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Comparative analysis of the conformation, aggregation, interaction and fibril morphologies of human #, # and # synuclein proteins Manish Kumar Jain, Priyanka Singh, Sneha Roy, and Rajiv Bhat Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00343 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018
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Biochemistry
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Comparative analysis of the conformation, aggregation, interaction and fibril
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morphologies of human α, β, and γ synuclein proteins
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Manish Kumar Jain1, 2, Priyanka Singh1, 3, Sneha Roy1, 3, and Rajiv Bhat1
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School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110 067, India
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2
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University of Texas Medical Branch, Galveston, TX 77555, USA
Current Affiliation: Center for Addiction Research, Department of Pharmacology and Toxicology,
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3
Equal contribution
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Running Title: Biophysical studies of human α, β, and γ-synuclein proteins
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To whom correspondence should be addressed: Rajiv Bhat, School of Biotechnology, Jawaharlal Nehru
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University, New Delhi, 110 067, India; Email:
[email protected] 16 17 18
Keywords: α-syn, β-syn, γ-syn, Amyloid, Seeding, Aggregation, CD, ThT, ITC, SPR, TEM Abstract
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The human synucleins family is comprised of α-, β- and γ-syn proteins. α-syn has the highest
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propensity for aggregation, and its aggregated forms accumulate in Lewy bodies (LB) and Lewy neuritis
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(LN), which are involved in Parkinson’s disease (PD). β- and γ-syn are absent in LB and their exact role
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is still enigmatic. β-syn does not form aggregates under physiological conditions (pH 7.4), while γ-syn is
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associated with neural and non-neural diseases like breast cancer. Due to their similar regional
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distribution in brain, natively unfolded structure and high sequence homology, it has become important
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to study the effect of environment on their conformation, interactions, fibrillation and fibril
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morphologies. Our studies show that high temperature, low pH, and high concentrations increase the
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rate of fibrillation of α- and γ-syn while β-syn forms fibrils only at low pH. Fibril morphologies are
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strongly dependent on the immediate environment of the proteins. The high molar ratio of β-syn inhibits
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the fibrillation in α-, and γ-syn. However, preformed seed fibrils of β-, and γ-syn do not affect
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fibrillation of α-syn. Surface Plasmon Resonance (SPR) data shows that interaction between α-β, β-γ and
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α-γ syn are weak to moderate in nature and they can be physiologically significant to counteract several 1
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adverse conditions in the cells that trigger their aggregation. These studies could be helpful in
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understanding collective human synucleins behavior under various protein environments and in the
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modulation of the homeostasis between β-syn and healthy versus corrupt α- and γ-syn which can
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potentially affect PD pathology.
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Introduction
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Human synuclein family is comprised of small (~14kDa), natively unfolded α-, β- and γ-syn
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proteins1,2 which are expressed at high levels in the brain3-6. Human synucleins have acidic c-terminal3, 4
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and repetitive degenerative amino acid motif KTKEGV towards N-terminus5. α- and β-syn are abundant
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in the central nervous system and presynaptic nerve terminals3, whereas γ-syn is expressed mainly in the
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peripheral nervous system4, 6. These proteins are heat stable, relatively soluble and lack cysteine and
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tryptophan residues3, 5, 7.
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α-syn is located in the vicinity of the synaptic vesicles3, platelets8 and also plays a role in
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neurotransmission9. It is also known as the non-amyloid component precursor protein (NACP) or
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synelfin10, 11 and has three isoforms of 140, 126 and 112 residues resulting from alternative splicing12.
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The N-terminus of α-syn-140 isoform has the sites for six familial PD mutations (A30P, E46K, H50Q,
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G51D, A53T and A53E)13-17 and a lipid-binding domain of apolipoproteins5, 18. The central region of α-
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syn from residues 61–95 has highly aggregation-prone non-amyloid beta sequence (NAC)9, 19
16
20
10
. α-syn
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interacts with other proteins such as synphilin , tubulin , tau
and 14-3-3 proteins of chaperone
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family21. Human β-syn, a 134 amino acid long protein also known as phosphoneuroprotein-14 (PNP-
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14)3, 22, 23, is mainly expressed in brain3 and Sertoli cells of testis24 and shares 78% sequence homology
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to α-syn. A hydrophobic patch (residues 73-83) is absent in β-syn25. Human γ-syn is a 127 amino acids
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long protein, and is the smallest protein of the family sharing 60% sequence homology with α-syn25. γ-
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syn is also known as a breast cancer-specific gene 1 (BCSG-1)26, persyn27 and has only a single tyrosine
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at the C-terminus2. γ-syn is present in cell bodies, axons, spinal cord and sensory ganglia27, epidermis28,
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malignant breast cancer tissues26 and is involved in neural and non-neural disease. Surprisingly, it is
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highly expressed in the retina of Alzheimer’s disease (AD) patients29. Human synucleins are associated
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with PD, AD and other synucleinopathies such as multiple system atrophy, Hallervorden–Spatz
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diseases, LB disorders, and the LB variant of AD30-34. Synucleinopathies are caused by the depositions
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of fibrillated α-syn in LB in midbrain rich in cross-β structure35-38. However, β- and γ-syn are absent in
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LB and play a substantial role in hippocampal axon pathology in PD39. 2
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Biochemistry
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A large number of biophysical studies have been performed on α-syn previously. However, β-
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and γ-syn, the other two proteins of the family, have not been studied in detail so far. Also, their
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interaction with each other and their cross-seeding capabilities have not been investigated in detail. High
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sequence similarities amongst them, their presence in the same niche of brain, and homeostasis between
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them, may influence their aggregation and can modulate their functions in the presence of each other,
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which may make neurons vulnerable and lead to neurodegeneration. Due to the absence of a well folded
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structure, they are highly dynamic in nature and can modulate each other’s structure, function and
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aggregation propensity in the presence of ligands that could have significant bearing on PD
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pathogenesis. Therefore, it is of significant interest to study the various biophysical properties of these
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closely related proteins independently and in the presence of one another. Here, we present a
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comprehensive side-by-side comparative analysis and investigation of α-, β- and γ-syn and their
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interactions using a broad array of biophysical tools. We have characterized the structural properties of
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α-, β- and γ-syn under a broad range of pH, temperature, and chemical denaturant using Circular
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Dichroism (CD), Thioflavin T (ThT) fluorescence, and Transmission Electron Microscopy (TEM). Our
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results show that the three synucleins are more folded as well as more aggregation prone at low pH or
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high protein concentrations. The presence of high molar ratio of β-syn inhibited fibrillation propensity of
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α- and γ-syn. However, preformed seed fibrils of β- and γ-syn did not affect fibrillation of α-syn while
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seeds of α-syn accelerated fibrillation of γ-syn to a certain extent. SPR studies suggest binding between
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β- and α-syn and between β- and γ-syn in the micromolar range and an order of magnitude higher
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binding affinity between γ- and α-syn. These findings put together suggest that weak and dynamic
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interactions between synucleins can significantly affect their fibrillation properties which could have
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strong bearing on PD and other synucleinopathies.
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Materials and methods
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Streptomycin sulfate, pH standards, and ThT were purchased from Sigma-Aldrich Co. LLC., St.
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Louis, MO. Buffer components, di-hydrogen sodium phosphate and di-sodium hydrogen phosphate,
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acetic
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1thiogalactopyranoside (IPTG) were purchased either from Sisco Research Laboratory Pvt. Ltd., India
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or from Merck, USA. Water from Millipore water purification system (Milli-Q) was used to make all the
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solutions and buffers. A plasmid containing the full-length genes of the human α-, β-, and γ-syn were a
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generous gift from Prof. Peter T. Lansbury (Harvard Medical School, Cambridge, MA, USA).
acid,
ammonium
sulphate,
ethanol,
ampicillin,
kanamycin
and
isopropyl
β-D-
3
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Sequence alignments and evolutionary relationships Protein sequences were retrieved from NCBI protein sequence database according to their gene identifier. Multiple sequence alignment (MSA) has been done using Clustal X. Phylogenetic tree was made using Phylogeny.fr40. The degree of disorders in human synucleins (α, β, and γ) were predicted using IUPred41 (http://iupred.enzim.hu).
Protein expression and purification Expression and purification of human recombinant α-, β-, and γ-syn and H50A (α-syn) from E. coli were performed as described earlier with slight modifications42. Human α-, β- and γ-syn were expressed in E. coli BL21 (DE3) using plasmid vectors pT7-7, pET 30a and pET 29a respectively. Single colonies of BL21 (DE3) were taken out from the respective agar plates and transferred into 100 ml of Luria broth (LB). Cells were grown in LB in the presence of 100 µg/ml ampicillin for α- syn and 50 µg/ml kanamycin for β- and γ-syn. Cultures were grown at 37 ºC with shaking at 200 rpm. Expression of all the three proteins was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) when the absorbance (A600) of the cultures reached 0.9-1.5. After induction with IPTG, cultures
were grown for another four hours and the cells were harvested by centrifugation at 7000g for 20 min. Pellets were recovered and resuspended in 1.0 ml of Tris buffer (50mm Tris, 10 mM EDTA, 150mM NaCl, pH 8.0). At this step, samples were frozen at -80 ºC for further use. Protein purification has been performed through a non-chromatographic method42. Human synucleins are soluble at high temperature3, which is one of the important aspectsof this method. Frozen cell pellets tubes were directly transferred in a boiling water bath and boiled for 20 min. Samples were centrifuged at 14000g for 15 min and the supernatant was transferred to a fresh tube. After that, 140 µl of a 10% solution of streptomycin sulfate and 230 µl glacial acetic acid was added in per ml of supernatant and followed by centrifugation at 15000g for 15 min. The supernatant was again removed and then precipitated by adding ice cold supersaturated ammonium sulfate (1:1 v/v) to the supernatant. The precipitated protein pellet was collected by centrifuging the samples at 14000g. The pellet was washed with 1.0 ml of ammonium sulfate solution at 4 ºC in a 1:1 v/v ratio of protein versus ammonium sulfate and precipitated proteins were collected by centrifugation. The washed pellet was resuspended in 900 µl of 100 mM ammonium acetate. At this stage, there was a formation of a cloudy solution, which was again precipitated by adding an equal volume of ethanol at room temperature. Precipitation in ethanol was repeated twice to remove residual ammonium acetate. Recovered precipitate was air dried for 20 min 4
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Biochemistry
followed by its re-suspension in 10 mM phosphate buffer. At this stage, pellet was stored at -80 ºC after flash freezing in liquid N2 for further purification steps. Human synucleins are less soluble at acidic pH due to their acidic nature. The presence of residual acid during purification steps make the solution more acidic and less soluble. To compensate the acidity, samples were titrated using 1.0 M KOH to adjust the pH ~7.4, at which human synucleins become completely soluble. After that, proteins samples were dialyzed against 10 mM phosphate buffer by using 10 kDa ultrafilters (Microcon; prerinsed with phosphate buffer) at 4 ºC. The protein samples were then spun through 100 kDa ultrafilters (Microcon; prerinsed with phosphate buffer) at 4 ºC to sterilize and remove preformed oligomers and polymeric species. The purity of the monomeric proteins was determined by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein concentration was estimated by bovine serum albumin (BSA) standard curve as well as by molar extinction coefficient (ε) of the proteins, calculated using ProtParam43. The calculated molar extinction coefficient of human α-syn, β-syn, and H50A (α-syn) is 5960 M-1 cm-1 and for γ-syn is 1490 M-1 cm-1 at 280 nm. H50A mutant of α-syn was generated using Quick change® site-directed mutagenesis kit from Stratagene using the following primer and the introduced point mutation was further confirmed by DNA sequencing. H50A (α-syn) 5'-caaggagggagtggtggctggtgtggcaacagtg-3' 3'-gttcctccctcaccaccgaccacaccgttgtcac-5'
Gel filtration chromatography Gel filtration or size exclusion chromatography of human α-, β- and γ-syn was performed using an ÄKTA Prime 10 FPLC instrument and a Hi Prep 16/60 Sephracryl/S-200 HR column from GE Healthcare at 25 ºC. The column was equilibrated using 10 mM phosphate buffer, pH 7.4 with a flow rate of 0.5 ml/min. The pressure in the column was maintained at 0.30 MPa and sample were loaded using 2.0 ml loop. Gel filtration marker proteins (1mg/ml) of known molecular weight were applied to the column for molecular mass estimation of the recombinant protein. Standards were blue dextran (2 MDa), BSA (66 kDa), Ovalbumin (45 kDa), Carbonic anhydrase (29 kDa), Alpha-lactalbumin (14 kDa) and RNase A (13.8 kDa). The void volume of the column was estimated using blue dextran. Samples were filtered through a 0.22 µm filter and then degassed properly before loading, to avoid the problems caused by bubble formation. Protein absorbance was detected at 280 nm. α- and β-syn (2 mg/ml) and γ5
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syn (4 mg/ml) were used to observe their elution profile in 10 mM phosphate buffer, pH 7.4 in the presence of 8.0 M urea and 6.0 M guanidinium hydrochloride (GdmCl). Protein samples were incubated in 8.0 M urea and 6.0 M GdmCl at 25 ºC overnight. Molecular mass values for the human synucleins were interpolated from the calibration curve using the FPLC standards of known molecular weight.
UV- absorbance of human synucleins Tyrosine absorbance for human synucleins was measured using a 1.0 ml quartz cuvette with a path length of 10 cm in a Cary Varian 100 UV-Vis spectrometer attached with a peltier temperature controller device. Protein concentrations of 1.0 mg/ml were used for the absorption spectra and all the protein solutions were prepared in 10 mM phosphate buffer at pH 7.4. Buffer baseline was subtracted from the protein spectral data before plotting.
Intrinsic tyrosine fluorescence Fluorescence measurements were performed using a 400 µl, 1.0 cm path length quartz cell in a Cary Varian Eclipse fluorescence spectrophotometer attached with a peltier temperature controller device. Human α-syn, β-syn (100 µg/ml) and γ-syn (300 µg/ml) were prepared in 10 mM phosphate buffer at pH 7.4. Control buffer data were subtracted from the protein data before plotting. Protein samples were excited at 274 nm and emission spectra were recorded from 280- 400 nm at 25 ˚C with excitation and emission slit widths of 5 nm each.
ANS binding fluorescence 1-anilino-8-naphthalene sulfonate (ANS) binding fluorescence emission spectra were recorded from 400-600 nm with an excitation wavelength of 350 nm at 25 ºC and excitation and emission slit widths of 5 nm. Human α-syn, β-syn (100 µg/ml) and γ-syn (300 µg/ml) were used for the ANS binding studies and the final ratio of ANS to protein concentration was kept equal to 5. Buffer + ANS reads at various pH values were subtracted from protein + ANS data before plotting the final graph.
Circular dichroism (CD) measurements Far-UV CD spectra of α-syn and β-syn (100 µg/ml) and γ-syn (300 µg/ml) were recorded at 25 ºC from 190-260 nm with an interval of 1 nm at a scanning speed of 50 nm/min using a Jasco-815 CD spectropolarimeter (Jasco, Japan), equipped with a peltier device. A quartz cell of 1 mm pathlength 6
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Biochemistry
was used and averages of three scans were obtained and the data were plotted in mean residue ellipticity units expressed as deg cm2 dmol-1. Near UV-CD spectra of α-, β- and γ-syn (1 mg/ml) were recorded at 25 ºC from 360-260 nm in a 10 mm quartz cell. Buffer baselines were subtracted from the protein spectra and smoothing of data was done using Savitzky-Golay method. All the solutions were prepared in the 100 mM phosphate buffer at pH 7.4.
Differential scanning calorimetry (DSC) DSC experiments were performed with a VP-DSC instrument equipped with Thermovac degassing system from Micorcal LLC., Northampton, USA. Human α-, β- and γ-syn (75 µM) were prepared in 10 mM phosphate buffer, pH 7.4 and then dialyzed overnight in the same buffer using 10 kDa cutoff Slide-A-Lyzer cassettes from Thermo Scientific, U.S.A. The solutions were degassed for 1520 min prior to loading into the DSC cells. DSC thermal denaturation curves were recorded from 5-125 ºC at a scan rate of 60 ºC / h. Four scans were recorded for each protein in forward and reverse directions.
ThT fluorescence assay Fibril formation was monitored using thioflavin T (ThT)44. ThT (100 µM) stock solution was prepared in 20 mM phosphate buffer, pH 7.5 and its concentration was calculated using a molar extinction coefficient value of 35000 M-1cm-1 at 412 nm. Human α-, β- and γ-syn (1.0 mg/ml) were prepared in 20 mM phosphate buffer, 100 mM NaCl, pH 7.5 and were shaken at 37 °C in eppendorf tubes at 200 rpm continuously for several days to assess fibril formation. ThT was excited at 445 nm and emission was recorded at 480 nm with excitation and emission slit widths of 5 nm using a 400 µl stoppered fluorescence cuvette at 25 °C.
Surface Plasmon Resonance (SPR) The binding analysis was performed using BIAcore 3000 instrument at 25 ˚C (GE Healthcare). A CM5 sensor chip (GE Healthcare) was activated as recommended by the manufacturer using an equimolar mix of NHS (N-hydroxysuccinimide) and EDC (N-ethyl-N-(diethylaminopropyl) and carbodiimide and coupled with ligand in sodium acetate buffer pH 4.5 and then blocked with ethanolamine. The system was primed twice with the running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) and regenerated using one 30 sec pulse of regeneration buffer (50 mM 7
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NaOH, 2M NaCl) at flow rate 30 µl/min. For ligand immobilization, 100 µM of γ- and α-syn was injected for 150 sec at flow rate 5 µl / min over two different flow cells. The final coupling level of γand α-syn was around 3000 and 1600 resonance units (RU) respectively. One flow cell served as a reference surface which was blocked with ethanolamine to monitor possible non-specific binding of the analyte. The analytes were diluted in running buffer and injected over both flow cells for 2.5 min. Interaction of γ-syn with β- and α-syn, was performed by injecting different concentrations of β-syn (5– 30 µM) and α-syn (0.05-30 µM) diluted in running buffer over both flow cells. The association kinetics was monitored for 150 sec, followed by 150 sec long dissociation kinetics. Data were obtained by subtracting the response obtained from the control flow cell without immobilized ligand from that obtained in the sample cell containing the ligand. Similarly, the experiments were performed to study αand β-syn interactions, where α-syn was immobilized and β-syn (1-50 µM) was injected as an analyte. Dissociation constant values were evaluated using BIA evaluation 4.1 software (BIAcore) by fitting the data using 1:1 Langmuir binding model.
Cross-inhibition and cross-seeding fibrillation assays of human α-, β-, and γ-syn Cross-inhibition of fibrillation of human synuclein proteins has been performed at various stoichiometric ratios of the proteins. For this assay, target protein concentration was kept constant, while the other protein, which was used to see their inhibitory effect on the fibrillation of the target protein, was used in various stoichiometric ratios (1:4). Accordingly, 70-280 µM (1- 4 mg/ml) protein was used for the inhibition assays. Assays were preformed in 20 mM sodium phosphate, 100 mM NaCl, pH 7.5, buffer, supplemented with 10 µM ThT. Kinetics of fibril formation was measured using ThT fluorescence. Inhibition assay was set up in a 96- well, white, clear bottom plate (CorningTM) containing 100 µl of the desired sample in quadruplicate. The plate was incubated at 37 °C and 180 rpm in a fluorescence plate reader (Varioskan, Thermo-Fischer) and ThT fluorescence was measured with an excitation at 444 nm and emission at 485 nm. Data from four wells were averaged and plotting with Origin7 software. Respective standard errors of the mean (SEM) were included in each figure. For generating seed fibrils, human α-, β-, and γ-syn monomers with a concentration of 3 mg/ml in 20 mM sodium phosphate, 100 mM NaCl, pH 7.5, buffer were incubated at 37 °C, 200 rpm for 72 h in an orbital shaker. After 72 h, samples were centrifuged at 14000 rpm for 30 min. The supernatant was discarded and the fibrillar pellet was washed twice with phosphate buffer and then resuspended in 500 µl of 20 mM phosphate buffer, 100 mM NaCl, pH 7.4. Mechanical disruption of seed fibrils was done 8
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Biochemistry
through rigorous vortexing and the seeds sample was passed through a 25 gauge needle to prepare a homogenous mixture of seeds. To assess the cross-seeding effects, 5, 10 and 20% v/v seeds were added into monomeric α-, β-, and γ-syn. Cross-seeding effect on fibril kinetics was monitored using ThT fluorescence on a Varioskan fluorescence plate reader, as described for cross-inhibition experiments.
Transmission electron microscopy (TEM) TEM images of human α-, β-, γ-syn in respective conditions were collected using a JEOL TEM 2100 microscope operating at an accelerating voltage of 200 kV as described previously45.
Results Structural characteristics of human synucleins Purified recombinant human α-, β- and γ-syn proteins were run using 15% SDS-PAGE and proteins bands were observed around ~20 kDa in lane 4 (Fig.S1 A, B, and C). Multiple sequence alignment (MSA) of α-, β- and γ-syn from different vertebrates showed that N-terminus among different vertebrates is more conserved than the C-terminus (Fig. S2)46. MSA (Fig. S2) has been used to generate a phylogenetic tree (Fig S3)40 which has been created using Phylogeny.fr40. Phylogenetic analysis suggested that α- and β-syn have a close evolutionary relationship as compared with γ-syn47. In the phylogenetic tree, the horizontal lines or branches represent evolutionary lineages changing over time and the length of the branch represents a number of genetic changes over the time. Scale for this measurement (0.09) has been provided at the bottom of the tree (Fig. S3), showing the length of the branch that indicates an amount of genetic change of 0.09. The numbers next to each node represent a measure of support for the node. These numbers may lie between 0 (minimum support) and 1 (maximum support) and can also be represented as percentages. A high value at a particular node means the sequences next to the node are clustered together (Fig. S3). In the evolutionary lineage, γ-syn is distantly related to α- and β-syn (Fig. S3). The N-terminus of α-, β- and γ-syn is more conserved than the C-terminus46 (Fig. S4). Human α-, β- and γ-syn have 4, 4 and 1 tyrosine residues, respectively. In wild-type, human αsyn tyrosines are located at positions 39, 125, 133, and 136, whereas in β-syn, they are located at positions 39, 119, 127, and 130. However, in γ-syn, there is only one tyrosine at position 39 (Fig. S4). MSA of human synucleins showed that N-terminus tyrosine at position 39 in α-, β- and γ-syn are identically placed. While α- and β-syn have identically placed C-terminus tyrosine amino acids 9
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residues2, γ-syn lacks tyrosine-rich c-terminus2 (Fig S4). In the case of α- and β-syn, number and location of tyrosine amino acid residues are conserved. The C-terminus of human α-, β- and γ-syn has a greater tendency for disorder compared to N-terminus46 (Fig. S5) and is highly variable in terms of amino acids as well as sequence length46. In the case of γ-syn, there is only one tyrosine residue towards N-terminus, which is likely to be more ordered than the C-terminus of the sequence and tyrosine fluorescence in γ-syn is solely dependent on this tyrosine residue which could be the reason for the lower absorbance and fluorescence values of γ-syn (Fig. 1A and 1B).
Figure 1. Spectroscopic characterization of human synucleins (A) UV-absorbance spectra of human synucleins (1 mg/ ml) were recorded from 230-330 nm at a scan rate of 10 nm/min. (B) Fluorescence emission spectra of human α and β-syn (100µg/ml) and γ-syn (300µg/ml) were recorded from 280-400 nm using a λex of 274 nm, Ex and Em slit widths of 5 nm and a scan rate of 50 nm/min. (C) Far-UV CD spectra of human synucleins were recorded from 190-260 nm using 0.1 mm quartz cuvette and with protein concentration 100 µg / ml for α and β-syn and 300 µg / ml for γ-syn. (D) Near-UV CD spectra of human synucleins were recorded from 240-350 nm using 1.0 cm
10
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quartz cuvette with a protein concentration of 1.0 mg/ml. All the experiments were conducted in 10 mM phosphate buffer, pH 7.4 and 25 °C and buffer traces were subtracted from the protein data.
We have used tyrosine residue signature to assess their conformational changes. Free tyrosine was observed to have an absorption maximum at 274 nm and a fluorescence emission maximum at ~305 nm upon excitation at 274 nm. Human α- and β-syn exhibited a sharp absorbance peak at 274 nm while γsyn showed a broad peak at ~ 274 nm with far lower absorbance compared to α- and β-syn at a similar protein concentration of 1 mg/ml each (Fig. 1A). UV-absorbance is also affected by protein structure as well as the local environment of the aromatic amino acid48. β-syn is less compact in comparison with α-and γ-syn2. The differences in the compactness of human synucleins, the number of tyrosine residues in each and their location can also be responsible for the differences in their absorbance spectra. The α- and β-syn showed fluorescence emission maxima at ~305 nm while for γ-syn, it was at ~345 nm (Fig. 1B). Far-UV circular dichroism (CD) spectra of α-, βand γ-syn were recorded from 190-260 nm at pH 7.4, 25 °C. Far-UV CD spectra of these proteins are similar to the natively unfolded proteins with a characteristic minimum at ~200 nm (Fig. 1C) with a small helical structure evident by small negative ellipticity at 222 nm, which is higher in the case of γsyn as compared with α-, and β-syn. The near-UV CD, recorded from 250-350 nm at pH 7.5 and 25 °C, showed an absence of any tertiary structure (Fig. 1D).
pH and temperature-induced conformational changes in human synucleins Far-UV CD spectra of human synucleins were recorded at various pH conditions: 8.5 (Tris buffer), 7.5 and 6.5 (phosphate buffer), 5.5, 4.5 and 3.5 (acetate buffer), 2.5 and 1.5 (phosphate buffer) at 25 °C using 1 mm path length cell. Protein concentration was 100 µg/ml for α- and β-syn and 300 µg/ml for γ-syn. There was an increase in the negative ellipticity at 222 nm with a decrease in pH, which was associated with the decrease in negative ellipticity at 198 nm (Fig. 2 A, B and C). CD spectra of α-, β- and γ-syn also showed an increase in the negative ellipticity at 222 nm and a decrease in negative ellipticity at 199 nm with an increase in temperature (Fig. 2D, E, and F). The temperature-induced conformational changes were reversible (Fig. S6 A-C). Temperature-induced thermal denaturation at 222 and 199 nm of α-, β- and γ-syn led to an increase in negative ellipticity at 222 nm with a decrease at 199 nm with increasing temperature (Fig. S6). Thermal melting of α- and β-syn was reversible in nature (Fig. S7A and B), while it was not completely reversible in case of γ-syn at 199 nm (Fig. S7C). 11
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decreasing rapidly until the maximal measurable temperature of 125 ºC, which suggested that the disordered structure of α-syn is quite stable with no tertiary and limited secondary structure. The calorimetric traces showed reversibility even after a second heating and cooling cycle for all the three synucleins (Fig.S8A, B, and C). 1-anilino-8-naphthalene sulfonate (ANS) has been used to measure non-native partially folded conformations of globular proteins. ANS interacts with the solvent exposed hydrophobic patches of the proteins and leads to increased fluorescence with shifts to lower wavelengths49. With the decrease in pH, ANS binding to α-syn leads to a significant increase in ANS fluorescence intensity along with a slight blue shift (Fig. 2G). However, no significant change in ANS fluorescence emission was observed in the case of β-syn (Fig. 2H). Increasing ANS fluorescence intensity with decreasing pH in γ-syn was also associated with a larger blue shift in comparison with that of α-syn (Fig. 2I). The changes in ANS fluorescence of human α and γ-syn as a function of pH are due to the changes in their conformations at various pH, since intrinsic ANS fluorescence shows negligible pH dependence in the pH range used (Fig. S9). The control data of Fig. S9 A and B have been subtracted from the respective protein data and the resultant plots have been presented as Fig. 2G-I.
Effect of temperature and GdmCl on intrinsic tyrosine fluorescence Human α- and β-syn also showed a linear pattern of temperature-induced tyrosine quenching (Fig. 3A and B) similar to free L-tyrosine (Fig. S10A). γ-syn also exhibited non-linear, biphasic tyrosine fluorescence quenching with increasing temperature (Fig. 3C and inset). GdmCl, a strong protein denaturant, has no effect on the free-L tyrosine fluorescence (Fig. S10B). Intrinsic tyrosine fluorescence of α- and β-syn increased linearly with increasing GdmCl concentrations with more pronounced effects in β- as compared to α-syn, suggesting unfolding and conformational changes in α- and β- syn (Fig. 3D and E). However, intrinsic tyrosine fluorescence of γ-syn increased at 342 nm compared to 303 nm. Tyrosine fluorescence decreased sharply up to 3M GdmCl and no further decrease in tyrosine fluorescence was observed (Fig.3F). Size exclusion chromatography elution profile of the native α-, βand γ-syn at pH 7.4 showed that the majority of these protein fractions are in their monomeric form (larger peak) in comparison with higher order oligomeric species of the protein (smaller peak) (Fig. S11 B, C, and D). Calculated molecular mass of human synucleins on the basis of their elution profile patterns are ~ 45 kDa, which are larger than the molecular masses predicted from their amino acid sequences, ~14.4, ~14.2 and 13.0 kDa, respectively, which may be due to their large Stokes radius. α13
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syn has large Stokes radius in its monomeric, unstructured state50, 51. Size exclusion chromatogram of monomeric α-syn has been discussed previously52, 53.
Figure 3: Effect of temperature and denaturant (GdmCl) on the intrinsic tyrosine fluorescence of human synucleins Emission spectra of human α- (A), β- (B) and γ-syn (C) were recorded from 15-100 ˚C at a temperature difference of 5 ˚C. Inset panels showed the λEm max versus temperature. Intrinsic tyrosine fluorescence of α- (D), β- (E) and γ-syn (F) were recorded in the presence of GdmCl. Protein concentration for α- and β-syn (100 µg/ml) and 300 µg/ml for γ-syn were used. Buffer scans were subtracted from the protein data. Emission spectra were recorded from 280- 400 nm at 25 ˚C in 10 mM phosphate buffer, pH 7.4 with λex was 274 nm. Inset panels showed the effect of GdmCl on the intrinsic tyrosine fluorescence of α- and β- at 303 nm and γ-syn at 303 and 342 nm.
Hydrodynamic radii of α-, β- and γ-syn are larger in the presence of denaturants (8 M urea and 6 M GdmCl), as a result of further unfolding due to which they elute out first through the column in comparison to their native states55. α- and β-syn showed similar elution patterns, while γ-syn did not 14
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show a clear trend which may be due to fewer tyrosine amino acids or lesser structure (Fig. S11 B, C, and D).
Effect of pH, concentration, and temperature on the fibrillation of human synucleins Fibrillation of human synucleins at various pH values has been monitored using ThT dye, which is extensively used for the detection of the amyloid fibrils44. Upon excitation at 450 nm, ThT gives fluorescence at 480 nm after binding with cross-beta sheets of amyloid fibrils.
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Figure 4: Effect of pH, temperature and protein concentration on the fibrillation kinetics of human synucleins Fibrillation kinetics of human α- (A), β- (B), γ-syn (C) and H50A-α-syn (D) synucleins at 1 mg/ml were studied at different pH values and 25 °C. Phosphate buffer was used at pH 2.0, 6.0 and 7.4 while acetate buffer was used at pH 4.0 and 5.0. Fibrillation kinetics of human α- (E), β- (F) and γ- syn (G) were studied in presence of the different protein concentrations at 25 °C in 20 mM Phosphate buffer. Respective buffers of 20 mM strength, 100 mM NaCl, 0.02% Sodium azide, 37 °C and 200 rpm shaking were used for the fibrillation. ThT fluorescence was excited at 450 nm and the emission was plotted at 480 nm. Ex and Em slit widths were 2.5 nm. Fibrillation kinetics of human α- (H), β- (I) and γ-syn (J) were studied in 20 mM Phosphate buffer at 25, 37 and 45 ºC with a protein concentration of 1 mg/ml.
Fibrillation kinetics exhibit a peculiar sigmoidal pattern of amyloid formation characterized by an initial lag phase, followed by a log phase that ends with a saturation phase. The lag phase represents the nucleation, log phase corresponds to fibril elongation and mature fibrils are formed at saturation phase. Fibrillation studies at various pH values showed that fibrillation was accelerated in α-, β- and γ-syn with decreasing pH (Fig. 4A, B, and C). ThT fluorescence under control conditions (Buffer + ThT) has been also recorded under various pH values to assess the effect of pH on ThT fluorescence and observed to be insignificant in magnitude (Fig. S12). Fibrillation kinetics of α-syn have been reported previously with few pH values and have described the effect of preformed fibrils of α-syn on its aggregation53, 54 . We have carried out fibrillation kinetics of human α-, β-, and γ-syn proteins at various pH to study the effect of pH on their aggregation propensity. Our results show that ThT fluorescence of α-syn increases remarkably at pH 6, indicating the formation of large amounts of amyloids (Fig. 4A). Human β-syn does not fibrillate at physiological pH 7.4. However, at lower pH (9, ThT as 29
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a probe is reported to become highly unstable71. In pH dependence aggregation of human α-, β-, and γsyn, we have studied their fibrillation kinetics at pH 2 and 4 as well as at pH 9. We have not measured any ThT fluorescence above pH 9, where ThT becomes highly unstable. Since low pH controls of ThT were subtracted from synuclein fluorescence data, the pH effect on ThT fluorescence is minimized. We have seen different responses for α-, β-, and γ-syn aggregation at basic and acidic pH which suggests that aggregation patterns for α-, β-, and γ-syn are protein specific (Fig. 4 A, B and C). To corroborate the pH dependent aggregation of α-, β-, and γ-syn with fibril morphologies, we have monitored changes in fibril morphologies at the completion of ThT kinetics under various pH. For example, in the case of αsyn at pH 2, 4 and 9, we have seen different aggregation kinetics patterns (Fig. 4A) as well as different morphologies of the fibrils at specific pH values (Fig. 5 A-E). Side by side comparison of the pH dependent ThT kinetics (Fig. 4) and morphologies of the fibrils at a specific pH value for particular proteins varied significantly (Fig. 5). We have also monitored the changes in the unfolded peptide conformations of human synucleins under aggregation conditions at various pH values using CD spectroscopy and the data support well the ThT aggregation kinetics (Figs. S14, S15 and S16). Fibrils formed in human α-, β- and γ-syn at various pH values differ in their morphology, which suggests that fibril morphology is critically dependent on the environment in which they are formed. αsyn formed well-branched fibrils in the form of a network at pH 7, while at pH 2, it formed very small and thin fibers which became thick and short at pH 4 strongly suggest that changes in electrostatic interactions also significantly alter fibril morphologies in synucleins. Interestingly, β-syn did not form fibrils at pH 7.4, while at lower pH where the protein becomes protonated, it formed well-defined fibrils. γ-syn has a lesser tendency to form fibrils compared with α-syn, but its fibril forming propensity and morphology of the fibers were strongly dependent on protein environment. H50A α-syn also showed pH-dependent changes in fibrillation propensity and fibril morphology. Fibril morphologies in α- and γsyn also varied with protein concentration. However, β-syn fibril formation was not affected by increasing concentration and only amorphous aggregates were formed. Previously, it has been demonstrated that fibril morphologies are directly associated with different levels of cytotoxicity 45, 72. Due to its highly dynamic nature, it has been suggested that human α-syn could be involved in interactions with other proteins73. Further, it has been demonstrated that β- and γ-syn can inhibit α-syn fibrillation73 suggesting that interactions among synucleins are important and need to be investigated. Initial experiments using ITC indicated weak binding between α- and β-syn and between β- and γ-syn but negligible interaction between α- and γ-syn (Fig. S17). The data are qualitative in nature due to high 30
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heats of dilution of the proteins. Interactions between synucleins using SPR (Fig. 6 A-C) yielded micromolar binding affinity between β-γ syn and α-γ interactions while α-β syn interactions were weaker by nearly two orders of magnitude. Increasing amounts of β-syn added to α-syn inhibited fibrillation of α-syn. At a 1:4 molar ratio of α:β syn, fibrillation of α-syn was reduced with an increase in the lag phase and fewer fibrils were formed in comparison to α-syn alone (Fig. 7 A and D). The possibility of α- and β-syn interaction has been suggested earlier with the use of hen egg lysozyme which induced α-syn fibrillation2. The addition of γsyn to α-syn in different stoichiometric ratios increased the lag phase of fibrillation of α-syn (Fig. 7B) as well as the fibril morphology and the interactions observed between the two were of micromolar affinity. The effect of β-syn on the fibrillation of monomeric γ-syn was also prominent. The fibrillation propensity of γ-syn was significantly reduced at 1:4 molar ratio of γ:β syn (Fig. 7C) and fibril formation of γ-syn was reduced considerably (Fig. 7H). β-syn is present in the brain in the same stoichiometry with α-syn3 and it was observed that the same stoichiometric amounts of α- and β-syn were not able to inhibit the fibrillation of α-syn2. However, an excess amount of β-syn in comparison to α-syn significantly regulates the fibrillation of α-syn indicating that β-syn may act as a controller to check misfolding of αsyn in-vivo2. β-syn also inhibited the aggregation of α-syn in a transgenic mouse brain and transfected neurons74. These observations clearly suggest that the interactions between α-β-syn and β-γ-syn though weak in nature are crucial and decisive. It might be possible that elevated levels of β-syn can potentially reverse the factors which allow misfolding and fibrillation of α-syn in-vivo. The chaperone like properties of β-syn controlling the fibrillations of α-syn2 as well as γ-syn and the interaction data based on SPR measurements suggest that the interaction between α-β syn, α-γ syn, and γ-β syn are weak and possibly transient in nature and not due to any hydrophobic interaction. Recent studies using NMR paramagnetic relaxation enhancement experiments also suggest a weak and transient binding interaction in the µM range for α-β syn interaction which matches well with our SPR data75. Since hydrophobic region is absent in the β-syn, it is less likely to interfere in fibrillation of α- or γ-syn. The NMR data reveal that C-terminal acidic residues of β-syn interact with the ‘hotspot (HS)’ region of α-syn comprising residues 38-45 via specific interactions and thus prevent fibrillation of α-syn, while α-syn self-interactions were shown to be as a result of both HS-HS interactions and HS-C-terminal cross interactions comprising both nonspecific and specific interactions. Similar type of specific interactions are expected to occur between the three synucleins.
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A significant numbers of unstructured protein domains and entire disordered proteins have been characterized in terms of their functional roles and are known to be involved in many cellular signaling events76. Interaction between two intrinsically disordered proteins (IDPs) is still not understood well as they could have multiple binding sites and attain folding upon interaction with other partners. It has been shown that C-terminal domain of 4.1G protein and nuclear mitotic apparatus protein, which are IDPs, interact specifically in a dynamic equilibrium and their micromolar binding affinity has been measured using ITC77. The interaction between two unfolded proteins can modulate signal transduction and the specific binding events between two IDPs may or may not lead to structural transitions from disorder to ordered state76. Sometimes, IDPs remain disordered after binding with their partners and forms dynamic and fuzzy complexes78. The cytoplasmic domain of ζ chain of T cell receptor is intrinsically disordered in nature and dimerizes through specific interactions to modulate the immune response. During dimerization, there is no structural transition and it is distinct from non-specific aggregation76. Most of the proteins that participate in binding, signaling events, and in transcription events are enriched in intrinsically disordered regions79,80. Altered expression of IDPs is associated with many diseases also which suggests that their controlled regulation and homeostasis plays an essential role in signal fidelity80. Human α-, β-, and γ-syn are present in the same niche in the brain and their altered expression can influence their function by allowing them to interact differently under different stoichiometric ratios. Rat brain studies showed that α-syn is abundant in central catecholaminergic regions, β-syn is primarily localized in the somatic cholinergic neurons with weak expression in catecholaminergic neurons and γsyn is expressed in both cholinergic and catecholaminergic regions of the brain81. β- and γ -syn were also found in axon terminals of the hippocampus of PD patients39. In the case of neuronal damage, αand β-syn colocalize and accumulate rapidly in injured axons and accelerate axonal transport and cytoskeletal reorganization82. It is also reported that α- and γ-syn interact and influence the aggregation of synucleins83. Also, oxidized γ-syn at Met38 forms abnormal inclusions in amygdala and substantia nigra regions of brain and co-localized with α-syn84. On the basis of the interaction between α-β syn and γ-β syn and the inhibitory effect of monomeric β-syn on the aggregation of α- and γ-syn, it may be possible that human synuclein proteins can modulate various signaling activities due to their altered expression levels which can be detrimental to the neuronal health. Therefore, altered expression, availability and different levels of human synuclein proteins can affect the signal fidelity that can be one of the deterministic factors for the initiation of misfolding and aggregation of human α- or γ-syn. The interaction of β-syn to α- and γ- syn provides a clue that nature has developed a rescue system or a 32
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balancing system for α- and γ- syn aggregation in a way that β-syn can cross-talk with α- and γ- syn and can modulate their function by modulating their signals. Human α-, β-, and γ-syn are generally expressed in similar brain regions such as the thalamus, substantia nigra, caudate nucleus, amygdala, and the hippocampus85,
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homology69. The seeding experiments carried by us suggest that while preformed seeds of α-syn could accelerate the fibrillation in α-syn, cross-seeding by preformed seed fibrils of β-, and γ-syn did not induce any effect on the fibrillation of α-syn (Fig. 8 A-C) and no significant changes in the fibril morphology of α-syn in the presence of the seeds of β-, and γ-syn were seen (Fig. 8 H and I). These results suggest that β-, and γ-syn could be absent in LB and LN69 because of their inability to cross-seed α-syn. However, preformed seed fibrils of α-syn could cross seed γ-syn and enhance the fibrillation of γ-syn with a decrease in the lag phase. These changes were also associated with the changes in the fibril morphology of γ-syn (Fig. 8D, J and K). However, β-syn seeds did not affect γ-syn fibrillation much and fibril morphologies were similar to the fibrils of γ-syn alone (Fig. 8J and L). To summarize the findings, a schematic representation of the effects of pH, temperature, and concentration on the fibrillation propensity of human α- , β- and γ- syn have been shown in Fig. 9.
Figure 9: A schematic representation of the effect of the pH, concentration, and temperature on the fibrillation of human α-, and γ-syn (Fig. 9A) and β-syn (Fig. 9B) have been described in the figure.
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Conclusion Human α-, β- and γ-syn are highly homologous, natively unfolded proteins and are involved in both neural and non-neural human diseases including PD, AD, and other synucleinopathies. Low pH and high temperature shift the equilibrium from their natively unfolded state to partially folded intermediate states which accelerate fibril formation propensity of human synucleins. In native conditions, these proteins are unfolded with some residual amount of secondary structure that opens further under denaturing conditions. The immediate environment of human synucleins, such as pH, temperature, their concentration, protein-protein interactions, etc., are vital factors that affect their fibrillation and fibril morphologies. As cytotoxicity is highly influenced by fibril morphology, it is thus expected that fibrils formed by human synucleins under different cellular environments will have different levels of cytotoxicity. SPR data as well as cross-inhibition fibrillation assays suggest that the interaction between α-β syn, α-γ syn and γ-β syn are specific, weak and transient in nature. Fibrillation of γ-syn is strongly suppressed in the presence of 1:4 molar ratio of γ:β syn. Cross-seeding by β- and γ-syn is not able to accelerate the fibrillation of α-syn, while the preformed seeds of α-syn are able to enhance γ-syn fibril forming propensity. The ability of β-syn to interact either with α-syn or γ-syn, which can form fibrils at physiological conditions, suggests that the presence of these three proteins in the same niche of the brain could have a significant role in the modulation of their function by changing their homeostasis in the neuronal cytoplasm. It might be possible that β-syn plays a role of a rescue worker for α-, and γ-syn under certain stress conditions which would otherwise lead to their misfolding and fibrillation. The present studies, therefore, not only provide a comprehensive comparison of the various biophysical properties of α-, β-, and γ-syn, but also suggest the possible involvement of homeostasis between them towards modulating various signals under physiological versus disease conditions, and help in understanding their precise role in PD and other associated synucleinopathies.
Supporting Information 1. SDS-PAGE analysis of purified human α-, β- and γ-syn 2. Multiple sequence alignment of synucleins from vertebrates 3. Evolutionary relationship of synuclein proteins 4. Multiple sequence alignment of human synucleins 5. Prediction of disorderness in human synucleins using IUPred 6. Effect of temperature on the secondary structure of human synucleins 34
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7. Thermal denaturation of human α-, β- and γ-syn using CD spectroscopy 8. Thermal denaturation of human α-, β- and γ-syn using DSC 9. Effect of pH on the ANS fluorescence 10. Effect of temperature and GdmCl on the intrinsic tyrosine fluorescence of free L-tyrosine 11. Size exclusion chromatography of human α-, β- and γ-syn 12. Effect of pH on ThT fluorescence 13. Effect of temperature on ThT fluorescence 14. pH-dependent Far-UV CD structural changes in human β-syn under aggregation conditions 15. pH-dependent Far-UV CD structural changes in human γ-syn under aggregation conditions 16. pH-dependent Far-UV CD structural changes in human H50A (α-syn) under aggregation conditions 17. ITC isotherms of human α-β, α-γ, and β-γ syn interactions
Author Contributions: RB provided the lab space, chemicals, and infrastructure. MKJ and RB designed the experiments and analyzed the data. MKJ conducted most of the experiments. Cross-inhibition fibrillation experiments were done by PS, Cross-seeding experiments were performed by SR, and ITC experiments were repeated by SR and PS. SPR experiments were designed by MKJ and PS and conducted by PS. TEM images of cross-inhibition and cross-seeding fibrillation experiments were taken by PS. MKJ and RB wrote the manuscript.
Acknowledgement We thank Prof. Peter Lansbury, Harvard Medical School, Cambridge, MA, USA for providing the human α-, β- and γ-syn clone and Advanced Instrument Research facility, Jawaharlal Nehru University, New Delhi for access to CD and TEM. Authors thank Dr. L. M. F. Holthauzen, University of Texas Medical Branch, Galveston, for fruitful discussions on SPR data, Dr. Shalini Gupta and Dr. Shruti Khanna, IIT Delhi for access to Biacore 3000 SPR equipment and for technical help and discussions and Dr. Souvik Maiti, IGIB, Delhi for CM-5 Chip. Manish Kumar Jain acknowledges Council of Scientific and Industrial Research, India and Indian Council of Medical Research for the fellowship support. DBTBUILDER facility (BT/PR/5006/INF/22/153/2012) and DST-PURSE grant is also acknowledged.
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Abbreviations: AD; Alzheimer’s disease, ANS; 1-anilino-8-naphthalene sulfonate, BCSG-1; Breast cancer-specific gene 1, BSA; Bovine serum albumin, CD; Circular dichroism, DSC; Differential scanning calorimetry, DN; Dopaminergic neurons, DLB; Dementia with Lewy bodies, Ex; excitation, Em; emission, FPLC; Fast protein liquid chromatography, GdmCl; Guanidinium hydrochloride, h; hour, ITC; Isothermal titration calorimetry, IPTG; isopropyl β-D-1-thiogalactopyranoside, IDP; Intrinsically disordered protein, LB; Lewy body, LN; Lewy neutrites, Min; minute, MSA; Multiple sequence alignment, NACP; Non-amyloid component precursor protein, PNP; Phosphonucleoprotein, ThT; Thioflavin T, TEM; Transmission electron microscopy, Sec; second, SN; Substantia nigra, PD; Parkinson’s disease, αsyn/SNCA; α-synuclein,β-syn/SNCB; β-synuclein,γ-syn/SNCG; γ-synuclein.
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