"Spice of life": polyphenol curcumin by-products may act on Parkinson's relates protein aggregation. Paolo Ruzza, ICB-CNR
Parkinson's disease (PD) belongs to the group of protein misfolding diseases and it is characterized by the progressive degeneration of the dopaminergic neurons from the substantia nigra of the brain. Among the biochemical mechanisms proposed for the pathogenesis of PD, our attention has been focused on the intracellular accumulation and aggregation of misfolded proteins, in particular of α-synuclein; the oxidative stress caused by dopamine metabolites; and the alterations in the iron and glutathione levels in the substantia nigra. A main goal in the development of new therapeutic approaches for PD is the design of molecules able to target simultaneously different processes. Recent experimental findings indicate that various antioxidants, including polyphenols belonging to the curcumin family, have potent anti-fibrillogenic effects and destabilize α-synuclein fibrils in in vitro models. The rapid degradation of curcumin in aqueous solution at physiological pH, suggested us to evaluate the ability of its by-products dehydrozingerone (1), its O-methyl derivative (2), zingerone (3), and their C-2 symmetric dimers (biphenyl 4-6, respectively) (Fig. 1) to interact with α-synuclein and to modulate its aggregation. Dehydrozingerone and zingerone, as well as their dimers, show also potent antioxidant and anti-inflammatory activities in various models.
To investigate the interaction of these molecules with α-synuclein, we used the Synchrotron Radiation Circular Dichroism (SRCD) spectroscopy at the Diamond Light Source (UK) B23 beamline. In the SRCD spectroscopy, the traditional xenon arc lamp source is replaced with the more intense and bright light beam produced by the synchrotron, this permits to measure lower wavelength data containing more structural information, while the higher signal-to-noise requires smaller samples and enables measurements in absorbing buffers.
The far-UV SRCD spectrum of native α-synuclein displayed a negative band at about 195 nm, characteristic of the essentially unstructured nature of α-synuclein in aqueous environment. The addition of ligands (1-6) induced only small changes in the intensity of the negative band at 195 nm, indicating the absence of any folded α-synuclein conformation in presence of ligands.
The effects of ligand binding on α-synuclein aggregation were evaluated using both Congo red assay and SRCD spectra. Congo red strongly interacts with amyloid aggregates, showing characteristic changes in its absorption band. Experimental data showed that compounds 4 and 5 are able to inhibit significantly the aggregation of α-synuclein. To confirm this result, the secondary structures of α-synuclein alone and in the presence of compounds 4 and 5, at the end of the aggregation experiment, were evaluated by SRCD. The far-UV SRCD spectrum of α-synuclein alone displayed the presence of a β-sheet structure characteristic of aggregated protein, while in the presence of compound 4 or 5, the far-UV SRCD spectra of α-synuclein showed a broad negative band indicating the presence of a large amount of unstructured native α-synuclein conformation (Fig. 2).
This study confirms the suitability of SRCD spectroscopy for analyzing tiny samples quantitating ligand-protein interactions, and confirms the interaction between curcumin derivatives and α-synuclein with inhibition of its aggregation, providing insights on future molecules designed to inhibit α-synuclein aggregation.
Acknowledgements
We thank Diamond Light Source for access to beamline B23 (SM7153-1) that contributed to the results presented here. The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement nº 226716.
Publications: Marchiani, A.; Mammi, S.; Siligardi, G.; Hussain, R.; Tessari, I.; Bubacco, L.; Delogu, G.; Fabbri, D.; Dettori, A.M.; Sanna, D.; Dedola, S.; Serra, P.A.; Ruzza, P. Small molecules interacting with alpha-synuclein: antiaggregating and cytoprotective properties. Amino Acids 2013, 45, 327-338.
Ruzza, P. 'Spice of life': Indian spice turmeric polyphenol by-products may act on Parkinson's related α-synuclein aggregation. Diamond Light Source Annual Review 2013/14. 2014, 34-35.
Figure 1: Chemical structure of curcumin by-products tested. Dehydrozingerone (1), O-methyldehydrozingerone (2), zingerone (3), and their biphenyl dimers (4-6).
Figure 2: Far-UV SRCD spectra of α-synuclein alone or in the presence of 2.5 equiv. of compound 4 at 0 and 12 days of incubation at 37°C. The far-UV SRCD spectrum of α-synuclein in the presence of 2.5 equiv. of compound 4 at time zero is superimposable to that of α-synuclein at the same time. α-Synuclein solution was 100 µM in 20 mM phosphate buffer, pH 6.8. Samples were diluted with buffer to obtain a theoretical protein concentration of 4 µM.
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