synuclein occurs physiologically as a helically folded tetramer that resists aggregation
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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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synuclein occurs physiologically as a helically folded tetramer that resists aggregation

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 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
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
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
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
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
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
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
2D IEF/SDS-PAGE/Western blot analysis of crosslinked RBC lysate
  • Higher molecular weight oligomers have same isoelectric point as monomers
purification of endogenous synuclein from living human cells
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
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
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
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
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
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
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
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
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
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
STEM measurements of 3D5 expressed α-synuclein
  • Very similar molecular weight to RBC α-synuclein
comparison of synuclein from 3d5 and bacterial recombinant expression
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
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
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
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
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
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
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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