Exploring the theranostic potential of aegerolysin-based protein complexes in combating bladder cancer

The development of new methods designed to spatially detect specific membrane lipid pools in order to study their involvement in cellular functions, or to target these lipids for therapeutic purposes, is a rapidly growing approach in biomedicine. The main advantage of targeting lipids as opposed to proteins is that lipids are not prone to the development of resistance caused by alterations in various protein receptors. Sphingomyelin (SM) is the major sphingolipid in plasma membranes of vertebrates. Its content and association with other membrane lipids influence various cellular functions. In particular, SM associates with cholesterol to form membrane rafts. These membrane nanodomains serve as functional platforms in various processes in health and disease (e.g. immune signalling, host-pathogen interactions, cancer) through segregation of specific proteins within raft domains. Remodelling of membrane lipids can be also observed in the programmed cell death (apoptosis). During this process, lipids that are not associated with the outer leaflet of the plasma membrane, (e.g. phosphatidylserine or cardiolipin), are translocated to the cell surface to generate an “eat me” signal that leads to the recognition of apoptotic cells and their clearance by phagocytes. Selected aegerolysin proteins from edible oyster mushrooms (Pleurotus sp.) are excellent candidates for the detection of the above-mentioned membrane lipids. Through their specific interaction with cholesterol-complexed or free SM, or with cardiolipin, these proteins can serve as valuable tools for detection of these lipids in membranes of living mammalian cells. In addition, aegerolysins can assemble into larger pore-forming complexes in target membranes containing their lipid receptor when a specific protein partner, bearing the membrane-attack complex/perforin (MACPF) domain, is present. These cytolytic aegerolysin/MACPF complexes can be used to eliminate cells containing the aegerolysin membrane lipid receptor. Ostreolysin A6 (OlyA6) is a Pleurotus aegerolysin which specifically interacts with cholesterol-bound conformation of SM in membrane rafts, and is therefore an ideal non-toxic marker for visualizing the structure and dynamics of these domains in living cells. In addition, a native isolate from P. ostreatus containing the mixture of OlyA6 and its MACPF partnering protein molecules can specifically target and eliminate urothelial carcinoma cells. These cells are highly enriched in membrane rafts, in contrast to normal urothelial cells, which have a decreased amount of membrane rafts and protein plaques on their apical membranes that cover the membrane rafts and make them inaccessible to the OlyA6/MAPCF complex. The OlyA6 mutant E69A can interact with both cholesterol-sequestered and cholesterol-free SM. The last Pleurotus aegerolysin of interest, erylysin A (EryA), does not bind to SM/cholesterol membranes and therefore does not target membrane rafts, but can specifically sense cardiolipin. In this project, we aim to critically evaluate the potential of recombinant lipid-binding aegerolysin proteins OlyA6, OlyA6-E69A and EryA, alone or in combination with their MACPF protein partners, as theranostic tools in biomedicine. Cardiolipin-binding fluorescently labelled EryA will be assessed as a molecular marker of apoptotic mammalian cells, and the fluorescently labelled OlyA6 and its mutant E69A will be tested as complementary tools to detect and study cholesterol-associated and free SM in membranes of living mammalian cells, respectively. Finally, we will specifically label urothelial carcinoma cells with selected fluorescently labelled aegerolysins or induce their lysis by the aegerolysin/MACPF complexes. Using binding assays with artificial and biological membranes, fluorescence microscopy techniques and toxicity assays, we will select the best aegerolysin-based complex for further use in the treatment of urothelial carcinoma.