A Simple Fluorescence Affinity Assay to Decipher Uranyl-Binding to Native Proteins.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus.

Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet ß-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of ß-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined.

(R)-α-trifluoromethylalanine containing short peptide in the inhibition of amyloid peptide fibrillation.

The extracellular deposition of insoluble amyloid fibrils resulting from the aggregation of the amyloid-ß (Aß) is a pathological feature of neuronal loss in Alzheimer's disease (AD). Numerous small molecules have been reported to interfere with the process of Aß aggregation. Compounds containing aromatic structures, hydrophobic amino acids and/or the α-aminoisobutyric acid (Aib) as ß-sheet breaker elements have been reported to be effective inhibitors of Aß aggregation. We synthesized two peptides, one containing the Aib amino acid and the other including its trifluoromethylated analog (R)-α-Trifluoromethylalanine ((R)-Tfm-Alanine) and we evaluated the impact of these peptides on Aß amyloid formation. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy and transmission electron microscopy but also by circular dichroism, liquid state nuclear magnetic resonance (NMR) and NMR saturation transfer difference (STD) experiments to further characterize the effect of the two molecules on Aß structure and on the kinetics of depletion of monomeric, soluble Aß. Our results demonstrate that the peptide containing Aib reduces the quantity of aggregates containing ß-sheet structure but slightly inhibits Aß fibril formation, while the molecule including the trifluoromethyl (Tfm) group slows down the kinetics of Aß fibril formation, delays the random coil to ß-sheet structure transition and induces a change in the oligomerization pathway. These results suggest that the hydrophobic Tfm group has a better affinity with Aß than the methyl groups of the Aib and that this Tfm group is effective and important in preventing the Aß aggregation.