Abstract
Here we present Small Angle X-ray (SAXS) data of α-synuclein oligomers obtained by incubation with the ligand FN075. Data from complementary methods such as NMR and CD are also shown.
Aggregated α-synuclein is the major constituent of the Lewy Bodies regarded as the hallmark of Parkinson’s Disease and Dementia with Lewy Bodies (DLB) (Spillantini et al. 1997). The role of lewy bodies in the pathology of Parkinson’s Disease and DLB is however not well understood but in vitro experiments suggest that transient oligomeric species could be involved in cell toxicity (Giehm et al. 2011). The natural function of α-synuclein has also not been established (Drescher et al. 2012). The monomeric species of α-synuclein is intrinsically disordered meaning it does not have just one stable conformation in solution. The solution structure of an on pathway oligomer consisting of 16 monomers has been solved by decomposition of SAXS data from the evolving fibrillating solution (Giehm et al. 2011). NMR data have furthermore suggested that the C-terminal is exposed on oligomers obtained by incubation with the ligand FN075 (Horvath et al. 2012). In this study we aim at obtaining SAXS data from isolated stabilized oligomers, and to compare this with the in-equilibrium oligomers (Giehm et al. 2011).
Using NMR spectroscopy we have optimized the ratio between FN075 and α-synuclein resulting in high yield of oligomers and low, if any, unspecific aggregation. This enabled us to obtain SAXS data from the stabilized oligomer (MAX-lab, May 2012); data analysis is in progress. ITC experiments are furthermore planned to more accurately determine the stoichiometry between α-synuclein and FN075.
Horvath and co-workers have already shown that the FN075 stabilized oligomer is on pathway. We have shown that the in-equilibrium oligomer disrupts lipid membranes (Giehm et al 2011) and it is of interest to establish if the stabilized oligomer may be an earlier species in the fibrillation pathway or if the oligomer corresponds to the assumed cytotoxic species. Obtaining a solution structure will therefore also evaluate the mechanism of elongation originally suggested by Giehm and co-workers and potentially elaborate it.
Acknowledgements: We would like to thank Drug Research Academy, DANSCATT and The Danish Council for Independent Research for funding as well as EMBL Hamburg and Maxlab, Lund for beamtime
Drescher, M., et al. (2012). "Hunting the Chameleon: Structural Conformations of the Intrinsically Disordered Protein Alpha-Synuclein." Chembiochem 13(6): 761-768.
Giehm, L., et al. (2011). "Low-resolution structure of a vesicle disrupting alpha-synuclein oligomer that accumulates during fibrillation." Proceedings of the National Academy of Sciences of the United States of America 108(8): 3246-3251.
Horvath, I., et al. (2012). "Mechanisms of protein oligomerization: In-hibitor of functional amyloids templates a-synuclein fibrilla-tion." Journal of the American Chemical Society.
Spillantini, M. G., et al. (1997). "[alpha]-Synuclein in Lewy bodies." Nature 388(6645): 839-840.
Aggregated α-synuclein is the major constituent of the Lewy Bodies regarded as the hallmark of Parkinson’s Disease and Dementia with Lewy Bodies (DLB) (Spillantini et al. 1997). The role of lewy bodies in the pathology of Parkinson’s Disease and DLB is however not well understood but in vitro experiments suggest that transient oligomeric species could be involved in cell toxicity (Giehm et al. 2011). The natural function of α-synuclein has also not been established (Drescher et al. 2012). The monomeric species of α-synuclein is intrinsically disordered meaning it does not have just one stable conformation in solution. The solution structure of an on pathway oligomer consisting of 16 monomers has been solved by decomposition of SAXS data from the evolving fibrillating solution (Giehm et al. 2011). NMR data have furthermore suggested that the C-terminal is exposed on oligomers obtained by incubation with the ligand FN075 (Horvath et al. 2012). In this study we aim at obtaining SAXS data from isolated stabilized oligomers, and to compare this with the in-equilibrium oligomers (Giehm et al. 2011).
Using NMR spectroscopy we have optimized the ratio between FN075 and α-synuclein resulting in high yield of oligomers and low, if any, unspecific aggregation. This enabled us to obtain SAXS data from the stabilized oligomer (MAX-lab, May 2012); data analysis is in progress. ITC experiments are furthermore planned to more accurately determine the stoichiometry between α-synuclein and FN075.
Horvath and co-workers have already shown that the FN075 stabilized oligomer is on pathway. We have shown that the in-equilibrium oligomer disrupts lipid membranes (Giehm et al 2011) and it is of interest to establish if the stabilized oligomer may be an earlier species in the fibrillation pathway or if the oligomer corresponds to the assumed cytotoxic species. Obtaining a solution structure will therefore also evaluate the mechanism of elongation originally suggested by Giehm and co-workers and potentially elaborate it.
Acknowledgements: We would like to thank Drug Research Academy, DANSCATT and The Danish Council for Independent Research for funding as well as EMBL Hamburg and Maxlab, Lund for beamtime
Drescher, M., et al. (2012). "Hunting the Chameleon: Structural Conformations of the Intrinsically Disordered Protein Alpha-Synuclein." Chembiochem 13(6): 761-768.
Giehm, L., et al. (2011). "Low-resolution structure of a vesicle disrupting alpha-synuclein oligomer that accumulates during fibrillation." Proceedings of the National Academy of Sciences of the United States of America 108(8): 3246-3251.
Horvath, I., et al. (2012). "Mechanisms of protein oligomerization: In-hibitor of functional amyloids templates a-synuclein fibrilla-tion." Journal of the American Chemical Society.
Spillantini, M. G., et al. (1997). "[alpha]-Synuclein in Lewy bodies." Nature 388(6645): 839-840.
| Originalsprog | Engelsk |
|---|---|
| Publikationsdato | 31 maj 2012 |
| Status | Udgivet - 31 maj 2012 |
| Begivenhed | SAS2012: International Small-Angle Scattering Conference - Sydney, Australien Varighed: 18 nov. 2012 → 23 nov. 2012 Konferencens nummer: 15 |
Konference
| Konference | SAS2012 |
|---|---|
| Nummer | 15 |
| Land/Område | Australien |
| By | Sydney |
| Periode | 18/11/2012 → 23/11/2012 |