Diphenylalanine as a Reductionist Model for the Mechanistic Characterization of β-Amyloid Modulators

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, on the basis of 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 that 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 a 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.

Coassembly of Complementary Peptide Nucleic Acid into Crystalline Structures by Microfluidics

“…Next, we utilized a microfluidics platform which allows the injection of two separate solutions through inlets 1 and 2 (Figure 3a), as we have previously described. [21,22] A microfluidics platform allows rapid adjustment of the environment inside the device due to the small volumes used. Moreover, this system allowed us to regulate the dimensions of individual coassemblies as the addition of each of the di-PNA building blocks could be rapidly and finely tuned.…”

“…The low flow rate is similar to that used in previous work, in order to prevent shear stress applied on the assemblies by the solution. [21,22] The length of a single coassembled crystal was measured at different timepoints to determine the growth dynamics ( Figure 3b and Video S1, Supporting Information). Alternatively, shrinkage was accomplished by exclusively injecting a single building block solution of either one of the components.…”

“…Next, we utilized a microfluidics platform which allows the injection of two separate solutions through inlets 1 and 2 ( Figure a), as we have previously described . A microfluidics platform allows rapid adjustment of the environment inside the device due to the small volumes used.…”

“…Introducing the two solutions into the device at equal flow rates of 5 µL h −1 resulted in growth of the assemblies. The low flow rate is similar to that used in previous work, in order to prevent shear stress applied on the assemblies by the solution . The length of a single coassembled crystal was measured at different timepoints to determine the growth dynamics (Figure b and Video S1, Supporting Information).…”

Go with the Flow—Microfluidics Approaches for Amyloid Research

“…[45] This model peptidew as later used for characterization of Ab polypeptides modulators and holds much potentiali nn ano-biotechnology and martials science applications. [46][47][48][49]…”