Fluorescence labeling methods influence the aggregation process of α-syn in vitro differently.

In a previous study, the coexistence of different aggregation pathways of insulin and ß-amyloid (Aß) peptides was demonstrated by correlative stimulated emission depletion (STED) microscopy and atomic force microscopy (AFM). This had been explained by suboptimal proteins labeling strategies that generate heterogeneous populations of aggregating species. However, because of the limited number of proteins considered, the failure of the fluorescent labeling that occurs in a large portion of the aggregating fibrils observed for insulin and Aß peptides, could not be considered a general phenomenon valid for all molecular systems. Here, we investigated the aggregation process of α-synuclein (α-syn), an amyloidogenic peptide involved in Parkinson's disease, which is significantly larger (MW ∼14 kDa) than insulin and Aß, previously investigated. The results showed that an unspecific labeling procedure, such as that previously adopted for shorter proteins, reproduced the coexistence of labeled/unlabeled fibers. Therefore, a site-specific labeling method was developed to target a domain of the peptide scarcely involved in the aggregation process. Correlative STED-AFM illustrated that all fibrillar aggregates derived from the aggregation of α-syn at the dye-to-protein ratio of 1 : 22 were fluorescent. These results, demonstrated here for the specific case of α-syn, highlight that the labeling artifacts can be avoided by careful designing the labeling strategy for the molecular system under investigation. The use of a label-free correlative microscopy technique would play a crucial role in the control of the setting of these conditions.

Milk-derived bioactive peptides protect against oxidative stress in a Caco-2 cell model.

Milk and milk-derived products are a relevant source of bioactive peptides, which are also potential components of functional foods. Bioactive peptides exert multiple actions including an antioxidant role. In the present paper, four synthetic peptides (NPYVPR, AVPYPQR, KVLPVPEK, and ARHPHPHLSFM), corresponding to milk-derived peptides were studied. Although with different features, as revealed by RP-HPLC chromatography and MS analysis, the obtained peptides were shown to be taken up by Caco-2 cells arranged in an epithelial monolayer formation. The four peptides were all able to preserve cell viability against induced oxidative stress indicating that they might have a role in the control of oxidative stress. Therefore, an estimation of total thiols and glutathione content was performed after cell treatment with oxidants like hydrogen peroxide (H2O2) or tert-butyl hydroperoxide (TbOOH). The peptides were able to prevent the decrease of both total thiols and glutathione induced by H2O2 or TbOOH, and, in addition, they showed a protective effect on the thiol-related antioxidant enzymes thioredoxin reductase and glutathione reductase. Finally, they caused a decrease of ROS production induced by TbOOH in Caco-2 cells. The reported results highlight the relevant antioxidant role played by bioactive peptides in cells, which adds to other previously known properties.