Comparative analysis of the conformation, aggregation, interaction

Subscriber access provided by University of Winnipeg Library

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

1

Comparative analysis of the conformation, aggregation, interaction and fibril

2

morphologies of human α, β, and γ synuclein proteins

3

Manish Kumar Jain1, 2, Priyanka Singh1, 3, Sneha Roy1, 3, and Rajiv Bhat1

4 1

5

School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110 067, India

6 7

2

8

University of Texas Medical Branch, Galveston, TX 77555, USA

Current Affiliation: Center for Addiction Research, Department of Pharmacology and Toxicology,

9 10

3

Equal contribution

11 12

Running Title: Biophysical studies of human α, β, and γ-synuclein proteins

13 14

To whom correspondence should be addressed: Rajiv Bhat, School of Biotechnology, Jawaharlal Nehru

15

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

19

The human synucleins family is comprised of α-, β- and γ-syn proteins. α-syn has the highest

20

propensity for aggregation, and its aggregated forms accumulate in Lewy bodies (LB) and Lewy neuritis

21

(LN), which are involved in Parkinson’s disease (PD). β- and γ-syn are absent in LB and their exact role

22

is still enigmatic. β-syn does not form aggregates under physiological conditions (pH 7.4), while γ-syn is

23

associated with neural and non-neural diseases like breast cancer. Due to their similar regional

24

distribution in brain, natively unfolded structure and high sequence homology, it has become important

25

to study the effect of environment on their conformation, interactions, fibrillation and fibril

26

morphologies. Our studies show that high temperature, low pH, and high concentrations increase the

27

rate of fibrillation of α- and γ-syn while β-syn forms fibrils only at low pH. Fibril morphologies are

28

strongly dependent on the immediate environment of the proteins. The high molar ratio of β-syn inhibits

29

the fibrillation in α-, and γ-syn. However, preformed seed fibrils of β-, and γ-syn do not affect

30

fibrillation of α-syn. Surface Plasmon Resonance (SPR) data shows that interaction between α-β, β-γ and

31

α-γ syn are weak to moderate in nature and they can be physiologically significant to counteract several 1

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 42

32

adverse conditions in the cells that trigger their aggregation. These studies could be helpful in

33

understanding collective human synucleins behavior under various protein environments and in the

34

modulation of the homeostasis between β-syn and healthy versus corrupt α- and γ-syn which can

35

potentially affect PD pathology.

36 37

Introduction

38

Human synuclein family is comprised of small (~14kDa), natively unfolded α-, β- and γ-syn

39

proteins1,2 which are expressed at high levels in the brain3-6. Human synucleins have acidic c-terminal3, 4

40

and repetitive degenerative amino acid motif KTKEGV towards N-terminus5. α- and β-syn are abundant

41

in the central nervous system and presynaptic nerve terminals3, whereas γ-syn is expressed mainly in the

42

peripheral nervous system4, 6. These proteins are heat stable, relatively soluble and lack cysteine and

43

tryptophan residues3, 5, 7.

44

α-syn is located in the vicinity of the synaptic vesicles3, platelets8 and also plays a role in

45

neurotransmission9. It is also known as the non-amyloid component precursor protein (NACP) or

46

synelfin10, 11 and has three isoforms of 140, 126 and 112 residues resulting from alternative splicing12.

47

The N-terminus of α-syn-140 isoform has the sites for six familial PD mutations (A30P, E46K, H50Q,

48

G51D, A53T and A53E)13-17 and a lipid-binding domain of apolipoproteins5, 18. The central region of α-

49

syn from residues 61–95 has highly aggregation-prone non-amyloid beta sequence (NAC)9, 19

16

20

10

. α-syn

50

interacts with other proteins such as synphilin , tubulin , tau

and 14-3-3 proteins of chaperone

51

family21. Human β-syn, a 134 amino acid long protein also known as phosphoneuroprotein-14 (PNP-

52

14)3, 22, 23, is mainly expressed in brain3 and Sertoli cells of testis24 and shares 78% sequence homology

53

to α-syn. A hydrophobic patch (residues 73-83) is absent in β-syn25. Human γ-syn is a 127 amino acids

54

long protein, and is the smallest protein of the family sharing 60% sequence homology with α-syn25. γ-

55

syn is also known as a breast cancer-specific gene 1 (BCSG-1)26, persyn27 and has only a single tyrosine

56

at the C-terminus2. γ-syn is present in cell bodies, axons, spinal cord and sensory ganglia27, epidermis28,

57

malignant breast cancer tissues26 and is involved in neural and non-neural disease. Surprisingly, it is

58

highly expressed in the retina of Alzheimer’s disease (AD) patients29. Human synucleins are associated

59

with PD, AD and other synucleinopathies such as multiple system atrophy, Hallervorden–Spatz

60

diseases, LB disorders, and the LB variant of AD30-34. Synucleinopathies are caused by the depositions

61

of fibrillated α-syn in LB in midbrain rich in cross-β structure35-38. However, β- and γ-syn are absent in

62

LB and play a substantial role in hippocampal axon pathology in PD39. 2

ACS Paragon Plus Environment

Page 3 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

63

A large number of biophysical studies have been performed on α-syn previously. However, β-

64

and γ-syn, the other two proteins of the family, have not been studied in detail so far. Also, their

65

interaction with each other and their cross-seeding capabilities have not been investigated in detail. High

66

sequence similarities amongst them, their presence in the same niche of brain, and homeostasis between

67

them, may influence their aggregation and can modulate their functions in the presence of each other,

68

which may make neurons vulnerable and lead to neurodegeneration. Due to the absence of a well folded

69

structure, they are highly dynamic in nature and can modulate each other’s structure, function and

70

aggregation propensity in the presence of ligands that could have significant bearing on PD

71

pathogenesis. Therefore, it is of significant interest to study the various biophysical properties of these

72

closely related proteins independently and in the presence of one another. Here, we present a

73

comprehensive side-by-side comparative analysis and investigation of α-, β- and γ-syn and their

74

interactions using a broad array of biophysical tools. We have characterized the structural properties of

75

α-, β- and γ-syn under a broad range of pH, temperature, and chemical denaturant using Circular

76

Dichroism (CD), Thioflavin T (ThT) fluorescence, and Transmission Electron Microscopy (TEM). Our

77

results show that the three synucleins are more folded as well as more aggregation prone at low pH or

78

high protein concentrations. The presence of high molar ratio of β-syn inhibited fibrillation propensity of

79

α- and γ-syn. However, preformed seed fibrils of β- and γ-syn did not affect fibrillation of α-syn while

80

seeds of α-syn accelerated fibrillation of γ-syn to a certain extent. SPR studies suggest binding between

81

β- and α-syn and between β- and γ-syn in the micromolar range and an order of magnitude higher

82

binding affinity between γ- and α-syn. These findings put together suggest that weak and dynamic

83

interactions between synucleins can significantly affect their fibrillation properties which could have

84

strong bearing on PD and other synucleinopathies.

85 86

Materials and methods

87

Streptomycin sulfate, pH standards, and ThT were purchased from Sigma-Aldrich Co. LLC., St.

88

Louis, MO. Buffer components, di-hydrogen sodium phosphate and di-sodium hydrogen phosphate,

89

acetic

90

1thiogalactopyranoside (IPTG) were purchased either from Sisco Research Laboratory Pvt. Ltd., India

91

or from Merck, USA. Water from Millipore water purification system (Milli-Q) was used to make all the

92

solutions and buffers. A plasmid containing the full-length genes of the human α-, β-, and γ-syn were a

93

generous gift from Prof. Peter T. Lansbury (Harvard Medical School, Cambridge, MA, USA).

acid,

ammonium

sulphate,

ethanol,

ampicillin,

kanamycin

and

isopropyl

β-D-

3

ACS Paragon Plus Environment

Biochemistry 1 2 94 3 95 4 5 96 6 7 97 8 9 98 10 99 11 12 100 13 14 101 15 16 102 17 103 18 19 104 20 21 105 22 106 23 24 107 25 26 108 27 28 109 29 110 30 31 111 32 33 112 34 35 113 36 114 37 38 115 39 40 116 41 117 42 43 118 44 45 119 46 47 120 48 121 49 50 122 51 52 123 53 54 124 55 56 57 58 59 60

Page 4 of 42

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

ACS Paragon Plus Environment

Page 5 of 42 1 2 125 3 126 4 5 127 6 7 128 8 9 129 10 130 11 12 131 13 14 132 15 16 133 17 134 18 19 135 20 21 136 22 137 23 24 138 25 26 139 27 28 140 29 141 30 31 142 32 33 143 34 35 144 36 145 37 38 146 39 40 147 41 148 42 43 149 44 45 150 46 47 151 48 152 49 50 153 51 52 154 53 54 155 55 56 57 58 59 60

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

ACS Paragon Plus Environment

Biochemistry 1 2 156 3 157 4 5 158 6 7 159 8 9 160 10 161 11 12 162 13 14 163 15 16 164 17 165 18 19 166 20 21 167 22 168 23 24 169 25 26 170 27 28 171 29 172 30 31 173 32 33 174 34 35 175 36 176 37 38 177 39 40 178 41 179 42 43 180 44 45 181 46 47 182 48 183 49 50 184 51 52 185 53 54 186 55 56 57 58 59 60

Page 6 of 42

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

ACS Paragon Plus Environment

Page 7 of 42 1 2 187 3 188 4 5 189 6 7 190 8 9 191 10 192 11 12 193 13 14 194 15 16 195 17 196 18 19 197 20 21 198 22 199 23 24 200 25 26 201 27 28 202 29 203 30 31 204 32 33 205 34 35 206 36 207 37 38 208 39 40 209 41 210 42 43 211 44 45 212 46 47 213 48 214 49 50 215 51 52 216 53 54 217 55 56 57 58 59 60

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

ACS Paragon Plus Environment

Biochemistry 1 2 218 3 219 4 5 220 6 7 221 8 9 222 10 223 11 12 224 13 14 225 15 16 226 17 227 18 19 228 20 21 229 22 230 23 24 231 25 26 232 27 28 233 29 234 30 31 235 32 33 236 34 35 237 36 238 37 38 239 39 40 240 41 241 42 43 242 44 45 243 46 47 244 48 245 49 50 246 51 52 247 53 54 248 55 56 57 58 59 60

Page 8 of 42

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

ACS Paragon Plus Environment

Page 9 of 42 1 2 249 3 250 4 5 251 6 7 252 8 9 253 10 254 11 12 255 13 14 256 15 16 257 17 258 18 19 259 20 21 260 22 261 23 24 262 25 26 263 27 28 264 29 265 30 31 266 32 33 267 34 35 268 36 269 37 38 270 39 40 271 41 272 42 43 273 44 45 274 46 47 275 48 276 49 50 277 51 52 278 53 54 279 55 56 57 58 59 60

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

ACS Paragon Plus Environment

Biochemistry 1 2 280 3 281 4 5 282 6 7 283 8 9 284 10 285 11 12 286 13 14 287 15 16 288 17 289 18 19 290 20 21 291 22 292 23 24 293 25 26 294 27 28 295 29 296 30 31 297 32 33 298 34 35 299 36 300 37 38 301 39 40 302 41 303 42 43 304 44 45 305 46 47 306 48 307 49 50 308 51 52 309 53 54 55 56 57 58 59 60

Page 10 of 42

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

ACS Paragon Plus Environment

Page 11 of 42 1 2 310 3 311 4 5 312 6 7 313 8 9 314 10 315 11 12 316 13 14 317 15 16 318 17 319 18 19 320 20 21 321 22 322 23 24 323 25 26 324 27 28 325 29 326 30 31 327 32 33 328 34 35 329 36 330 37 38 331 39 40 332 41 333 42 43 334 44 45 335 46 47 336 48 337 49 50 338 51 52 339 53 54 340 55 56 57 58 59 60

Biochemistry

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

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 42

12

ACS Paragon Plus Environment

Page 13 of 42 1 2 372 3 373 4 5 374 6 7 375 8 9 376 10 377 11 12 378 13 14 379 15 16 380 17 381 18 19 382 20 21 383 22 384 23 24 385 25 26 386 27 28 387 29 388 30 31 389 32 33 390 34 35 391 36 392 37 38 393 39 40 394 41 395 42 43 396 44 45 397 46 47 398 48 399 49 50 400 51 52 401 53 54 402 55 56 57 58 59 60

Biochemistry

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

ACS Paragon Plus Environment

Biochemistry 1 2 403 3 404 4 5 405 6 7 406 8 9 407 10 408 11 12 409 13 14 410 15 16 411 17 412 18 19 413 20 21 414 22 415 23 24 416 25 26 417 27 28 418 29 419 30 31 420 32 33 421 34 35 422 36 423 37 38 424 39 40 425 41 426 42 43 427 44 45 428 46 47 429 48 430 49 50 431 51 52 432 53 54 433 55 56 57 58 59 60

Page 14 of 42

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

ACS Paragon Plus Environment

Page 15 of 42 1 2 434 3 435 4 5 436 6 7 437 8 9 438 10 439 11 12 440 13 14 441 15 16 442 17 443 18 19 444 20 21 445 22 446 23 24 447 25 26 448 27 28 449 29 450 30 31 451 32 33 452 34 35 453 36 454 37 38 455 39 40 456 41 457 42 43 458 44 45 459 46 47 460 48 461 49 50 462 51 52 463 53 54 464 55 56 57 58 59 60

Biochemistry

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.

15

ACS Paragon Plus Environment

Biochemistry 1 2 465 3 4 466 5 467 6 7 468 8 9 469 10 470 11 12 471 13 14 472 15 16 473 17 474 18 19 475 20 21 476 22 477 23 24 478 25 26 479 27 28 480 29 481 30 31 482 32 33 483 34 35 484 36 485 37 38 486 39 40 487 41 488 42 43 489 44 45 490 46 47 491 48 492 49 50 493 51 52 494 53 54 495 55 56 57 58 59 60

Page 16 of 42

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

ACS Paragon Plus Environment

Biochemistry 1 2 897 3 898 4 5 899 6 7 900 8 9 901 10 902 11 12 903 13 14 904 15 16 905 17 906 18 19 907 20 21 908 22 909 23 24 910 25 26 911 27 28 912 29 913 30 31 914 32 33 915 34 35 916 36 917 37 38 918 39 40 919 41 920 42 43 921 44 45 922 46 47 923 48 924 49 50 925 51 52 926 53 54 927 55 56 57 58 59 60

Page 30 of 42

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

ACS Paragon Plus Environment

Page 31 of 42 1 2 928 3 929 4 5 930 6 7 931 8 9 932 10 933 11 12 934 13 14 935 15 16 936 17 937 18 19 938 20 21 939 22 940 23 24 941 25 26 942 27 28 943 29 944 30 31 945 32 33 946 34 35 947 36 948 37 38 949 39 40 950 41 951 42 43 952 44 45 953 46 47 954 48 955 49 50 956 51 52 957 53 54 55 56 57 58 59 60

Biochemistry

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.

31

ACS Paragon Plus Environment

Biochemistry 1 2 958 3 959 4 5 960 6 7 961 8 9 962 10 963 11 12 964 13 14 965 15 16 966 17 967 18 19 968 20 21 969 22 970 23 24 971 25 26 972 27 28 973 29 974 30 31 975 32 33 976 34 35 977 36 978 37 38 979 39 40 980 41 981 42 43 982 44 45 983 46 47 984 48 985 49 50 986 51 52 987 53 54 988 55 56 57 58 59 60

Page 32 of 42

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

ACS Paragon Plus Environment

Page 33 of 42 1 2 989 3 990 4 5 991 6 7 992 8 9 993 10 994 11 12 995 13 14 996 15 16 997 17 998 18 19 999 20 211000 22 1001 23 241002 25 261003 27 281004 29 1005 30 311006 32 331007 34 351008 361009 37 381010 39 401011 41 1012 42 431013 44 451014 46 471015 48 1016 49 501017 51 521018 53 541019 55 56 57 58 59 60

Biochemistry

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,

86

and share high sequence

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.

33

ACS Paragon Plus Environment

Biochemistry 1 2 1020 3 1021 4 5 1022 6 7 1023 8 9 1024 10 1025 11 121026 13 141027 15 161028 171029 18 191030 20 211031 22 1032 23 241033 25 261034 27 281035 29 1036 30 311037 32 331038 34 351039 361040 37 381041 39 401042 41 1043 42 431044 44 451045 46 471046 48 1047 49 501048 51 521049 53 541050 55 56 57 58 59 60

Page 34 of 42

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

ACS Paragon Plus Environment

Page 35 of 42 1 2 1051 3 1052 4 5 1053 6 7 1054 8 9 1055 10 1056 11 121057 13 141058 15 161059 171060 18 191061 20 211062 22 1063 23 241064 25 261065 27 281066 29 1067 30 311068 32 331069 34 351070 361071 37 381072 39 401073 41 1074 42 431075 44 451076 46 471077 48 1078 49 501079 51 521080 53 541081 55 56 57 58 59 60

Biochemistry

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.

35

ACS Paragon Plus Environment

Biochemistry 1 2 1082 3 1083 4 5 1084 6 7 1085 8 9 1086 10 1087 11 121088 13 141089 15 161090 171091 18 191092 20 211093 22 1094 23 1095 24 251096 261097 271098 281099 291100 301101 31 1102 32 1103 33 341104 351105 361106 371107 381108 391109 401110 41 1111 42 1112 43 441113 451114 461115 471116 481117 491118 50 1119 51 521120 531121 541122 55 56 57 58 59 60

Page 36 of 42

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.

References: [1] Arawaka, S., Saito, Y., Murayama, S., and Mori, H. (1998) Lewy body in neurodegeneration with brain iron accumulation type 1 is immunoreactive for alpha-synuclein, Neurology 51, 887-889. [2] Uversky, V. N., Li, J., Souillac, P., Millett, I. S., Doniach, S., Jakes, R., Goedert, M., and Fink, A. L. (2002) Biophysical properties of the synucleins and their propensities to fibrillate: inhibition of alpha-synuclein assembly by beta- and gamma-synucleins, J Biol Chem 277, 11970-11978. [3] Jakes, R., Spillantini, M. G., and Goedert, M. (1994) Identification of two distinct synucleins from human brain, FEBS Lett 345, 27-32. [4] Clayton, D. F., and George, J. M. (1999) Synucleins in synaptic plasticity and neurodegenerative disorders, J Neurosci Res 58, 120-129. [5] George, J. M., Jin, H., Woods, W. S., and Clayton, D. F. (1995) Characterization of a novel protein regulated during the critical period for song learning in the zebra finch, Neuron 15, 361-372. [6] Akopian, A. N., and Wood, J. N. (1995) Peripheral nervous system-specific genes identified by subtractive cDNA cloning, J Biol Chem 270, 21264-21270. [7] Nakajo, S., Omata, K., Aiuchi, T., Shibayama, T., Okahashi, I., Ochiai, H., Nakai, Y., Nakaya, K., and Nakamura, Y. (1990) Purification and characterization of a novel brain-specific 14-kDa protein, J Neurochem 55, 2031-2038. [8] Hashimoto, M., Yoshimoto, M., Sisk, A., Hsu, L. J., Sundsmo, M., Kittel, A., Saitoh, T., Miller, A., and Masliah, E. (1997) NACP, a synaptic protein involved in Alzheimer's disease, is differentially regulated during megakaryocyte differentiation, Biochem Biophys Res Commun 237, 611-616. [9] Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., de Silva, H. A., Kittel, A., and Saitoh, T. (1995) The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system, Neuron 14, 467-475. [10] Ueda, K., Fukushima, H., Masliah, E., Xia, Y., Iwai, A., Yoshimoto, M., Otero, D. A., Kondo, J., Ihara, Y., and Saitoh, T. (1993) Molecular cloning of cDNA encoding an unrecognized component of amyloid in Alzheimer disease, Proc Natl Acad Sci U S A 90, 11282-11286. [11] Maroteaux, L., Campanelli, J. T., and Scheller, R. H. (1988) Synuclein: a neuron-specific protein localized to the nucleus and presynaptic nerve terminal, J Neurosci 8, 2804-2815. [12] Beyer, K. (2006) Alpha-synuclein structure, posttranslational modification and alternative splicing as aggregation enhancers, Acta Neuropathol 112, 237-251. 36

ACS Paragon Plus Environment

Page 37 of 42 1 2 1123 3 1124 4 1125 5 1126 6 1127 7 1128 8 1129 9 1130 10 111131 121132 131133 141134 151135 161136 17 1137 18 1138 19 201139 211140 221141 231142 241143 251144 26 1145 27 1146 28 291147 301148 311149 321150 331151 341152 351153 36 1154 37 381155 391156 401157 411158 421159 431160 441161 45 1162 46 1163 47 1164 48 491165 501166 511167 521168 531169 54 55 56 57 58 59 60

Biochemistry

[13] Appel-Cresswell, S., Vilarino-Guell, C., Encarnacion, M., Sherman, H., Yu, I., Shah, B., Weir, D., Thompson, C., Szu-Tu, C., Trinh, J., Aasly, J. O., Rajput, A., Rajput, A. H., Jon Stoessl, A., and Farrer, M. J. (2013) Alphasynuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease, Mov Disord 28, 811-813. [14] Kasten, M., and Klein, C. (2013) The many faces of alpha-synuclein mutations, Mov Disord 28, 697-701. [15] Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease, Nat Genet 18, 106-108. [16] Payton, J. E., Perrin, R. J., Clayton, D. F., and George, J. M. (2001) Protein-protein interactions of alphasynuclein in brain homogenates and transfected cells, Brain Res Mol Brain Res 95, 138-145. [17] Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honore, A., Rozas, N., Pieri, L., Madiona, K., Durr, A., Melki, R., Verny, C., Brice, A., and French Parkinson's Disease Genetics Study, G. (2013) G51D alphasynuclein mutation causes a novel parkinsonian-pyramidal syndrome, Ann Neurol 73, 459-471. [18] Clayton, D. F., and George, J. M. (1998) The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease, Trends Neurosci 21, 249-254. [19] Kawamata, H., McLean, P. J., Sharma, N., and Hyman, B. T. (2001) Interaction of alpha-synuclein and synphilin-1: effect of Parkinson's disease-associated mutations, J Neurochem 77, 929-934. [20] Jensen, P. H., Hager, H., Nielsen, M. S., Hojrup, P., Gliemann, J., and Jakes, R. (1999) alpha-synuclein binds to Tau and stimulates the protein kinase A-catalyzed tau phosphorylation of serine residues 262 and 356, J Biol Chem 274, 25481-25489. [21] Ostrerova, N., Petrucelli, L., Farrer, M., Mehta, N., Choi, P., Hardy, J., and Wolozin, B. (1999) alpha-Synuclein shares physical and functional homology with 14-3-3 proteins, J Neurosci 19, 5782-5791. [22] Nakajo, S., Tsukada, K., Omata, K., Nakamura, Y., and Nakaya, K. (1993) A new brain-specific 14-kDa protein is a phosphoprotein. Its complete amino acid sequence and evidence for phosphorylation, Eur J Biochem 217, 1057-1063. [23] Tobe, T., Nakajo, S., Tanaka, A., Mitoya, A., Omata, K., Nakaya, K., Tomita, M., and Nakamura, Y. (1992) Cloning and characterization of the cDNA encoding a novel brain-specific 14-kDa protein, J Neurochem 59, 1624-1629. [24] Nakajo, S., Tsukada, K., Kameyama, H., Furuyama, Y., and Nakaya, K. (1996) Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay, Brain Res 741, 180-184. [25] Goedert, M. (2001) Alpha-synuclein and neurodegenerative diseases, Nat Rev Neurosci 2, 492-501. [26] Ji, H., Liu, Y. E., Jia, T., Wang, M., Liu, J., Xiao, G., Joseph, B. K., Rosen, C., and Shi, Y. E. (1997) Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing, Cancer Res 57, 759-764. [27] Buchman, V. L., Hunter, H. J., Pinon, L. G., Thompson, J., Privalova, E. M., Ninkina, N. N., and Davies, A. M. (1998) Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system, J Neurosci 18, 9335-9341. [28] Ninkina, N. N., Privalova, E. M., Pinon, L. G., Davies, A. M., and Buchman, V. L. (1999) Developmentally regulated expression of persyn, a member of the synuclein family, in skin, Exp Cell Res 246, 308-311. [29] Surguchov, A., Palazzo, R. E., and Surgucheva, I. (2001) Gamma synuclein: subcellular localization in neuronal and non-neuronal cells and effect on signal transduction, Cell Motil Cytoskeleton 49, 218-228. [30] Spillantini, M. G., Crowther, R. A., Jakes, R., Cairns, N. J., Lantos, P. L., and Goedert, M. (1998) Filamentous alpha-synuclein inclusions link multiple system atrophy with Parkinson's disease and dementia with Lewy bodies, Neurosci Lett 251, 205-208. [31] Spillantini, M. G., Schmidt, M. L., Lee, V. M., Trojanowski, J. Q., Jakes, R., and Goedert, M. (1997) Alphasynuclein in Lewy bodies, Nature 388, 839-840. [32] Wakabayashi, K., Matsumoto, K., Takayama, K., Yoshimoto, M., and Takahashi, H. (1997) NACP, a presynaptic protein, immunoreactivity in Lewy bodies in Parkinson's disease, Neurosci Lett 239, 45-48. 37

ACS Paragon Plus Environment

Biochemistry 1 2 1170 3 1171 4 1172 5 1173 6 1174 7 1175 8 1176 9 101177 111178 121179 131180 141181 151182 161183 17 1184 18 1185 19 201186 211187 221188 231189 241190 251191 26 1192 27 1193 28 291194 301195 311196 321197 331198 341199 351200 36 1201 37 1202 38 391203 401204 411205 421206 431207 441208 45 1209 46 1210 47 1211 48 491212 501213 511214 521215 531216 541217 55 56 57 58 59 60

Page 38 of 42

[33] Trojanowski, J. Q., Goedert, M., Iwatsubo, T., and Lee, V. M. (1998) Fatal attractions: abnormal protein aggregation and neuron death in Parkinson's disease and Lewy body dementia, Cell Death Differ 5, 832837. [34] Lucking, C. B., and Brice, A. (2000) Alpha-synuclein and Parkinson's disease, Cell Mol Life Sci 57, 1894-1908. [35] Goedert, M. (1999) Filamentous nerve cell inclusions in neurodegenerative diseases: tauopathies and alphasynucleinopathies, Philos Trans R Soc Lond B Biol Sci 354, 1101-1118. [36] Spillantini, M. G., and Goedert, M. (2000) The alpha-synucleinopathies: Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, Ann N Y Acad Sci 920, 16-27. [37] Galvin, J. E., Lee, V. M., and Trojanowski, J. Q. (2001) Synucleinopathies: clinical and pathological implications, Arch Neurol 58, 186-190. [38] Trojanowski, J. Q., and Lee, V. M. (2003) Parkinson's disease and related alpha-synucleinopathies are brain amyloidoses, Ann N Y Acad Sci 991, 107-110. [39] Galvin, J. E., Uryu, K., Lee, V. M., and Trojanowski, J. Q. (1999) Axon pathology in Parkinson's disease and Lewy body dementia hippocampus contains alpha-, beta-, and gamma-synuclein, Proc Natl Acad Sci U S A 96, 13450-13455. [40] Dereeper, A., Guignon, V., Blanc, G., Audic, S., Buffet, S., Chevenet, F., Dufayard, J. F., Guindon, S., Lefort, V., Lescot, M., Claverie, J. M., and Gascuel, O. (2008) Phylogeny.fr: robust phylogenetic analysis for the nonspecialist, Nucleic Acids Res 36, W465-469. [41] Dosztanyi, Z., Csizmok, V., Tompa, P., and Simon, I. (2005) IUPred: web server for the prediction of intrinsically unstructured regions of proteins based on estimated energy content, Bioinformatics 21, 3433-3434. [42] Volles, M. J., and Lansbury, P. T., Jr. (2007) Relationships between the sequence of alpha-synuclein and its membrane affinity, fibrillization propensity, and yeast toxicity, J Mol Biol 366, 1510-1522. [43] Wilkins, M. R., Gasteiger, E., Bairoch, A., Sanchez, J. C., Williams, K. L., Appel, R. D., and Hochstrasser, D. F. (1999) Protein identification and analysis tools in the ExPASy server, Methods Mol Biol 112, 531-552. [44] Naiki, H., Higuchi, K., Hosokawa, M., and Takeda, T. (1989) Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1, Anal Biochem 177, 244-249. [45] Jain, M. K., and Bhat, R. (2014) Modulation of human alpha-synuclein aggregation by a combined effect of calcium and dopamine, Neurobiol Dis 63, 115-128. [46] Yuan, J., and Zhao, Y. (2013) Evolutionary aspects of the synuclein super-family and sub-families based on large-scale phylogenetic and group-discrimination analysis, Biochem Biophys Res Commun 441, 308-317. [47] Lavedan, C. (1998) The synuclein family, Genome Res 8, 871-880. [48] Lakowicz, J. R. (1999) Principles of fluorescence spectroscopy, 2nd ed., Kluwer Academic/Plenum, New York. [49] Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas, A. F., and Gilmanshin, R. I. (1991) Study of the "molten globule" intermediate state in protein folding by a hydrophobic fluorescent probe, Biopolymers 31, 119-128. [50] Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Harper, J. D., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Accelerated oligomerization by Parkinson's disease linked alpha-synuclein mutants, Ann N Y Acad Sci 920, 42-45. [51] Kim, J. (1997) Evidence that the precursor protein of non-A beta component of Alzheimer's disease amyloid (NACP) has an extended structure primarily composed of random-coil, Mol Cells 7, 78-83. [52] Kim, T. D., Ryu, H. J., Cho, H. I., Yang, C. H., and Kim, J. (2000) Thermal behavior of proteins: heat-resistant proteins and their heat-induced secondary structural changes, Biochemistry 39, 14839-14846. [53] Buell, A. K., Galvagnion, C., Gaspar, R., Sparr, E., Vendruscolo, M., Knowles, T. P., Linse, S., and Dobson, C. M. (2014) Solution conditions determine the relative importance of nucleation and growth processes in alpha-synuclein aggregation, Proc Natl Acad Sci U S A 111, 7671-7676. [54] Uversky, V. N., Li, J., and Fink, A. L. (2001) Evidence for a partially folded intermediate in alpha-synuclein fibril formation, J Biol Chem 276, 10737-10744. 38

ACS Paragon Plus Environment

Page 39 of 42 1 2 1218 3 1219 4 1220 5 1221 6 1222 7 1223 8 1224 9 101225 111226 121227 131228 141229 151230 161231 17 1232 18 1233 19 201234 211235 221236 231237 241238 251239 26 1240 27 1241 28 1242 29 301243 311244 321245 331246 341247 351248 36 1249 37 381250 391251 401252 411253 421254 431255 441256 45 1257 46 1258 47 1259 48 491260 501261 511262 521263 531264 54 55 56 57 58 59 60

Biochemistry

[55] Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) A soluble alpha-synuclein construct forms a dynamic tetramer, Proc Natl Acad Sci U S A 108, 17797-17802. [56] George, J. M. (2002) The synucleins, Genome Biol 3, REVIEWS3002. [57] Perrin, R. J., Woods, W. S., Clayton, D. F., and George, J. M. (2000) Interaction of human alpha-Synuclein and Parkinson's disease variants with phospholipids. Structural analysis using site-directed mutagenesis, J Biol Chem 275, 34393-34398. [58] Hashimoto, M., Hsu, L. J., Sisk, A., Xia, Y., Takeda, A., Sundsmo, M., and Masliah, E. (1998) Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease, Brain Res 799, 301-306. [59] Paik, S. R., Lee, J. H., Kim, D. H., Chang, C. S., and Kim, Y. S. (1998) Self-oligomerization of NACP, the precursor protein of the non-amyloid beta/A4 protein (A beta) component of Alzheimer's disease amyloid, observed in the presence of a C-terminal A beta fragment (residues 25-35), FEBS Lett 421, 7376. [60] Szabo, A. G., Lynn, K. R., Krajcarski, D. T., and Rayner, D. M. (1978) Tyrosinate fluorescence maxima at 345 nm in proteins lacking tryptophan at pH 7, FEBS Lett 94, 249-252. [61] Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) alpha-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation, Nature 477, 107-110. [62] Eliezer, D., Kutluay, E., Bussell, R., Jr., and Browne, G. (2001) Conformational properties of alpha-synuclein in its free and lipid-associated states, J Mol Biol 307, 1061-1073. [63] Waudby, C. A., Camilloni, C., Fitzpatrick, A. W., Cabrita, L. D., Dobson, C. M., Vendruscolo, M., and Christodoulou, J. (2013) In-cell NMR characterization of the secondary structure populations of a disordered conformation of alpha-synuclein within E. coli cells, PLoS One 8, e72286. [64] Burre, J., Vivona, S., Diao, J., Sharma, M., Brunger, A. T., and Sudhof, T. C. (2013) Properties of native brain alpha-synuclein, Nature 498, E4-6; discussion E6-7. [65] Baldwin, R. L. (1986) Temperature dependence of the hydrophobic interaction in protein folding, Proc Natl Acad Sci U S A 83, 8069-8072. [66] Kim, T. D., Paik, S. R., Yang, C. H., and Kim, J. (2000) Structural changes in alpha-synuclein affect its chaperone-like activity in vitro, Protein Sci 9, 2489-2496. [67] Fink, A. L. (2006) The aggregation and fibrillation of alpha-synuclein, Acc Chem Res 39, 628-634. [68] Giasson, B. I., Uryu, K., Trojanowski, J. Q., and Lee, V. M. (1999) Mutant and wild type human alphasynucleins assemble into elongated filaments with distinct morphologies in vitro, J Biol Chem 274, 76197622. [69] Biere, A. L., Wood, S. J., Wypych, J., Steavenson, S., Jiang, Y., Anafi, D., Jacobsen, F. W., Jarosinski, M. A., Wu, G. M., Louis, J. C., Martin, F., Narhi, L. O., and Citron, M. (2000) Parkinson's disease-associated alphasynuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs, J Biol Chem 275, 34574-34579. [70] Yamin, G., Munishkina, L. A., Karymov, M. A., Lyubchenko, Y. L., Uversky, V. N., and Fink, A. L. (2005) Forcing nonamyloidogenic beta-synuclein to fibrillate, Biochemistry 44, 9096-9107. [71] Hackl, E. V., Darkwah, J., Smith, G., and Ermolina, I. (2015) Effect of acidic and basic pH on Thioflavin T absorbance and fluorescence, Eur Biophys J 44, 249-261. [72] Ahsan, N., Mishra, S., Jain, M. K., Surolia, A., and Gupta, S. (2015) Curcumin Pyrazole and its derivative (N-(3Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-Synuclein, Sci Rep 5, 9862. [73] Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A., and Lansbury, P. T., Jr. (1996) NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded, Biochemistry 35, 13709-13715. 39

ACS Paragon Plus Environment

Biochemistry 1 2 1265 3 1266 4 1267 5 1268 6 1269 7 1270 8 1271 9 1272 10 111273 121274 131275 141276 151277 161278 17 1279 18 1280 19 201281 211282 221283 231284 241285 251286 26 1287 27 1288 28 1289 29 301290 311291 321292 331293 341294 351295 36 371296 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 40 of 42

[74] Hashimoto, M., Rockenstein, E., Mante, M., Mallory, M., and Masliah, E. (2001) beta-Synuclein inhibits alpha-synuclein aggregation: a possible role as an anti-parkinsonian factor, Neuron 32, 213-223. [75] Janowska, M. K., Wu, K. P., and Baum, J. (2015) Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that colocalizes with alpha-synuclein, Sci Rep 5, 15164. [76] Sigalov, A. B., Zhuravleva, A. V., and Orekhov, V. Y. (2007) Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form, Biochimie 89, 419-421. [77] Wu, S., Wang, D., Liu, J., Feng, Y., Weng, J., Li, Y., Gao, X., Liu, J., and Wang, W. (2017) The Dynamic Multisite Interactions between Two Intrinsically Disordered Proteins, Angew Chem Int Ed Engl 56, 7515-7519. [78] Tompa, P., and Fuxreiter, M. (2008) Fuzzy complexes: polymorphism and structural disorder in proteinprotein interactions, Trends Biochem Sci 33, 2-8. [79] Galea, C. A., Wang, Y., Sivakolundu, S. G., and Kriwacki, R. W. (2008) Regulation of cell division by intrinsically unstructured proteins: intrinsic flexibility, modularity, and signaling conduits, Biochemistry 47, 7598-7609. [80] Babu, M. M., van der Lee, R., de Groot, N. S., and Gsponer, J. (2011) Intrinsically disordered proteins: regulation and disease, Curr Opin Struct Biol 21, 432-440. [81] Li, J., Henning Jensen, P., and Dahlstrom, A. (2002) Differential localization of alpha-, beta- and gammasynucleins in the rat CNS, Neuroscience 113, 463-478. [82] Quilty, M. C., Gai, W. P., Pountney, D. L., West, A. K., and Vickers, J. C. (2003) Localization of alpha-, beta-, and gamma-synuclein during neuronal development and alterations associated with the neuronal response to axonal trauma, Exp Neurol 182, 195-207. [83] Surgucheva, I., Sharov, V. S., and Surguchov, A. (2012) gamma-Synuclein: seeding of alpha-synuclein aggregation and transmission between cells, Biochemistry 51, 4743-4754. [84] Surgucheva, I., Newell, K. L., Burns, J., and Surguchov, A. (2014) New alpha- and gamma-synuclein immunopathological lesions in human brain, Acta Neuropathol Commun 2, 132. [85] Lavedan, C., Leroy, E., Dehejia, A., Buchholtz, S., Dutra, A., Nussbaum, R. L., and Polymeropoulos, M. H. (1998) Identification, localization and characterization of the human gamma-synuclein gene, Hum Genet 103, 106-112. [86] Murphy, D. D., Rueter, S. M., Trojanowski, J. Q., and Lee, V. M. (2000) Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons, J Neurosci 20, 3214-3220.

40

ACS Paragon Plus Environment

Page 41 of 42 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

44x23mm (600 x 600 DPI)

ACS Paragon Plus Environment

Biochemistry 1 2 3 4 5 6

Page 42 of 42

ACS Paragon Plus Environment