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Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes
Accepted Manuscript Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes
Exiquio Maldonado Vidaurri, Abelardo Chavez-Montes, Marsela Garza Tapia, Rocio Castro-Rios, Azucena Gonzalez-Horta PII: DOI: Reference:
S0141-8130(18)30409-4 doi:10.1016/j.ijbiomac.2018.08.049 BIOMAC 10293
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23 January 2018 8 August 2018 9 August 2018
Please cite this article as: Exiquio Maldonado Vidaurri, Abelardo Chavez-Montes, Marsela Garza Tapia, Rocio Castro-Rios, Azucena Gonzalez-Horta , Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes. Biomac (2018), doi:10.1016/j.ijbiomac.2018.08.049
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ACCEPTED MANUSCRIPT DIFFERENTIAL INTERACTION OF -SYNUCLEIN N-TERMINAL SEGMENT WITH MITOCHONDRIAL MODEL MEMBRANES
Exiquio Maldonado Vidaurri1, Abelardo Chavez-Montes2, Marsela Garza Tapia3, Rocio Castro-
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Rios3, Azucena Gonzalez-Horta1*
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Laboratory of Genomic Science, Faculty of Biological Sciences, Universidad Autonoma de Nuevo
Leon, 66451 San Nicolas de los Garza, N.L. Mexico 2
66451 San Nicolas de los Garza, N.L. Mexico 3
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Department of Chemistry, Faculty of Biological Sciences, Universidad Autonoma de Nuevo Leon,
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Department of Analytical Chemistry, Faculty of Medicine, Universidad Autonoma de Nuevo Leon,
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66451 San Nicolas de los Garza, N.L. Mexico
Azucena Gonzalez-Horta
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*Corresponding author:
Laboratory of Genomic Science
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Faculty of Biological Science
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Universidad Autonoma de Nuevo Leon
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66451 Monterrey, N.L. MEXICO Email: [email protected] Phone: +52 8183294110
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ACCEPTED MANUSCRIPT ABSTRACT Alpha-synuclein (-syn) is an intrinsically-disordered protein that has been associated with Parkinson’s disease through its deposition in an amyloid fibril form within Lewy Body. Several lines of evidence suggest that the physical association of -syn with the mitochondrial membranes may cause membrane damage and mitochondrial dysfunction, playing an important role in disease
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progression. Although there is strong evidence that the N-terminus part of -syn is essential for
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membrane affinity, cooperative formation of helical domains and regulation of mitochondria membrane permeability, the amino acids involve in this membrane binding is still controversial.
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Fluorescence spectroscopy, circular dichroism and Langmuir monolayer technique were used to elucidate this recognition process of mitochondrial membrane system by synthetic peptides derived
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from -syn N-terminal segment. The results obtained in this work show that the first 15 amino acid
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of the -syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16-30 residues interacts only with the
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phospholipid polar headgroup, confirming that the binding affinity of the N-terminus is nonuniform.
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Key words: -synuclein; mitochondria; monolayer technique
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ACCEPTED MANUSCRIPT 1. INTRODUCTION Parkinson’s disease (PD) is among the most common neurodegenerative diseases, and is characterized by the loss of dopaminergic neurons in the substantia nigra of the brain, which results in impaired motor functions, displayed as compromised postural reflexes, muscular rigidity and bradykinesia in PD patients [1,2]. In most PD cases, the main pathological hallmark is the presence
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in the brain of Lewy bodies and Lewy neurites which are the aggregated form (amyloid fibrils) of
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-synuclein (-syn) a highly conserved presynaptic 140 amino acid protein [3,4]. Native -syn is a small protein, 14kDa, in which three regions can be distinguished: The N-terminal region consisting
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of five degenerate 11-mer repeats of KTKEGV of overall positive charge, a highly hydrophobic central stretch of 12 amino acids referred as non-amyloid component (NAC) and the C-terminal 40
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residues which are highly enriched in negatively charged amino acid [5] (Figure 1). The N-terminal domain is responsible for lipid binding interactions, the NAC region for oligomerization and the C-
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terminal domain possibly presents a chaperone-like activity [6,7]. Although the exact function of
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-syn remains unknown, four principal roles have been proposed including (i) a vesicle fusion and neurotransmitter release, (ii) an intracellular trafficking of lipids, (iii) regulation of cell death [8]
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(iv) and alterations of mitochondrial biology [9-11]. The interaction of -syn with mitochondria is
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thought to contribute to dopaminergic neuronal death in PD. This interaction is mediated by the N terminus of -syn (residues 1-60), whereas the negatively charged C terminus remains unfolded and
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can potentially interact with other proteins [12]. It has been reported that the middle of the Nterminal region (residues 10-30) which contains the KAKEGVVAAAE repeats is involved in mitochondrial binding [13], which induce morphological changes to mitochondria that lead to dysfunction [14]. However, it was also reported that deletion of just residues 2-11 suppress mitochondrial binding [15]. In this work, we have used different synthetic peptides and biophysical techniques to further explore the interaction of the N-terminal segment of -syn with mitochondrial membranes, paying special attention to the differential behavior presented for the first 15 residues
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ACCEPTED MANUSCRIPT and 16-30 residues of -syn N-terminal region in this interaction. The results advance our understanding of -synuclein’s interaction with mitochondrial membranes.
2. MATERIALS AND METHODS Materials
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2.1.
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DOPC (1,2-oleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-oleoyl-sn-glycero-3-
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phosphoethanolamine), and CL (cardiolipin), specified as 99% pure, were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. Recombinant human -
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synuclein and synthetic peptides with a purity 95% were purchased from Sigma-Aldrich (Saint
2.2.
Methods
2.2.1. Peptide synthesis and purification
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Louis, MO).
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The peptides studied in this work was synthesized by Fmoc Chemistry, purified by HPLC and
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analyzed by mass spectrometry by Sigma-Aldrich (Supplementary Fig S1-S3). The 15-residue peptides, named P1 (NH2-WMDVFMKGLSKAKEGV-CONH2), P3 (NH2-
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WVAAEKTKQGVAEAAG-CONH2) were designed taking as a model the sequence of the first 30
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residues of the N-terminal segment of human -syn. We include a tryptophan residue in the native sequence of P1 and P3 with the purpose of using it as an intrinsic probe to characterize structure and
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lipid-peptide interactions by fluorescence spectroscopy. The C-terminal carboxyl of the peptides was amidated to better mimic the interactions occurring at that region of native -synuclein. We also synthetized a peptide corresponding to the complete N-terminal segment of human -synuclein named N-term, with the modification of tryptophan at 39-position instead of tyrosine. These fragments were selected to cover sequence domains that have been previously shown to be critical for the regulation of mitochondrial membrane interaction (Figure 1C).
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ACCEPTED MANUSCRIPT 2.2.2. Lipid/peptide samples Lipid/peptide samples were prepared by different methods depending on the experiment. To mimic the mitochondrial inner membrane (MMI), we used a lipid composition that contains DOPC:DOPE:CL in a molar ratio 45:28:22 [16], the mixture was prepared in chloroform/methanol (2:1 v/v) at [10 mg/mL] final concentration, then the solvent was removed by drying under nitrogen
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flow and then 2 h in speed vac. For the circular dichroism experiments, lipid/peptide suspension
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were prepared by hydrating dry lipid/peptide films for 2 h in 5mM Hepes buffer at 25ºC in a termomixer with cycles (2 min vortex 1400 rpm every 10 min) and then sonicated in a Branson UP
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200S tip sonifier to obtain small unilamellar vesicles (SUVs). To prepare the samples for the binding experiments, large unilamellar vesicles (LUVs) were prepared by extrusion of the lipid
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suspension, equilibrated at 25ºC, through 0.1 m (pore diameter) polycarbonate membranes in a mini-extruder (Avanti Polar Lipids, Alabaster, AL USA). To analyze quenching of peptide
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tryptophan fluorescence by brominated lipids, liposomes were prepared containing different weight
(mol/mol).
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2.2.3. Circular dichroism
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percentages of Br(6,7)-PC or Br(11,12)-PC, with or without peptide, at peptide/lipid ratio 1:45
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Far-UV circular dichroism (CD) spectra were recorded in a J-1100 spectropolarimeter (JASCO, Easton, MD) equipped with a Xenon lamp and a Koolance Peltier-type holder for temperature
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control. All spectra were recorded in a 0.5 mL thermostated quartz cell with an optical path length of 0.1 cm. Each spectrum was registered over the 190-240 nm at a scan rate of 50 nm/min and represents an average of 3 scans with a full-scale sensitivity of 50 mdeg. The samples contained 10 M of peptide in buffer Hepes 5 mM pH 7.4 and 2 mM of SUVs. Corresponding buffer scans were subtracted from sample scans before analysis and presentation of the data. Helix content of peptides was assumed to be directly proportional to mean residue ellipticity (MRE) at 222 nm due at this wavelength, the contribution of random coil structure is relatively small [17]. At 222 nm, the -
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ACCEPTED MANUSCRIPT helical content was calculated using the formula previously reported [18]: % helicity = [222 - coil / helix - coil] 100 where helix = -4000 x (1 - 2.5/n) + 100 x ; and coil= (640 – 45) where 222 is the measured mean residue ellipticity at 222nm, helix and coil are mean residue ellipticities at 222 nm of idealized a-helix and random coil proteins, n is the number of amino acids for each peptide and
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is the temperature (ºC).
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2.2.4. Intrinsic fluorescence
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Tryptophan fluorescence emission spectra of peptides were recorded using 280 nm as the excitation wavelength in a LS-45 spectrofluorometer (Perkin-Elmer Inc.). Emission spectra were registered at
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25ºC from 300 to 500 nm with 1 cm cells using a scanning speed of 1 nm/s. The slit widths were 10 nm for the excitation and emission beams.
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2.2.5. Quenching of Trp by Brominated Phosphatidylcholines
Collisional quenching of Trp by brominated phospholipids was introduced to assess the localization
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of this residue in bilayers [19]. Liposomes were prepared containing different weight percentages
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of Br(6,7)-PC or Br(11,12)-PC, with or without peptide, at peptide/lipid ratio 1:45 (mol/mol). After 30 min at 25ºC, emission spectra were recorded, averaging 3 spectra. The differences in the quenching
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of Trp fluorescence by Br(6,7)-PC and Br(11,12)-PC were used to calculate the probability for location
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of the fluorophore in the membrane using the parallax method [20]. The depth of the Trp residue was calculated as follows: Zcf = Lc1 + [(-ln(F1/F2)/C – L21]/2L21 where Zcf represents the distance
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of the fluorophore from the center of the bilayer, Lc1 is the distance of the shallow quencher from the center of the bilayer, L21 is the distance between the shallow and deep quencher, F1 is the fluorescence intensity in the presence of the shallow quencher, F2 is the fluorescence intensity in the presence of the deep quencher, and C is the concentration of quencher in molecules /Å2. 2.2.6. Peptides interfacial adsorption Interfacial adsorption of each peptide was assayed using a Stainless-steel trough low volume with a fully automated microtensiometer (TROUGH SX, Kibron Inc. Helsinki, Finland). The
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ACCEPTED MANUSCRIPT microbalance was filled with 1.3 mL of Tris 5 mM pH 7.4 containing 100 mM NaCl, thermostated at 25ºC and subjected to continuous stirring. After the injection of a small volume of methanolic peptide solution into the subphase, changes in surface pressure were monitored over time (t isotherms). Injections of equivalent volumes of pure methanol did not produce any detectable
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change in surface pressure.
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2.2.7. Insertion of peptides into preformed monolayers
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Insertion of -syn peptides into MMI monolayers was followed using the same microbalance by monitoring changes in surface pressure () over time after peptide injection into the subphase.
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Monomolecular films of the lipid (0.1 mg/mL chloroform: methanol 3:1 v/v) were spread on top of buffer Tris 5 mM pH 7.4 NaCl 100 mM. After 5 min for solvent evaporation, 10 L of the protein
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or the synthetic peptides solution, prepared in methanol at [1 mg/mL] final concentration, were injected in the subphase (1.3 mL) and pressure increases produced were continuously recorded as a
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function of time. The surface pressure at the time of injection is indicated in the figure legends.
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Control experiments were run in parallel to ensure that the injection of 10 L of methanol in which protein and peptides were dissolved did not induce by itself any surface pressure change. The data
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were analyzed with the FilmWareX 3.57 program (Kibron Inc). The accuracy of the system under
2.2.8. Statistics
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our experimental conditions was 0.25 mN/m for surface pressure.
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Unless otherwise indicated, results have been presented as representative experiments after repeated examination of three different samples from at least two different batches of peptide.
3. RESULTS The secondary structure of -synuclein peptides was analyzed by CD spectroscopy. Figure 2 shows the far-UV CD spectra of the three synthetic peptides (N-term, P1, P3) containing the amino-acid sequence of the N-terminal half of -synuclein with the sequence indicated in Fig. 1. In an aqueous
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ACCEPTED MANUSCRIPT solution, the three peptides adopt a random coil conformation, revealed by the single minimum at 200 nm. Upon binding membranes mimicking mitochondrial lipid composition, the peptides present an -helical conformation with the characteristic double minima at 208 and 222 nm in the CD spectrum. The -helical content of the peptides upon lipid binding was estimated from the mean residue ellipticity (MRE) at 222 nm as explained in the Materials and Methods. Binding of N-term
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peptide resulted in an -helical content of 63%, P1 and P3 peptides adopted a comparable amount
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of -helix (74% and 73%, respectively). Therefore, the similarity of the CD spectra suggests that all peptides were fully bound to the SUVs used and in a predominantly -helical conformation.
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The fluorescence emission spectra of -synuclein N-terminal peptides in buffer and in the presence of different amounts of DOPC/DOPE/CL (45:28:22 molar) liposomes is shown in Figure 3A. In
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buffer, all peptides show the peak of tryptophan at 353 nm. For N-term and P1 peptides in going
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from water to liposomes, fluorescence intensity slightly increases and undergoes a blue shift from 353 nm to 330 nm, as the lipid to protein molar ratio increases, which implies that in the presence
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of liposomes, the tryptophan residue of both peptides moves to a more hydrophobic environment.
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P3 peptide had a blue shift of 13 nm (Fig. 3B) with a reduction in peak fluorescence intensity therefore could be due to a relatively shallow depth in the liposomes. To analyze peptide behavior
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in terms of lipid-peptide binding in more detail, we evaluated quenching of peptide tryptophan fluorescence by brominated lipids. Figure 4 shows fluorescence emission spectra of P1 and P3 in
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PC vesicles containing different weight percentages of brominated PC bearing bromines either at the (6,7) or at the (11,12) positions of its acyl chains. Increasing amounts of both brominated lipid probes produce progressive quenching of peptide fluorescence as a result of peptide association with membranes. Quantitative comparison of quenching by the two brominated lipids allows estimation of the relative position of the peptide tryptophan in the bilayers, provided that the location in the membrane of the two quenching groups is known. It can be observed that the extent of quenching of P1 fluorescence by the Br(11,12) isomer is clearly higher than that of quenching by
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ACCEPTED MANUSCRIPT the Br(6,7) isomer (Fig. 4 A, B). While the extent of quenching of P3 fluorescence was clearly higher by Br(6,7) isomer (Fig. 4 C, D). Applying the parallax analysis to these data, it was possible to estimate that the P1 peptide tryptophan is located in a region 10 1Å from the center of the bilayer while the tryptophan residue of P3 resides at a distance of 15 1Å from the center of the
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bilayer. These results suggest a deeper bilayer penetration for P1 and an association only with the
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membrane surface for P3 peptide. All these results show the ability of the peptides used in this work
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to interact and insert in DOPC/DOPE/CL bilayers but with a different extent, adopting an -helical conformation. It has been suggested that -syn in the presence of vesicles adopts a two-helix
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antiparallel arrangement around small unilamellar vesicles [21] and becomes even more extended when it is in contact with less curved lipid bilayers [22], also it was reported that -synuclein is in
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an -helical conformation with its helical axis parallel to the air-water interface [23] to analyze the
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interfacial conformation of these peptides, we used the Langmuir-balance technique as a simple model to mimic the interface of the mitochondrial membrane considering that the physicochemical
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properties of the thin aqueous layer that exists at the interface of the cell membrane are different to the bulk aqueous phase in the cytoplasm [24,25]. The compression isotherms of pure peptides films
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can provide valuable information on the conformational tendency of each peptide as a function of
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surface pressure. Two important data can be obtained from these graphs. First, the area per residue where the compression begins to induce an increase in surface pressure, gives an idea of the space
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occupied by each peptide molecule in the most extended orientation at the air-liquid interface. Moreover, the maximum pressure at which the peptide film can be compressed indicates the degree of stability of the peptide in the more compressed conformation. Figure 5 shows the surface behavior of pure peptide monolayers subjected to compression. Both P1 and P3 peptides are able to form stable monolayers on saline subphase, due their intrinsic amphipathic character. The lift-off area for P1 peptide was 350 A2 per molecule (Fig. 5A), while the one for P3 peptide was around 40 A2 per molecule (Fig. 5B). It therefore seems that P3 could adopt a more perpendicular
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ACCEPTED MANUSCRIPT conformation. Monolayers of the P3 peptide collapse at approximately 15 mN/m, while P1 monolayers could sustain higher surface pressures around 30 mN/m. The isotherm of the peptide corresponding to the N-terminal segment of -syn presents a plateau around 15 mN/m before reaching higher surface pressures (Fig. 6). This plateau indicates that the peptide undergoes a
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change in interfacial conformation. It is very probable that the plateau is due to the expulsion from
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the interface of the segment corresponding to 16-30 residues which, as observed in the isotherm of P3 peptide, cannot withstand pressures higher than 15mN/m. This result suggests that only the first
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15 amino acids of -syn remain associated to the interface at high surface pressure, those enriched
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in cardiolipin as would be the contact sites or the mitochondrial crests. The current model for the structure and disposition of -syn in phospholipid monolayers assumes that the N-terminal segment
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of the protein is responsible for the association with phospholipids [27, 28] and also Shen et al. (2014) have shown that the N-terminus of -syn is essential for the regulation of mitochondrial
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membrane permeability so, to investigated the ability of each peptide to interact with the
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mitochondrial inner membrane (MMI), we formed monolayers composed of (DOPC:DOPE:CL 45:28:22 molar ratio) at the air-water interface and then injected the peptides of -syn (4 M) in
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the buffered subphase. P1 and P3 peptides were able to perturb and insert into MMI monolayer
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prepared at different initial surface pressures (i). Figure 7A illustrates that injection of P1 into the subphase underneath the DOPC:DOPE:CL monolayer promotes an instantaneous pressure increase
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() as a consequence of the association of the peptide with the interfacial film. Very similar behavior was observed for P3 peptide (Fig. 7B) however, the interface association of P1 peptide with the monolayer film, produce higher increase in surface pressure than P3 peptide. These could be due the absence of one K residue on P3, suggesting that in the association and stabilization of the peptide with the interfacial films, an electrostatic component is participating. Devi et al. (2008), reported similar results showing that the N-terminal 32-amino acid region of -syn, which contains evenly spaced positive residues, is critical for mitochondrial targeting. In Figure 8 has been plotted
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ACCEPTED MANUSCRIPT the increase in surface pressure () versus the initial surface pressure of the preexisting phospholipid monolayer (i) after injection of a given amount of each peptide. For both peptides, the value always decreased as i increase, as would be expected if lipid molecules are densely packed, preventing peptide insertion. From these plots one can calculate the critical insertion
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pressure (c), which represents the maximum surface pressure at which the peptides are still able to
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insert. This parameter depends on the monolayer composition as well as on the relative affinity of
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the peptides to associate with the interface film [29]. It is generally assumed that the lateral packing in a lipid bilayer can be roughly mimicked by a monolayer compressed to 30 mN/m. Therefore,
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molecules with c values higher than 30mN/m are generally considered as competent to interact with and insert into lipid membranes [30]. The critical pressures calculated for the insertion of P1 in
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MMI monolayer was around 42 mN/m, while the c for P3 was around 36 mN/m so both peptides
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exceed the physiologic membrane pressure. However, these differential c values demonstrated
DISCUSSION
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4.
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again that -syn fragment comprising 1-15 residues has deeper association with the monolayer.
Binding of -synuclein with biomembranes has been extensively studied. Typically, experiments
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make use of planar bilayers, liposome vesicles and micelles containing physiologically relevant phospholipids [31]. Although several distinct binding modes have been shown for -syn, there is a
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consensus that upon binding to a membrane, -syn forms an amphipathic -helical structure involving the N-terminal and NAC regions [32]. As monitored with CD spectroscopy, lipid binding induced a coil-to-helix transition wherein the -helical content of -synuclein increased from 2 to 71% [33], which is comparable to the observations reported in the present study for the -syn Nterminal peptides. Membrane binding of monomeric -syn appears to involve two steps: first, anchoring by the N-terminal residues 3-25, followed by a coil-to-helix transition of residues 26-97 which act as a membrane sensor and which determine the affinity of -syn binding; the C-terminal 11
ACCEPTED MANUSCRIPT domain exhibits only weak interactions with the membrane surface [34]. The results obtained in this work show that the first 15 amino acid of the -syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16-30 residues interacts only with the phospholipid polar headgroup, suggesting that the mitochondrial
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targeting signal proposed by Devi et al. (2008) and Zigoneanu et al. (2012), relies only to the first
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15 amino acids. These results agree with the observations made by Robotta et al. (2012), which revealed that the binding affinity of the N-terminus is nonuniform. Our results not only imply that
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the binding of -syn to inner mitochondrial membranes is initiated in the N-terminal part of -syn
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but also suggest that the binding affinity of regions close to the N-terminus is stronger than that of regions distal from the N-terminus sequence. For many years cardiolipin (CL) was assumed to be
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associated exclusively with the mitochondrial inner membrane where, as measured in bovine heart mitochondria, it represents 25% of the total phospholipids. More recently CL has also been
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identified in the mitochondrial outer (4%) especially at the contact sites connecting the outer
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membrane with the inner one due the non-bilayer hexagonal structure of this lipid [35], so it is possible that -syn mitochondrial membrane association at contact sites could be modulated by the
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first fifteen residues of the N-terminal segment of the protein, responding to hydrophobic and
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electrostatic forces as shown by the ability of P1 peptide to remain associated to the interface at higher lateral pressures like those caused by cardiolipin, since this lipid increases lateral interaction
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between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentration [36]. A recent report demonstrates that cardiolipin translocate to the outer mitochondrial membrane in response to the presence of -syn on mitochondrial membranes [37], so it is possible that while the first 15 amino acids allow the anchoring of the protein to the contact sites, the following residues of the N-terminal segment participate in cardiolipin translocation although future experiments are required to analyze the reorganization of mitochondrial phospholipids.
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5. CONCLUSION Taken together with literature data, our results show that the affinity of the N-terminal segment of -syn with mitochondria is nonuniform. Apparently, the segment corresponding to the first 15
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amino acids of -syn has deeper bilayer penetration on liposomes that mimic inner mitochondrial
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membrane and remains associated to the interface at high surface pressure. Although, our current
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understanding of the role that synuclein and mitochondria play in PD pathogenesis is limited, it is possible that this segment of the protein be the first fraction that interacts with mitochondria and
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disrupt its normal morphology and function. Future studies will examine morphological changes or
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any alteration in the cardiolipin levels originated by this lipid-peptide interaction.
ACKNOWLEDGMENT
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This research was financially supported by the Consejo Nacional de Ciencia y Tecnologia
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(CONACYT) Project No. C.B. 2014 236834.
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CONFLICTS OF INTEREST
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None declared.
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ACCEPTED MANUSCRIPT [23] C. Wang, N. Shah, G. Thakur, F. Zhou, M. R.M. Leblanc, a-synuclein in a-helical conformation at air-water
interface:
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accumulation/aggregation, Chem. Commun 46 (2010) 6702-6704. [24] A. Chattopadhyay, E. London, Parallax Method for Direct Measurement of Membrane Penetration Depth Utilizing Fluorescence Quenching by Spin-Labeled Phospholipids, Biochemistry 26 (1987)
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[26] A. Chaari, H. Horchani, F. Frikha, R. Verger, Y. Gargouri, M. Ladjimi, Surface behavior of asynuclein and its interaction with phospholipids using the Langmuir monolayer technique: A
Macromolecules 58 (2013) 190-198.
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comparison between monomeric and fibrillar -synuclein. International Journal of Biological
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[27] D. Ottolini, T. Calí, I. Szabo, M. Brini, Alpha-synuclein at the intracellular side: functional and
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dysfunctional implications. Journal of Biological Chemistry 398 (2017) 77-100. [28] N. Lorenzen, L. Lemminger, J.N. Pedersen, S.B. Nielsen, D.E. Otzen, The N-terminus of a-
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synuclein is essential for both monomeric and oligomeric interactions with membranes, FEBS Letters 588 (2014) 497-502.
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[29] A. Serrano, M. Ryan, T. Weaver, J. Perez-Gil, Critical structure-function determinants within the N-terminal region of pulmonary surfactant protein SP-B, Biophysical Journal 90 (2006) 238-249.
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[30] H. Brockman, Lipid monolayers: why use half a membrane to characterize protein-membrane interactions? Current Opinion in Structural Biology 9 (1999) 438-443.
[31] K. Beyer, Mechanistic aspects of Parkinson’s disease: alpha-synuclein and the biomembranes, Cell Biochem Biophys 47 (2007) 285-299. [32] G. Fusco, A. De Simone, T. Gopinath, V. Vostrikov, M. Vendruscolo, C.M. Dobson, et al. Direct observation of the three regions in alpha-synuclein that determine its membrane-bound behavior, Nat Commun 5 (2014) 3827.
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ACCEPTED MANUSCRIPT [33] T. Alderson, J.L. Markley, Biophysical characterization of -synuclein and its controversial structure, Intrinsecally Disordered Proteins 1 (2013) e26255-1- e26255-22. [34] T. Bartels, L.S. Ahlstrom, A. Leftin, F. Kamp, Ch. Haass, M. Brown, K. Beyer, The N-terminus of the Intrinsically Disordered Protein -synuclein Triggers Membrane Binding and Helix Folding, Biophysical Journal 99 (2010) 2116-2124.
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[35] S. Ghio, F. Kamp, R. Cauchi, A. Giese, N. Vassallo, Interaction of a-synuclein with biomembranes
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in Parkinson’s disease – role of cardiolipin, Progress in Lipid Research 61 (2016) 73-82.
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[36] S. Nichols-Smith, S.Y. The, T.L. Kyhl, Thermodynamic and mechanical properties of model mitochondrial membranes, Biochimica et Biophysica Acta 1663 (2004) 82-88.
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[37] T. Ryan, V. Bamm, M.G. Stykel, C.L. Coackley, K.M. Humphries, R. Jamieson-Williams, R. Ambasudhan, D.D. Mosser, S.A. Lipton, G. Harauz, S.D. Ryan, Cardiolipin exposure on the outer
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mitochondrial membrane modulates -synuclein, Nature Communications 9 (2018) 817.
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ACCEPTED MANUSCRIPT LEGENDS OF FIGURES
Figure 1. (Top) Alpha-synuclein primary amino acid sequence with aromatic (blue), acidic (red) and lysine (green) residues highlighted. (Middle) Schematic representation of three different regions
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of alpha-synuclein. (Bottom) Sequence of -synuclein-derivated peptides studied in this work.
Figure 2.
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Secondary structure of -synuclein derived peptides. Far-UV CD spectra at 25ºC of -synuclein
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synthetic peptides (10 M) in buffer Hepes 5 mM pH 7.4 (dashed line) and in the presence of 2 mM
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DOPC/DOPE/CL (45:28:22 molar) SUVs (solid line).
Figure 3
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(A) Fluorescence emission spectra of -synuclein synthetic peptides in buffer () Hepes 50 mM
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pH 7.4 containing 150 mM NaCl and in the presence of liposomes composed of DOPC/DOPE/CL (45:28:22 molar) 1 mM (), 2 mM (), 4 mM (). The spectra were obtained upon excitation at
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280 nm. Peptide concentration was 10 M. The temperature was maintained at 25ºC with a
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circulating water bath. (B) Wavelength of maximum emission plotted versus lipid to peptide molar ratio for N-term (), P1 () and P3 () peptides. Lines in (B) are visual guides to show the trend in
Figure 4
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the experimental data.
Quenching of the fluorescence of -synuclein-derivated peptides by brominated phospholipids. Fluorescence emission spectra at 25ºC of P1 and P3 peptides in DOPC/DOPE/CL (45:28:22 molar) bilayers containing 0 (), 20% () and 40% () (w/w) of Br(6,7)-PC (left panel) or Br(11,12)-PC
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ACCEPTED MANUSCRIPT (right panel). The excitation wavelength was 280 nm and the fluorescence intensity is given in arbitrary units.
Figure 5 Compression isotherms of alpha-synuclein derived peptides P1 (A) and P3 (B) monolayers. The
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subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM, thermostated at 25ºC and peptide
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concentration at the interface was 4 M.
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Figure 6
Compression isotherms of the peptide corresponding to the complete sequence of the N-terminal
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segment of -synuclein. The subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM,
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thermostated at 25ºC.
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Figure 7
Insertion of -syn derived peptides into DOPC:DOPE:CL (45:28:22 molar ratio) monolayers.
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Insertion kinetics of 4 M of P1 injected underneath phospholipid monolayers preformed at
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different pressures. Initial pressure (i) was 10 (), 20 (), 27(), 38 () and 40 () mN/m, and
mM.
Figure 8
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the through was maintained at 25ºC and the subphase was composed of Tris 5 mM pH 7.4 NaCl 100
Determination of the critical pressure for insertion of -syn derived peptides P1 () and P3 () into DOPC:DOPE:CL (45:28:22 molar ratio) films. Increase in surface pressure () vs. initial pressure (i) of the film upon injection of each peptide. The subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM maintained at 25ºC. The peptide concentration in all the experiments was 4 M. The
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ACCEPTED MANUSCRIPT lines represent the linear regression that best fits the experimental data. The critical insertion
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Exiquio Maldonado Vidaurri, Abelardo Chavez-Montes, Marsela Garza Tapia, Rocio Castro-Rios, Azucena Gonzalez-Horta PII: DOI: Reference:
S0141-8130(18)30409-4 doi:10.1016/j.ijbiomac.2018.08.049 BIOMAC 10293
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
23 January 2018 8 August 2018 9 August 2018
Please cite this article as: Exiquio Maldonado Vidaurri, Abelardo Chavez-Montes, Marsela Garza Tapia, Rocio Castro-Rios, Azucena Gonzalez-Horta , Differential interaction of α-synuclein N-terminal segment with mitochondrial model membranes. Biomac (2018), doi:10.1016/j.ijbiomac.2018.08.049
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ACCEPTED MANUSCRIPT DIFFERENTIAL INTERACTION OF -SYNUCLEIN N-TERMINAL SEGMENT WITH MITOCHONDRIAL MODEL MEMBRANES
Exiquio Maldonado Vidaurri1, Abelardo Chavez-Montes2, Marsela Garza Tapia3, Rocio Castro-
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Rios3, Azucena Gonzalez-Horta1*
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Laboratory of Genomic Science, Faculty of Biological Sciences, Universidad Autonoma de Nuevo
Leon, 66451 San Nicolas de los Garza, N.L. Mexico 2
66451 San Nicolas de los Garza, N.L. Mexico 3
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Department of Chemistry, Faculty of Biological Sciences, Universidad Autonoma de Nuevo Leon,
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Department of Analytical Chemistry, Faculty of Medicine, Universidad Autonoma de Nuevo Leon,
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66451 San Nicolas de los Garza, N.L. Mexico
Azucena Gonzalez-Horta
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*Corresponding author:
Laboratory of Genomic Science
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Faculty of Biological Science
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Universidad Autonoma de Nuevo Leon
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66451 Monterrey, N.L. MEXICO Email: [email protected] Phone: +52 8183294110
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ACCEPTED MANUSCRIPT ABSTRACT Alpha-synuclein (-syn) is an intrinsically-disordered protein that has been associated with Parkinson’s disease through its deposition in an amyloid fibril form within Lewy Body. Several lines of evidence suggest that the physical association of -syn with the mitochondrial membranes may cause membrane damage and mitochondrial dysfunction, playing an important role in disease
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progression. Although there is strong evidence that the N-terminus part of -syn is essential for
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membrane affinity, cooperative formation of helical domains and regulation of mitochondria membrane permeability, the amino acids involve in this membrane binding is still controversial.
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Fluorescence spectroscopy, circular dichroism and Langmuir monolayer technique were used to elucidate this recognition process of mitochondrial membrane system by synthetic peptides derived
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from -syn N-terminal segment. The results obtained in this work show that the first 15 amino acid
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of the -syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16-30 residues interacts only with the
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phospholipid polar headgroup, confirming that the binding affinity of the N-terminus is nonuniform.
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Key words: -synuclein; mitochondria; monolayer technique
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ACCEPTED MANUSCRIPT 1. INTRODUCTION Parkinson’s disease (PD) is among the most common neurodegenerative diseases, and is characterized by the loss of dopaminergic neurons in the substantia nigra of the brain, which results in impaired motor functions, displayed as compromised postural reflexes, muscular rigidity and bradykinesia in PD patients [1,2]. In most PD cases, the main pathological hallmark is the presence
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in the brain of Lewy bodies and Lewy neurites which are the aggregated form (amyloid fibrils) of
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-synuclein (-syn) a highly conserved presynaptic 140 amino acid protein [3,4]. Native -syn is a small protein, 14kDa, in which three regions can be distinguished: The N-terminal region consisting
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of five degenerate 11-mer repeats of KTKEGV of overall positive charge, a highly hydrophobic central stretch of 12 amino acids referred as non-amyloid component (NAC) and the C-terminal 40
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residues which are highly enriched in negatively charged amino acid [5] (Figure 1). The N-terminal domain is responsible for lipid binding interactions, the NAC region for oligomerization and the C-
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terminal domain possibly presents a chaperone-like activity [6,7]. Although the exact function of
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-syn remains unknown, four principal roles have been proposed including (i) a vesicle fusion and neurotransmitter release, (ii) an intracellular trafficking of lipids, (iii) regulation of cell death [8]
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(iv) and alterations of mitochondrial biology [9-11]. The interaction of -syn with mitochondria is
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thought to contribute to dopaminergic neuronal death in PD. This interaction is mediated by the N terminus of -syn (residues 1-60), whereas the negatively charged C terminus remains unfolded and
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can potentially interact with other proteins [12]. It has been reported that the middle of the Nterminal region (residues 10-30) which contains the KAKEGVVAAAE repeats is involved in mitochondrial binding [13], which induce morphological changes to mitochondria that lead to dysfunction [14]. However, it was also reported that deletion of just residues 2-11 suppress mitochondrial binding [15]. In this work, we have used different synthetic peptides and biophysical techniques to further explore the interaction of the N-terminal segment of -syn with mitochondrial membranes, paying special attention to the differential behavior presented for the first 15 residues
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ACCEPTED MANUSCRIPT and 16-30 residues of -syn N-terminal region in this interaction. The results advance our understanding of -synuclein’s interaction with mitochondrial membranes.
2. MATERIALS AND METHODS Materials
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2.1.
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DOPC (1,2-oleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-oleoyl-sn-glycero-3-
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phosphoethanolamine), and CL (cardiolipin), specified as 99% pure, were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and used without further purification. Recombinant human -
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synuclein and synthetic peptides with a purity 95% were purchased from Sigma-Aldrich (Saint
2.2.
Methods
2.2.1. Peptide synthesis and purification
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Louis, MO).
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The peptides studied in this work was synthesized by Fmoc Chemistry, purified by HPLC and
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analyzed by mass spectrometry by Sigma-Aldrich (Supplementary Fig S1-S3). The 15-residue peptides, named P1 (NH2-WMDVFMKGLSKAKEGV-CONH2), P3 (NH2-
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WVAAEKTKQGVAEAAG-CONH2) were designed taking as a model the sequence of the first 30
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residues of the N-terminal segment of human -syn. We include a tryptophan residue in the native sequence of P1 and P3 with the purpose of using it as an intrinsic probe to characterize structure and
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lipid-peptide interactions by fluorescence spectroscopy. The C-terminal carboxyl of the peptides was amidated to better mimic the interactions occurring at that region of native -synuclein. We also synthetized a peptide corresponding to the complete N-terminal segment of human -synuclein named N-term, with the modification of tryptophan at 39-position instead of tyrosine. These fragments were selected to cover sequence domains that have been previously shown to be critical for the regulation of mitochondrial membrane interaction (Figure 1C).
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ACCEPTED MANUSCRIPT 2.2.2. Lipid/peptide samples Lipid/peptide samples were prepared by different methods depending on the experiment. To mimic the mitochondrial inner membrane (MMI), we used a lipid composition that contains DOPC:DOPE:CL in a molar ratio 45:28:22 [16], the mixture was prepared in chloroform/methanol (2:1 v/v) at [10 mg/mL] final concentration, then the solvent was removed by drying under nitrogen
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flow and then 2 h in speed vac. For the circular dichroism experiments, lipid/peptide suspension
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were prepared by hydrating dry lipid/peptide films for 2 h in 5mM Hepes buffer at 25ºC in a termomixer with cycles (2 min vortex 1400 rpm every 10 min) and then sonicated in a Branson UP
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200S tip sonifier to obtain small unilamellar vesicles (SUVs). To prepare the samples for the binding experiments, large unilamellar vesicles (LUVs) were prepared by extrusion of the lipid
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suspension, equilibrated at 25ºC, through 0.1 m (pore diameter) polycarbonate membranes in a mini-extruder (Avanti Polar Lipids, Alabaster, AL USA). To analyze quenching of peptide
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tryptophan fluorescence by brominated lipids, liposomes were prepared containing different weight
(mol/mol).
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2.2.3. Circular dichroism
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percentages of Br(6,7)-PC or Br(11,12)-PC, with or without peptide, at peptide/lipid ratio 1:45
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Far-UV circular dichroism (CD) spectra were recorded in a J-1100 spectropolarimeter (JASCO, Easton, MD) equipped with a Xenon lamp and a Koolance Peltier-type holder for temperature
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control. All spectra were recorded in a 0.5 mL thermostated quartz cell with an optical path length of 0.1 cm. Each spectrum was registered over the 190-240 nm at a scan rate of 50 nm/min and represents an average of 3 scans with a full-scale sensitivity of 50 mdeg. The samples contained 10 M of peptide in buffer Hepes 5 mM pH 7.4 and 2 mM of SUVs. Corresponding buffer scans were subtracted from sample scans before analysis and presentation of the data. Helix content of peptides was assumed to be directly proportional to mean residue ellipticity (MRE) at 222 nm due at this wavelength, the contribution of random coil structure is relatively small [17]. At 222 nm, the -
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ACCEPTED MANUSCRIPT helical content was calculated using the formula previously reported [18]: % helicity = [222 - coil / helix - coil] 100 where helix = -4000 x (1 - 2.5/n) + 100 x ; and coil= (640 – 45) where 222 is the measured mean residue ellipticity at 222nm, helix and coil are mean residue ellipticities at 222 nm of idealized a-helix and random coil proteins, n is the number of amino acids for each peptide and
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is the temperature (ºC).
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2.2.4. Intrinsic fluorescence
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Tryptophan fluorescence emission spectra of peptides were recorded using 280 nm as the excitation wavelength in a LS-45 spectrofluorometer (Perkin-Elmer Inc.). Emission spectra were registered at
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25ºC from 300 to 500 nm with 1 cm cells using a scanning speed of 1 nm/s. The slit widths were 10 nm for the excitation and emission beams.
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2.2.5. Quenching of Trp by Brominated Phosphatidylcholines
Collisional quenching of Trp by brominated phospholipids was introduced to assess the localization
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of this residue in bilayers [19]. Liposomes were prepared containing different weight percentages
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of Br(6,7)-PC or Br(11,12)-PC, with or without peptide, at peptide/lipid ratio 1:45 (mol/mol). After 30 min at 25ºC, emission spectra were recorded, averaging 3 spectra. The differences in the quenching
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of Trp fluorescence by Br(6,7)-PC and Br(11,12)-PC were used to calculate the probability for location
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of the fluorophore in the membrane using the parallax method [20]. The depth of the Trp residue was calculated as follows: Zcf = Lc1 + [(-ln(F1/F2)/C – L21]/2L21 where Zcf represents the distance
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of the fluorophore from the center of the bilayer, Lc1 is the distance of the shallow quencher from the center of the bilayer, L21 is the distance between the shallow and deep quencher, F1 is the fluorescence intensity in the presence of the shallow quencher, F2 is the fluorescence intensity in the presence of the deep quencher, and C is the concentration of quencher in molecules /Å2. 2.2.6. Peptides interfacial adsorption Interfacial adsorption of each peptide was assayed using a Stainless-steel trough low volume with a fully automated microtensiometer (TROUGH SX, Kibron Inc. Helsinki, Finland). The
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ACCEPTED MANUSCRIPT microbalance was filled with 1.3 mL of Tris 5 mM pH 7.4 containing 100 mM NaCl, thermostated at 25ºC and subjected to continuous stirring. After the injection of a small volume of methanolic peptide solution into the subphase, changes in surface pressure were monitored over time (t isotherms). Injections of equivalent volumes of pure methanol did not produce any detectable
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change in surface pressure.
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2.2.7. Insertion of peptides into preformed monolayers
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Insertion of -syn peptides into MMI monolayers was followed using the same microbalance by monitoring changes in surface pressure () over time after peptide injection into the subphase.
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Monomolecular films of the lipid (0.1 mg/mL chloroform: methanol 3:1 v/v) were spread on top of buffer Tris 5 mM pH 7.4 NaCl 100 mM. After 5 min for solvent evaporation, 10 L of the protein
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or the synthetic peptides solution, prepared in methanol at [1 mg/mL] final concentration, were injected in the subphase (1.3 mL) and pressure increases produced were continuously recorded as a
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function of time. The surface pressure at the time of injection is indicated in the figure legends.
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Control experiments were run in parallel to ensure that the injection of 10 L of methanol in which protein and peptides were dissolved did not induce by itself any surface pressure change. The data
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were analyzed with the FilmWareX 3.57 program (Kibron Inc). The accuracy of the system under
2.2.8. Statistics
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our experimental conditions was 0.25 mN/m for surface pressure.
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Unless otherwise indicated, results have been presented as representative experiments after repeated examination of three different samples from at least two different batches of peptide.
3. RESULTS The secondary structure of -synuclein peptides was analyzed by CD spectroscopy. Figure 2 shows the far-UV CD spectra of the three synthetic peptides (N-term, P1, P3) containing the amino-acid sequence of the N-terminal half of -synuclein with the sequence indicated in Fig. 1. In an aqueous
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ACCEPTED MANUSCRIPT solution, the three peptides adopt a random coil conformation, revealed by the single minimum at 200 nm. Upon binding membranes mimicking mitochondrial lipid composition, the peptides present an -helical conformation with the characteristic double minima at 208 and 222 nm in the CD spectrum. The -helical content of the peptides upon lipid binding was estimated from the mean residue ellipticity (MRE) at 222 nm as explained in the Materials and Methods. Binding of N-term
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peptide resulted in an -helical content of 63%, P1 and P3 peptides adopted a comparable amount
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of -helix (74% and 73%, respectively). Therefore, the similarity of the CD spectra suggests that all peptides were fully bound to the SUVs used and in a predominantly -helical conformation.
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The fluorescence emission spectra of -synuclein N-terminal peptides in buffer and in the presence of different amounts of DOPC/DOPE/CL (45:28:22 molar) liposomes is shown in Figure 3A. In
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buffer, all peptides show the peak of tryptophan at 353 nm. For N-term and P1 peptides in going
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from water to liposomes, fluorescence intensity slightly increases and undergoes a blue shift from 353 nm to 330 nm, as the lipid to protein molar ratio increases, which implies that in the presence
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of liposomes, the tryptophan residue of both peptides moves to a more hydrophobic environment.
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P3 peptide had a blue shift of 13 nm (Fig. 3B) with a reduction in peak fluorescence intensity therefore could be due to a relatively shallow depth in the liposomes. To analyze peptide behavior
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in terms of lipid-peptide binding in more detail, we evaluated quenching of peptide tryptophan fluorescence by brominated lipids. Figure 4 shows fluorescence emission spectra of P1 and P3 in
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PC vesicles containing different weight percentages of brominated PC bearing bromines either at the (6,7) or at the (11,12) positions of its acyl chains. Increasing amounts of both brominated lipid probes produce progressive quenching of peptide fluorescence as a result of peptide association with membranes. Quantitative comparison of quenching by the two brominated lipids allows estimation of the relative position of the peptide tryptophan in the bilayers, provided that the location in the membrane of the two quenching groups is known. It can be observed that the extent of quenching of P1 fluorescence by the Br(11,12) isomer is clearly higher than that of quenching by
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ACCEPTED MANUSCRIPT the Br(6,7) isomer (Fig. 4 A, B). While the extent of quenching of P3 fluorescence was clearly higher by Br(6,7) isomer (Fig. 4 C, D). Applying the parallax analysis to these data, it was possible to estimate that the P1 peptide tryptophan is located in a region 10 1Å from the center of the bilayer while the tryptophan residue of P3 resides at a distance of 15 1Å from the center of the
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bilayer. These results suggest a deeper bilayer penetration for P1 and an association only with the
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membrane surface for P3 peptide. All these results show the ability of the peptides used in this work
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to interact and insert in DOPC/DOPE/CL bilayers but with a different extent, adopting an -helical conformation. It has been suggested that -syn in the presence of vesicles adopts a two-helix
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antiparallel arrangement around small unilamellar vesicles [21] and becomes even more extended when it is in contact with less curved lipid bilayers [22], also it was reported that -synuclein is in
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an -helical conformation with its helical axis parallel to the air-water interface [23] to analyze the
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interfacial conformation of these peptides, we used the Langmuir-balance technique as a simple model to mimic the interface of the mitochondrial membrane considering that the physicochemical
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properties of the thin aqueous layer that exists at the interface of the cell membrane are different to the bulk aqueous phase in the cytoplasm [24,25]. The compression isotherms of pure peptides films
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can provide valuable information on the conformational tendency of each peptide as a function of
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surface pressure. Two important data can be obtained from these graphs. First, the area per residue where the compression begins to induce an increase in surface pressure, gives an idea of the space
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occupied by each peptide molecule in the most extended orientation at the air-liquid interface. Moreover, the maximum pressure at which the peptide film can be compressed indicates the degree of stability of the peptide in the more compressed conformation. Figure 5 shows the surface behavior of pure peptide monolayers subjected to compression. Both P1 and P3 peptides are able to form stable monolayers on saline subphase, due their intrinsic amphipathic character. The lift-off area for P1 peptide was 350 A2 per molecule (Fig. 5A), while the one for P3 peptide was around 40 A2 per molecule (Fig. 5B). It therefore seems that P3 could adopt a more perpendicular
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ACCEPTED MANUSCRIPT conformation. Monolayers of the P3 peptide collapse at approximately 15 mN/m, while P1 monolayers could sustain higher surface pressures around 30 mN/m. The isotherm of the peptide corresponding to the N-terminal segment of -syn presents a plateau around 15 mN/m before reaching higher surface pressures (Fig. 6). This plateau indicates that the peptide undergoes a
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change in interfacial conformation. It is very probable that the plateau is due to the expulsion from
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the interface of the segment corresponding to 16-30 residues which, as observed in the isotherm of P3 peptide, cannot withstand pressures higher than 15mN/m. This result suggests that only the first
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15 amino acids of -syn remain associated to the interface at high surface pressure, those enriched
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in cardiolipin as would be the contact sites or the mitochondrial crests. The current model for the structure and disposition of -syn in phospholipid monolayers assumes that the N-terminal segment
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of the protein is responsible for the association with phospholipids [27, 28] and also Shen et al. (2014) have shown that the N-terminus of -syn is essential for the regulation of mitochondrial
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membrane permeability so, to investigated the ability of each peptide to interact with the
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mitochondrial inner membrane (MMI), we formed monolayers composed of (DOPC:DOPE:CL 45:28:22 molar ratio) at the air-water interface and then injected the peptides of -syn (4 M) in
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the buffered subphase. P1 and P3 peptides were able to perturb and insert into MMI monolayer
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prepared at different initial surface pressures (i). Figure 7A illustrates that injection of P1 into the subphase underneath the DOPC:DOPE:CL monolayer promotes an instantaneous pressure increase
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() as a consequence of the association of the peptide with the interfacial film. Very similar behavior was observed for P3 peptide (Fig. 7B) however, the interface association of P1 peptide with the monolayer film, produce higher increase in surface pressure than P3 peptide. These could be due the absence of one K residue on P3, suggesting that in the association and stabilization of the peptide with the interfacial films, an electrostatic component is participating. Devi et al. (2008), reported similar results showing that the N-terminal 32-amino acid region of -syn, which contains evenly spaced positive residues, is critical for mitochondrial targeting. In Figure 8 has been plotted
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ACCEPTED MANUSCRIPT the increase in surface pressure () versus the initial surface pressure of the preexisting phospholipid monolayer (i) after injection of a given amount of each peptide. For both peptides, the value always decreased as i increase, as would be expected if lipid molecules are densely packed, preventing peptide insertion. From these plots one can calculate the critical insertion
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pressure (c), which represents the maximum surface pressure at which the peptides are still able to
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insert. This parameter depends on the monolayer composition as well as on the relative affinity of
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the peptides to associate with the interface film [29]. It is generally assumed that the lateral packing in a lipid bilayer can be roughly mimicked by a monolayer compressed to 30 mN/m. Therefore,
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molecules with c values higher than 30mN/m are generally considered as competent to interact with and insert into lipid membranes [30]. The critical pressures calculated for the insertion of P1 in
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MMI monolayer was around 42 mN/m, while the c for P3 was around 36 mN/m so both peptides
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exceed the physiologic membrane pressure. However, these differential c values demonstrated
DISCUSSION
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4.
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again that -syn fragment comprising 1-15 residues has deeper association with the monolayer.
Binding of -synuclein with biomembranes has been extensively studied. Typically, experiments
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make use of planar bilayers, liposome vesicles and micelles containing physiologically relevant phospholipids [31]. Although several distinct binding modes have been shown for -syn, there is a
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consensus that upon binding to a membrane, -syn forms an amphipathic -helical structure involving the N-terminal and NAC regions [32]. As monitored with CD spectroscopy, lipid binding induced a coil-to-helix transition wherein the -helical content of -synuclein increased from 2 to 71% [33], which is comparable to the observations reported in the present study for the -syn Nterminal peptides. Membrane binding of monomeric -syn appears to involve two steps: first, anchoring by the N-terminal residues 3-25, followed by a coil-to-helix transition of residues 26-97 which act as a membrane sensor and which determine the affinity of -syn binding; the C-terminal 11
ACCEPTED MANUSCRIPT domain exhibits only weak interactions with the membrane surface [34]. The results obtained in this work show that the first 15 amino acid of the -syn N-terminal segment mainly participate in the anchoring, perturbing the membrane hydrophobic region, while the peptide corresponding to 16-30 residues interacts only with the phospholipid polar headgroup, suggesting that the mitochondrial
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targeting signal proposed by Devi et al. (2008) and Zigoneanu et al. (2012), relies only to the first
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15 amino acids. These results agree with the observations made by Robotta et al. (2012), which revealed that the binding affinity of the N-terminus is nonuniform. Our results not only imply that
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the binding of -syn to inner mitochondrial membranes is initiated in the N-terminal part of -syn
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but also suggest that the binding affinity of regions close to the N-terminus is stronger than that of regions distal from the N-terminus sequence. For many years cardiolipin (CL) was assumed to be
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associated exclusively with the mitochondrial inner membrane where, as measured in bovine heart mitochondria, it represents 25% of the total phospholipids. More recently CL has also been
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identified in the mitochondrial outer (4%) especially at the contact sites connecting the outer
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membrane with the inner one due the non-bilayer hexagonal structure of this lipid [35], so it is possible that -syn mitochondrial membrane association at contact sites could be modulated by the
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first fifteen residues of the N-terminal segment of the protein, responding to hydrophobic and
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electrostatic forces as shown by the ability of P1 peptide to remain associated to the interface at higher lateral pressures like those caused by cardiolipin, since this lipid increases lateral interaction
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between lipids within monolayer leaflets, while simultaneously decreasing the cohesive energy of membranes at physiologically relevant concentration [36]. A recent report demonstrates that cardiolipin translocate to the outer mitochondrial membrane in response to the presence of -syn on mitochondrial membranes [37], so it is possible that while the first 15 amino acids allow the anchoring of the protein to the contact sites, the following residues of the N-terminal segment participate in cardiolipin translocation although future experiments are required to analyze the reorganization of mitochondrial phospholipids.
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5. CONCLUSION Taken together with literature data, our results show that the affinity of the N-terminal segment of -syn with mitochondria is nonuniform. Apparently, the segment corresponding to the first 15
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amino acids of -syn has deeper bilayer penetration on liposomes that mimic inner mitochondrial
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membrane and remains associated to the interface at high surface pressure. Although, our current
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understanding of the role that synuclein and mitochondria play in PD pathogenesis is limited, it is possible that this segment of the protein be the first fraction that interacts with mitochondria and
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disrupt its normal morphology and function. Future studies will examine morphological changes or
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any alteration in the cardiolipin levels originated by this lipid-peptide interaction.
ACKNOWLEDGMENT
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This research was financially supported by the Consejo Nacional de Ciencia y Tecnologia
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(CONACYT) Project No. C.B. 2014 236834.
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CONFLICTS OF INTEREST
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None declared.
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ACCEPTED MANUSCRIPT LEGENDS OF FIGURES
Figure 1. (Top) Alpha-synuclein primary amino acid sequence with aromatic (blue), acidic (red) and lysine (green) residues highlighted. (Middle) Schematic representation of three different regions
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of alpha-synuclein. (Bottom) Sequence of -synuclein-derivated peptides studied in this work.
Figure 2.
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Secondary structure of -synuclein derived peptides. Far-UV CD spectra at 25ºC of -synuclein
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synthetic peptides (10 M) in buffer Hepes 5 mM pH 7.4 (dashed line) and in the presence of 2 mM
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DOPC/DOPE/CL (45:28:22 molar) SUVs (solid line).
Figure 3
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(A) Fluorescence emission spectra of -synuclein synthetic peptides in buffer () Hepes 50 mM
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pH 7.4 containing 150 mM NaCl and in the presence of liposomes composed of DOPC/DOPE/CL (45:28:22 molar) 1 mM (), 2 mM (), 4 mM (). The spectra were obtained upon excitation at
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280 nm. Peptide concentration was 10 M. The temperature was maintained at 25ºC with a
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circulating water bath. (B) Wavelength of maximum emission plotted versus lipid to peptide molar ratio for N-term (), P1 () and P3 () peptides. Lines in (B) are visual guides to show the trend in
Figure 4
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the experimental data.
Quenching of the fluorescence of -synuclein-derivated peptides by brominated phospholipids. Fluorescence emission spectra at 25ºC of P1 and P3 peptides in DOPC/DOPE/CL (45:28:22 molar) bilayers containing 0 (), 20% () and 40% () (w/w) of Br(6,7)-PC (left panel) or Br(11,12)-PC
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ACCEPTED MANUSCRIPT (right panel). The excitation wavelength was 280 nm and the fluorescence intensity is given in arbitrary units.
Figure 5 Compression isotherms of alpha-synuclein derived peptides P1 (A) and P3 (B) monolayers. The
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subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM, thermostated at 25ºC and peptide
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concentration at the interface was 4 M.
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Figure 6
Compression isotherms of the peptide corresponding to the complete sequence of the N-terminal
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segment of -synuclein. The subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM,
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thermostated at 25ºC.
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Figure 7
Insertion of -syn derived peptides into DOPC:DOPE:CL (45:28:22 molar ratio) monolayers.
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Insertion kinetics of 4 M of P1 injected underneath phospholipid monolayers preformed at
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different pressures. Initial pressure (i) was 10 (), 20 (), 27(), 38 () and 40 () mN/m, and
mM.
Figure 8
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the through was maintained at 25ºC and the subphase was composed of Tris 5 mM pH 7.4 NaCl 100
Determination of the critical pressure for insertion of -syn derived peptides P1 () and P3 () into DOPC:DOPE:CL (45:28:22 molar ratio) films. Increase in surface pressure () vs. initial pressure (i) of the film upon injection of each peptide. The subphase was composed of Tris 5 mM pH 7.4 NaCl 100 mM maintained at 25ºC. The peptide concentration in all the experiments was 4 M. The
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ACCEPTED MANUSCRIPT lines represent the linear regression that best fits the experimental data. The critical insertion
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pressure (c) was calculated by extrapolation of the plot at = 0 mN/m.
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