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[ACS Nano] Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid


Abstract:

The phenomenon of protein aggregation into amyloid fibrils is associated with a large number of major diseases of unrelated etiology. Unraveling the mechanism of amyloid self-assembly and identifying therapeutic directions to control this process are of utmost importance. Research in this field has been hampered by several challenges, including reproducibility, low protein purification yields and the inherent aggregation propensity of amyloidogenic proteins, making them extremely difficult to study. Herein, based on the similarity in the assembly mechanism, as well as the physical, chemical and biological characteristics, of diphenylalanine nanostructures and aromatic amino acid containing amyloid fibrils, we report a simple, yet robust peptide-based platform which could be used for screening of small molecules, potentially capable of interfering with the aggregation process, and for mechanistic exploration of their mode of action. The system was validated using four small-molecule inhibitors and the effect was examined via turbidity assay, Thioflavin T fluorescence and electron microscopy. The aggregation profile of diphenylalanine was very similar to that of β-amyloid polypeptide in the presence of the modulators. Rosmarinic acid emerged as an extremely potent inhibitor and a destabilizer of the aggregates. The effect of stoichiometric variation of rosmarinic acid on the process of destabilization was also probed using microfluidic technique. Finally, the formation of equimolar complexes of diphenylalanine and inhibitors was detected using mass spectrometry. This approach not only provides a system for high throughput screening of possible inhibitor molecules from larger libraries of modulators, but is also highly useful for understanding the mechanistic aspects of the interactions leading to the process of inhibition.

Sayanti Brahmachari, Zohar A. Arnon, Anat Frydman-Marom, Lihi Adler-Abramovich, and Ehud Gazit ACS Nano, Just Accepted Manuscript DOI: 10.1021/acsnano.7b01662 Publication Date (Web): June 2, 2017 Copyright © 2017 American Chemical Society

Link: http://pubs.acs.org/doi/abs/10.1021/acsnano.7b01662

#06052017 #Biologicalapplication

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