Valorization of Apple Peels through the Study of the Effects on the Amyloid Aggregation Process of κ-Casein.

Waste valorization represents one of the main social challenges when promoting a circular economy and environmental sustainability. Here, we evaluated the effect of the polyphenols extracted from apple peels, normally disposed of as waste, on the amyloid aggregation process of κ-casein from bovine milk, a well-used amyloidogenic model system. The effect of the apple peel extract on protein aggregation was examined using a thioflavin T fluorescence assay, Congo red binding assay, circular dichroism, light scattering, and atomic force microscopy. We found that the phenolic extract from the peel of apples of the cultivar "Fuji", cultivated in Sicily (Caltavuturo, Italy), inhibited κ-casein fibril formation in a dose-dependent way. In particular, we found that the extract significantly reduced the protein aggregation rate and inhibited the secondary structure reorganization that accompanies κ-casein amyloid formation. Protein-aggregated species resulting from the incubation of κ-casein in the presence of polyphenols under amyloid aggregation conditions were reduced in number and different in morphology.

Inhibition of Aß1-42 Fibrillation by Chaperonins: Human Hsp60 Is a Stronger Inhibitor than Its Bacterial Homologue GroEL.

Alzheimer's disease is a chronic neurodegenerative disease characterized by the accumulation of pathological aggregates of amyloid beta peptide. Many efforts have been focused on understanding peptide aggregation pathways and on identification of molecules able to inhibit aggregation in order to find an effective therapy. As a result, interest in neuroprotective proteins, such as molecular chaperones, has increased as their normal function is to assist in protein folding or to facilitate the disaggregation and/or clearance of abnormal aggregate proteins. Using biophysical techniques, we evaluated the effects of two chaperones, human Hsp60 and bacterial GroEL, on the fibrillogenesis of Aß1-42. Both chaperonins interfere with Aß1-42 aggregation, but the effect of Hsp60 is more significant and correlates with its more pronounced flexibility and stronger interaction with ANS, an indicator of hydrophobic regions. Dose-dependent ThT fluorescence kinetics and SAXS experiments reveal that Hsp60 does not change the nature of the molecular processes stochastically leading to the formation of seeds, but strongly delays them by recognition of hydrophobic sites of some peptide species crucial for triggering amyloid formation. Hsp60 reduces the initial chaotic heterogeneity of Aß1-42 sample at high concentration regimes. The understanding of chaperone action in counteracting pathological aggregation could be a starting point for potential new therapeutic strategies against neurodegenerative diseases.

Investigation on different chemical stability of mitochondrial Hsp60 and its precursor.

In the large class of molecules that maintain protein homeostasis, called molecular chaperones, chaperonins constitute a subclass that specifically assist the correct folding of newly synthesized proteins. Among them, Hsp60 is composed of a double heptameric ring structure with a large central cavity where the unfolded protein binds via hydrophobic interactions and is supported, in this function, by the co-chaperonin Hsp10. Hsp60 is typically located in the mitochondria, but in some pathological situations, such as cancers and chronic inflammatory diseases, Hsp60 accumulates in the cytoplasm. In these cases, cytoplasmatic Hsp60 is a mixture of mitochondrial Hsp60 secreted from mitochondria upon stress, and its precursor, called naïve Hsp60, never entered into the organella. The difference between the naïve and mitochondrial Hsp60s resides in the absence of the mitochondrial import signal (MIS) in the mitochondrial form, but information on their different structure and stability is still lacking. We present here a study on the stability against a chemical denaturant, of the different cytoplasmic Hsp60 species. By combining Circular Dichroism and Small Angle X-ray Scattering as experimental biophysical techniques to investigate Hsp60, we find that naïve and mitochondrial Hsp60 (mtHsp60) forms differ in their stability. Furthermore, specific responses from the two forms are discussed in terms of the biological environment they are working in, thus opening new questions on their biological function.