1. 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

2. 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 1 Dobson, C. M.(2001)

3. 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 2 http://en.wikipedia.org/wiki/File:Lewy_Koerperchen.JPG T Ulmer et al. Journal of Biological Chemistry 280, 9595-9603 (2005)

4. 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 3

5. 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 4

6. 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 5

7. 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 6 Native gel electrophoresis results

8. In vitro crosslinking used to preserve assembled state Tetramer bands observed in crosslinked SDS- PAGE 7 SDS-PAGE results

9. 2D IEF/SDS-PAGE/Western blot analysis of crosslinked RBC lysate Higher molecular weight oligomers have same isoelectric point as monomers 8

10. Purification of endogenous α -synuclein from living human cells RBC in lysate were precipitated with (NH 4 ) 2 SO 4 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 9

11. 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 10

12. 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 11

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14. 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 13 http://www.ap-lab.com/circular_dichroism.htm

15. 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 14

16. 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 15

17. 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 16

18. Examining post-translational modifications using mass spectrometry MALDI-TOF used to analyze –(A) Recombinant α Syn –(B) Purified α Syn from human RBC Predicted theoretical mass = 14,502 kDa N- α -acetylation found on monomer from human RBC 17

19. 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 18

20. Comparison of stably transfected and RBC α -synuclein Similar migration and CD spectra between both types of expression/purification 19

21. STEM measurements of 3D5 expressed α - synuclein Very similar molecular weight to RBC α -synuclein 20

22. 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 21

23. 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 22 http://upload.wikimedia.org/wikipedia/commons/f/f8/Otto-schema.png

24. 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 23

25. 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 24

26. 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 25

27. 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 26

28. 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 27

29. Questions? 28