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α -Synuclein occurs physiologically as a helically folded tetramer that resists aggregation. Tim Bartels, Joanna G. Choi, & Dennis J. Selkoe. Christopher O’Brien CHEM 645 Project Presentation 11/09/11. Protein misfolding and disease. Protein misfolding is the cause of a number of diseases

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

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Synuclein occurs physiologically as a helically folded tetramer that resists aggregation l.jpg

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

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

Christopher O’Brien

CHEM 645 Project Presentation

11/09/11


Protein misfolding and disease l.jpg

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

Dobson, C. M.(2001)


Synuclein role in disease l.jpg

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

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

T Ulmeret al. Journal of Biological Chemistry280, 9595-9603 (2005)


Synuclein natively unfolded protein l.jpg

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


New methods for determination of native state of synuclein l.jpg

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


Native gel electrophoresis l.jpg

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


Native gel electrophoresis results l.jpg

Native gel electrophoresis results

  • 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


Sds page results l.jpg

SDS-PAGE results

  • In vitro crosslinking used to preserve assembled state

  • Tetramer bands observed in crosslinked SDS-PAGE


2d ief sds page western blot analysis of crosslinked rbc lysate l.jpg

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


Scanning transmission electron microscopy stem l.jpg

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


Sedimentation equilibrium analytical ultracentrifugation se auc l.jpg

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


Circular dichroism looking at secondary structure l.jpg

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

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


Cd spectra of rbc synuclein l.jpg

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


Analysis of cd spectra with lipidex treatment l.jpg

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


Quantitative elemental phosphate analysis l.jpg

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


Examining post translational modifications using mass spectrometry l.jpg

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


Purification of stably transfected synuclein from human neuroblastoma cells l.jpg

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


Comparison of stably transfected and rbc synuclein l.jpg

Comparison of stably transfected and RBC α-synuclein

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


Stem measurements of 3d5 expressed synuclein l.jpg

STEM measurements of 3D5 expressed α-synuclein

  • Very similar molecular weight to RBC α-synuclein


Comparison of synuclein from 3d5 and bacterial recombinant expression l.jpg

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


Surface plasmon resonance spr l.jpg

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

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


Spr results l.jpg

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


Thioflavin t tht fluorescence l.jpg

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


Tht fluorescence results l.jpg

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


Conclusions l.jpg

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


Possible future directions l.jpg

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


Questions l.jpg

Questions?


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