Novel insights on the interaction of lactoferrin with proton pumping ATPases towards understanding its antifungal and anticancer activities

• Author(s): Pereira, Cátia Sofia Santos
• Date: 2021
• Persistent ID: https://hdl.handle.net/1822/78614
• Origin: RepositóriUM - Universidade do Minho
• Subject(s): Cancer; Fungi; Lactoferrin; Pma1p; V-ATPase; Cancro; Fungos; Lactoferrina
• Document Type: Doctoral thesis
• Language: English
• Contributor(s): Universidade do Minho; Côrte-Real, Manuela; Rodrigues, L. R.

Description

Lactoferrin (Lf) is a naturally occurring iron-binding protein and one of the most well-known milk bioactive compounds. In addition to milk, it is also found in different fluids of mammals including several mucosal surfaces. A multitude of biological activities, such as antimicrobial, antiviral, immunomodulatory and anticancer, have been ascribed to this protein, revealing its multifunctional character. Among them, its antifungal and anticancer activities stand out, as toxicity against a wide collection of yeasts and filamentous fungi, and a broad range of cancer types has been demonstrated. However, the molecular mechanisms underlying these activities are still poorly elucidated, which limits Lf applications as an anticancer and antifungal agent. In the present thesis, we sought to improve the current knowledge on the mechanisms through which Lf exerts its cytotoxicity against fungal and cancer cells, aiming to contribute to its rational, targeted and more efficient application. Since previous studies have identified proton pumping ATPases as Lf molecular targets, we focused on exploring the interaction between Lf and these proton pumps, as well as on the consequences of these interactions. A combination of biochemical, genetic and computational approaches was employed to attain the envisioned goals. Our work revealed a novel effect of bovine Lf (bLf) against yeast, that was further validated in cancer cells, through perturbation of ergosterol/cholesterol-rich lipid rafts, membrane microdomains that function as platforms for signalling and protein trafficking. These results reinforced the advantages of using yeast as a simple unicellular eukaryotic complementary model to elucidate the mechanisms underlying the anticancer activity of Lf. These alterations were intimately related with the bLf inhibitory effect towards the proton pump Pma1p in yeast, and V-ATPase in both yeast and cancer cells. Specifically, we found that lipid rafts composition, as well as Pma1p-lipid rafts association are critical for bLf yeast killing activity. Regarding bLf anticancer activity, we showed that bLf-driven lipid rafts disruption is associated with cellular trafficking perturbations, downregulation of components of the PI3K/AKT signalling pathway and inhibition of glycolysis. The computational approach herein developed allowed to predict a mechanism of Lf-induced V-ATPase inhibition, and to identify critical residues for their interaction. Altogether, the work developed in this thesis uncovered novel cellular and molecular events triggered by Lf in the context of its antifungal and anticancer activities that have great potential to pave the way for future Lf-based applications.