears to be mediatedby six to seven imperfect repeats in its N-terminalregion that are homologous to the α-helical region ofapolipoproteins.15
Recently, the role of α-syn has been associated alsowith fatty acids (FAs). α-Syn is implicated in FAuptake and metabolism. In fact, its gene ablationincreases docosahexaenoic acid (DHA) incor-poration.16–18 In addition, the protein exhibits aninhibitory activity of phospholipase D both in vitroand in vivo, indicating a role in lipid-mediated signaltransduction.19 α-Syn seems to interact with unsat-urated and polyunsaturated fatty acids (PUFAs), butwhether this interaction involves free FAs, as in thecase of FA binding proteins20 ormicelles,21,22 has notbeen elucidated yet. This interaction promotes theoligomerization of α-syn. Using electrophoreticmethods, Perrin et al. have shown that α-syn formsmultimers in vitro upon exposure to vesicles contain-ing PUFA acyl groups and that this process occurs atphysiological concentrations.23 Since exposure ofneuronal cell lines to PUFAs increases the levels of α-syn oligomers, Sharon et al. suggested that α-syncould interact with PUFAs in vivo to promote theformation of highly soluble oligomers that precedethe insoluble aggregates associated with neurode-generation.24,25 The exact mechanism responsible forPUFA-dependent multimerization and the specificrole played by the aggregative state of the FA inmodulating this process are still unknown.Here,we have investigated the interaction ofα-syn
with DHA using several biochemical and biophysi-cal techniques, considering the significance of thisFA for the role of α-syn inside the cell. DHA is adietary essential ω3 PUFA and an absolute require-ment for the development of the human centralnervous system and the continuous maintenance ofbrain cell functions. The highest level of DHA isfound in the synaptic plasma membrane and in thesynaptic vesicles.26–28 In fact, up to 60% of all FAsesterified in neuronal plasma membrane phospholi-pids consist of DHA.27–29 Moreover, DHA is crucialto maintain cellular activity by modulating mem-
brane order, gene transcription, cell signaling, andcaspase activation.30 Epidemiological studies haveassociated lowω3 PUFA consumptionwith high riskof developing Alzheimer's disease and PD. Howev-er, the link between ω3 PUFA intake and the risk ofdeveloping PD is not yet clear,31,32 but a recent studyprovided the preclinical evidence that highω3 PUFAconsumption exerts a neuroprotective action againstMPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyri-dine)-induced toxicity.32 Interestingly, DHA levelshave been shown to be elevated in those brain areascontaining α-syn inclusions in PD patients.33,34
In the present study, we show that upon bindingto DHA, α-syn acquires an α-helical conformationas assessed by circular dichroism (CD) measure-ments, in accordance with previous results.22 Sincethe aggregation state of FAs (monomer, micelle,vesicle, and oil droplet)35 has been shown to deeplyaffect both the molecular interaction with proteinsand their resulting conformation, several measure-ments were carried out to clarify the state adoptedby DHA in the presence of α-syn. We concludedthat DHA forms oil droplets with a spread sizedistribution under our experimental conditions. Theprotein significantly modifies the physical state ofDHA, reducing the size of the droplets andlowering its critical aggregative concentration(CAC). Moreover, proteolysis mapping of theprotein–FA complex was performed to identify theregion(s) of α-syn directly involved in the interac-tion, considering that the peptide bonds hidden byFAs or engaged in the interaction are expected to beless prone to proteolysis. The results of this studyallowed a better comprehension of the role of DHAin the structural transition of α-syn and encouragefurther investigations of the implication of lipids inamyloid formation in vivo.
Results
α-Syn acquires an α-helical conformation in thepresence of DHA
The secondary-structure content of α-syn inphosphate-buffered saline (PBS) buffer, pH 7.4,and at different DHA concentrations (0–500 μM)was evaluated by far-UV CD. In Fig. 1, theellipticity of α-syn samples at 222 nm is shown asa function of the molar ratio of DHA and α-syn. Asimilar trend is obtained when both 5 μM (○) and15 μM (●) α-syn are used, and a correspondingincrement of the ellipticity is observed when theamount of DHA is increased. The recorded spectrabetween 250 and 197 nm are reported (Fig. 1, inset),and the appearance of the two typical minima at222 and 208 nm, in the presence of DHA, indicatesthe acquisition of an α-helical secondary structure.The presence of an isodichroic point at 203 nm inthe titration experiments (Fig. 1, inset) suggests asimple two-state conformational transition betweenrandom coil and α-helix. For DHA/protein molarratios higher than ∼60, the addition of FA does not
Fig. 2. Characterization of the physical state of DHA.(a) Turbidimetric analysis at 400 nm of samples containingDHA up to 500 μM, in the absence (filled circles) and in thepresence of α-syn (2.5 and 5 μM, open triangles and opencircles, respectively). The filled square indicates the opticaldensity value upon addition of α-syn to a 500 μM DHAsuspension, to a final protein concentration of 5 μM. (b)Ratio between the first and the third vibronic band (I1/I3)of the pyrene emission spectrum as a function of DHAconcentration in the absence and in the presence of α-syn(5 μM). Inset: Iex/I1 ratio as a function of DHA concentra-tion in the absence and in the presence of α-syn (5 μM).
Fig. 1. DHA-induced structural changes in α-synmonitored by far-UV CD spectroscopy. CD signals at222 nm are plotted against the molar ratio of DHA andα-syn. Two different concentrations of α-syn were used:5 μM, open circles; 15 μM, filled circles. Inset: Far-UV CDspectra of α-syn in the presence of DHA. The spectra wereobtained at a protein concentration of 5 μM in PBS buffercontaining increasing concentrations of DHA (0–500 μM,as indicated in the labels).
96 α-Syn and DHA
result in any further increase in the helix content,estimated to be ∼70%.36 From the graph in Fig. 1, itcan be estimated that∼35 DHAmolecules per α-synmolecule are required for such complete structuraltransition.
α-Syn effect on DHA aggregates
Here, we have conducted systematic measure-ments to understand the effect of the protein onthe FA and the type of interaction that occursbetween α-syn and DHA. The physical state andthe self-aggregation process of DHA are stronglydependent on its concentration, ionic strength, andmore severely on pH. Indeed, DHA can formmicelles (pH 9–11), vesicles (pH 8–9), and oildroplets (below pH 8).37,38Turbidity methods have been used to measure the
change of the scattered light caused by the formationof large aggregates, such as oil droplets and vesicles.These measurements allow the calculation of acritical concentration for aggregate formation.39
The optical density at 400 nm of solutions containingincreasing concentrations (0–500 μM) of DHA in PBSat pH 7.4wasmeasured (Fig. 2a, filled circles). Belowa DHA concentration of 82±18 μM, there is noappreciable variation of turbidity at 400 nm. Whenthe concentration above this value is increased, asudden increase of optical density is observed. Thisphenomenon indicates the formation of large FAaggregates. The same measurements were con-ducted in the presence of α-syn (5 μM), and nosignificant increase of turbidity was observed up to500 μM DHA (Fig. 2a, open circles), suggesting thatthe protein is able to alter the DHA assembly.Moreover, addition of α-syn to a 500 μM DHAsuspension to a final protein concentration of 5 μM,
causes a sharp decrease of the optical density at400 nm (Fig. 2a, filled square). If the turbidity ismeasured in the presence of 2.5 μM α-syn (Fig. 2a,open triangles), there is no variation of the opticaldensity up to 240 ±25 μM DHA. Above thisconcentration, the turbidity starts to increase as afunction of DHA concentration, showing that theamount of α-syn is not sufficient to prevent theformation of large lipid aggregates.To better understand the effect of α-syn on the
aggregation properties of DHA, we used pyrene as afluorescent probe. We evaluated the ratio betweenthe first and the third vibronic band (I1∼374 nm,I3∼383 nm) of the pyrene emission spectrum.40 Thevalue of this ratio is correlated to the polarity of theenvironment, and low values correspond to anonpolar surrounding.41,42 The I1/I3 ratio decreaseswhen the amount of DHA is increased. A sigmoidal
97α-Syn and DHA
curve fits the experimental data points (Fig. 2b, filledcircles), indicating the induction of a more hydro-phobic microenvironment. The critical aggregationconcentration of the FA, calculated at the inflectionpoint of the plot,40 is 131±9 μM. In the presence ofα-syn (5 μM) (Fig. 2b, open circles), the I1/I3 ratioshows a steeper decrease with an inflection point at47±13 μM (Fig. 2b, open circles). This indicates thatthe final aggregation state is reached at lower DHAconcentrations but does not imply that the nonpolarenvironments that result in the absence and in thepresence of α-syn are the same.
Fig. 3. Negative staining (a) and cryo (b) EM images of sam(+α-syn) of the protein. Oil droplets are indicated by arrows. (absence (dark gray bars) and in the presence (white bars)distribution contribution of the measured particles.
The pyrene excimer emission can be detected bythe broad band centered at 470 nm. Pyrene can forman excited state dimer (excimer) only at concentra-tions higher than 1 μM or when it is dissolved inhydrophobic micro-domains.43 The ratio (Iex/I1)between the maximum emission intensity of theexcimer (Iex) and the I1 band of pyrene can be used toevaluate the efficiency of excimer formation. Weplotted the Iex/I1 ratio as a function of DHAconcentration (Fig. 2b, inset) in the absence (filledcircles) and in the presence (open circles) of α-syn.When the DHA amount is increased, the Iex/I1 ratio
ples of DHA in the absence (−α-syn) and in the presencec) DLS measurements of a sample of DHA (250 μM) in theof α-syn. The percentages close to the bars indicate the
Fig. 4. 1H–15N correlation (HSQC) spectra of (a) α-synin PBS, pH 7.4, and (b) α-syn in the presence of saturatingconcentrations of DHA. Conformational averaging or fastamide proton exchange at 25 °C is responsible for the lossof several peaks in the spectrum of the free protein. Theslower tumbling rate of the lipid vesicles obtained after theaddition of DHA restricts the motion of the vesicle-interacting residues, resulting in line broadening and infurther loss of peaks.
98 α-Syn and DHA
increases and a maximum value is reached above aDHA concentration of 100 μM. In the presence of theprotein, the Iex/I1 ratio follows a similar trend up to100 μM. Above such concentration, the ratio remainsroughly constant (Fig. 2b, inset, open circles). Thisdifferent behavior can derive from the fact that, in theabsence of the protein, pyrene is dissolved in a largerhydrophobic volume, while α-syn forces DHA toform only aggregates with a small hydrophobicvolume that may also imply reduced mobility of thepyrene molecules.38
Morphological analysis and size determinationwere conducted via electron microscopy (EM) (cryoand negative staining) and dynamic light scattering(DLS) (Fig. 3). EM pictures of the DHA suspensionwere taken in the absence (Fig. 3a, left) and in thepresence (Fig. 3a, right) of α-syn. In the first case, theimage clearly indicates the presence of aggregateswith a heterogeneous distribution of sizes, while inthe second case, smaller and more regular specieswere observed. Figure 3b (left) shows a cryo-EMpicture obtained from a DHA suspension preparedat pH 7.4. Vaguely bordered particles are visible,compatible with the presence of oil droplets.44 In thepresence of α-syn (Fig. 3b, right), DHA formssimilar oil droplets with reduced diameter. Thesize distribution of DHA samples in the presence ofα-syn was estimated from rotationally averagedimages of individual particles. The resulting distri-bution shows a particle diameter of 13.3±4.4 nm fornegative staining specimen and 26.5±2.4 nm forcryo specimen, as calculated from the center of theGaussian curve (Fig. S1).DLS measurements show that, in a 250 μM DHA
sample, 95% of the distribution contribution derivesfrom particles with an average diameter around517 nm (Fig. 3b, dark gray bars), while in thepresence of 5 μM α-syn, a high percentage of thespecies (99.2%) have a smaller diameter (34.2 nm)(Fig. 3c, white bars). This last peak is quite broad(width at half height of 31.5 nm), indicating that theDHA aggregates undergo a resizing toward smallerspecies, but they still have a broad distribution.
NMR structural characterization
To compare the conformational properties of freeand DHA-bound α-syn, we recorded 1H–15N hetero-nuclear single quantum coherence (HSQC) spectra ofthe protein alone and in the presence of saturatingconcentration of DHA (α-syn/DHA molar ratio of1:100). The spectrum of α-syn (Fig. 4a) exhibits adense cluster of cross-peaks over a narrow range, inagreement with the fact that the protein is largelyunfolded at pH 7.4. As already described,45 at 25 °C,the number of visible peaks (∼70) is lower thanexpected (135). This behavior can be attributed toconformational exchange in the first 100 residues ofα-syn46 or to fast chemical exchange between amidegroups and the solvent.47 After the addition of DHA,many peaks disappear (Fig. 4b). The positions of theremaining peaks are not changed, indicating that thecorresponding residues do not bind lipids and
continue to be unfolded and mobile in the presenceof DHA. Interestingly, the spectrum obtained in thepresence of DHA is quite similar to those previouslyrecorded in the presence of acidic small unilamellarvesicles.48 The remaining peaks correspond to the∼40 C-terminal residues. The disappearance of themajority of peaks indicates that under the experi-mental conditions used here, α-syn promotes theformation of DHA aggregates at least as large assmall unilamellar vesicles. A similar interpretationwas given for the reduction of signal intensity in the1D spectrumofα-syn in the presence of linolenic acidand DHA.49
99α-Syn and DHA
Mapping of the complex between α-syn and DHA
A combination of proteolytic digestion and massspectrometry (MS) was used to directly define theregion(s) of interaction of α-syn with DHA. Thisstrategy relies on the consideration that regions ofthe protein normally available to proteases exhibitlimited accessibility when involved in interactionwith lipids.50,51 Proteinase K,52 a protease thatdisplays broad substrate specificity and, thus, isparticularly suitable for this study, was used.Moreover, additional experiments were conductedusing also trypsin, which specifically hydrolyzespeptide bonds involving Lys and Arg residues. Bothproteases retain proteolytic activity in the presenceof DHA (data not shown). Proteolysis experimentswere conducted in the absence or in the presence of
Fig. 5. Proteolysis of α-syn by proteinase K analyzed by SDconducted at a protein concentration of 5 μM in PBS in the aDHA (50 and 250 μM) using an E/S ratio of 1:1000 by weigh0 min (lane 1), 5 min (lanes 2, 4, and 6), and 30 min (lanes 3, 5,of apomyoglobin was loaded onto the gel as a marker of moreported on the left. RP-HPLC chromatograms correspondinmixture of α-syn and proteinase K after 5 and 60 min of incuba250 μM DHA (c and d). The identity of α-syn fragments was
increasing concentrations of DHA, and the proteol-ysis patterns were compared.In Fig. 5 (top), the SDS-PAGE analysis of the
digestion by proteinase K is reported. In the absenceof DHA, the protein is easily degraded in smallfragments (lanes 2 and 3), as expected for anunfolded polypeptide chain. If the proteolysis isconducted in the presence of 50 μM DHA (DHA/protein molar ratio of 10), a slight protection of theprotein from proteolysis is observed (lanes 4 and 5),while in the presence of 250 μMDHA (molar ratio of50), α-syn is rather resistant to proteolysis and largefragments are formed (lanes 6 and 7). Specifically, inthe proteolytic mixture corresponding to 30 min ofincubation of α-syn with the enzyme, two mainbandswere found at 7.5 and 9.0 kDa. The proteolysismixtures were analyzed also by reverse-phase high-
S-PAGE (top) and RP-HPLC (bottom). The reactions werebsence or in the presence of increasing concentrations oft. Aliquots from the proteolysis mixture corresponding toand 7) of incubation were analyzed. A partial BrCN digestlecular weights, and the position of the relative bands isg to the analysis of aliquots taken from the proteolysistion in the absence of DHA (a and b) and in the presence ofestablished by ESI–MS (Table S1).
Fig. 6. Far-UV CD spectra of the proteolysis mixture ofα-syn with proteinase K. The reaction was conducted at aprotein concentration of 5 μM in PBS in the presence ofDHA (250 μM) with an E/S ratio of 1:1000 by weight.Spectra were recorded up to 2 h at intervals duringincubation.
100 α-Syn and DHA
performance liquid chromatography (RP-HPLC)(Fig. 5, bottom), and the identities of the proteinfragments were obtained by MS analysis of theisolated fractions (Table S1). In Fig. 5a and b, thechromatograms relative to 5 min and 1 h reactionconducted in the absence of DHA are shown. Severalpeaks are detected in the chromatogram, corre-sponding to fragments that span almost all theregions of the α-syn sequence. After a 1 h incubationwith the protease, the peaks relative to α-syn andlarge fragments disappear from the RP-HPLCchromatogram (Fig. 5b). Nevertheless, the C-termi-nal fragments 90–140 and 93–140 remain undigestedin the proteolysis mixture.We relate the resistance toproteolysis of the C-terminal tail of α-syn to itsunusual amino acid sequence (see Discussion).
Fig. 7. Amino acid sequence of human α-syn in one-letter sstructure (bottom). Three regions can be described: 1–60, contamphipathic character (dark gray); the highly hydrophobic nacidic tail (light gray). Gray and black arrows indicate the mainthe presence of DHA.
In the presence of 250 μM DHA (Fig. 5c and d),α-syn is quite resistant to proteinase K and onlyfew minor peaks are visible in the chromatogram ofa protein sample digested for 5 min (Fig. 5c). Thesechromatographic peaks correspond to N-terminalfragments 1–89 and 1–92, as well as to the comple-mentary C-terminal fragments 90–140 and 93–140,indicating that the protein is initially cleaved at thepeptide bonds Ala89-Ala90 and Thr92-Gly93. Afterprolonged incubation with the protease (1 h, Fig.5d), the predominant product of proteolysis isfragment 1–72, which remains undigested evenafter 3 h of reaction (data not shown). The C-terminal fragments 90–140 and 93–140 are quiteresistant to further proteolysis, since the additionalfragments, 95–140 and 126–140, are produced to aminor extent. We also analyzed the proteolysismixture after 24 h of incubation (Fig. S2). In theabsence of DHA (Fig. 5a), only peaks correspondingto very short peptides are present. In the presence of250 μM DHA, although many of the cleavagesoccurred during the prolonged incubation, the peakscorresponding to fragment 1–72 and species close toit (retention time, 28–29 min) are still visible. This is aclear indication of the protective effect exerted byDHA and that this protein region still maintainsresidual structure that limits proteolytic events.Considering that α-syn contains 15 lysine residues
in the N-terminal region, α-syn was also digestedwith trypsin in the absence and in the presence ofDHA. The RP-HPLC analyses of these proteolysismixtures are shown in Fig. S3. Despite the numeroussites of possible tryptic attack, the initial cleavages inthe presence of DHA occur at the level of peptidebonds 102–103, 97–98, and 96–97, followed byfission of bond 80–81. The fragment 1–80 is stillpresent in the reaction mixture even after 24 h ofincubation, indicating that this is the most resistantto proteolysis (Fig. S3c). Also, fragment 103–140remains undigested in the proteolysis mixture withtrypsin, but this can be explained by the fact that thisfragment does not contain basic residues amenableto tryptic attack.The rationale of the limited proteolysis approach
implies that the protein species resistant to proteo-lytic degradation should be folded and rigid. Far-UV CD spectra of the proteolysis mixture of α-syn
ymbols (top).53 Schematic representation of α-syn primaryaining part of the seven 11-residue imperfect repeats withon-amyloid-β component domain (61–95) (gray); and thecleavage sites by proteinase K and trypsin, respectively, in
101α-Syn and DHA
with proteinase K in the presence of 250 μM DHAwere recorded during incubation (up to 2 h) toestablish if the proteolytic products maintain theα-helical structure (Fig. 6). The CD spectra showthe presence of a regular α-helix, albeit the helicalcontent of the protein species in solution is reducedfrom 70% to 40%.36
Thus, combining the results of RP-HPLC (Fig. 5)and far-UV CD (Fig. 6), it can be concluded that, inthe presence of 250 μM DHA, the initial proteolyticevents occur in the region 89–102, while subsequentcleavages lead to the truncation of the previouslyformed N-terminal fragments at their C-terminalend (Fig. 7).
Discussion
There is ample evidence that natively unfoldedproteins, such as α-syn, are not simple random coilsbut rather form transient ordered structures thatcould be stabilized by the interactions with naturalligands or cofactors, which are able to affect their netcharge or hydrophobicity.54–56 It has been proposedthat α-syn could interact with free FAs20 or,alternatively, with FAs in a micellar or bilayerstate.21,49 In several reports, it has been suggestedthat PUFAs are strictly connected to neurologicaldisorders, including depression, Alzheimer's dis-ease, and PD.28–32 Specifically, alterations in PUFAlevels have been related to aggregation of α-syn.21–25
Our data show that α-syn interacts with the poly-unsaturated FA DHA, acquiring an α-helical con-formation and strongly affecting the self-associationprocess of the lipid. Several experiments were alsoconducted using palmitic and oleic acid (Figs. S4, S5,and S6). While oleic acid seems to interact with α-syn similarly to DHA, the saturated FA palmitic aciddoes not (Fig. S4). Our data, in agreement withprevious studies,20,22,49 suggest that the specificphysical state of the FA containing at least oneunsaturation is essential to trigger the conforma-tional transition in α-syn. Nevertheless, we havefocused our studies on the interaction of DHA withα-syn, considering that this PUFA seems to haveimplications for the role of α-syn in the nervoussystem.26–32The species formed by DHA and the transition
occurring in the presence of α-syn were character-ized by several techniques to reach a unifying pictureof the α-syn/DHA interaction. DHA self-aggrega-tion is strongly dependent on its concentration, aswell as on ionic strength and temperature. Also, ithas been shown that pH defines precisely the speciesformed by DHA.37 In fact, in the presence of morethan 95% water and above a critical concentration,micelles, vesicles, and oil droplets form as a functionof the ratio between ionized and non-ionized FA.57
At room temperature and under neutral conditions(pH 7.4), an increase in concentration of DHA leadsto the formation of oil droplets of different sizes.37
Indeed, when allowed to form spontaneously,38 FAaggregates are generally highly polydispersed and
form an ensemble of morphologically distinctspecies in dynamic equilibrium. Under the condi-tions used in our experiments, DHA aggregates spana wide range of diameters from 50 to more than600 nm and exhibit features compatible with theformation of lipid droplets, which, at variance fromvesicles, does not present bilayers, with a criticalaggregate concentration in the 100 to 130 μM range(Figs. 2 and 3). Addition of the protein causes a deepvariation in both size and properties of DHA oildroplets. This product of DHA self-assembly showsa smaller diameter and a reduced hydrophobicvolume (Figs. 2 and 3). Therefore, α-syn seems toexert a double effect, inhibiting the formation ofgiant aggregates and lowering the DHA concentra-tion necessary to form aggregates. Of interest is thatthis phenomenon is correlated to the availability ofthe protein since, when all the protein molecules arebound to the FA, the excess DHA is not prevented toform aggregates of various sizes. The ability of α-synto interfere with lipid packing in vesicles,58 aswell asto distort micelles,59 has already been observed.Moreover, it was also suggested that the proteinmayplay a role in modulating both the organization oflipid membrane components and the size of thesynaptic vesicles.60 Lipid aggregates are very de-formable because of the flexibility of the hydrocar-bon chains. The shape and the dimension of theaggregate can adjust to maximize the interactionwith the protein. In DHA, the presence of six doublebonds confers high fluidity to its self-assemblyproducts.61
Of note, the DHA concentrations used here aresuperphysiological, since the concentration of thefree DHA molecules in the cytoplasm is in thenanomolar order.62 However, its abundance in brainphospholipids suggests that, upon local phospholi-pase-mediated release, its concentration can rapidlybecome micromolar, thus reaching a thresholduseful to induce aggregated forms of the FA.63,64
In addition, brain trauma ischemia has beenassociated with up to 6-fold increase in concentra-tion of free DHA in human cerebrospinal fluid.65
Upon binding to DHA, α-syn adopts an α-helicalsecondary structure. The interaction between α-synand membrane-like systems (micelles and vesicles)has been intensively studied using NMR14,59,66 andlimited proteolysis experiments.51,66 High-resolu-tion NMR measurements indicated that, in thepresence of SDS micelles, α-syn forms two antipar-allel amphipathic helices, wrapping around themicelle, while the C-terminal domain (residues100–140) remains unstructured. On the other hand,in vesicular systems, the formation of an extendedhelical structure over 90 residues in length has beenrecently proposed.67–69 The differences in size andcurvature of SDS micelles and vesicles can explainthe different structures adopted by α-syn.70,71 Atvariance, other authors suggested that vesicle-bound α-syn may have a similar structure to themicelle-bound form.72
Limited proteolysis experiments evidenced thatsynthetic lipid vesicles and SDS micelles lead to
Fig. 8. Scheme summarizing the conformational transition of α-syn in the presence of DHA. The DHA self-assemblyprocess in the presence of the protein and the early and subsequent proteolytic events by proteinase K (PK) are alsoshown. DHAm, FA monomer; DHAod, oil droplet; U, unfolded free protein; Im, protein in the presence of non-saturatingconcentration of DHA; Fod, protein in the presence of oil droplets; Iod, species in equilibriumwith the lipid-bound protein;CAC, critical aggregative concentration. The products of proteolysis show high affinity for DHA oil droplets.
102 α-Syn and DHA
different protection ofα-syn fromproteases.51,66,73 Inthe presence of SDS micelles, the ∼100 N-terminalresidues of α-syn display resistance to pro-teolysis.51,73 In the case of vesicles, proteolysisseems to occur on the population of free moleculesin equilibrium with the lipid-bound form.66 Theinteractions with vesicles are weaker than those withSDSmicelles, and the protein dissociatesmore easily,becoming accessible to proteases in its lipid-freeform.66,73 It was also observed that the equilibriumbetween lipid-bound and lipid-free α-syn is depen-dent on lipid composition, and α-syn exhibits ahigher affinity for negatively charged surfaces oflipid. Here, proteolysis experiments were conductedin the presence of DHA oil droplets, a completelydifferent physical state from micelles and vesicles.When bound to these DHA aggregates, α-synappears to be quite resistant to proteolysis, asexpected from the fact that the protein has adoptedan α-helical conformation (see Fig. 5). At non-saturating concentrations (molar ratio DHA/proteinb50), DHA slows down α-syn proteolysis, but thefragmentation pattern is not perturbed. This clearlysuggests that two different populations of confor-mers of α-syn are present in equilibrium, one free insolution and the other one bound to the lipid. Here,proteolysis experiments indicate that the C-terminaltail of α-syn is rather resistant to proteolysis when
the protein is digested in the presence or absence ofDHA. On the other hand, NMR data show that theC-terminal region of α-syn is unfolded even in thepresence of DHA. Indeed, fragments 90–140 and93–140 remain undigested even after 1 h reactionwith proteinase K (see Fig. 5). Analogous resistanceto proteolysis of the C-terminal tail of α-syn waspreviously observed by us in the presence ofmicellar SDS.51 We hypothesized that the C-terminal tail of α-syn in the presence of thedetergent is sufficiently rigid to resist proteolysis,despite the lack of regular secondary structure ofthe 40 C-terminal residues. However, herewith wemay consider also an alternative view for explainingthe resistance to proteolysis of the C-terminal tail ofα-syn. In fact, proteinase K displays a very broadsubstrate specificity (or no specificity at all), but itpreferentially cleaves polypeptide chains at the levelof aliphatic or hydrophobic amino acid residues. Itssubstrate recognition site is given by the peptidesegments 99–104 and 132–136 forming a hydro-phobic cleft given by Tyr104 and Leu133.74,75 The40-residue C-terminal tail of α-syn has a peculiaramino acid sequence containing up to 14 carbox-ylate groups of Glu and Asp residues and 5 Proresidues (see Fig. 7). This highly hydrophilic,negatively charged C-terminal tail may not beeasily accommodated at the hydrophobic binding
103α-Syn and DHA
site of proteinase K, thusmaking it rather resistant toproteolysis. Moreover, also the numerous Pro resi-dues of the tail may hinder proteolysis, consideringthat a Pro residue flanking the scissile peptide bondinhibits proteolysis and a variety of Pro-containingpeptides act as inhibitors of proteinase K.75,76
Above a DHA/protein molar ratio of 50, all α-synmolecules seem to be bound to the FA, as assessed byCD measurements and HSQC spectra. In order tocorrelate limited proteolysis data with the structureand dynamics of a protein substrate, it is importantto rank proteolysis events into initial and subsequentcleavages77 since the latter ones occur on a perturbedprotein substrate that does not necessarily retain theoverall structure and dynamics of the intact protein.In the presence of DHA, α-syn is initially cleaved byproteinase K and trypsin in the 89–102 region,suggesting that this chain segment is sufficientlyflexible or unfolded to be protease-sensitive.50,78,79The initial proteolytic N-terminal fragments aresubsequently trimmed at their C-terminal regionand they correspond to fragments 1–72 and 1–80,obtained by proteolysis with proteinase K andtrypsin, respectively. Since these fragments areresistant to further proteolysis and accumulate inthe reaction mixture, they should retain a rigidconformation. The presence of proteolysis-resistantregions together with the observation that theymaintain an α-helical structure leads to the suppo-sition that these species did not derive from thedigestion of unfolded α-syn in equilibrium with thelipid-bound one. From our experience, if proteolysisoccurred on the minor population of free moleculesin equilibrium with those bound, the pattern shouldbe similar to that obtained in the absence of the lipid.In Fig. 8, a scheme summarizing our results is
reported. The free DHA molecules are in rapidequilibrium with their aggregated states (oil dro-plets, od).38 At DHA concentrations lower than theCAC, there is an equilibrium between free unfolded(U) and the partially lipid-bound (Im) α-syn. In thiscondition, proteolysis preferentially occurs on theunfolded α-syn species, resulting in a patternanalogous to that obtained in the absence of DHA.Above the CAC and in the presence of saturatingconcentration of DHA (α-syn/DHA molar ratioN50), α-syn binds to DHA aggregates and acquiresan α-helical conformation (Fod). We suggest that thebound α-syn may be involved in rapid dissociationequilibrium that leads to a short-lived free α-syn(Iod), still containing some residual secondarystructure. Consequentially, this partially structuredα-syn is a different substrate for the protease'sdigestion, when compared to the free α-syn (U). Inthe Fod state, the protein segment 89–103 is flexibleand unfolded enough to be protease-sensitive. Lateproteolysis events by proteinase K and trypsinproduce fragments corresponding to the first 70 or80 residues, which remain structured and show highaffinity for the lipid.In conclusion, the results of this study may clarify
the debated question on the type of interactionbetween α-syn and DHA, providing clear evidence
for the formation of DHA oil droplets in the presenceof the protein. FA aggregates can act as carriers forproteins, suggesting a more important role of lipidsin neurodegeneration. Furthermore, though α-synacquires an α-helical conformation, proteolysisexperiments showed that a region of the polypeptidechain remains accessible to proteases. This meansthat this region is not characterized by a persistentstructure and it is conceivable that it could beinvolved in other interactions. Indeed, consideringthat prolonged exposure of α-syn to FAs inducesoligomerization of the protein,23,24 it would be ofutmost importance to define the exact mechanismresponsible for PUFA-dependent multimerizationand the specific role played by the aggregative stateof FAs in modulating this process.
Materials and Methods
Materials
Proteinase K from Tritirachium album and porcinetrypsin were purchased from Sigma (St. Louis, MO,USA). All other chemicals were of analytical reagentgrade and were obtained from Sigma or Fluka (Buchs,Switzerland).
Expression and purification of recombinanthuman α-syn
Human α-syn was expressed in Escherichia coli BL21(DE3) cell line transfectedwith the pET28b/α-syn plasmid.Overexpression of the protein was achieved by growingcells in LB medium at 37 °C to an A600 of 0.6 followed byinduction with 0.5 mM isopropyl β-thiogalactopyranosidefor 4 h. Toobtain 15N-labeled protein forNMRexperiments,the transfected cells were grown in M9 minimal mediumsupplemented with 1 g/l [15N]ammonium chloride.The purification of the recombinant protein was
conducted following a procedure previously described80
with minor modifications, that is, boiling the cell homog-enate for 15 min. The soluble fraction, containing α-syn,was treated with 55% ammonium sulfate. The pellet wasthen resuspended, dialyzed, loaded into a 6-ml Resource Qcolumn (Amersham Biosciences, Uppsala, Sweden), andeluted with a NaCl gradient from 0 to 500 mM. Furtherpurification was obtained by RP-HPLC, and the identityand integrity of the eluted material were assessed by wayof MS. We operated under conditions designed to avoidprotein aggregation within the time frame of the experi-ments, performing the measurements immediately aftersample preparation and using very low protein concentra-tions. The presence of aggregates was checked by nativeand SDS PAGE electrophoresis (Fig. S7).
Circular dichroism
Protein concentrations were determined by absorptionmeasurements at 280 nm using a double-beam Perkin-Elmer (Norwalk, CT, USA) Lambda-20 spectrophotometer.The molar absorptivity at 280 nm for α-syn was 5960 cm−1
M−1, as evaluated from its amino acid composition by themethod ofGill andvonHippel.81 CD spectrawere recordedon a Jasco (Tokyo, Japan) J-710 spectropolarimeter. Far-UV
104 α-Syn and DHA
CDspectrawere recorded using a 1-mmpath-length quartzcell and a protein concentration of 5–15 μM. The meanresidue ellipticity [θ] (deg·cm2·dmol−1) was calculatedfrom the formula [θ]=(θobs/10)·(MRW/lc), where θobs isthe observed ellipticity in degrees, MRW is the meanresiduemolecular weight of the protein, l is the optical pathlength in centimeters, and c is the protein concentration ingrams per milliliter. The spectra were recorded in PBS(8 mM Na2HPO4, 137 mM NaCl, 2 mM KH2PO4, and2.7 mM KCl), pH 7.4, in the absence or in the presence ofDHA in the 5 to 500 μM range.
Determination of DHA critical aggregate concentrationbased on turbidity measurements and pyrenefluorescence
Aliquots of DHA were stored at a concentration of76 mM in 100% ethanol at −80 °C. Air was evacuated withhelium gas to prevent oxidation. The aggregation of DHA,in the absence or in the presence of α-syn (2.5–5 μM), wasanalyzed by turbidity measurement at 400 nm of differentsamples containing increasing amounts ofDHA (0–500μM)in PBS, pH 7.4.39 The analyses were carried out with aPerkin-Elmer (Norwalk, CT, USA) Lambda-25 spectropho-tometer using a 10-mm path-length quartz cuvette.The critical concentration for aggregate formation of
DHAwas determined by the pyrene 1:3 ratiomethod.40,42,82
Two series of pyrene fluorescence emission measurements(1 μM)were obtained, increasing the FA concentration from0 to 500μMin the absence or in the presence ofα-syn (5μM).Fluorescence emission spectra were recorded in PBS(pH 7.4) using an excitation wavelength of 335 nm, andthe intensity of themaxima corresponding to the first (I1) andthe third (I3) vibronic band, located near 373 and 384 nm,respectively, was measured. A Perkin-Elmer LS-50B spec-trofluorimeter was employed, and a 2×10 mm path-lengthquartz cuvette was used. The critical aggregate concentra-tion was obtained from the inflection point of the pyrene 1:3ratio plots, which can be described by a decreasing sigmoidof the Boltzmann type.40 The fluorescence emission ofpyrene was recorded also up to 600 nm to monitor theincrease of the signal near 470 nm (band Iex), ascribed to thepyrene excimer emission,83 and the ratio Iex/I1 was plottedagainst DHA concentration.
Transmission electron microscopy
To evaluate the morphology and the size of the speciesderiving from the self-assembly of DHA, we examinedaliquots of the samples by means of transmission electronmicroscopy (TEM; negative staining and cryo). For thenegative staining, a drop of the sample solution was placedon a Butvar-coated copper grid (400 square mesh) (TAABLaboratories Equipment Ltd., Berks, UK), dried, andnegatively stained with a drop of uranyl acetate solution(1%, w/v). TEM pictures were taken on a Tecnai G2 12Twin instrument (FEI Company, Hillsboro, OR, USA),operating at an excitation voltage of 100 kV. For the cryo-EM, 3.5 μl of a DHA suspension was mounted onto laceycarbon grids (TAAB Laboratories Equipment Ltd.), blottedto make thin aqueous films under controlled roomtemperature and humidity conditions (∼95%), followedbyplunging into liquid ethaneusing aVitrobot™ apparatus(FEI Company). The grids were examined at the tempera-ture of liquid nitrogen using a cryo-holder (model 626,Gatan, Pleasanton, CA, USA) and a Tecnai G2 12 Twinmicroscope operated at an accelerating voltage of 100 kV.
The size distribution of DHA samples in the presence ofα-syn was obtained with the following procedure: 247(negative staining) and 24 (cryo) particles were manuallyextracted from micrographs using the semiautomaticprocedure implemented in the BOXER program of theEMAN software package.84 Only clearly defined sphericaland isolated particles were selected and boxed in 48×48pixel images. The particles were subsequently centeredand aligned by cross-correlating the individual images to arotationally averaged image. Once aligned, all imageswere rotationally averaged to obtain one-dimensionalradial profile. Center alignment and rotational averagingwere obtained using the SPIDER image processingsystem.85 The intensity profiles were fitted by a piecewisefunction that starts as a constant value (particle intensity)followed by half-period cosine drop and again by aconstant (background intensity). The particle half-lengthwas taken as a pixel position at the midpoint of the cosineintensity drop. Mean particle diameter was obtained froma Gaussian fit to the histogram distribution while thediameter error was estimated from the Gaussian fullwidth at half maximum. Intensity profile fits andhistogram analysis were performed with the MATHE-MATICA software package (Wolfram Research Inc.,Champaign, IL, USA).
Dynamic Light Scattering
DLS measurements were carried out with a ZetasizerNano-S instrument (Malvern Instrument, UK). Thisapparatus, which uses the backscattering detection(scattering angle θ=173°) and an avalanche photodiodedetector (APD), is equipped with a Helium–Neon lasersource (wavelength 633 nm; power 4.0 mW), and athermostated sample chamber controlled by a thermoelec-tric Peltier. DLS measurements were performed at 25 °Cin PBS pH 7.4 in duplicate. During every measurement 15runs were collected.
NMR analysis
NMR experiments were performed on a BrukerAvance DMX600 spectrometer equipped with a gradienttriple-resonance probe. HSQC spectra were recorded on a100-μM α-syn sample at 25 °C in 20 mM phosphatebuffer (pH 7.4), in the absence or presence of 10 mMDHA. The experiments consisted of 256 increments of512 time points each, acquired with 24 to 128 transientseach. Spectra were processed to obtain matrices of512×512 real points. Prior to Fourier transformation,90° shifted sine and sine square functions were used inthe f2 and f1 dimension, respectively.
Proteolysis of the complex between α-syn and DHA
Proteolysis experiments of the complex between α-synand DHA were carried out at room temperature usingproteinase K52 at an enzyme-to-substrate (E/S) ratio of1:1000 (by weight) and trypsin at an E/S ratio of 1:50(by weight). The reactions were conducted in PBS,pH 7.4, in the absence or in the presence of differentconcentrations (50 and 250 μM) of DHA. The α-synconcentration was 5 μM. The reactions were quenched atspecified times by acidification with trifluoroacetic acid(TFA) in water (4%, v/v). The proteolysis mixtures wereanalyzed by RP-HPLC and SDS-PAGE according to
105α-Syn and DHA
Schägger and von Jagow.86 The HPLC analyses wereconducted using a Vydac C18 column (4.6 mm×250 mm;The Separations Group, Hesperia, CA, USA), eluted witha gradient of acetonitrile/0.085% TFA versus water/0.1%TFA from 5% to 25% in 5 min, from 25% to 28% in 13 min,from 28% to 39% in 3 min, and from 39% to 45% in21 min. The effluent was monitored by recording theabsorbance at 226 nm. The sites of cleavage along the 140-residue chain of α-syn were identified by MS analyses ofthe protein fragments purified by RP-HPLC. Massdeterminations were obtained with an electrosprayionization (ESI) mass spectrometer with a Micro Q-Tofanalyzer from Waters (Manchester, UK). The measure-ments were conducted at a capillary voltage of 2.5–3 kVand a cone voltage of 30–35 V. The molecular masses ofprotein samples were estimated using the Masslynxsoftware 4.1 (Waters).
Acknowledgements
We thank Dr. Ivan Micetic for assistance with theanalysis of TEM images, Dr. Micaela Pivato forpreparing cryo-EM specimen, Dr. Marco Bisaglia forperforming NMR measurements, and Mr. GiuseppeTognon for technical assistance. We gratefullyacknowledge the financial support of the ItalianMinistry of University and Research (PRIN-2006)and of the University of Padua (PA-2008).
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2009.09.008
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