FEBS Letters 589 (2015) 798–804

journal homepage: www.FEBSLetters .org

Insight into conformational modification of alpha-synuclein in thepresence of neuronal whole cells and of their isolated membranes

http://dx.doi.org/10.1016/j.febslet.2015.02.0120014-5793/� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

⇑ Corresponding author.E-mail address: empedone@unina.it (E. Pedone).

1 These authors contributed equally to this work.

Giovanni Smaldone a,1, Donatella Diana a,1, Loredano Pollegioni b,c, Sonia Di Gaetano a,Roberto Fattorusso d, Emilia Pedone a,⇑a Istituto di Biostrutture e Bioimmagini, C.N.R. via Mezzocannone, 16, 80134 Napoli, Italyb DBSV – Dept. of Biotechnology and Life Sciences – Università degli Studi dell’Insubria, via J.H. Dunant 3, 21100 Varese, Italyc The Protein Factory, Centro Interuniversitario di Biotecnologie Proteiche, Politecnico di Milano, ICRM CNR Milano, Università degli studi dell’Insubria, Milano, Italyd Dipartimento di Scienze e Tecnologie Ambientali, Biologiche e Farmaceutiche, Seconda Università di Napoli via Vivaldi, 43, 81100 Caserta, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 November 2014Revised 30 January 2015Accepted 10 February 2015Available online 18 February 2015

Edited by Jesus Avila

Keywords:Alpha synucleinNMR spectroscopyCD analysesProtein–membranes interaction

A change in the conformational plasticity of a-Synuclein (a-Syn) is hypothesised to be a key step inthe pathogenic mechanism of Parkinson’s disease (PD). Here, we report the study of extracellular a-Syn interaction with whole cells and membranes isolated from the neuronal SH-SY5Y cells,exploiting NMR and CD spectroscopies. In addition, the crosslinking agent DSG was used to freezethe conformational and oligomeric state of a-Syn in the presence of cells. These data, in aquasi-physiological environment, confirm the protein monomeric state with a propensity to adopta transient alpha helical following interaction with biological membranes.� 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction

a-Synuclein (a-Syn) is a 140-residue protein that is highlyexpressed in the central nervous system and is the major con-stituent of Lewy bodies (LBs), the pathological hallmark of Parkin-son’s disease (PD) [1].

The protein can be subdivided in three main regions accordingto its primary sequence: (1) the N-terminal region (1–60) known tointeract with lipid vesicles [2], and including the well-conserved,imperfect KTKEGV repeats; (2) the hydrophobic NAC (non-amyloid–b component) region (aa 61–95) responsible for proteinaggregation [3] and (3) the negatively charged C-terminal region,spanning amino acids 96–140, which plays an important role incounteracting the aggregation [4].

a-Syn is mainly detected as a cytosolic protein, nonetheless aportion of it is recovered bound to membranes. In particular it issuggested that binding to membrane phospholipids controls a-Syn structure, physiology and pathogenesis [5]. It is well knownthat a-Syn has a preference to bind highly curved, negativelycharged membranes that incorporate synaptic vesicle lipids [6].

Following the loss of membrane curvature generated from theendocytosis of neurotransmitter, a-Syn is likely induced to dissoci-ate from the membrane into the cytosol [6,7].

a-Syn exists predominantly as disordered monomeric proteinprone to associate with negative charged small unilamellar vesicles(SUVs) in order to adopt essentially helical conformation in aqueoussolution [6,8]. The presence of a-Syn as a disordered monomericform has been widely reported in different cellular systems[1]. Indetail, as reported in Fusco et al. [9], the N-terminal alpha helix(residues 6–25) seems to act as an anchor to the membrane and isonly marginally affected by lipid composition while the region26–97 results to act as a membrane ‘‘sensor’’ modulating thestrength of the interactions in a lipid-specific manner. AvailableNMR/EPR structures of a-Syn on micelles are indicative of amonomeric horseshoe-like broken-helix conformation [10]. Othertechniques, on the other hand, suggest that a-Syn monomers adoptan extended helical structure in the membrane [10].

In addition, aSyn has long been thought to occur normally as anatively unfolded monomer of �14.5 kDa [11,12] defined as a hubprotein because involved in several interactions with many part-ners, representing a crucial node in different pathways. A debatehas recently begun over its oligomeric state [11] focusing the dis-cussion on the existence of a-Syn as a putative alpha helical tetra-mer as physiologically relevant protein structure [13,14] or as a

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mostly monomeric and disordered state [4]. In cell-NMR spec-troscopy has been used to observe directly the structure and thedynamics of this protein within Escherichia coli cells [15] revealingstill another time a disordered monomeric form as that present inaqueous solution.

Despite being a cytosolic protein, a small amount of a-Syn hasbeen detected in human plasma and cerebrospinal fluid [16]. Thisform, released by a non-classical exocytosis, seems to represent aphysiological event that could designate extracellular Syn as abiomarker for related diseases [17]. In this study, to get furtherinsight into a-Syn structure when bound to cellular membranes,circular dichroism, NMR spectroscopy and confocal microscopyhave been used to characterise a-Syn conformational behaviour,not only in the presence of intact neuronal cells SH-SY5Y, a cellularsystem widely used to study PD [18], but also of membranesobtained from the same cells. In addition, the oligomeric state ofa-Syn was monitored in the presence of cells by using thecrosslinking agent DSG.

2. Materials and methods

2.1. Expression and purification of a-Syn and TAT-a-Syn

Recombinant a-Syn and TAT-a-Syn were expressed and purifiedfrom periplasmic space content as reported in Caldinelli et al. [19].The pET5a-a-Syn plasmid was a gift of Prof. Bartels (Boston, Mas-sachusetts, United states of America). The recombinant proteinwas expressed and purified according to the procedure previouslyreported [20].

For NMR studies, cell cultures were growth in a minimum mediacontaining Na2HPO4 6 g/L, KH2PO4 3 g/L, NaCl 0.5 g/L and 15NH4Cl1 g/L completed adding MgSO4 1 mM, Thiamine hydrochloride1 mM, CaCl2 0.1 mM and Glucose 0.2% [21]. Protein expressionand purification protocols for 15N labelled samples have been per-formed as previously reported [19,22]. The purity of all the proteinswas evaluated by using 15% SDS–PAGE. The molecular mass wasanalysed by ESI-Quadrupole mass spectroscopy. For NMR experi-ments proteins were extensively dialyzed against PBS at pH 7.4.

2.2. Cell culture and membrane recovery

SH-SY5Y cells (ATCC, USA) were grown in DMEM supplementedwith 10% fetal bovin serum (FBS), 1% glutamine, 100 U/mL peni-cillin and 100 lg/mL streptomycin (Euroclone, MI, Italy), at 37 �Cin a humidified atmosphere with 5% CO2. The viability of the cellswas checked by Trypan blue reactive after each CD and NMRexperiment.

Membranes used in NMR experiments were isolated from SH-SY5Ycells. Cells were detached from the flask with trypsin andwashed twice with phosphate buffered saline. Then the cells weretransferred into homogenisation buffer containing PBS and homo-genised by means of a pellet pestle (Sigma). Particulate matter wasremoved by centrifuging at 2500 rpm for 15 min. The supernatantwas then centrifuged at 28000 rpm for 1 h at 4 �C. The pellet waswashed and centrifuged at 28000 rpm for 30 min at 4 �C. 200 llof PBS was added to the pellet, and the membrane was resuspend-ed by 20 passages through a 25 gauge needle.

2.3. Confocal microscopy analyses of a-Syn and TAT-a-Syn in presenceof SH-SY5Y cells

15 lM of a-Syn and TAT-a-Syn were labelled with Fluoresceinisothiocyanate (FITC, Sigma). The reaction was conducted in PBSpH 7.4 with a tenfold molar excess of FITC for 16 h at 4 �C in slowagitation. The excess of FITC was removed by extensive dialysis at4 �C in PBS. FITC-labelled-a-Syn and FITC-labelled-TAT-a-Syn

(15 lM) were incubated with 105 SH-SY5Y cells on a 35 mm glassbottom dishes (P35G-1.5-10-C Case, MatTek corporation) 16 h at37 �C in a humidified atmosphere with 5% CO2 [23]. Thereafter,the cells were washed in PBS and analysed with confocalmicroscopy. The confocal images were acquired using a Leica TCSSP5 Confocal Laser Scanning Microscopy (CLSM) (Leica Microsys-tems, Wetzlar, Germany). Leica Application Suite software wasused for confocal images processing.

2.4. Circular dichroism studies

CD spectra of a-Syn were recorded with a J-810 spectropolarime-ter equipped with a Peltier temperature control system (Model PTC-423-S, Jasco Europe, Cremella, LC, Italy). Far-ultraviolet (far-UV)measurements (195–260 nm) were carried out at 20 �C using a0.1 cm optical path length cell and a protein concentration of15 lM. CD spectra, recorded with a time constant of 4 s, a 2 nmbandwidth, and a scan rate of 10 nm min�1, were signal-averagedover at last three scans. The baseline was corrected by subtractingthe complete buffer spectrum.

CD spectra in the presence of SH-SY5Y cells were registeredusing 3 � 105 cells in PBS buffer. In this case the baseline was cor-rected by subtracting the spectrum of the cells alone at the sametime of incubation. All CD experiments were performed in thesame experimental conditions using the recombinant a-Syn with-out any tag.

Further CD spectra were performed after 16 h of incubation atroom temperature in the presence of cells and adding a 25-foldmolar excess of DSG (Di(N-succinimidyl) glutarate, Sigma Aldrich)[24]. The reaction mixture was incubated at room temperature for30 min. Finally the reaction was quenched for 15 min using 25 mMTris Base. The samples were centrifuged at 1200 rpm for 5 min toremove cells and were loaded onto a Superdex 200 gel filtrationcolumn (HiLoad 10/30; GE Healthcare Bio-Sciences AB, Uppsala,Sweden) pre-equilibrated with PBS (pH 7.4). The peak, eluted at�14 ml, was recovered and analysed by CD.

MREs (deg dmol�1 cm�2) were calculated using the equationspreviously reported [25].

2.5. NMR sample preparation and analyses

NMR data were collected on a Varian INOVA 600-MHz NMRspectrometer equipped with a cold-probe and at room tem-perature. 2D [1H–15N] heteronuclear single quantum coherence(HSQC) correlation spectra were acquired by using a fast versionof sensitivity enhanced HSQC pulse-sequence. Data were collectedusing a spectral width of 5400 Hz with 1024 complex points inthe direct dimension and a spectral width of 1600-Hz with 64 com-plex points in the nitrogen dimension. Data were processed usingSparky (Goddard, T.D.; Kneller, D.G., SPARKY 3. University of California:San Francisco.) and analysed using Neasy, a tool of CARA soft-ware.[26] Intensity ratios were computed as I0 � I/I0, where I0 isthe signal intensity in the reference spectra and I is the intensityin the presence of whole cells or of isolated membranes.

The 15N relaxation parameters R1, R2 and [1H–15N] NOE weredetermined at 600 MHz. 15N R1 data were collected using relax-ation delay times in the following order (in seconds): 0.01, 0.05,0.09, 0.36, 0.73, 1.01, 1.31, 1.80. 15N R2 data were measured usinga pulse sequence employing a CPMG (Carr Purcell Meiboom Gill)pulse train [27] and the relaxation delays (in seconds) were 0.01,0.03, 0.05, 0.07, 0.09, 0.13, 0.17, 0.21, 0.25. {1H–15N} steady stateNOE values are reported as the ratio of peak heights in paired spec-tra collected with and without an initial period (4s) of protonsaturation during the 5-s recycle delay.

NMR samples were prepared in a final volume of 500uL. Fornatural membrane interaction analyses 15N-labelled a-Syn

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(150 lM) was mixed with membrane derived from 80 � 106 SH-SY5Y cells as previously reported [22]. Further experiments wereconducted on 15N-labelled a-Syn (150 lM) after incubation of16 h at room temperature in the presence of 3 � 106 SH-SY5Yintact cells.

3. Results

3.1. Expression and purification of a-Syn and TAT-a-Syn

a-Syn and TAT-a-Syn were expressed and purified as elsewheredescribed [19]. The periplasmic contents were loaded onto a Ni–NTA affinity column (GE Healthcare, USA) and the recombinantproteins were eluted increasing imidazole concentration(300 mM). The proteins was obtained in an homogenous form asshown by SDS–PAGE and the yield of the proteins was respectively15 mg/L for a-Syn and 10 mg/L for TAT-a-Syn.

For further experiments, proteins were extensively dialyzedagainst PBS buffer (pH 7.4).

3.2. a-Syn characterisation in the presence of intact cells

a-Syn CD spectrum is typical of an intrinsically disordered pro-tein with a negative peak at 198 nm (Fig. 1). 15 lM of a-Syn wasincubated with 3 � 105 SH-SY5Y cells in PBS buffer at pH 7.4 and

Fig. 1. (A) Far-UV CD spectra of a-Syn in the presence of whole SH-SY5Y cells at differealone at the same time of incubation. (B) a-helix relative percentage. Data are the mean

CD spectra were recorded at different time of incubation (Fig. 1Aand B). Even if a partial structuring is observed already after onehour of incubation, an increase up to about 15% in alpha helix,characterised by two negative bands at 208 and 222 nm in theCD spectrum, occurs after 16 h incubation. The same results wereobtained also with the untagged protein: this demonstrating thatthe His-tag at the N-terminus did not affect the conformationalmodification in the presence of the cells. Confocal microscopyresults, in which FITC-labelled-a-Syn and FITC-labelled-TAT-a-Syn were incubated with SH-SY5Y cells (Fig. 2), demonstrate thatin our experimental conditions a-Syn (lacking the TAT transportsequence) does not cross the cellular membrane to a significantextent, i.e. an uptake is apparent for the TAT-a-Syn only followinga 16 h of incubation with SH-SY5Y cells. Similarly, CD data of a-Synincubated with SH-SY5Y cells are representative of a protein pre-sent on the surface of cytoplasmic membrane (Fig. 3). In additiona-Syn was incubated with SH-SY5Y cells and after 16 h of incuba-tion at RT a specific lysine crosslinking, di(N-succinimidyl) glu-tarate (DSG), was added. After removing cells by centrifugation,samples were loaded onto a Superdex 200 gel filtration columnpre-equilibrated with PBS (pH 7.4) and the peak with a retentiontime of �14 mL was collected and analysed by CD spectroscopy.As shown in Fig. 3 a-Syn, after incubation with cells and additionof DSG, retains an alpha helical conformation of about 15%. Never-theless a-Syn in presence of SH-SY5Y, without the crosslinking

nt time of incubation. Data were obtained by subtracting the spectrum of the cellsof three different measurements.

Fig. 2. Uptake of SH-SY5Y cells incubated at 37 �C in a humidified atmosphere with 5% CO2 with TAT-a-Syn (A–C) and a-Syn (D–F) for 16 h. Fluorescence (A and D),transmitted light (B and E) and merge (C and F) images. Magnification bar: 20 lm.

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agent, partially loses its secondary structure displaying no morethan 5% a-helix (Fig. 3A and B). This structuration is underlinedby the gel permeation profile showing a more compact form fora-Syn when compared with the protein no treated with DSG thatpresents a retention time of 15.1 ml versus 14.2 mL (Fig. 3C).

In addition, SDS–PAGE analysis of a-Syn samples treated withDSG in the presence or not of cells indicated a role of the crosslink-ing in stabilizing the monomeric form of a-Syn only due to theinteraction with SH-SY5Y membranes (Fig. 3D).

3.3. NMR results

The structural switch of a-Syn from the free to membranebound state has been examined by NMR spectroscopy in presenceof whole living neuronal cells and in presence of isolated naturalmembranes.

Under these conditions, the a-Syn backbone resonances wereidentified and assigned by comparison with spectra of the proteinin aqueous solution. Fig. S1 shows a selected region of the 2D[1H–15N] HSQC spectra under different experimental conditions;intensities of several resonances are strongly reduced both in thepresence of whole cells (Fig. S1A) and isolated membranes(Fig. S1B), indicating a-syn interaction to the cellular membranes.

Indeed, in the presence of whole cells the average a-Syn amidesignal intensity (Fig. 4A) displays significant broadening effectsover most of the sequence, particularly in the N-terminal domain(residues 1–100) of the protein. Within the N-terminal lipid bind-ing region of the protein three distinct regions, encompassingamino acids from D2 to K12, from S42 to V55 and from V71 toK80, undergo the highest signal attenuations. A much smallerbroadening effect is observed for residues in the C-terminal region,except for those included between S129 and Y136.

To further investigate the a-Syn transition to membranebound state, the NMR experiments have been performed by usingisolated cellular membranes. As showed in Fig. 4B, the regions

most affected upon interaction with the isolated cellular mem-branes correspond to those identified in the presence of intactcells.

Furthermore, a-Syn backbone dynamics was explored in pres-ence of isolated membranes, by measuring 15N relaxation rate con-stants R1 and R2 and heteronuclear {1H–15N} steady state NOEs.The relaxation data, shown in Fig. S2, indicate that a-Syn backbonedynamics is quite uniformly rigid throughout the N-terminal lipid-binding domain of the protein. On the other hand, a high degree offlexibility of the C-terminal tail is clearly evidenced by both{1H–15N} NOE and R2 and to a lesser extent by R1 values. Three dis-tinct regions, encompassing M5-A11, G47-A56, T72-V77, exhibitsignificant R2 increases.

4. Discussion

a-Syn is a versatile protein, presenting different states i.e.cytosolic, membrane-bound and aggregated forms [9]. It has beenshown to be an intrinsically disordered protein [12] in differentconditions, nonetheless in other environments further plausibleoligomeric structured form have been hypothesised [13]. Themembrane-associated state of a-Syn, in equilibrium with itscytosolic form, has been widely studied because it results to playa key role in the physiology and in PD insurgence [28]. Literaturedata strongly suggest that the bound state is regulated by differentproperties such as the curvature and charge but it is not affected bythe compositions [9,29].

In the last years it is emerging a relevant role also for the extra-cellular form of a-Syn as demonstrated by the proinflammatoryresponses of glial cells [30,31] following the treatment with extra-cellular a-Syn. Therefore, extracellular a-Syn may have significantimplications in pathogenesis resulting in all three major patho-logical features of neurodegenerative disease, namely proteinaggregates, neurodegeneration, and neuroinflammation [17]. Thus,the release of a-Syn is a physiological event, which also contributes

Fig. 3. (A) Far-UV CD spectra of purified a-Syn after 16 h incubation at room temperature in the presence of SH-SY5Y cells and the addition of DSG. (B) a-helix relativepercentage. Data are the mean of three different measurements. (C) Superdex S200 10/30 analyses of a-Syn incubated with SH-SY5Y cells and treated with DSG, after removalof the cells. (D) SDS–PAGE analyses of a-Syn incubated with DSG in the presence of SH-SY5Y cells. Lane (1) a-Syn; lane (2) molecular weight marker; lane (3) a-Syn treatedwith DSG; lane (4) supernatant of SH-SY5Y treated with DSG; lane (5) a-Syn incubated 16 h with SH-SY5Y cells and treated with DSG, after removal of the cells.

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to the spreading of Syn-derived pathology, indicating extracellularSyn as a biomarker for related diseases [17].

Very recently an exchange of a-Syn between the brain andperipheral tissues has been demonstrated being able to cross theblood–brain-barrier (BBB). In this context this study focuses onthe conformational plasticity of the extracellular a-Syn form inthe presence of neuronal whole cells and their isolated mem-branes. SH-SY5Y cells [18] are used to analyse the propensity ofa-Syn to increase its a-helical content. Our data well corroboratedwith the observation, obtained both using whole cells and isolatedmembranes, that lipid bilayers determine an a-Syn’s conforma-tional equilibrium towards structural states preferentially in a-he-lices, as clearly indicated by CD results. Furthermore, the NMRcharacterisation identifies the N-terminal lipid binding region ofthe protein as that involved in the interaction with membranephospholipids. In particular, regions encompassing amino acidsfrom D2 to K12, from S42 to V55 and from V71 to K80 are indicatedas major players in the membrane recognition, likely folded ashelices upon membrane interaction. Accordingly, the three a-synregions above identified are characterised by a more rigid back-bone dynamics within the N-terminal region that is globally muchless flexible than a-syn C-terminus.

Similar results have been observed for a-Syn in the presence ofmodel membrane systems [32,33]. In the presence of SH-SY5Y cells,the CD-estimated percentage of alpha-helix results to be smallerwhether compared to that determined in other systems (15% versus60%) [29], but it is in agreement with the extent of a-syn residuesmostly involved in membrane interaction, as determined by the

NMR chemical shift perturbation data. Such differences in helicalcontent of a-syn upon membrane interaction could be ascribed tothe heterogeneous composition of cellular membranes respect toartificial model membranes used [5,20]. In addition, the use of thecrosslinking agent DSG has allowed to freeze the conformationaland oligomeric state of a-Syn in the presence of cells, further con-firming the protein monomeric state with a propensity to adoptan alpha helical conformation as showed in CD spectra. This confor-mation is lost after the removal of the cells suggesting that a-Synassumes an alpha helical conformation to interact with SH-SY5Ycells and it returns to be a disordered protein in its membrane freestate. a-Syn binds to the cytoplasmic membrane in its unstructuredconformation and its content in a-helix increases following interac-tion with the phospholipids.

Overall, NMR characterisation of a-syn binding to whole cells andto isolated membranes showed similar results that can be easily inte-grated. Whole cells represent a simple and suitable handle systembut they bear some disadvantages such as being labile and lesshomogeneous when compared to the isolated membranes. At thesame time, isolated membranes, extracted by the same cells, allowlonger experimental investigations, such NMR relaxation analysis,and hamper possible cell internalisation of membrane ligands andtherefore can provide further structural details in defining mem-brane binding modes of extracellular molecular partners.

Altogether our results corroborate the hypothesis that, as a typi-cal IDP, upon binding with membranes, a-Syn retains a certaindegree of flexibility [34] and does not assume considerable amountsof secondary structures.

Fig. 4. (A) Intensity ratios for all a-Syn residues (I0 � I/I0 P 0.45 ± 0.21) extracted from 2D [15N, 1H] HSQC experiments performed in SH-SY5Y suspension. (B) Intensity ratiosfor all a-Syn residues (I0 � I/I0 P 0.40 ± 0.18) extracted from 2D [15N, 1H] HSQC experiments performed in the presence of isolated natural membranes.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.febslet.2015.02.012.

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