Concanavalin A aggregation and toxicity on cell cultures.
A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which-as a consequence-turns out to be non-toxic.
Dipartimento di Scienze Fisiche ed Astronomiche, Università di Palermo and CNISM-MeSIAM, 90123 Palermo, Italy. valeria.vetri@fisica.unipa.it
Valeria
V
Vetri
Rita
R
Carrotta
Pasquale
P
Picone
Marta
M
Di Carlo
Valeria
V
Militello
D000682:Amyloid / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival; D003208:Concanavalin A / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D018625:Microscopy, Atomic Force; D009474:Neurons / Q000378:metabolism; D020836:Protein Structure, Quaternary; D017550:Spectroscopy, Fourier Transform Infrared
D000682:Amyloid; D003208:Concanavalin A