Extrinsic factors affecting amyloid aggregation

Protein conformational diseases (PCD) include a range of degenerative disorders in which specific peptides or proteins misfold and aberrantly self-assemble, eventually forming amyloid-like fibrils, which constitute the hallmark of many neurodegenerative diseases. Plenty of works demonstrated that amyloid aggregation is strongly influenced by several extrinsic factors, such as high concentrations of macromolecules or the presence of proteases and chaperons. Molecular chaperones have been recognised as key players in the avoidance of amyloid fibril formation and, in particular, recent evidences demonstrate that low levels of chaperone heat-shock protein 70 kDa (Hsp70) are strictly related to the formation of intra-neuronal inclusions associated with Parkinson‘s (PD) and polyglutamine (polyQ) diseases. Human Hsp70 is composed of two major functional domains connected by a conserved interdomain linker: the 44-kDa N-terminal nucleotide-binding domain (NBD), with ATPase activity, followed by the 30-kDa substrate-binding domain (SBD) that contains a C-terminal lid subdomain (LS). Using a battery of Hsp70 variants, including full-length Hsp70 and SBD truncated variants, we have been able to discover an interaction between the LS of SBD and the interdomain linker, which we propose could play an important role in the allosteric communication between NBD and SBD. Therefore, we analyzed the anti-amyloidogenic activity of Hsp70, using two model proteins: alpha-synuclein (AS), whose deposition in the brain is associated with PD and the polyQ protein ataxin-3 (AT3), the causative agent of the Machado-Joseph disease (MCD). We demonstrated Hsp70 is able to interact and stabilize pre-fibrillar species formed during amyloid aggregation and that the binding mechanism of these species is different from that of the monomeric protein. Plenty of evidence supports the idea that protein aggregation observed in in vitro experimental conditions is different from that naturally occurring in in vivo systems. This is also related with the fact that the high concentration of macromolecules present in the intra- and extra-cellular compartments, a condition known as molecular crowding (MC), strongly affects protein folding and aggregation. Here, we successfully employed Escherichia coli as in vivo model for studying the aggregation mechanism of the polyQ protein ataxin-3 (AT3) in the presence of MC. In particular we investigated the relationship between the aggregation pathway and cytotoxicity and we were able to characterize the kinetic of formation of aggregated toxic and non-toxic species of AT3. Our future efforts will be aimed to investigate in vivo Hsp70 action by analyzing structural and physiological features of AT3 aggregated species formed in the intracellular environment of E. coli that co-express Hsp70 under different conditions.