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The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation
Accepted Manuscript The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation Niloofar Rezaeian, Niloofar Shirvanizadeh, Soheila Mohammadi, Maryam Nikkhah, Seyed Shahriar Arab PII:
S0003-9861(17)30505-2
DOI:
10.1016/j.abb.2017.09.015
Reference:
YABBI 7562
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 24 July 2017 Revised Date:
5 September 2017
Accepted Date: 20 September 2017
Please cite this article as: N. Rezaeian, N. Shirvanizadeh, S. Mohammadi, M. Nikkhah, S.S. Arab, The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation, Archives of Biochemistry and Biophysics (2017), doi: 10.1016/j.abb.2017.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation Niloofar. Rezaeian a, Niloofar. Shirvanizadeh, Soheila. Mohammadi, Maryam. Nikkhah b*, Seyed Shahriar. Arab c* Department of Bioscience, University of Azad, Tehran, Iran
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Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat modares
University, Tehran, Iran c
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Department of Biophysics, Faculty of Biological Sciences, University of Tarbiat modares,
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Tehran, Iran
Correspondence: Maryam Nikkhah
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran
Fax: +98 21 82884718
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Tel: +98 21 82884734
Email: [email protected] Seyed Shahriar Arab
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Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran
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Tel: +98 21 82884717
Fax: +98 21 82884718
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ABSTRACT
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Parkinson̕ s disease is characterized by accumulation of inclusion bodies in dopaminergic neurons, where insoluble and fibrillar α-synuclein makes up the major component of these inclusion bodies. So far, several strategies have been applied in order to suppress α-synuclein aggregation and toxicity in Parkinson̕ s disease. In the present study, a new datebase has been established by segmentation of all the proteins deposited in protein Data Bank. The database data
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base was searched for the sequences which adopt β structure and are identical or very similar to the regions of α-synuclein which are involved in aggregation. The adjacent β strands of the found
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sequences were chosen as the peptide inhibitors of α-synuclein aggregation. Two of the predicted peptides, namely KISVRV and GQTYVLPG, were experimentally proved to be efficient in suppressing aggregation of α-synuclein in vitro. Moreover, KISVRV exhibited the ability to disrupt oligomers of α-syn which are assumed to be the pathogenic species in Parkinson’s disease.
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Keywords: peptide inhibitor; amyloid fibril formation; α-synuclein; parkinson̕ s disease
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1. Introduction
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Many neurodegenerative diseases are characterized by the formation and accumulation of misfolded proteins into amyloid oligomers or fibrils [1-3]. The prevalence of neurodegenerative diseases is growing with a rapidly aging population. Parkinson’s disease (PD) which is the second most common neurodegenerative disease manifests with movement disorders such as resting tremor, muscular rigidity, bradykinesia, and postural instability [3-7] . The main problem of finding cures for PD is that the symptoms only manifest when major part of dopaminergic neurons in the substantia nigra pars compacta has been lost in the midbrain [8-10]. The two pathological hallmarks of PD are the loss of dopaminergic neurons and the presence of intracellular protein aggregation of α-synuclein (α-syn): Lewy bodies (LBs) and Lewy neurites (LNs) in the substantia nigra region of the brain in PD patients [11-16]. α-syn is a 140-amino acid intrinsically disordered protein which is abundant in the human brain, found mainly at the tips of neurons in presynaptic terminals [3-5, 11, 15]. α-syn primary structure is usually divided into three distinct regions: 1. Residues 1-60: an amphipathic N-terminal region in which five missense mutations have been identified (A30P, E46K, H50Q, G51D and A53T) that are involved in the familial form of PD. 2. Residues 61-95: A central hydrophobic region which is involved in protein aggregation. 3. Residues 96-140: a highly acidic and proline-rich region which has no distinct structural propensity [11, 15, 17-23]. During the course of aggregation, α-syn undergoes conformational changes from disordered monomers to dimers and then to oligomers that may assemble into protofibril [3, 24]. The aggregated proteins in the fibrils have the characteristic conformation of β-sheets that are packed perpendicular to the fiber axis. Several studies have identified key regions within the α-syn sequence that are involved in protein aggregation [3, 25]. A central hydrophobic region of α-syn (61–95 residues), has a high propensity for formation of β-sheet rich structures, and is known to drive the amyloid fibril formation. Within this core region, residues 66-74, 68-78 and 71-82 were identified as the key initiating sequences in amyloid fibril formation [1, 21, 26-28]. In addition, the five missense point mutations in the amphipathic N-terminus region of α-syn (1-60 residues) have been shown to accelerate the rate of α-Syn fibrillation and have been associated with pathogenesis of PD [29-37]. The relation between α-syn mutations and the altered kinetics of fibrillation has inspired the design of peptide inhibitors that block α-syn aggregation and fibrillation [1, 28, 30, 32, 38-44]. Considering the cytotoxicity of the oligomers which form during the process of amyloid formation, compounds that inhibit the formation of fibrils and/or oligomers might lead to appropriate therapeutic approaches for PD treatment [40, 42, 45-52]. Inhibition of amyloid fibril formation by several naturally occurring compounds has already been proven. These compounds usually affect more than one protein. Some polyphenolic compounds suppress the formation of amyloid fibrils but their effects are rather non-specific. For example, curcumin can inhibit the aggregation of multiple amyloidogenic proteins including Aβ, α-syn,
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egg-white lysozyme, transthyretin (TTR) and human islet amyloid polypeptide (hIAPP) [53]. Compared to the chemical drugs, peptide drugs offer several advantages, such as high specificity, minimization of drug-drug interactions, low accumulation in tissues, low toxicity and great biological diversity. In spite of the mentioned advantages, the application of therapeutic peptides has been limited by their rapid decomposition in biological fluids and tissues and their low bioavailability [54]. However, there are strategies to increase the stability of the peptids against proteolytic degradation like replacing some or all of the L-amino acids with D-amino acids and chemical modifications of the constituting amino acids [55]. By exploiting different strategies several peptides have been found to prevent α-syn aggregation; Some examples are as followings: designing an N-Methylated peptide inhibitor of α-syn aggregation guided by SolidState NMR [1], synthesizing a library of overlapping 7-mer peptides spanning the entire α-syn sequence [28], design of a hexapeptide based on the mutations (V66S, V66P, T72P, V74E, V74G, and T75P) [38, 56], designing the PQQ-modified α-syn36–46 peptide by analyzing the peptide sequences of unmodified α-Syn proteolytic products [48], and screening peptide ligands against α-syn by in silico panning [56].
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1. Materials and Methods
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In this study we have developed a novel database (ProDA) which is able to search for any peptide with defined properties in PDB. The database was searched to find sequences which fulfill two requirements: first, being the same or similar to two regions of α-syn which are involved in aggregation and second, existing as a strand of a beta sheet. The adjacent strands of the found peptides were further assessed and among them, considering some structural requirements, two peptides were chosen as the inhibitors. The biomimetically designed peptides not only could arrest the aggregation of α-syn efficiently but also are able to disrupt oligomers of α-syn which are critically involved in the pathogenesis of PD.
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2.1.Design of the peptides
ProDA database was generated by fragmentation of all the deposited proteins in PDB into fragments of 4-20 amino acids in length. The deposited peptides in ProDA could be searched for their length, secondary structures, the number and type of amino acids, hydrophobicity and accessible surface area. This database offers many applications, including peptide design. We aimed to design peptides which are able to bind residues 70 to 75 and 46 to 53 of α-syn. We have searched through ProDA for protein fragments that possess only β structure and have identical or very similar sequences to the target regions of α-syn. The search resulted in finding several fragments involved in parallel and antiparallel β-sheets. The fragments with antiparallel β-sheet were then selected. In antiparallel β-sheet the hydrogen bonds are perpendicular to the strands, and narrowly spaced bond pairs alternate with widely spaced pairs. In addition, the successive β-
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strands alternate directions so that the N-terminus of one strand is adjacent to the C-terminus of the next. This arrangement provides the strongest inter-strand stability. Finally, the adjacent strands of the selected fragments were taken as the peptide inhibitors. The scrambled peptides which have the same amino acid composition and the same beta propensity as peptide inhibitors have been considered as negative controls. 2.2. protein expression and purification
2.3. Amyloid fibril formation
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The α-syn protein was expressed in Escherichia coli (E.Coli C41). α-syn expression was induced by the addition of 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After induction for 4 hours, the bacteria were harvested by centrifugation (6000 RPM, 20 min, 4 ˚C). The cells were disrupted using a UP400S Ultrasonic instrument (Hielscer Uiltrasoincs GmbH, Teltow, Germany), and the supernatant was collected by centrifugation (13000 RPM, 30 min, 4 ˚C). The supernatant was loaded onto a His 60 Ni Superflow Resin (Clontech, Takara Bio Company) column and eluted with elution buffer (imidazole 300mM, NaCl 300mM, NaH2PO4 50Mm, pH 8). The eluted fractions were analyzed by SDS-PAGE, and the fractions containing αsyn protein were dialyzed against phosphate-buffered saline (PBS) (1.76 mM KH2PO4, 10mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4). The protein concentration was determined by a UV-Visible spectrophotometer ( PerkinElmer, Lambda 25 ).
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Purified wild-type α-syn was incubated at final concentration of 75µM, either alone or with peptide inhibitors and the scrambled peptides, (synthesized by Mimotopes Corporation Australia) at a molar ratio of 1:1, 1:5 and 1:10 for (α-syn: KISVRV, VSRKIV) and 1:1, 1:5, 1:10 and 1:40 for (α-syn: GQTYVLPG, VGPTQGLY) with 0.02% NaN3 as an antiseptic agent at 37 ˚C in a Turbo Thermo Shaker (TMS-200, China) with continuous agitation at 850 RPM. Amyloid fibril formation was monitored by fluorescence enhancement of Thioflavin T (ThT) solution (Sigma-Aldrich Company): Aliquots of 5µL were removed from the incubated samples and added to 95 µL of 25 µM ThT in PBS buffer. Fluorescence emission was measured with excitation wavelength at 440 nm and emission at 485 nm using a Cytation3 Cell Imaging MultiMode Reader (BioTek Instruments, Winooski, VT). Each sample was assayed in triplicate, and each experiment was repeated three times. 2.4. The effects of the peptide inhibitor on different steps of the fibrillation pathway Monomers, oligomers and fibrils of α-syn were isolated by size exclusion chromatography. Purified α-syn at final concentration of 75µM was incubated at 37 ˚C with continuous mixing at 850 RPM. The samples at t=0, 48 and 144 hour of incubation were taken and loaded onto a Sephadex G-75 column (0.7×45 cm) in PBS buffer pH 7.4. One milliliter fractions were collected and assayed for protein content by measuring the absorbance at 280 nm. It should be
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noted that at first, the column was calibrated using a series of molecular weight standards: bovine serum albumin (BSA) 66 kDa, ovalbumin (OA) 44 kDa, chymotrypsinogen A (CTA) 27.5 kDa, myoglobin (MG) 17.5 kDa, and ubiquitin (ub) 8 kDa. The purified monomer, oligomer and fibrils of α-syn were incubated alone or in the presence of peptide inhibitor and/or scrambled peptide (molar ratio of KISVRV, VSRKIV: α-syn 1:1, 5:1, 10:1) for 6 days with continuous shaking (850 RPM) at 37 ˚C. The presence of amyloid fibrils was estimated by ThT fluorescence every 24 hour. 2.5. Assessment of the capability of the peptide inhibitor to split oligomers
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Isolated α-syn oligomers by SEC were incubated alone or in the presence of peptide inhibitor and scrambled peptide (molar ratio of KISVRV, VSRKIV: α-syn 1:1, 5:1, 10:1) under continuous shaking (850 RPM) at 37 ˚C. The amyloid fibril formation was monitored by ThT fluorescence measurements. Samples at t=0, 48 and 96 hour during incubation were taken and loaded onto the SEC column. Then, one milliliter fractions were collected and protein absorption was monitored at 280 nm.
2.6. Electron microscopy
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The α-syn protein (75µM) either alone or with peptide inhibitors and theirs coresponding scrambled peptides at a molar ratio of 1:10 (α-syn: KISVRV, VSRKIV) and 1:40 (α-syn: GQTYVLPG, VGPTQGLY) with 0.02% NaN3 were incubated for 144 h at 37 ˚C under continuous shaking (850 RPM). 10µl of the samples were adsorbed onto formvar carbon coated grid Cu Mesh 300 for 2 min. The excess fluid was drained with filter paper and the samples were stained for 90 s with 2% uranyl acetate. The grid was air-dried and examined with a Zeiss EM10C electron microscope at 80 KV.
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3. Results and Discussion: Considerable evidence demonstrates that the conversion of soluble monomeric α-syn to insoluble amyloid oligomers or fibrils in neuronal cells is the main cause of PD [3,11-16,28,32]. Aggregation is the significant process where the α-syn protein loses its function and gains toxicity. This suggests that prevention of α-syn fibrillation may be an appropriate therapeutic strategy for PD. In this study, we attempted to design peptides that can specifically interact with the regions of α-syn that are responsible for its self-aggregation. Here, we have utilized the new ProDA database for design of peptides targeting two different regions of α-syn. The potency of the designed peptide inhibitors were analyzed in vitro.
3.1. Biomimetic design of peptide inhibitors
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A central hydrophobic region of α-syn (61–95 residues) has been identified to have a critical role in aggregation and formation of amyloid fibrils [1, 21, 26-28]. The occurrence of 12 amino acid sequence in the hydrophobic region (residues 71–82) has been reported to play an indispensable role in oligomerization and formation of amyloid fibrils [21, 57-59]. Actually, many researchers have used this region as a starting point for the design of inhibitors. Additionally, among the known point mutations in the α-syn gene associated with early onset of PD, four (E46K, H50Q, G51D, A53T) are located between residues 46 and 53. This region is clearly important in modulating amyloid formation such that toxicity associated with the α-syn protein becomes increased with the mutations that decrease α-helix propensity or increase βsheet propensity, and either increase the rate or the number of oligomers that are formed [29, 6062]. Given the interests in the above mentioned regions, in this study, we have chosen residues 70 to 75 (VVTGVT) and 46 to 53 (EGVVHGVA) of α-syn for designing the peptide inhibitors. The selected regions of α-syn have the propensity to form β-sheet in spite of their helix structures (Fig. 1A and C and Table 1); these sequences are known as Chameleon sequences [63-66]. At first we, have developed a database called ProDA which is able to search for any peptide with defined properties in PDB. We have searched through ProDA database for identical or similar sequences to target regions of α-syn with anti-parallel β structures (as mentioned in methods) and subsequently selected residues 71 to 76 ( VVTGLT) of 1KMT (RhoGDI) and residues 85 to 91 (YVVHGVY) of 3I0Y (putative polyketide cyclase) (Fig. 2). The adjacent strand of the selected sequence in 1KMT composed of residues 113 to 118 (KISFRV) and the adjacent strand of the selected sequence in 3I0Y composed of residues 103 to 110 (GQTYVLPG) were chosen as the peptide inhibitors.) . As these sequences are located on the edge of the beta sheets, they are expected to interact with the target sequence only from one side. This may offer an advantages in a way that upon the interaction of the peptide with α-syn, a dead end forms that doesn’t permit the beta sheet to extend. To increase the β formation propensity of KISFRV, phenylalanine was replaced by valine which has the highest propensity to form β-sheet among all the amino acids.
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We expect that the designed peptides to be able to bind to aggregation prone regions of α-syn and make deadlocks that do not allow the other α-syn proteins to join and promote amyloid formation. The effect of the peptide inhibitors on amyloids formation were investigated using the ThT fluorescence measurements and Transmission Electron Microscopy (TEM) analysis.
3.2. KISVRV and GQTYVLPG prevent the formation of amyloid fibrils The formation of amyloid fibrils was monitored by the increase in ThT fluorescence. ThT is a fluorescent dye that is widely used to visualize and quantify the presence of misfolded protein aggregates called amyloid. The binding of ThT to β-sheet-rich structures such as those in amyloid aggregates is accompanied by a characteristic increase in the fluorescence intensity in the proximity of 485 nm. The process of α-syn aggregation exhibits three distinct phases including lag phase, rapid growth phase and stationary phase. In order to characterize the
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effect of KISVRV and VSRKIV on α-syn aggregation, we tested three different concentrations of peptide inhibitors and scrambled peptides. Fig. 3, shows the effect of VSRKIV on the amyloid fibril formation at three different concentrations (molar ratio of 1:1, 1:5, and 1:10 of α-syn: VSRKIV). By incubation of α-syn under aggregation condition, after 24 h of lag time, a rapid increase in fluorescence intensity was observed, suggesting the accumulation of α-syn amyloid fibrils. After 144 h of incubation at 37 ˚C, the maximum value of the fluorescence intensity was observed. Additionally, Fig. 3A, B and C, show that the scrambled peptide (VSRKIV) could not affect the kinetics of amyloid fibrils formation and the intensity of fluorescence at 485nm has been increased. The inhibitory effect of KISVRV peptide on the aggregation of α-syn was demonstrated at all the three different molar ratios of α-syn: KISVRV. Fig. 3, demonstrates that after 144 h of incubation in the presence of KISVRV, no increase in the ThT fluorescence of αsyn was observed. This observation suggests that KISVRV could arrest amyloid fibril formation. It should be noted that the incubation of peptide inhibitor alone at 37 ˚C for 6 days has not resulted in any increase in the ThT fluorescence (fig. 3D). TEM observation also revealed the amyloid fibrils formation of α-syn, incubated under aggregation conditions alone (Fig. 3E, a) or in the presence of the scrambled peptide VSRKIV after 144h of incubation (Fig. 3E, b). By the presence of KISVRV in the aggregation process, no typical fibrillar amyloids were detected after 144 h of incubation under amyloid forming conditions (Fig. 3E, c). Similarly we have analyzed the inhibitory effect of GQTYVLPG peptide by ThT binding and TEM microscopy over the time course of 144 h of incubation under amyloid forming conditions. Fig. 4A, shows the effects of GQTYVLPG peptide and the scrambled peptide at molar ratio of 1:40 (α-syn: GQTYVLPG, VGPTQGLY). It should be noted that incubation of the peptide alone at 37 ˚C for 6 days resulted in no increase in the ThT fluorescence intensity (fig. 4B). The incubation of α-syn in the presence of scrambled peptide indicated significant increase in ThT fluorescence intensity and the presence of amyloid fibrils was confirmed by TEM (fig. 4C). The images demonstrate that no fibrils can be visualized by the incubations of α-syn in the presence of the GQTYVLPG peptide even after extending the incubation time to 6 days (Fig. 4C, c). Presumably, GQTYVLPG peptide could interact with α-syn and consequently inhibit fibrillation. It should be noted that lower ratios of α-syn: GQTYVLPG led to incomplete or no prevention of aggregation. In Fig. 5, the efficiency of KISVRV at molar ratios of 1:1, 1:5 and 1:10 and GQTYVLPG at molar ratios of 1:1, 1:5, 1:10 and 1:40 (protein: peptides) in inhibition of amyloid formation after 6 days of incubation under amyloid forming condition have been compared. As mentioned earlier the selected peptide inhibitors existed in anti-parallel β-sheets of two naturally occurring proteins and expected to create dead ends preventing the other α-syn proteins to join. These peptides were against two different regions of α-syn. The KISVRV peptide that could arrest α-syn fibrillation in very low concentration was designed against residues 70 to 75 of α-syn. This sequence is part of the hydrophobic region that according to previous studies it has critical role in α-syn fibrillation process [1, 21, 26-28]. The GQTYVLPG peptide that could inhibit α-syn fibrillation at higher concentration was designed based on residues 46 to 53 of α-
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syn. It has been demonstrated that five identified missense mutations that are involved in the familial form of PD, are located in the amphipathic region (residues 1 to 60) [11, 15, 17-23]. Due to the different role of these regions in aggregation of α-syn, the peptides designed by the same logic have resulted in different results. A variety of small peptides that arrest α-syn aggregation and diminish its toxic effects has already been described. It should be noted that the majority of these peptides merely inhibits the formation of mature fibrils of α-syn and has no or negligible effects on oligomeric species [1, 28, 38, 42, 56]. A β-syn derived peptide has been reported to prevent oligomers and fibrils formation of α-syn [67]. Here in, The KISVRV peptide which inhibited the formation of both the oligomers and insoluble fibrillar aggregates of α-syn exhibits another remarkable property. This peptide is able to remove the oligomers after being produced (as shown below). This suggests a therapeutic application of this peptide for PD patients.
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3.3. Disruption α-syn oligomers by KISVRV peptide inhibitor
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To investigate the effect of the KISVRV peptide inhibitor on the progress of amyloid formation starting from monomer, oligomer and fibrils of α-syn, SEC was used to fractionate the aggregation products of α-syn (Fig. 6). As can be seen in Fig. 6, two distinguished peaks were observed that can be related to fibrils and prefibrils (pink line). The oligomers of α-syn formed upon 2 days of incubation were resulted in the appearance of two peaks and a single peak corresponding to monomeric species was observed at t=0 (blue line). The effect of KISVRV peptide on the aggregation process started from monomers, oligomers or fibrils of α-syn was monitored by ThT fluorescence measurements. Interestingly, not only the progress of amyloid fibril formation from oligomeric species was diminished by the peptide inhibitors (KISVRV) but also significant decrease of ThT fluorescence is observed (Fig. 7). This finding suggests the ability of the peptide to dissolve the oligomers of α-syn. Fig. 8 shows the effect of peptide inhibitor at three different concentrations on amyloid fibrils incubated under aggregation condition for 6 days. It can be seen that the peptide could not split the amyloid fibrils and a slight increase in ThT fluorescence is observed over the time. The inhibition of the aggregation process from products of α-syn by peptide inhibitors, suggest the ability of the peptide to dissolve the oligomers of α-syn. To confirm this suggestion SEC of the oligomers incubated under aggregation condition in the presence of the peptide inhibitors at different time points was performed (fig. 9). The chromatogram at t=0 shows the peaks corresponding to oligomers (fig. 9A). After 48 h of incubation, three distinguished peaks were observed. The two peaks according to their elution volumes are corresponding to oligomeric species and the peak eluted at 30 ml is corresponding to monomers of α-syn. The results of SEC upon incubation for 4 days show a sharp peak corresponding to monomers (fig. 9C). These findings which suggest the ability of the peptide to dissolve the oligomers of α-syn are of great importance. In vitro studies have revealed that the
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protofibrils and prefibrillar oligomers, rather than mature fibrils of α-syn, are the pathogenic species in PD. Previous studies showed disruption of cells membranes, mitochondrial depolarization, disrupting microtubules, impairment of protein clearance pathways, oxidative stress, triggering lysosomal leakage and cell death can be increased by toxic oligomeric species of α-syn [24, 40, 68-72]. The correlation between the toxicity of oligomeric α-syn and the pathogenesis of PD inspired the design of peptide inhibitors that dissolve the toxic oligomers and block α-syn aggregation and fibrillation [73]. The presented results clearly demonstrated that the KISVRV peptide was effective not only in blocking the fibrillation of α-syn but also in dissolving oligomers, which may indicate a therapeutic application of this peptide for PD patients.
4. Conclusion
Acknowledgments
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In this research biomimetic design of two peptides which inhibit α-syn amyloid formation was done by using a new database. The designed peptides (KISVRV and GQTYVLPG) were experimentally proved to be efficient in blocking the formation of oligomers and fibrils of α-syn. Another remarkable property of the KISVRV peptide was the ability of dissolving the preformed oligomers which have been suggested to be highly cytotoxic. The developed method does not require any sophisticated instrument and seems to be cost effective, fast and straight forward compared to other strategies like spanning the whole sequence of α-syn or analyzing the proteolytic fragments of it. This work has demonstrated the potential of our approach for design of peptide inhibitors for amyloidogenic proteins aggregation.
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This work was supported by a grant from research council of Tarbiat Modares University.
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PD Parkinson̕ s disease α-syn α-synuclein ProDA Protein linker design assistant PDB Protein data bank IPTG Isopropyl β-D-1-thiogalactopyranoside Thioflavin T ThT PBS Phosphate-buffered saline LBs Lewy bodies LNs Lewy neurites TEM Transmission electron microscopy
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Legend to Figures Fig. 1. The secondary structure of (A) Residues 70 to 75 (VVTGVT) and (B) Residues 46 to 53 (EGVVHGVA). These images were generated by Yasara.
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Fig. 2. The secondary structures of (A) Residues 71 to 76 of 1KMT and the adjacent strand (residues 113 to 118) and (B) residues 85 to 91 of 3I0Y and the adjacent strand (residues 103 to 110).
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Fig. 3. Effect of KISVRV peptide inhibitor on the amyloid fibril formation of α-syn. The time courses of α-syn aggregation alone and in the presence of KISVRV and VSRKIV were monitored by ThT Fluorescence measurements (A) 1:1 (α-syn: KISVRV, VSRKIV); (B): 1:5 (αsyn: KISVRV, VSRKIV); (C) 1:10 (α-syn: KISVRV, VSRKIV); (D) KISVRV alone and (E) TEM images of α-syn in the presence and absence of KISVRV and VSRKIV after 144 h of incubation (a) at molar ratio of 1:0 α-syn: peptide; (b) at molar ratio of 1:10 (α-syn: VSRKIV); (c) at molar ratio of 1:10 (α-syn: KISVRV). Scale bar represents 12 nm for all micrographs.
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Fig. 4. Effect of GQTYVLPG (peptide inhibitor) on the amyloid fibril formation of α-syn: The time course of α-syn aggregation and its mixtures with GQTYVLPG and VGPTQGLY was monitored by ThT Fluorescence aasay analysis (A) at a molar ratio of 1:140 (α-syn: GQTYVLPG, VGPTQGLY); (B) GQTYVLPG alone and (C) TEM images of α-syn in the presence and absence of GQTYVLPG and VGPTQGLY after 144 h of incubation (a) no additive 75µM α-syn; (b) at molar ratio of 1:40 (α-syn: VGPTQGLY); (c) at molar ratio of 1:40 (α-syn: GQTYVLPG). Scale bar represents 10 nm for all micrographs. Fig. 5. The effect of peptides on α-syn aggregation: Samples were incubated for 6 days at 37 ˚C with shaking at 850 RPM. The amyloid fibril formation was measured by ThT fluorescence. Each experiment was conducted three times.*p<0.05; **p<0.01; ***p<0.001, Unpaired t-test.
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Fig. 6. Size exclusion chromatography of the aggregation products of α-syn at different time points: (pink line) SEC upon incubation of α-syn for 6 days, showing distinguished peaks corresponding to fibrils. (green line ) SEC upon incubation of α-syn for 2 days, showing distinct peaks corresponding to oligomers. (Blue line ) SEC at t=0, showing sharp peak corresponding to monomers of α-syn. The arrows indicate elution volumes of the protein standards [bovine serum albumin (BSA), 66 kDa (1); ovalbumin (OA), 44 kDa (2); chymotrypsinogen A, (CTA) 27.5 kDa (3); myoglobin (MG), 17.5 kDa (4); ubiquitin (ub) 8 kDa (5)]. Fig. 7. Effect of KISVRV peptide inhibitor on the amyloid fibril formation starting from α-syn oligomers: The time course of oligomers aggregation was monitored by ThT Fluorescence enhancement assay (A) 1:1 (α-syn: KISVRV, VSRKIV); (B) 1:5 (α-syn: KISVRV, VSRKIV) and (C) 1:10 (α-syn: KISVRV, VSRKIV).
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Fig. 8. Effect of KISVRV peptide inhibitor on the amyloid fibrils of α-syn as monitored by ThT Fluorescence enhancement assay (A) 1:1 (α-syn: KISVRV, VSRKIV); (B) 1:5 (α-syn: KISVRV, VSRKIV) and (C): 1:10 (α-syn: KISVRV, VSRKIV).
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Fig. 9. Size exclusion chromatography of α-syn oligomers: At molar ratio of 1:1 (α-syn: KISVRV) (A) SEC at t=0, showing clear peaks corresponding to oligomers; (B) SEC upon incubation for 2 days, showing distinguished peaks corresponding to oligomers and monomers of α-syn and (C) SEC upon 4 days of incubation, showing a sharp peak corresponding to monomers. The arrows indicate elution volumes of the standard proteins [ bovine serum albumin (BSA), 66 kDa (1); ovalbumin (OA), 44 kDa (2); chymotrypsinogen A, (CTA) 27.5 kDa (3); myoglobin (MG), 17.5 kDa (4); ubiquitin (ub) 8 kDa (5)].
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Legend of tables
Figures
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Fig. 1.
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Table. 1. The propensity of α-helix and β-sheet structures for residues 70 to75 and 46 to 53 of α-syn [75].
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Fig. 5.
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Tables Table. 1.
Helix 151 57 106 106 100 57 106 142 106 106 83 57 106 83
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Glu Gly Val Val His Gly Val Ala Val Val Thr Gly Val Thr
propensity
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46 47 48 49 50 51 52 53 70 71 72 73 74 75
Amino acid
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Beta 37 75 170 170 87 75 170 83 170 170 119 75 170 119
S0003-9861(17)30505-2
DOI:
10.1016/j.abb.2017.09.015
Reference:
YABBI 7562
To appear in:
Archives of Biochemistry and Biophysics
Received Date: 24 July 2017 Revised Date:
5 September 2017
Accepted Date: 20 September 2017
Please cite this article as: N. Rezaeian, N. Shirvanizadeh, S. Mohammadi, M. Nikkhah, S.S. Arab, The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation, Archives of Biochemistry and Biophysics (2017), doi: 10.1016/j.abb.2017.09.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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The inhibitory effects of biomimetically designed peptides on α-synuclein aggregation Niloofar. Rezaeian a, Niloofar. Shirvanizadeh, Soheila. Mohammadi, Maryam. Nikkhah b*, Seyed Shahriar. Arab c* Department of Bioscience, University of Azad, Tehran, Iran
b
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat modares
University, Tehran, Iran c
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a
Department of Biophysics, Faculty of Biological Sciences, University of Tarbiat modares,
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Tehran, Iran
Correspondence: Maryam Nikkhah
Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran
Fax: +98 21 82884718
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Tel: +98 21 82884734
Email: [email protected] Seyed Shahriar Arab
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Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran
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Tel: +98 21 82884717
Fax: +98 21 82884718
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ABSTRACT
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Parkinson̕ s disease is characterized by accumulation of inclusion bodies in dopaminergic neurons, where insoluble and fibrillar α-synuclein makes up the major component of these inclusion bodies. So far, several strategies have been applied in order to suppress α-synuclein aggregation and toxicity in Parkinson̕ s disease. In the present study, a new datebase has been established by segmentation of all the proteins deposited in protein Data Bank. The database data
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base was searched for the sequences which adopt β structure and are identical or very similar to the regions of α-synuclein which are involved in aggregation. The adjacent β strands of the found
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sequences were chosen as the peptide inhibitors of α-synuclein aggregation. Two of the predicted peptides, namely KISVRV and GQTYVLPG, were experimentally proved to be efficient in suppressing aggregation of α-synuclein in vitro. Moreover, KISVRV exhibited the ability to disrupt oligomers of α-syn which are assumed to be the pathogenic species in Parkinson’s disease.
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Keywords: peptide inhibitor; amyloid fibril formation; α-synuclein; parkinson̕ s disease
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1. Introduction
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Many neurodegenerative diseases are characterized by the formation and accumulation of misfolded proteins into amyloid oligomers or fibrils [1-3]. The prevalence of neurodegenerative diseases is growing with a rapidly aging population. Parkinson’s disease (PD) which is the second most common neurodegenerative disease manifests with movement disorders such as resting tremor, muscular rigidity, bradykinesia, and postural instability [3-7] . The main problem of finding cures for PD is that the symptoms only manifest when major part of dopaminergic neurons in the substantia nigra pars compacta has been lost in the midbrain [8-10]. The two pathological hallmarks of PD are the loss of dopaminergic neurons and the presence of intracellular protein aggregation of α-synuclein (α-syn): Lewy bodies (LBs) and Lewy neurites (LNs) in the substantia nigra region of the brain in PD patients [11-16]. α-syn is a 140-amino acid intrinsically disordered protein which is abundant in the human brain, found mainly at the tips of neurons in presynaptic terminals [3-5, 11, 15]. α-syn primary structure is usually divided into three distinct regions: 1. Residues 1-60: an amphipathic N-terminal region in which five missense mutations have been identified (A30P, E46K, H50Q, G51D and A53T) that are involved in the familial form of PD. 2. Residues 61-95: A central hydrophobic region which is involved in protein aggregation. 3. Residues 96-140: a highly acidic and proline-rich region which has no distinct structural propensity [11, 15, 17-23]. During the course of aggregation, α-syn undergoes conformational changes from disordered monomers to dimers and then to oligomers that may assemble into protofibril [3, 24]. The aggregated proteins in the fibrils have the characteristic conformation of β-sheets that are packed perpendicular to the fiber axis. Several studies have identified key regions within the α-syn sequence that are involved in protein aggregation [3, 25]. A central hydrophobic region of α-syn (61–95 residues), has a high propensity for formation of β-sheet rich structures, and is known to drive the amyloid fibril formation. Within this core region, residues 66-74, 68-78 and 71-82 were identified as the key initiating sequences in amyloid fibril formation [1, 21, 26-28]. In addition, the five missense point mutations in the amphipathic N-terminus region of α-syn (1-60 residues) have been shown to accelerate the rate of α-Syn fibrillation and have been associated with pathogenesis of PD [29-37]. The relation between α-syn mutations and the altered kinetics of fibrillation has inspired the design of peptide inhibitors that block α-syn aggregation and fibrillation [1, 28, 30, 32, 38-44]. Considering the cytotoxicity of the oligomers which form during the process of amyloid formation, compounds that inhibit the formation of fibrils and/or oligomers might lead to appropriate therapeutic approaches for PD treatment [40, 42, 45-52]. Inhibition of amyloid fibril formation by several naturally occurring compounds has already been proven. These compounds usually affect more than one protein. Some polyphenolic compounds suppress the formation of amyloid fibrils but their effects are rather non-specific. For example, curcumin can inhibit the aggregation of multiple amyloidogenic proteins including Aβ, α-syn,
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egg-white lysozyme, transthyretin (TTR) and human islet amyloid polypeptide (hIAPP) [53]. Compared to the chemical drugs, peptide drugs offer several advantages, such as high specificity, minimization of drug-drug interactions, low accumulation in tissues, low toxicity and great biological diversity. In spite of the mentioned advantages, the application of therapeutic peptides has been limited by their rapid decomposition in biological fluids and tissues and their low bioavailability [54]. However, there are strategies to increase the stability of the peptids against proteolytic degradation like replacing some or all of the L-amino acids with D-amino acids and chemical modifications of the constituting amino acids [55]. By exploiting different strategies several peptides have been found to prevent α-syn aggregation; Some examples are as followings: designing an N-Methylated peptide inhibitor of α-syn aggregation guided by SolidState NMR [1], synthesizing a library of overlapping 7-mer peptides spanning the entire α-syn sequence [28], design of a hexapeptide based on the mutations (V66S, V66P, T72P, V74E, V74G, and T75P) [38, 56], designing the PQQ-modified α-syn36–46 peptide by analyzing the peptide sequences of unmodified α-Syn proteolytic products [48], and screening peptide ligands against α-syn by in silico panning [56].
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1. Materials and Methods
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In this study we have developed a novel database (ProDA) which is able to search for any peptide with defined properties in PDB. The database was searched to find sequences which fulfill two requirements: first, being the same or similar to two regions of α-syn which are involved in aggregation and second, existing as a strand of a beta sheet. The adjacent strands of the found peptides were further assessed and among them, considering some structural requirements, two peptides were chosen as the inhibitors. The biomimetically designed peptides not only could arrest the aggregation of α-syn efficiently but also are able to disrupt oligomers of α-syn which are critically involved in the pathogenesis of PD.
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2.1.Design of the peptides
ProDA database was generated by fragmentation of all the deposited proteins in PDB into fragments of 4-20 amino acids in length. The deposited peptides in ProDA could be searched for their length, secondary structures, the number and type of amino acids, hydrophobicity and accessible surface area. This database offers many applications, including peptide design. We aimed to design peptides which are able to bind residues 70 to 75 and 46 to 53 of α-syn. We have searched through ProDA for protein fragments that possess only β structure and have identical or very similar sequences to the target regions of α-syn. The search resulted in finding several fragments involved in parallel and antiparallel β-sheets. The fragments with antiparallel β-sheet were then selected. In antiparallel β-sheet the hydrogen bonds are perpendicular to the strands, and narrowly spaced bond pairs alternate with widely spaced pairs. In addition, the successive β-
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strands alternate directions so that the N-terminus of one strand is adjacent to the C-terminus of the next. This arrangement provides the strongest inter-strand stability. Finally, the adjacent strands of the selected fragments were taken as the peptide inhibitors. The scrambled peptides which have the same amino acid composition and the same beta propensity as peptide inhibitors have been considered as negative controls. 2.2. protein expression and purification
2.3. Amyloid fibril formation
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The α-syn protein was expressed in Escherichia coli (E.Coli C41). α-syn expression was induced by the addition of 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). After induction for 4 hours, the bacteria were harvested by centrifugation (6000 RPM, 20 min, 4 ˚C). The cells were disrupted using a UP400S Ultrasonic instrument (Hielscer Uiltrasoincs GmbH, Teltow, Germany), and the supernatant was collected by centrifugation (13000 RPM, 30 min, 4 ˚C). The supernatant was loaded onto a His 60 Ni Superflow Resin (Clontech, Takara Bio Company) column and eluted with elution buffer (imidazole 300mM, NaCl 300mM, NaH2PO4 50Mm, pH 8). The eluted fractions were analyzed by SDS-PAGE, and the fractions containing αsyn protein were dialyzed against phosphate-buffered saline (PBS) (1.76 mM KH2PO4, 10mM Na2HPO4, 2.7 mM KCl, 137 mM NaCl, pH 7.4). The protein concentration was determined by a UV-Visible spectrophotometer ( PerkinElmer, Lambda 25 ).
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Purified wild-type α-syn was incubated at final concentration of 75µM, either alone or with peptide inhibitors and the scrambled peptides, (synthesized by Mimotopes Corporation Australia) at a molar ratio of 1:1, 1:5 and 1:10 for (α-syn: KISVRV, VSRKIV) and 1:1, 1:5, 1:10 and 1:40 for (α-syn: GQTYVLPG, VGPTQGLY) with 0.02% NaN3 as an antiseptic agent at 37 ˚C in a Turbo Thermo Shaker (TMS-200, China) with continuous agitation at 850 RPM. Amyloid fibril formation was monitored by fluorescence enhancement of Thioflavin T (ThT) solution (Sigma-Aldrich Company): Aliquots of 5µL were removed from the incubated samples and added to 95 µL of 25 µM ThT in PBS buffer. Fluorescence emission was measured with excitation wavelength at 440 nm and emission at 485 nm using a Cytation3 Cell Imaging MultiMode Reader (BioTek Instruments, Winooski, VT). Each sample was assayed in triplicate, and each experiment was repeated three times. 2.4. The effects of the peptide inhibitor on different steps of the fibrillation pathway Monomers, oligomers and fibrils of α-syn were isolated by size exclusion chromatography. Purified α-syn at final concentration of 75µM was incubated at 37 ˚C with continuous mixing at 850 RPM. The samples at t=0, 48 and 144 hour of incubation were taken and loaded onto a Sephadex G-75 column (0.7×45 cm) in PBS buffer pH 7.4. One milliliter fractions were collected and assayed for protein content by measuring the absorbance at 280 nm. It should be
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noted that at first, the column was calibrated using a series of molecular weight standards: bovine serum albumin (BSA) 66 kDa, ovalbumin (OA) 44 kDa, chymotrypsinogen A (CTA) 27.5 kDa, myoglobin (MG) 17.5 kDa, and ubiquitin (ub) 8 kDa. The purified monomer, oligomer and fibrils of α-syn were incubated alone or in the presence of peptide inhibitor and/or scrambled peptide (molar ratio of KISVRV, VSRKIV: α-syn 1:1, 5:1, 10:1) for 6 days with continuous shaking (850 RPM) at 37 ˚C. The presence of amyloid fibrils was estimated by ThT fluorescence every 24 hour. 2.5. Assessment of the capability of the peptide inhibitor to split oligomers
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Isolated α-syn oligomers by SEC were incubated alone or in the presence of peptide inhibitor and scrambled peptide (molar ratio of KISVRV, VSRKIV: α-syn 1:1, 5:1, 10:1) under continuous shaking (850 RPM) at 37 ˚C. The amyloid fibril formation was monitored by ThT fluorescence measurements. Samples at t=0, 48 and 96 hour during incubation were taken and loaded onto the SEC column. Then, one milliliter fractions were collected and protein absorption was monitored at 280 nm.
2.6. Electron microscopy
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The α-syn protein (75µM) either alone or with peptide inhibitors and theirs coresponding scrambled peptides at a molar ratio of 1:10 (α-syn: KISVRV, VSRKIV) and 1:40 (α-syn: GQTYVLPG, VGPTQGLY) with 0.02% NaN3 were incubated for 144 h at 37 ˚C under continuous shaking (850 RPM). 10µl of the samples were adsorbed onto formvar carbon coated grid Cu Mesh 300 for 2 min. The excess fluid was drained with filter paper and the samples were stained for 90 s with 2% uranyl acetate. The grid was air-dried and examined with a Zeiss EM10C electron microscope at 80 KV.
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3. Results and Discussion: Considerable evidence demonstrates that the conversion of soluble monomeric α-syn to insoluble amyloid oligomers or fibrils in neuronal cells is the main cause of PD [3,11-16,28,32]. Aggregation is the significant process where the α-syn protein loses its function and gains toxicity. This suggests that prevention of α-syn fibrillation may be an appropriate therapeutic strategy for PD. In this study, we attempted to design peptides that can specifically interact with the regions of α-syn that are responsible for its self-aggregation. Here, we have utilized the new ProDA database for design of peptides targeting two different regions of α-syn. The potency of the designed peptide inhibitors were analyzed in vitro.
3.1. Biomimetic design of peptide inhibitors
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A central hydrophobic region of α-syn (61–95 residues) has been identified to have a critical role in aggregation and formation of amyloid fibrils [1, 21, 26-28]. The occurrence of 12 amino acid sequence in the hydrophobic region (residues 71–82) has been reported to play an indispensable role in oligomerization and formation of amyloid fibrils [21, 57-59]. Actually, many researchers have used this region as a starting point for the design of inhibitors. Additionally, among the known point mutations in the α-syn gene associated with early onset of PD, four (E46K, H50Q, G51D, A53T) are located between residues 46 and 53. This region is clearly important in modulating amyloid formation such that toxicity associated with the α-syn protein becomes increased with the mutations that decrease α-helix propensity or increase βsheet propensity, and either increase the rate or the number of oligomers that are formed [29, 6062]. Given the interests in the above mentioned regions, in this study, we have chosen residues 70 to 75 (VVTGVT) and 46 to 53 (EGVVHGVA) of α-syn for designing the peptide inhibitors. The selected regions of α-syn have the propensity to form β-sheet in spite of their helix structures (Fig. 1A and C and Table 1); these sequences are known as Chameleon sequences [63-66]. At first we, have developed a database called ProDA which is able to search for any peptide with defined properties in PDB. We have searched through ProDA database for identical or similar sequences to target regions of α-syn with anti-parallel β structures (as mentioned in methods) and subsequently selected residues 71 to 76 ( VVTGLT) of 1KMT (RhoGDI) and residues 85 to 91 (YVVHGVY) of 3I0Y (putative polyketide cyclase) (Fig. 2). The adjacent strand of the selected sequence in 1KMT composed of residues 113 to 118 (KISFRV) and the adjacent strand of the selected sequence in 3I0Y composed of residues 103 to 110 (GQTYVLPG) were chosen as the peptide inhibitors.) . As these sequences are located on the edge of the beta sheets, they are expected to interact with the target sequence only from one side. This may offer an advantages in a way that upon the interaction of the peptide with α-syn, a dead end forms that doesn’t permit the beta sheet to extend. To increase the β formation propensity of KISFRV, phenylalanine was replaced by valine which has the highest propensity to form β-sheet among all the amino acids.
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We expect that the designed peptides to be able to bind to aggregation prone regions of α-syn and make deadlocks that do not allow the other α-syn proteins to join and promote amyloid formation. The effect of the peptide inhibitors on amyloids formation were investigated using the ThT fluorescence measurements and Transmission Electron Microscopy (TEM) analysis.
3.2. KISVRV and GQTYVLPG prevent the formation of amyloid fibrils The formation of amyloid fibrils was monitored by the increase in ThT fluorescence. ThT is a fluorescent dye that is widely used to visualize and quantify the presence of misfolded protein aggregates called amyloid. The binding of ThT to β-sheet-rich structures such as those in amyloid aggregates is accompanied by a characteristic increase in the fluorescence intensity in the proximity of 485 nm. The process of α-syn aggregation exhibits three distinct phases including lag phase, rapid growth phase and stationary phase. In order to characterize the
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effect of KISVRV and VSRKIV on α-syn aggregation, we tested three different concentrations of peptide inhibitors and scrambled peptides. Fig. 3, shows the effect of VSRKIV on the amyloid fibril formation at three different concentrations (molar ratio of 1:1, 1:5, and 1:10 of α-syn: VSRKIV). By incubation of α-syn under aggregation condition, after 24 h of lag time, a rapid increase in fluorescence intensity was observed, suggesting the accumulation of α-syn amyloid fibrils. After 144 h of incubation at 37 ˚C, the maximum value of the fluorescence intensity was observed. Additionally, Fig. 3A, B and C, show that the scrambled peptide (VSRKIV) could not affect the kinetics of amyloid fibrils formation and the intensity of fluorescence at 485nm has been increased. The inhibitory effect of KISVRV peptide on the aggregation of α-syn was demonstrated at all the three different molar ratios of α-syn: KISVRV. Fig. 3, demonstrates that after 144 h of incubation in the presence of KISVRV, no increase in the ThT fluorescence of αsyn was observed. This observation suggests that KISVRV could arrest amyloid fibril formation. It should be noted that the incubation of peptide inhibitor alone at 37 ˚C for 6 days has not resulted in any increase in the ThT fluorescence (fig. 3D). TEM observation also revealed the amyloid fibrils formation of α-syn, incubated under aggregation conditions alone (Fig. 3E, a) or in the presence of the scrambled peptide VSRKIV after 144h of incubation (Fig. 3E, b). By the presence of KISVRV in the aggregation process, no typical fibrillar amyloids were detected after 144 h of incubation under amyloid forming conditions (Fig. 3E, c). Similarly we have analyzed the inhibitory effect of GQTYVLPG peptide by ThT binding and TEM microscopy over the time course of 144 h of incubation under amyloid forming conditions. Fig. 4A, shows the effects of GQTYVLPG peptide and the scrambled peptide at molar ratio of 1:40 (α-syn: GQTYVLPG, VGPTQGLY). It should be noted that incubation of the peptide alone at 37 ˚C for 6 days resulted in no increase in the ThT fluorescence intensity (fig. 4B). The incubation of α-syn in the presence of scrambled peptide indicated significant increase in ThT fluorescence intensity and the presence of amyloid fibrils was confirmed by TEM (fig. 4C). The images demonstrate that no fibrils can be visualized by the incubations of α-syn in the presence of the GQTYVLPG peptide even after extending the incubation time to 6 days (Fig. 4C, c). Presumably, GQTYVLPG peptide could interact with α-syn and consequently inhibit fibrillation. It should be noted that lower ratios of α-syn: GQTYVLPG led to incomplete or no prevention of aggregation. In Fig. 5, the efficiency of KISVRV at molar ratios of 1:1, 1:5 and 1:10 and GQTYVLPG at molar ratios of 1:1, 1:5, 1:10 and 1:40 (protein: peptides) in inhibition of amyloid formation after 6 days of incubation under amyloid forming condition have been compared. As mentioned earlier the selected peptide inhibitors existed in anti-parallel β-sheets of two naturally occurring proteins and expected to create dead ends preventing the other α-syn proteins to join. These peptides were against two different regions of α-syn. The KISVRV peptide that could arrest α-syn fibrillation in very low concentration was designed against residues 70 to 75 of α-syn. This sequence is part of the hydrophobic region that according to previous studies it has critical role in α-syn fibrillation process [1, 21, 26-28]. The GQTYVLPG peptide that could inhibit α-syn fibrillation at higher concentration was designed based on residues 46 to 53 of α-
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syn. It has been demonstrated that five identified missense mutations that are involved in the familial form of PD, are located in the amphipathic region (residues 1 to 60) [11, 15, 17-23]. Due to the different role of these regions in aggregation of α-syn, the peptides designed by the same logic have resulted in different results. A variety of small peptides that arrest α-syn aggregation and diminish its toxic effects has already been described. It should be noted that the majority of these peptides merely inhibits the formation of mature fibrils of α-syn and has no or negligible effects on oligomeric species [1, 28, 38, 42, 56]. A β-syn derived peptide has been reported to prevent oligomers and fibrils formation of α-syn [67]. Here in, The KISVRV peptide which inhibited the formation of both the oligomers and insoluble fibrillar aggregates of α-syn exhibits another remarkable property. This peptide is able to remove the oligomers after being produced (as shown below). This suggests a therapeutic application of this peptide for PD patients.
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3.3. Disruption α-syn oligomers by KISVRV peptide inhibitor
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To investigate the effect of the KISVRV peptide inhibitor on the progress of amyloid formation starting from monomer, oligomer and fibrils of α-syn, SEC was used to fractionate the aggregation products of α-syn (Fig. 6). As can be seen in Fig. 6, two distinguished peaks were observed that can be related to fibrils and prefibrils (pink line). The oligomers of α-syn formed upon 2 days of incubation were resulted in the appearance of two peaks and a single peak corresponding to monomeric species was observed at t=0 (blue line). The effect of KISVRV peptide on the aggregation process started from monomers, oligomers or fibrils of α-syn was monitored by ThT fluorescence measurements. Interestingly, not only the progress of amyloid fibril formation from oligomeric species was diminished by the peptide inhibitors (KISVRV) but also significant decrease of ThT fluorescence is observed (Fig. 7). This finding suggests the ability of the peptide to dissolve the oligomers of α-syn. Fig. 8 shows the effect of peptide inhibitor at three different concentrations on amyloid fibrils incubated under aggregation condition for 6 days. It can be seen that the peptide could not split the amyloid fibrils and a slight increase in ThT fluorescence is observed over the time. The inhibition of the aggregation process from products of α-syn by peptide inhibitors, suggest the ability of the peptide to dissolve the oligomers of α-syn. To confirm this suggestion SEC of the oligomers incubated under aggregation condition in the presence of the peptide inhibitors at different time points was performed (fig. 9). The chromatogram at t=0 shows the peaks corresponding to oligomers (fig. 9A). After 48 h of incubation, three distinguished peaks were observed. The two peaks according to their elution volumes are corresponding to oligomeric species and the peak eluted at 30 ml is corresponding to monomers of α-syn. The results of SEC upon incubation for 4 days show a sharp peak corresponding to monomers (fig. 9C). These findings which suggest the ability of the peptide to dissolve the oligomers of α-syn are of great importance. In vitro studies have revealed that the
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protofibrils and prefibrillar oligomers, rather than mature fibrils of α-syn, are the pathogenic species in PD. Previous studies showed disruption of cells membranes, mitochondrial depolarization, disrupting microtubules, impairment of protein clearance pathways, oxidative stress, triggering lysosomal leakage and cell death can be increased by toxic oligomeric species of α-syn [24, 40, 68-72]. The correlation between the toxicity of oligomeric α-syn and the pathogenesis of PD inspired the design of peptide inhibitors that dissolve the toxic oligomers and block α-syn aggregation and fibrillation [73]. The presented results clearly demonstrated that the KISVRV peptide was effective not only in blocking the fibrillation of α-syn but also in dissolving oligomers, which may indicate a therapeutic application of this peptide for PD patients.
4. Conclusion
Acknowledgments
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In this research biomimetic design of two peptides which inhibit α-syn amyloid formation was done by using a new database. The designed peptides (KISVRV and GQTYVLPG) were experimentally proved to be efficient in blocking the formation of oligomers and fibrils of α-syn. Another remarkable property of the KISVRV peptide was the ability of dissolving the preformed oligomers which have been suggested to be highly cytotoxic. The developed method does not require any sophisticated instrument and seems to be cost effective, fast and straight forward compared to other strategies like spanning the whole sequence of α-syn or analyzing the proteolytic fragments of it. This work has demonstrated the potential of our approach for design of peptide inhibitors for amyloidogenic proteins aggregation.
Abbraviations
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This work was supported by a grant from research council of Tarbiat Modares University.
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PD Parkinson̕ s disease α-syn α-synuclein ProDA Protein linker design assistant PDB Protein data bank IPTG Isopropyl β-D-1-thiogalactopyranoside Thioflavin T ThT PBS Phosphate-buffered saline LBs Lewy bodies LNs Lewy neurites TEM Transmission electron microscopy
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Legend to Figures Fig. 1. The secondary structure of (A) Residues 70 to 75 (VVTGVT) and (B) Residues 46 to 53 (EGVVHGVA). These images were generated by Yasara.
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Fig. 2. The secondary structures of (A) Residues 71 to 76 of 1KMT and the adjacent strand (residues 113 to 118) and (B) residues 85 to 91 of 3I0Y and the adjacent strand (residues 103 to 110).
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Fig. 3. Effect of KISVRV peptide inhibitor on the amyloid fibril formation of α-syn. The time courses of α-syn aggregation alone and in the presence of KISVRV and VSRKIV were monitored by ThT Fluorescence measurements (A) 1:1 (α-syn: KISVRV, VSRKIV); (B): 1:5 (αsyn: KISVRV, VSRKIV); (C) 1:10 (α-syn: KISVRV, VSRKIV); (D) KISVRV alone and (E) TEM images of α-syn in the presence and absence of KISVRV and VSRKIV after 144 h of incubation (a) at molar ratio of 1:0 α-syn: peptide; (b) at molar ratio of 1:10 (α-syn: VSRKIV); (c) at molar ratio of 1:10 (α-syn: KISVRV). Scale bar represents 12 nm for all micrographs.
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Fig. 4. Effect of GQTYVLPG (peptide inhibitor) on the amyloid fibril formation of α-syn: The time course of α-syn aggregation and its mixtures with GQTYVLPG and VGPTQGLY was monitored by ThT Fluorescence aasay analysis (A) at a molar ratio of 1:140 (α-syn: GQTYVLPG, VGPTQGLY); (B) GQTYVLPG alone and (C) TEM images of α-syn in the presence and absence of GQTYVLPG and VGPTQGLY after 144 h of incubation (a) no additive 75µM α-syn; (b) at molar ratio of 1:40 (α-syn: VGPTQGLY); (c) at molar ratio of 1:40 (α-syn: GQTYVLPG). Scale bar represents 10 nm for all micrographs. Fig. 5. The effect of peptides on α-syn aggregation: Samples were incubated for 6 days at 37 ˚C with shaking at 850 RPM. The amyloid fibril formation was measured by ThT fluorescence. Each experiment was conducted three times.*p<0.05; **p<0.01; ***p<0.001, Unpaired t-test.
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Fig. 6. Size exclusion chromatography of the aggregation products of α-syn at different time points: (pink line) SEC upon incubation of α-syn for 6 days, showing distinguished peaks corresponding to fibrils. (green line ) SEC upon incubation of α-syn for 2 days, showing distinct peaks corresponding to oligomers. (Blue line ) SEC at t=0, showing sharp peak corresponding to monomers of α-syn. The arrows indicate elution volumes of the protein standards [bovine serum albumin (BSA), 66 kDa (1); ovalbumin (OA), 44 kDa (2); chymotrypsinogen A, (CTA) 27.5 kDa (3); myoglobin (MG), 17.5 kDa (4); ubiquitin (ub) 8 kDa (5)]. Fig. 7. Effect of KISVRV peptide inhibitor on the amyloid fibril formation starting from α-syn oligomers: The time course of oligomers aggregation was monitored by ThT Fluorescence enhancement assay (A) 1:1 (α-syn: KISVRV, VSRKIV); (B) 1:5 (α-syn: KISVRV, VSRKIV) and (C) 1:10 (α-syn: KISVRV, VSRKIV).
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Fig. 8. Effect of KISVRV peptide inhibitor on the amyloid fibrils of α-syn as monitored by ThT Fluorescence enhancement assay (A) 1:1 (α-syn: KISVRV, VSRKIV); (B) 1:5 (α-syn: KISVRV, VSRKIV) and (C): 1:10 (α-syn: KISVRV, VSRKIV).
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Fig. 9. Size exclusion chromatography of α-syn oligomers: At molar ratio of 1:1 (α-syn: KISVRV) (A) SEC at t=0, showing clear peaks corresponding to oligomers; (B) SEC upon incubation for 2 days, showing distinguished peaks corresponding to oligomers and monomers of α-syn and (C) SEC upon 4 days of incubation, showing a sharp peak corresponding to monomers. The arrows indicate elution volumes of the standard proteins [ bovine serum albumin (BSA), 66 kDa (1); ovalbumin (OA), 44 kDa (2); chymotrypsinogen A, (CTA) 27.5 kDa (3); myoglobin (MG), 17.5 kDa (4); ubiquitin (ub) 8 kDa (5)].
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Legend of tables
Figures
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Fig. 1.
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Table. 1. The propensity of α-helix and β-sheet structures for residues 70 to75 and 46 to 53 of α-syn [75].
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Tables Table. 1.
Helix 151 57 106 106 100 57 106 142 106 106 83 57 106 83
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Glu Gly Val Val His Gly Val Ala Val Val Thr Gly Val Thr
propensity
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46 47 48 49 50 51 52 53 70 71 72 73 74 75
Amino acid
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#
Beta 37 75 170 170 87 75 170 83 170 170 119 75 170 119