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α-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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a-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein Maki Shirasaka a, Kazuo Kuwata a, b, Ryo Honda a, * a b
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan Department of Gene and Development, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 September 2019 Accepted 14 October 2019 Available online xxx
Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as a-synuclein (aS) in Parkinson’s disease, amyloid-b in Alzheimer’s disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show aS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric aS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between aS and monomeric PrP nor fibrillar PrP. These results suggested that aS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric aS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of aS against PrP amyloidogenesis, suggesting a possible involvement of aS in the pathology of TSEs. © 2019 Elsevier Inc. All rights reserved.
1. Introduction Amyloid formation is a key pathological event in protein misfolding diseases, by which soluble, typically monomeric, proteins are converted into insoluble, filamentous, and toxic aggregates that deposit in tissues [1]. The amyloid deposits are composed mainly of a specific disease-associated amyloidogenic protein, such as amyloid-b (Ab) in Alzheimer’s disease, a-synuclein (aS) in Parkinson’s disease, and prion protein (PrP) in transmissible spongiform encephalopathy. This prominent pathological feature and other experimental evidence led to the prevailing view that amyloid formation occurs by misfolding of a single defined amyloidogenic protein. However, recent evidence has indicated that several amyloidogenic proteins can interact with a different disease-associated protein to dramatically impact on the development of amyloid deposits [2]. For example, one of the authors recently reported that Ab accelerates amyloidogenesis of many amyloidogenic proteins [3]. Cross-seeding is also a prominent example of the cross-
* Corresponding author. E-mail address: [email protected] (R. Honda).
interactions, by which an amyloid fibril composed of one specific protein can promote amyloidogenesis of a different amyloidogenic protein [4]. Thus, the mechanism of amyloid formation can be elucidated by studying cross-interactions between heterologous amyloidogenic proteins. The goal of this study is to examine if and how aS would affect amyloidogenesis of PrP, in particular in the native (monomeric) state. aS in a fibrillar state was recently shown to accelerate misfolding of PrP and propagate transmissible spongiform encephalopathy (TSE) through cross-seeding [5]. Direct interaction between fibrillar aS and PrP was confirmed by a different group using surface plasmon resonance (SPR) [6]. Moreover, oligomeric aS was also recently shown to interact with PrP to induce cognitive failure in Parkinson’s disease [7], although controversy remains [8]. These results clearly demonstrate the close link between PrP and aggregated aS (i.e., oligomeric aS and fibrillar aS); however, it remains elusive how monomeric aS would affect misfolding of PrP. This question is of importance because the major population of aS exists as a monomer at a high concentration (c.a. 50 mM) in a brain tissue [9], in which PrP coexists with aS. Moreover, monomeric aS has a chaperon-like activity against aggregation of a number of different
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proteins, including insulin, albumin, lysozyme in vitro [10e12], and SNARE in vivo [13]. Here we show that monomeric aS can also suppress amyloid formation of PrP. We also address the mechanism underlying the chaperon-like activity of aS against PrP aggregation. 2. Result 2.1. Expression and purification of monomeric recombinant asynuclein As we previously established a protocol of generating recombinant PrP [14], we initially focused on the generation of recombinant aS. After the expression of aS using a bacterial system, the boiling procedure [15] and reverse-phase chromatography were employed for purification of aS (Fig. 1A). The purified aS was highly homologous, as judged by SDS-PAGE (Fig. 1B), and represented an intrinsically disordered structure characterized by a negative minimum at 195 nm in the near-UV circular dichroism (Fig. 1C). Consistent with previous reports [16], in the presence of SDS micelles, aS underwent a large structural change into an a-helical state characterized by a negative CD minimum at 207 nm. Moreover, in size-exclusion chromatography (SEC), the purified aS was eluted as a single peak corresponding to molecular mass of 60 kDa (Fig. 1D). This size was larger than that predicted from the amino acid sequence (14 kDa), consistent with the intrinsically disordered structure of aS [11]. Overall, the biophysical characteristics of the recombinant aS were in good agreement with those of the previously reported aS.
aS remained in the supernatant fraction and was not cosedimented with fibrillar PrP. Thus, aS can neither interact with fibrillar PrP nor inhibit growing of fibrillar PrP. 2.4. Monomeric or oligomeric a-synuclein did not interact with prion protein monomer The above experiments had implicated that aS suppresses the nucleation step of amyloidogenesis, suggesting two possible scenarios: (a) aS interacts with PrP monomer; and (b) aS interacts with nucleus or other transiently accumulated intermediates, such as partially unfolded states (Fig. 4A). As the latter possibility is difficult
2.2. Monomeric a-synuclein retarded prion protein amyloid formation To examine effects of aS on the amyloidogenesis of PrP, we employed a ThT-fluorescence assay previously described [3]. Here, monomeric PrP was partially unfolded by 3 M guanidine hydrochloride and subjected to continuous agitation in a 96-well plate reader to initiate spontaneous amyloid formation. Formation of amyloid fibrils was monitored by fluorescence of thioflavin-T (ThT), a dye specific for amyloid fibrils [17]. Under the experimental condition, PrP alone represented significant increase in ThT fluorescence after 10 h of lag time, suggesting the formation of amyloid fibrils (Fig. 2A). By contrast, aS alone did not produce any amyloid fibril (data not shown) and remained monomeric even after 72 h of agitation (Fig. 1D). Intriguingly, in the presence of aS, the lag time of PrP amyloid formation was significantly prolonged to 35e50 h (Fig. 2A). In a transmission electron microscopy, the amyloid fibrils generated in the presence of aS were indistinguishable from pure amyloid fibrils of PrP (Fig. 2B). Thus, aS can retard amyloidogenesis of PrP. 2.3. Monomeric a-synuclein did not interact with prion protein amyloid fibrils As the mechanism of amyloid formation is typically described by two consecutive processes, nucleation and growth [1], we next sought to determine which process is retarded by aS. To this end, we performed a seeded-growth experiment, in which pre-formed amyloid fibrils of PrP (termed “seeds”) was mixed with monomeric PrP to initiate rapid growth of the seed fibrils (Fig. 3A). In contrast to the previous result, in the presence of a high concentration of seed fibrils (10%), aS did not suppress amyloid formation of PrP. To further examine an interaction between aS and seed fibrils of PrP, we performed a co-sedimentation assay [18]. Here, fibrillar PrP was co-incubated with aS and sedimented by centrifugation to examine co-sedimentation of aS. As shown in Fig. 3B, all
Fig. 1. Preparations of monomeric and oligomeric a-synuclein (aS). (A) A HPLC profile of an E. coli lysate expressing recombinant aS. aS was eluted with a liner gradient (0e60% acetonitrile in 0.1% TFA, right axis) as a single peak at 56% acetonitrile. (B) A Coomassie-stained SDS-PAGE gel of purified aS showing a homogeneous band at 15 kDa. (C) Far-UV circular dichroism spectra for purified aS without or with 10 mM SDS. aS at a concentration of 10 mM was dissolved in the 10 mM sodium phosphate (pH 7.4). (D) Size exclusion chromatography profiles of aS in monomeric or 4hydroxynonenal (HNE)-modified oligomeric state. A size exclusion chromatography profile of aS after 72 h agitation is also shown.
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Fig. 2. (A) The upper panel represents a representative kinetics of prion protein (PrP) amyloid formation in the presence of 0, 5, and 50 mM monomeric a-synuclein (aS). The reaction was followed by change in Thioflavin-T (ThT) fluorescence. Lower panel represents the half-times (t1/2) of reaction derived from the upper panel. Error bars represent standard deviations of 4 independent trials. *P < 0.01, **P < 0.001, n.s. P 0.01. P-values from unpaired t-test. (B) Representative transmission electron micrographs of (left) pure PrP amyloid fibrils and (right) PrP amyloid fibrils generated in the presence of 50 mM aS (magnification, 25,000; scale bar, 100 nm).
to access using currently available equilibrium methods, we tested the former possibility using SPR. Here, PrP monomer was immobilized on the sensor-chip surface of SPR and flowed over with a solution containing aS. As shown in Fig. 4B, no interaction between monomeric PrP and aS was detected. We also tested the possibility that oligomeric aS, which presents as a minor population in a soluble aS preparation, might interact with PrP monomer [7]. We stabilized aS oligomers using a lipid peroxidation product (4hydroxy-2-nonenal) [19] (Fig. 1C). However, as shown in Fig. 4B, no interaction was detected between PrP and aS oligomers. This result was in sharp contrast to the case of Ab oligomers, which strongly bound to the immobilized PrP (Fig. 4B) [20]. Thus, neither monomeric nor oligomeric aS can interact with PrP. This result indicated that aS interacts with an intermediate transiently accumulated during PrP amyloid formation (scenario (b), in Fig. 4A).
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Fig. 3. (A) A representative kinetics of prion protein (PrP) amyloid formation in the presence of 0, 5, and 50 mM monomeric a-synuclein (aS). The reaction was seeded with 1 or 10% (v/v) pre-formed PrP amyloid fibrils. Error bars represent standard deviations of 3 independent trials *P < 0.01, **P < 0.001, n.s. P 0.01. P-values from unpaired ttest. (B) PrP amyloid fibrils with or without 50 mM aS were centrifuged and separated into supernatant (S) and pellet (P) fractions. Each fraction was analyzed using 15% SDSPAGE gel stained with Coomassie blue. aS did not co-precipitate with PrP amyloid fibrils.
3. Discussion Protein misfolding diseases are a group of devastating disorders characterized by amyloid-like deposits, for which no effective treatment is currently available. Understanding the basic principles of amyloid formation is critical step for rational design of therapeutics. Recent evidence has indicated that a number of amyloidogenic proteins, such as Ab, aS and PrP, cross-interact during the development of amyloid deposits [2,3,5,21,22]. It is therefore crucial to elucidate the mechanism underlying the crossinteractions between amyloidogenic proteins in order to provide novel insights into protein misfolding diseases.
Please cite this article as: M. Shirasaka et al., a-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.120
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amyloid formation, aS might suppress spontaneous generation, but not propagation, of infectious prion. Finally, our result supports the recent notion that aS oligomer did not bind to PrP [8]. Controversy remains whether PrP acts as a receptor of aS oligomer to trigger cognitive failure in Parkinson’s disease pathology [7,8]. Our SPR analysis did not detect interaction between PrP and an aS oligomer that was generated using a lipid peroxidation product (Fig. 4B). Intriguingly, an Ab oligomer, which partly shares structural and functional similarities with the aS oligomer (including extensive b-sheet structure and toxicity [27]), bound to PrP. Thus, PrP has a binding specificity for Ab oligomers, arguing against the notion that PrP serves as a general receptor of bsheet rich conformers [28]. 4. Material and methods 4.1. Expression and purification of recombinant a-synuclein
Fig. 4. (A) Three possible targets of a-synuclein (aS) to suppress prion protein (PrP) amyloid formation; (a) monomeric PrP, (b) nucleus and other transiently accumulated intermediates, and (c) PrP amyloid fibrils. (B) Representative surface plasmon resonance sensorgrams for the interactions between PrP, aS, and Ab oligomer. PrP was immobilized on a carboxymethylated dextran surface at a density of 10,000 RU. aS and Ab in monomeric or oligomer state at 1 mM monomer equivalent concentration was flowed over the surface.
Here we report the first in vitro evidence that aS can suppress amyloidogenesis of PrP. We found aS monomer, but not oligomeric and fibrillar aS, was sufficient to suppress amyloidogenesis of PrP. It should be emphasis that our finding is irrelevant to the crossseeding between fibrillar aS and PrP recently reported, which accelerates amyloidogenesis of PrP [5]. Rather, our finding can be linked to prior research showing the chaperone-like activity of monomeric aS. Monomeric aS can suppress misfolding of a number of different proteins both in vivo and in vitro [10e13,23,24]. Accordingly, our study serves to expand the set of client proteins involved in the chaperon-like activity of aS. It has been hypothesized that monomeric aS binds to the hydrophobic region of partially unfolded client proteins to prevent further structural change toward misfolded aggregates [11,24]. This hypothesis is also consistent with our current observations, in which aS neither bind to fully folded PrP (Fig. 4B) nor fibrillar PrP (Fig. 3), suggesting a possible interaction between aS and a partially unfolded state of PrP (Fig. 4A). Thus, aS chaperone might suppress misfoldings of PrP and other client proteins via a common mechanism. A further study using a sophisticated kinetic method is required to prove the interaction between aS and partially folded proteins [25]. Our results suggest a possible involvement of aS in TSE pathology. It was recently shown that knockout of aS gene did not affect the incubation period of prion propagation in mice brain [26]. This result is consistent with our current in vitro observations, in which aS failed to inhibit PrP aggregation in the presence of a large amount of seed fibrils (Fig. 3A). Since aS chaperon interacts with partially folded proteins and suppresses the nucleation step of
A pT7-7 vector encoding wild-type human aS (residues 1-140), which was created by Paleologou et al. [29], was purchased from Addgene (code 36046). ECOS™ competent E.coli BL21 (DE3) (Nippongene, code 312-06534) was transformed with this construct and cultured in LB medium containing 100 mg/mL ampicillin at 37 C. Protein expression was induced by 1 mM IPTG (Carbosynth, code EI05931) when the culture reached OD ¼ 0.6e0.8. After 4 h of induction period, the bacteria were harvested by centrifugation at 9000 g for 7 min and resuspended with 10 mL lysis buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA, and 1 mM PMSF] per gram weight pellet. The bacteria were then frozen-and-thawed, sonicated for 1 min on ice using an ultrasonic processor with 100% amplitude (VCX 130, SONICS), and centrifuged at 30,000 g for 30 min to remove cell debris. The cell lysate was boiled for 20 min using Hot Plate Stirrer (PC-420, Corning), centrifuged at 30,000 g for 30 min to remove aggregated proteins [15]. The supernatant enriched with aS was supplemented with 1e2% streptomycin sulfate (Nacalai Tesqu, code 32237-72), incubated for 30 min on ice, and centrifuged at 30,000 g for 30 min to remove nuclei acids [30]. The supernatant was finally applied to a COSMOSIL 5C4-AR-300 reverse-phase HPLC column (Nacalai Tesque, code 38048-01) equipped with AKTApurifier (GE healthcare), and eluted with a linear gradient of 0e60% AcCN in 0.1% TFA [16]. A fraction enriched with aS (approx. 56% AcCN) was collected, lyophilized, and dialyzed extensively against MillQ water. The dialyzed solution was lyophilized again and stored at 20 C until use. The purified proteins were initially dissolved with MillQ water or 6 M guanidine hydrochloride (GuHCl) at a concentration of 100 mM. The protein concentration was determined by UV absorbance using an extinction coefficient of 5140 cm1M1 at 280 nm. 4.2. Expression and purification of prion protein A recombinant human PrP (residues 23-230) was expressed using an E.coli expression system and purified to homogeneity according to the previously published protocol [14]. 4.3. Thioflavin-T fluorescence assay and amyloid formation Thioflavin-T fluorescence assay and amyloid formation were performed according to a previously published protocol [3]. Briefly, pre-cooled PrP solution was mixed with aS solution such that the final mixture contained 25 mM PrP, 0e50 mM aS, 25 mM HEPES (pH 7.4), 3 M GuHCl, 0.02% NaN3, and 25 mM ThT. 100-ml of the mixture was immediately transferred to a pre-cooled 96-well plate and subjected to the repeated cycle of shaking at 37 C. The time courses of ThT fluorescence were continuously recorded with the excitation
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and emission wavelengths at 445 and 485 nm, respectively. The half-time of amyloid formation was determined by fitting to a sigmoidal function. The seeded-growth experiment was performed by adding pre-formed PrP amyloid fibrils to the above-described reaction mixture, as described previously [14]. 4.4. Circular dichroism, size exclusion chromatography, and transmission electron microscopy Near-UV circular dichroism spectra were acquired using a Chirascan-plus CD spectrometer (Applied Photophysics) with a 1 mm path length quartz cuvette at 25 C. SEC experiments were performed using a Superdex 75 10/300 column (GE healthcare) on the AKTApurifier. A HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) was used as a running buffer at the flow rate 0.5 mL/min. For TEM analysis, amyloid fibrils were negatively stained with 2% phosphotungstate and examined by JEM-2100F (JEOL, Japan). 4.5. Co-sedimentation assay PrP amyloid fibrils generated in the presence of 50 mM aS via the above described procedure were centrifuged at 20,600 g for 60 min. Approximately 70% of PrP amyloid fibrils were pelleted by centrifugation, as judged by measuring ThT fluorescence of the supernatant. The pellet was resuspended with Laemmli sample buffer containing 7.5 M urea, boiled at 95 C for 30 min, and loaded onto the SDS-PAGE gel. The supernatant was mixed with the same Laemmli sample buffer and analyzed similarly. 4.6. Surface plasmon resonance analysis and preparations of asynuclein and amyloid-b oligomers Direct interactions between aS, Ab, and PrP were analyzed using a BIACORE T200 (GE Healthcare). The recombinant PrP was immobilized into a CM5 sensorchip using an amine-coupling kit at a density of 10,000 response units. An adjacent flow cell was blocked with ethanolamine and utilized as a reference surface. The surface was injected over with aS or Ab at a protein concentration of 1 mM with a flow rate of 40 mL/min at 20 C. HBS buffer containing 0.005% P20 was used as a running buffer. To prepare an Ab oligomer, a lyophilized Ab42 (Peptide Institute, code 4349-v) was initially dissolved with 10 mM NaOH at a concentration of approximately 400 mM, and diluted four-fold with a precooled 50 mM sodium phosphate buffer (pH 7.4). After adjusting the protein concentration to 100 mM using UV absorbance (ε280 ¼ 1290 M1 cm1) [31], the solution was incubated at 37 C for 3 h and applied to a 30 kDa filter device to remove residual monomers. The non-filtered fraction was enriched in oligomers, as judged by SEC, and used for SPR analysis without further purification. To prepare an aS oligomer, recombinant aS at the concentration of 100 mM was mixed with 3 mM 4-HNE (Cayman Chemical, Item No. 32100) in HBS buffer and incubated at 37 C for 2 days. The HNE-modified aS samples were purified by SEC and the oligomeric fraction was used for SPR analysis. Author contributions statement R.H. designed this study, collected data, analyzed data, and wrote the manuscript. M.S. collected data. Kazuo Kuwata supervised this study. Declaration of competing interest The authors declare no competing financial interests.
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Acknowledgments This study was supported by JSPS KAKENHI, Japan Grant Number 16J04145 (to R.H.). Funding was also received from the Japan Agency for Medical Research and Development, Japan (AMED; 17ek0109075h0003 to Kazuo Kuwata) and from the Japanese Ministry of Health, Labor, and Welfare Research Committee on Surveillance and Infection Control of Prion Disease (to Kazuo Kuwata). References [1] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333e366, https://doi.org/10.1146/ annurev.biochem.75.101304.123901. PubMed PMID: WOS: 000239807600014; English. [2] R. Morales, I. Moreno-Gonzalez, C. Soto, Cross-seeding of misfolded proteins: implications for etiology and pathogenesis of protein misfolding diseases, PLoS Pathog. 9 (9) (2013) e1003537. [3] H. Ryo, Amyloid-b peptide induces prion protein amyloid formation: evidence for its widespread amyloidogenic effect, Angew. Chem. Int. Ed. 57 (21) (2018) 6086e6089, https://doi.org/10.1002/anie.201800197. [4] B. O’Nuallain, A.D. Williams, P. Westermark, et al., Seeding specificity in amyloid growth induced by heterologous fibrils, J. Biol. Chem. 279 (17) (2004) 17490e17499. [5] E. Katorcha, N. Makarava, Y.J. Lee, et al., Cross-seeding of prions by aggregated alpha-synuclein leads to transmissible spongiform encephalopathy, PLoS Pathog. 13 (8) (2017 Aug 10), https://doi.org/10.1371/journal.ppat.1006563 e1006563, PubMed PMID: 28797122; eng. [6] S. Auli c, L. Masperone, J. Narkiewicz, et al., a-Synuclein amyloids hijack prion protein to gain cell entry, facilitate cell-to-cell spreading and block prion replication, Sci. Rep. 7 (1) (2017) 10050. [7] D.G. Ferreira, M. Temido-Ferreira, H.V. Miranda, et al., alpha-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B, Nat. Neurosci. (2017 Sep 25), https://doi.org/10.1038/nn.4648. PubMed PMID: 28945221; eng. [8] P. La Vitola, M. Beeg, C. Balducci, et al., Cellular prion protein neither binds to alpha-synuclein oligomers nor mediates their detrimental effects, Brain : J. Neurol. 142 (2) (2019) 249e254. [9] F.X. Theillet, A. Binolfi, B. Bekei, et al., Structural disorder of monomeric alphasynuclein persists in mammalian cells, Nature (2016 Jan 25), https://doi.org/ 10.1038/nature16531. PubMed PMID: 26808899; Eng. [10] J.M. Souza, B.I. Giasson, V.M. Lee, et al., Chaperone-like activity of synucleins, FEBS Lett. 474 (1) (2000 May 26) 116e119, https://doi.org/10.1016/s00145793(00)01563-5. PubMed PMID: 10828462; eng. [11] T.D. Kim, S.R. Paik, C.-H. Yang, et al., Structural changes in a-synuclein affect its chaperone-like activity in vitro, Protein Sci. 9 (12) (2000) 2489e2496. [12] K.M. Manda, D. Yedlapudi, S. Korukonda, et al., The chaperone-like activity of a-synuclein attenuates aggregation of its alternatively spliced isoform, 112synuclein in vitro: plausible cross-talk between isoforms in protein aggregation, PLoS One 9 (6) (2014) e98657. [13] S. Chandra, G. Gallardo, R. Fernandez-Chacon, et al., Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration, Cell 123 (3) (2005 Nov 4) 383e396, https://doi.org/10.1016/j.cell.2005.09.028. PubMed PMID: 16269331; eng. [14] R.P. Honda, K. Kuwata, The native state of prion protein (PrP) directly inhibits formation of PrP-amyloid fibrils in vitro, Sci. Rep. 7 (1) (2017) 562, https:// doi.org/10.1038/s41598-017-00710-x, 2017//. [15] P.H. Weinreb, W. Zhen, A.W. Poon, et al., NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded, Biochemistry 35 (43) (1996) 13709e13715. [16] D. Eliezer, E. Kutluay, R. Bussell Jr., et al., Conformational properties of alphasynuclein in its free and lipid-associated states, J. Mol. Biol. 307 (4) (2001 Apr 6) 1061e1073, https://doi.org/10.1006/jmbi.2001.4538. PubMed PMID: 11286556; eng. [17] H. Naiki, K. Higuchi, M. Hosokawa, et al., Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1, Anal. Biochem. 177 (2) (1989 Mar) 244e249. PubMed PMID: 2729542; eng. [18] K. Nieznanski, K. Surewicz, S. Chen, et al., Interaction between prion protein and Abeta amyloid fibrils revisited, ACS Chem. Neurosci. 5 (5) (2014 May 21) 340e345, https://doi.org/10.1021/cn500019c. PubMed PMID: 24669873; PubMed Central PMCID: PMCPmc4030797. eng. [19] Z. Qin, D. Hu, S. Han, et al., Effect of 4-hydroxy-2-nonenal modification on asynuclein aggregation, J. Biol. Chem. 282 (8) (2007) 5862e5870. n, D.A. Gimbel, H.B. Nygaard, et al., Cellular prion protein mediates [20] J. Laure impairment of synaptic plasticity by amyloid-b oligomers, Nature 457 (7233) (2009) 1128e1132. [21] E. Masliah, E. Rockenstein, I. Veinbergs, et al., b-Amyloid peptides enhance asynuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer’s disease and Parkinson’s disease, Proc. Natl. Acad. Sci. 98 (21) (2001) 12245e12250.
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a-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein Maki Shirasaka a, Kazuo Kuwata a, b, Ryo Honda a, * a b
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan Department of Gene and Development, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan
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a b s t r a c t
Article history: Received 23 September 2019 Accepted 14 October 2019 Available online xxx
Protein misfolding diseases are a group of devastating disorders characterized by structural conversion of a soluble protein into an amyloid-like aggregate. Typically, the structural conversion occurs by misfolding of a single disease-associated protein, such as a-synuclein (aS) in Parkinson’s disease, amyloid-b in Alzheimer’s disease, and prion protein (PrP) in transmissible spongiform encephalopathies (TSEs). However, accumulating evidence has implicated that cross-interactions between heterologous amyloidogenic proteins dramatically impact on amyloidogenesis and disease pathology. Here we show aS in a monomeric state can suppress amyloidogenesis of PrP in vitro. Thioflavin-T assays and transmission electron miscopy revealed that monomeric aS inhibits the nucleation step of amyloidogenesis without inhibiting the growing step. Surface plasmon resonance and co-sedimentation assays neither detected interaction between aS and monomeric PrP nor fibrillar PrP. These results suggested that aS suppress amyloidogenesis of PrP by binding to a transiently accumulated intermediate, such as a partially unfolded state. Moreover, we found that oligomeric aS, which was recently suggested to interact with PrP, also did not interact with PrP. Taken together, our study revealed a chaperon-like activity of aS against PrP amyloidogenesis, suggesting a possible involvement of aS in the pathology of TSEs. © 2019 Elsevier Inc. All rights reserved.
1. Introduction Amyloid formation is a key pathological event in protein misfolding diseases, by which soluble, typically monomeric, proteins are converted into insoluble, filamentous, and toxic aggregates that deposit in tissues [1]. The amyloid deposits are composed mainly of a specific disease-associated amyloidogenic protein, such as amyloid-b (Ab) in Alzheimer’s disease, a-synuclein (aS) in Parkinson’s disease, and prion protein (PrP) in transmissible spongiform encephalopathy. This prominent pathological feature and other experimental evidence led to the prevailing view that amyloid formation occurs by misfolding of a single defined amyloidogenic protein. However, recent evidence has indicated that several amyloidogenic proteins can interact with a different disease-associated protein to dramatically impact on the development of amyloid deposits [2]. For example, one of the authors recently reported that Ab accelerates amyloidogenesis of many amyloidogenic proteins [3]. Cross-seeding is also a prominent example of the cross-
* Corresponding author. E-mail address: [email protected] (R. Honda).
interactions, by which an amyloid fibril composed of one specific protein can promote amyloidogenesis of a different amyloidogenic protein [4]. Thus, the mechanism of amyloid formation can be elucidated by studying cross-interactions between heterologous amyloidogenic proteins. The goal of this study is to examine if and how aS would affect amyloidogenesis of PrP, in particular in the native (monomeric) state. aS in a fibrillar state was recently shown to accelerate misfolding of PrP and propagate transmissible spongiform encephalopathy (TSE) through cross-seeding [5]. Direct interaction between fibrillar aS and PrP was confirmed by a different group using surface plasmon resonance (SPR) [6]. Moreover, oligomeric aS was also recently shown to interact with PrP to induce cognitive failure in Parkinson’s disease [7], although controversy remains [8]. These results clearly demonstrate the close link between PrP and aggregated aS (i.e., oligomeric aS and fibrillar aS); however, it remains elusive how monomeric aS would affect misfolding of PrP. This question is of importance because the major population of aS exists as a monomer at a high concentration (c.a. 50 mM) in a brain tissue [9], in which PrP coexists with aS. Moreover, monomeric aS has a chaperon-like activity against aggregation of a number of different
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Please cite this article as: M. Shirasaka et al., a-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.120
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proteins, including insulin, albumin, lysozyme in vitro [10e12], and SNARE in vivo [13]. Here we show that monomeric aS can also suppress amyloid formation of PrP. We also address the mechanism underlying the chaperon-like activity of aS against PrP aggregation. 2. Result 2.1. Expression and purification of monomeric recombinant asynuclein As we previously established a protocol of generating recombinant PrP [14], we initially focused on the generation of recombinant aS. After the expression of aS using a bacterial system, the boiling procedure [15] and reverse-phase chromatography were employed for purification of aS (Fig. 1A). The purified aS was highly homologous, as judged by SDS-PAGE (Fig. 1B), and represented an intrinsically disordered structure characterized by a negative minimum at 195 nm in the near-UV circular dichroism (Fig. 1C). Consistent with previous reports [16], in the presence of SDS micelles, aS underwent a large structural change into an a-helical state characterized by a negative CD minimum at 207 nm. Moreover, in size-exclusion chromatography (SEC), the purified aS was eluted as a single peak corresponding to molecular mass of 60 kDa (Fig. 1D). This size was larger than that predicted from the amino acid sequence (14 kDa), consistent with the intrinsically disordered structure of aS [11]. Overall, the biophysical characteristics of the recombinant aS were in good agreement with those of the previously reported aS.
aS remained in the supernatant fraction and was not cosedimented with fibrillar PrP. Thus, aS can neither interact with fibrillar PrP nor inhibit growing of fibrillar PrP. 2.4. Monomeric or oligomeric a-synuclein did not interact with prion protein monomer The above experiments had implicated that aS suppresses the nucleation step of amyloidogenesis, suggesting two possible scenarios: (a) aS interacts with PrP monomer; and (b) aS interacts with nucleus or other transiently accumulated intermediates, such as partially unfolded states (Fig. 4A). As the latter possibility is difficult
2.2. Monomeric a-synuclein retarded prion protein amyloid formation To examine effects of aS on the amyloidogenesis of PrP, we employed a ThT-fluorescence assay previously described [3]. Here, monomeric PrP was partially unfolded by 3 M guanidine hydrochloride and subjected to continuous agitation in a 96-well plate reader to initiate spontaneous amyloid formation. Formation of amyloid fibrils was monitored by fluorescence of thioflavin-T (ThT), a dye specific for amyloid fibrils [17]. Under the experimental condition, PrP alone represented significant increase in ThT fluorescence after 10 h of lag time, suggesting the formation of amyloid fibrils (Fig. 2A). By contrast, aS alone did not produce any amyloid fibril (data not shown) and remained monomeric even after 72 h of agitation (Fig. 1D). Intriguingly, in the presence of aS, the lag time of PrP amyloid formation was significantly prolonged to 35e50 h (Fig. 2A). In a transmission electron microscopy, the amyloid fibrils generated in the presence of aS were indistinguishable from pure amyloid fibrils of PrP (Fig. 2B). Thus, aS can retard amyloidogenesis of PrP. 2.3. Monomeric a-synuclein did not interact with prion protein amyloid fibrils As the mechanism of amyloid formation is typically described by two consecutive processes, nucleation and growth [1], we next sought to determine which process is retarded by aS. To this end, we performed a seeded-growth experiment, in which pre-formed amyloid fibrils of PrP (termed “seeds”) was mixed with monomeric PrP to initiate rapid growth of the seed fibrils (Fig. 3A). In contrast to the previous result, in the presence of a high concentration of seed fibrils (10%), aS did not suppress amyloid formation of PrP. To further examine an interaction between aS and seed fibrils of PrP, we performed a co-sedimentation assay [18]. Here, fibrillar PrP was co-incubated with aS and sedimented by centrifugation to examine co-sedimentation of aS. As shown in Fig. 3B, all
Fig. 1. Preparations of monomeric and oligomeric a-synuclein (aS). (A) A HPLC profile of an E. coli lysate expressing recombinant aS. aS was eluted with a liner gradient (0e60% acetonitrile in 0.1% TFA, right axis) as a single peak at 56% acetonitrile. (B) A Coomassie-stained SDS-PAGE gel of purified aS showing a homogeneous band at 15 kDa. (C) Far-UV circular dichroism spectra for purified aS without or with 10 mM SDS. aS at a concentration of 10 mM was dissolved in the 10 mM sodium phosphate (pH 7.4). (D) Size exclusion chromatography profiles of aS in monomeric or 4hydroxynonenal (HNE)-modified oligomeric state. A size exclusion chromatography profile of aS after 72 h agitation is also shown.
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Fig. 2. (A) The upper panel represents a representative kinetics of prion protein (PrP) amyloid formation in the presence of 0, 5, and 50 mM monomeric a-synuclein (aS). The reaction was followed by change in Thioflavin-T (ThT) fluorescence. Lower panel represents the half-times (t1/2) of reaction derived from the upper panel. Error bars represent standard deviations of 4 independent trials. *P < 0.01, **P < 0.001, n.s. P 0.01. P-values from unpaired t-test. (B) Representative transmission electron micrographs of (left) pure PrP amyloid fibrils and (right) PrP amyloid fibrils generated in the presence of 50 mM aS (magnification, 25,000; scale bar, 100 nm).
to access using currently available equilibrium methods, we tested the former possibility using SPR. Here, PrP monomer was immobilized on the sensor-chip surface of SPR and flowed over with a solution containing aS. As shown in Fig. 4B, no interaction between monomeric PrP and aS was detected. We also tested the possibility that oligomeric aS, which presents as a minor population in a soluble aS preparation, might interact with PrP monomer [7]. We stabilized aS oligomers using a lipid peroxidation product (4hydroxy-2-nonenal) [19] (Fig. 1C). However, as shown in Fig. 4B, no interaction was detected between PrP and aS oligomers. This result was in sharp contrast to the case of Ab oligomers, which strongly bound to the immobilized PrP (Fig. 4B) [20]. Thus, neither monomeric nor oligomeric aS can interact with PrP. This result indicated that aS interacts with an intermediate transiently accumulated during PrP amyloid formation (scenario (b), in Fig. 4A).
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Fig. 3. (A) A representative kinetics of prion protein (PrP) amyloid formation in the presence of 0, 5, and 50 mM monomeric a-synuclein (aS). The reaction was seeded with 1 or 10% (v/v) pre-formed PrP amyloid fibrils. Error bars represent standard deviations of 3 independent trials *P < 0.01, **P < 0.001, n.s. P 0.01. P-values from unpaired ttest. (B) PrP amyloid fibrils with or without 50 mM aS were centrifuged and separated into supernatant (S) and pellet (P) fractions. Each fraction was analyzed using 15% SDSPAGE gel stained with Coomassie blue. aS did not co-precipitate with PrP amyloid fibrils.
3. Discussion Protein misfolding diseases are a group of devastating disorders characterized by amyloid-like deposits, for which no effective treatment is currently available. Understanding the basic principles of amyloid formation is critical step for rational design of therapeutics. Recent evidence has indicated that a number of amyloidogenic proteins, such as Ab, aS and PrP, cross-interact during the development of amyloid deposits [2,3,5,21,22]. It is therefore crucial to elucidate the mechanism underlying the crossinteractions between amyloidogenic proteins in order to provide novel insights into protein misfolding diseases.
Please cite this article as: M. Shirasaka et al., a-Synuclein chaperone suppresses nucleation and amyloidogenesis of prion protein, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.10.120
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amyloid formation, aS might suppress spontaneous generation, but not propagation, of infectious prion. Finally, our result supports the recent notion that aS oligomer did not bind to PrP [8]. Controversy remains whether PrP acts as a receptor of aS oligomer to trigger cognitive failure in Parkinson’s disease pathology [7,8]. Our SPR analysis did not detect interaction between PrP and an aS oligomer that was generated using a lipid peroxidation product (Fig. 4B). Intriguingly, an Ab oligomer, which partly shares structural and functional similarities with the aS oligomer (including extensive b-sheet structure and toxicity [27]), bound to PrP. Thus, PrP has a binding specificity for Ab oligomers, arguing against the notion that PrP serves as a general receptor of bsheet rich conformers [28]. 4. Material and methods 4.1. Expression and purification of recombinant a-synuclein
Fig. 4. (A) Three possible targets of a-synuclein (aS) to suppress prion protein (PrP) amyloid formation; (a) monomeric PrP, (b) nucleus and other transiently accumulated intermediates, and (c) PrP amyloid fibrils. (B) Representative surface plasmon resonance sensorgrams for the interactions between PrP, aS, and Ab oligomer. PrP was immobilized on a carboxymethylated dextran surface at a density of 10,000 RU. aS and Ab in monomeric or oligomer state at 1 mM monomer equivalent concentration was flowed over the surface.
Here we report the first in vitro evidence that aS can suppress amyloidogenesis of PrP. We found aS monomer, but not oligomeric and fibrillar aS, was sufficient to suppress amyloidogenesis of PrP. It should be emphasis that our finding is irrelevant to the crossseeding between fibrillar aS and PrP recently reported, which accelerates amyloidogenesis of PrP [5]. Rather, our finding can be linked to prior research showing the chaperone-like activity of monomeric aS. Monomeric aS can suppress misfolding of a number of different proteins both in vivo and in vitro [10e13,23,24]. Accordingly, our study serves to expand the set of client proteins involved in the chaperon-like activity of aS. It has been hypothesized that monomeric aS binds to the hydrophobic region of partially unfolded client proteins to prevent further structural change toward misfolded aggregates [11,24]. This hypothesis is also consistent with our current observations, in which aS neither bind to fully folded PrP (Fig. 4B) nor fibrillar PrP (Fig. 3), suggesting a possible interaction between aS and a partially unfolded state of PrP (Fig. 4A). Thus, aS chaperone might suppress misfoldings of PrP and other client proteins via a common mechanism. A further study using a sophisticated kinetic method is required to prove the interaction between aS and partially folded proteins [25]. Our results suggest a possible involvement of aS in TSE pathology. It was recently shown that knockout of aS gene did not affect the incubation period of prion propagation in mice brain [26]. This result is consistent with our current in vitro observations, in which aS failed to inhibit PrP aggregation in the presence of a large amount of seed fibrils (Fig. 3A). Since aS chaperon interacts with partially folded proteins and suppresses the nucleation step of
A pT7-7 vector encoding wild-type human aS (residues 1-140), which was created by Paleologou et al. [29], was purchased from Addgene (code 36046). ECOS™ competent E.coli BL21 (DE3) (Nippongene, code 312-06534) was transformed with this construct and cultured in LB medium containing 100 mg/mL ampicillin at 37 C. Protein expression was induced by 1 mM IPTG (Carbosynth, code EI05931) when the culture reached OD ¼ 0.6e0.8. After 4 h of induction period, the bacteria were harvested by centrifugation at 9000 g for 7 min and resuspended with 10 mL lysis buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA, and 1 mM PMSF] per gram weight pellet. The bacteria were then frozen-and-thawed, sonicated for 1 min on ice using an ultrasonic processor with 100% amplitude (VCX 130, SONICS), and centrifuged at 30,000 g for 30 min to remove cell debris. The cell lysate was boiled for 20 min using Hot Plate Stirrer (PC-420, Corning), centrifuged at 30,000 g for 30 min to remove aggregated proteins [15]. The supernatant enriched with aS was supplemented with 1e2% streptomycin sulfate (Nacalai Tesqu, code 32237-72), incubated for 30 min on ice, and centrifuged at 30,000 g for 30 min to remove nuclei acids [30]. The supernatant was finally applied to a COSMOSIL 5C4-AR-300 reverse-phase HPLC column (Nacalai Tesque, code 38048-01) equipped with AKTApurifier (GE healthcare), and eluted with a linear gradient of 0e60% AcCN in 0.1% TFA [16]. A fraction enriched with aS (approx. 56% AcCN) was collected, lyophilized, and dialyzed extensively against MillQ water. The dialyzed solution was lyophilized again and stored at 20 C until use. The purified proteins were initially dissolved with MillQ water or 6 M guanidine hydrochloride (GuHCl) at a concentration of 100 mM. The protein concentration was determined by UV absorbance using an extinction coefficient of 5140 cm1M1 at 280 nm. 4.2. Expression and purification of prion protein A recombinant human PrP (residues 23-230) was expressed using an E.coli expression system and purified to homogeneity according to the previously published protocol [14]. 4.3. Thioflavin-T fluorescence assay and amyloid formation Thioflavin-T fluorescence assay and amyloid formation were performed according to a previously published protocol [3]. Briefly, pre-cooled PrP solution was mixed with aS solution such that the final mixture contained 25 mM PrP, 0e50 mM aS, 25 mM HEPES (pH 7.4), 3 M GuHCl, 0.02% NaN3, and 25 mM ThT. 100-ml of the mixture was immediately transferred to a pre-cooled 96-well plate and subjected to the repeated cycle of shaking at 37 C. The time courses of ThT fluorescence were continuously recorded with the excitation
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and emission wavelengths at 445 and 485 nm, respectively. The half-time of amyloid formation was determined by fitting to a sigmoidal function. The seeded-growth experiment was performed by adding pre-formed PrP amyloid fibrils to the above-described reaction mixture, as described previously [14]. 4.4. Circular dichroism, size exclusion chromatography, and transmission electron microscopy Near-UV circular dichroism spectra were acquired using a Chirascan-plus CD spectrometer (Applied Photophysics) with a 1 mm path length quartz cuvette at 25 C. SEC experiments were performed using a Superdex 75 10/300 column (GE healthcare) on the AKTApurifier. A HBS buffer (10 mM HEPES, pH 7.4, 150 mM NaCl) was used as a running buffer at the flow rate 0.5 mL/min. For TEM analysis, amyloid fibrils were negatively stained with 2% phosphotungstate and examined by JEM-2100F (JEOL, Japan). 4.5. Co-sedimentation assay PrP amyloid fibrils generated in the presence of 50 mM aS via the above described procedure were centrifuged at 20,600 g for 60 min. Approximately 70% of PrP amyloid fibrils were pelleted by centrifugation, as judged by measuring ThT fluorescence of the supernatant. The pellet was resuspended with Laemmli sample buffer containing 7.5 M urea, boiled at 95 C for 30 min, and loaded onto the SDS-PAGE gel. The supernatant was mixed with the same Laemmli sample buffer and analyzed similarly. 4.6. Surface plasmon resonance analysis and preparations of asynuclein and amyloid-b oligomers Direct interactions between aS, Ab, and PrP were analyzed using a BIACORE T200 (GE Healthcare). The recombinant PrP was immobilized into a CM5 sensorchip using an amine-coupling kit at a density of 10,000 response units. An adjacent flow cell was blocked with ethanolamine and utilized as a reference surface. The surface was injected over with aS or Ab at a protein concentration of 1 mM with a flow rate of 40 mL/min at 20 C. HBS buffer containing 0.005% P20 was used as a running buffer. To prepare an Ab oligomer, a lyophilized Ab42 (Peptide Institute, code 4349-v) was initially dissolved with 10 mM NaOH at a concentration of approximately 400 mM, and diluted four-fold with a precooled 50 mM sodium phosphate buffer (pH 7.4). After adjusting the protein concentration to 100 mM using UV absorbance (ε280 ¼ 1290 M1 cm1) [31], the solution was incubated at 37 C for 3 h and applied to a 30 kDa filter device to remove residual monomers. The non-filtered fraction was enriched in oligomers, as judged by SEC, and used for SPR analysis without further purification. To prepare an aS oligomer, recombinant aS at the concentration of 100 mM was mixed with 3 mM 4-HNE (Cayman Chemical, Item No. 32100) in HBS buffer and incubated at 37 C for 2 days. The HNE-modified aS samples were purified by SEC and the oligomeric fraction was used for SPR analysis. Author contributions statement R.H. designed this study, collected data, analyzed data, and wrote the manuscript. M.S. collected data. Kazuo Kuwata supervised this study. Declaration of competing interest The authors declare no competing financial interests.
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Acknowledgments This study was supported by JSPS KAKENHI, Japan Grant Number 16J04145 (to R.H.). Funding was also received from the Japan Agency for Medical Research and Development, Japan (AMED; 17ek0109075h0003 to Kazuo Kuwata) and from the Japanese Ministry of Health, Labor, and Welfare Research Committee on Surveillance and Infection Control of Prion Disease (to Kazuo Kuwata). References [1] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333e366, https://doi.org/10.1146/ annurev.biochem.75.101304.123901. PubMed PMID: WOS: 000239807600014; English. [2] R. Morales, I. Moreno-Gonzalez, C. Soto, Cross-seeding of misfolded proteins: implications for etiology and pathogenesis of protein misfolding diseases, PLoS Pathog. 9 (9) (2013) e1003537. [3] H. Ryo, Amyloid-b peptide induces prion protein amyloid formation: evidence for its widespread amyloidogenic effect, Angew. Chem. Int. Ed. 57 (21) (2018) 6086e6089, https://doi.org/10.1002/anie.201800197. [4] B. O’Nuallain, A.D. Williams, P. Westermark, et al., Seeding specificity in amyloid growth induced by heterologous fibrils, J. Biol. Chem. 279 (17) (2004) 17490e17499. [5] E. Katorcha, N. Makarava, Y.J. Lee, et al., Cross-seeding of prions by aggregated alpha-synuclein leads to transmissible spongiform encephalopathy, PLoS Pathog. 13 (8) (2017 Aug 10), https://doi.org/10.1371/journal.ppat.1006563 e1006563, PubMed PMID: 28797122; eng. [6] S. Auli c, L. Masperone, J. Narkiewicz, et al., a-Synuclein amyloids hijack prion protein to gain cell entry, facilitate cell-to-cell spreading and block prion replication, Sci. Rep. 7 (1) (2017) 10050. [7] D.G. Ferreira, M. Temido-Ferreira, H.V. Miranda, et al., alpha-synuclein interacts with PrPC to induce cognitive impairment through mGluR5 and NMDAR2B, Nat. Neurosci. (2017 Sep 25), https://doi.org/10.1038/nn.4648. PubMed PMID: 28945221; eng. [8] P. La Vitola, M. Beeg, C. Balducci, et al., Cellular prion protein neither binds to alpha-synuclein oligomers nor mediates their detrimental effects, Brain : J. Neurol. 142 (2) (2019) 249e254. [9] F.X. Theillet, A. Binolfi, B. Bekei, et al., Structural disorder of monomeric alphasynuclein persists in mammalian cells, Nature (2016 Jan 25), https://doi.org/ 10.1038/nature16531. PubMed PMID: 26808899; Eng. [10] J.M. Souza, B.I. Giasson, V.M. Lee, et al., Chaperone-like activity of synucleins, FEBS Lett. 474 (1) (2000 May 26) 116e119, https://doi.org/10.1016/s00145793(00)01563-5. PubMed PMID: 10828462; eng. [11] T.D. Kim, S.R. Paik, C.-H. Yang, et al., Structural changes in a-synuclein affect its chaperone-like activity in vitro, Protein Sci. 9 (12) (2000) 2489e2496. [12] K.M. Manda, D. Yedlapudi, S. Korukonda, et al., The chaperone-like activity of a-synuclein attenuates aggregation of its alternatively spliced isoform, 112synuclein in vitro: plausible cross-talk between isoforms in protein aggregation, PLoS One 9 (6) (2014) e98657. [13] S. Chandra, G. Gallardo, R. Fernandez-Chacon, et al., Alpha-synuclein cooperates with CSPalpha in preventing neurodegeneration, Cell 123 (3) (2005 Nov 4) 383e396, https://doi.org/10.1016/j.cell.2005.09.028. PubMed PMID: 16269331; eng. [14] R.P. Honda, K. Kuwata, The native state of prion protein (PrP) directly inhibits formation of PrP-amyloid fibrils in vitro, Sci. Rep. 7 (1) (2017) 562, https:// doi.org/10.1038/s41598-017-00710-x, 2017//. [15] P.H. Weinreb, W. Zhen, A.W. Poon, et al., NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded, Biochemistry 35 (43) (1996) 13709e13715. [16] D. Eliezer, E. Kutluay, R. Bussell Jr., et al., Conformational properties of alphasynuclein in its free and lipid-associated states, J. Mol. Biol. 307 (4) (2001 Apr 6) 1061e1073, https://doi.org/10.1006/jmbi.2001.4538. PubMed PMID: 11286556; eng. [17] H. Naiki, K. Higuchi, M. Hosokawa, et al., Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1, Anal. Biochem. 177 (2) (1989 Mar) 244e249. PubMed PMID: 2729542; eng. [18] K. Nieznanski, K. Surewicz, S. Chen, et al., Interaction between prion protein and Abeta amyloid fibrils revisited, ACS Chem. Neurosci. 5 (5) (2014 May 21) 340e345, https://doi.org/10.1021/cn500019c. PubMed PMID: 24669873; PubMed Central PMCID: PMCPmc4030797. eng. [19] Z. Qin, D. Hu, S. Han, et al., Effect of 4-hydroxy-2-nonenal modification on asynuclein aggregation, J. Biol. Chem. 282 (8) (2007) 5862e5870. n, D.A. Gimbel, H.B. Nygaard, et al., Cellular prion protein mediates [20] J. Laure impairment of synaptic plasticity by amyloid-b oligomers, Nature 457 (7233) (2009) 1128e1132. [21] E. Masliah, E. Rockenstein, I. Veinbergs, et al., b-Amyloid peptides enhance asynuclein accumulation and neuronal deficits in a transgenic mouse model linking Alzheimer’s disease and Parkinson’s disease, Proc. Natl. Acad. Sci. 98 (21) (2001) 12245e12250.
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