α-Synuclein occurs physiologically as a helically folded tetramer that resists

aggregationTim Bartels, Joanna G. Choi, & Dennis J. Selkoe

Christopher O’BrienCHEM 645 Project Presentation

11/09/11

Protein misfolding and disease

• Protein misfolding is the cause of a number of diseases– Neurodegenerative diseases

are a well-studied subset

• Misfolding of proteins is often the first step in an aggregation pathway– Formation of amyloid fibrils

• α-synuclein is associated with the pathogenesis of Parkinson’s disease

2Dobson, C. M.(2001)

α-synuclein – role in disease

• Implicated in molecular events leading to Parkinson’s disease– Lewy bodies characteristic of

Parkinson’s disease contain aggregates of α-synuclein

• Three point mutations are known to lead to familial forms of Parkinson’s disease:– A53T– A30P– E46K

• Forms amyloid-like fibrils

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http://en.wikipedia.org/wiki/File:Lewy_Koerperchen.JPG

T Ulmer et al. Journal of Biological Chemistry 280, 9595-9603 (2005)

α-synuclein – natively unfolded protein?

• Commonly believed to be natively unfolded monomer – Approximately 14 kDa

• Crystal structure of micelle-bound α-synuclein shown to the left– Solved by NMR– Helical secondary structure

only acquired with lipid binding

• Protein obtained by recombinant bacterial overexpression

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New methods for determination of native state of α-synuclein

• Look at cell lines that endogenously express α-synuclein– M17D (dopaminergic human neuroblastoma)– HEK293– HeLa– COS-7

• Other sources of native α-synuclein– Frontal cortex of wild-type mice– Human red blood cells

• The above were examined using native gel electrophoresis

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Native gel electrophoresis

• Non-denaturing conditions (no SDS)– Proteins expected to remain folded in native state

• Charged denaturing agent not used– Proteins may migrate in gel differently based on molecular

mass, charge in gel conditions, as well as hydrodynamic size and shape

• In this work, both BN-PAGE and CN-PAGE are used– CN-PAGE omits coomassie blue from sample preparation and

cathode buffer

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• BN-PAGE shows an approximately 45-50 kDa molecular weight for all endogenous α-synuclein

• CN-PAGE shows ~55-60 kDa, consistent with a tetramer

• Monoclonal and polyclonal α-synuclein antibodies used for detection in both systems

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Native gel electrophoresis results

• In vitro crosslinking used to preserve assembled state

• Tetramer bands observed in crosslinked SDS-PAGE

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SDS-PAGE results

2D IEF/SDS-PAGE/Western blot analysis of crosslinked RBC lysate

• Higher molecular weight oligomers have same isoelectric point as monomers

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Purification of endogenous α-synuclein from living human cells

• RBC in lysate were precipitated with (NH4)2SO4

• Three different purification methods– Hydrophobic interaction

chromatography– Anion exchange chromatography– Covalent chromatography

(activated thiopropyl sepharose)

• Subsequent size exclusion step for further purification

• Figure shows SDS-PAGE after three stages of purification

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Scanning transmission electron microscopy (STEM)

• Type of TEM– Electrons pass through thin sample

• Electron beam focused onto single spot and scanned over sample• Tobacco mosaic virus (TMV) rods included during preparation as

internal sizing standard• Measurements carried out at the Brookhaven National Laboratory

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Sedimentation equilibrium analytical ultracentrifugation (SE-AUC)

• Time-independent equilibrium concentration profile where sedimentation and diffusion are in equilibrium– Distributions are Boltzmann distributions– Insensitive to shape of molecules

• Results fitted using software SEDPHAT to calculate molecular weight– Determined from average of three concentrations– Error and standard deviation calculated using Monte-Carlo

simulations

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Circular dichroism – looking at secondary structure

• Measures the difference in adsorption of left and right polarized UV light

• Ideal spectra for alpha helix, beta sheet, and random coil shown to the right

• Spectra can be fit to multiple ideal spectra to determine content of different secondary structures in protein

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http://www.ap-lab.com/circular_dichroism.htm

CD spectra of RBC α-synuclein

• Minima at 222 and 208 nm and overall shape indicative of α-helical secondary structure

• Binding to lipid doesn’t appear to be required for folded structure

• Random coil conformation seen in recombinant monomer expressed from bacteria

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Analysis of CD spectra with Lipidex treatment

• Lipidex 1000 = a reagent used to strip proteins of bound lipids and fatty acids

• Used to confirm that lipid association is not required for helical structure

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Quantitative elemental phosphate analysis

• Used to verify lack of a significant phospholipid presence• Involves incubation with ammonium molybdate(VI) tetrahydrate

and 10% ascorbic acid • Absorbance at 280nm measured and compared to calibration

curve of 7 standards

• Results indicated only 0.25mol phosphate per mole α-synuclein present– Significant presence of phospholipids on purified native α-

synuclein unlikely

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Examining post-translational modifications using mass spectrometry

• MALDI-TOF used to analyze– (A) Recombinant αSyn– (B) Purified αSyn from

human RBC

• Predicted theoretical mass = 14,502 kDa

• N-α-acetylation found on monomer from human RBC

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Purification of stably transfected α-synuclein from human neuroblastoma

cells

• 3D5 cells, a M17D human neuroblastoma cell line was stably transfected to overexpress wild-type human α-synuclein– Endogenous α-synuclein from untransfected M17D cells also

analyzed• Cells were lysed by sonication• Similar purification performed as described for RBCs using anion

exchange chromatography

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Comparison of stably transfected and RBC α-synuclein

• Similar migration and CD spectra between both types of expression/purification

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STEM measurements of 3D5 expressed α-synuclein

• Very similar molecular weight to RBC α-synuclein

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Comparison of α-synuclein from 3D5 and bacterial recombinant expression

• Bacterial monomeric α-synuclein added extrinsically to parental M17D cell line prior to purification identical to 3D5

• Can conclude that α-helically folded α-synuclein doesn’t arise from artificial manipulation during lysis and purification

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Surface plasmon resonance (SPR)

• Used to measure adsorption of a material• Light beam used to excite surface plasmons (oscillating electrons

in metal film)– Light incident at an angle greater than the critical angle is

subject to total internal reflection– An evanescent wave interacts with surface plasmons and

excites them• This is dependent on proteins bound to surface

23http://upload.wikimedia.org/wikipedia/commons/f/f8/Otto-schema.png

SPR results

• RBC purified α-synuclein has a much greater resonance angle shift than recombinant monomer– Fit to two state model –

dissociation constant two orders of magnitude lower than recombinant monomer

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Thioflavin T (ThT) fluorescence

• Used to quantify formation of amyloid fibril growth– Binds to beta sheet-rich structures and displays enhanced

fluorescence with a red shift in its spectrum• Sample is incubated with ThT and fluorescence is measured from

460-550nm• Possibility of unwanted binding or spectroscopic change may

cause unreliable results in quantitative analysis

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ThT fluorescence results

• Purified cellular α-synuclein incubated over 10 days shows no evidence of fibril formation sufficient for ThT binding

• Recombinant monomer incubated for the same time shows amyloid fibril formation

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Conclusions

• Endogenous α-synuclein is natively an α-helical tetramer of about 58 kDa– This contradicts previously published work describing α-

synuclein as a natively unfolded 14 kDa monomer– Variable amounts of lower number oligomers also present in

cells• Based on the predominant native structure, it is likely that α-

synuclein tetramers undergo destabilization before non-native aggregation and fibrillar assemblies that are characteristic of Parkinson’s disease form– Consistent with the observation that partial unfolding of a

protein typically occurs before aggregation

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Possible future directions

• Further investigation into the native tetramer conformation of α-synuclein can be performed now that a protocol for its expression and purification is published– Closer examination of its mechanism of destabilization and

aggregation• Therapeutic compounds that could kinetically stabilize native

tetramers could also be investigated– Could lead to novel treatments for Parkinson’s and similar

diseases

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Questions?

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