ИСТИНА

Nowadays there are a number of serious diseases, which are caused by the accumulation of protein aggregates in the nervous tissue. For example, Parkinson's disease or prion diseases. This process activates cellular chaperone system, which stats to interact with the assembled aggregates. Under normal conditions chaperones regulate the folding of proteins and prevent their aggregation. However, the result of the interaction of this cellular system with a normal form of protein and its aggregates may be different and has not yet been fully studied [1]. During this work we investigated the role of chaperones in amyloidogenic transformation of proteins. To achieve our aims we used two model systems: sheep prion protein as a capable of aggregating agent and ATP-dependent bacterium chaperonin complex GroEL14/GroES7. We assumed the possibility of inhibition of chaperonin complex with amyloid proteins. To test our hypothesis we obtained various forms of prion protein (PrP): monomeric PrP, small oligomers of PrP, "protofibrils" and fibrils of PrP. The affiliation of molecules to specific form of prion protein was verified by dynamic light scattering (DLS). The amiloid fibrillation of prion protein was also indicated by the increase in ThT fluorescence intensity in its presence. We showed by ELISA that chaperonin complex GroEL14/GroES7 bound different forms of prion protein. Further we proved that chaperonin did not simply bind the monomeric PrP, but caused its aggregation and/or fibrillation. To confirm this fact we pre-incubated GroEL14/GroES7 complex with different forms of PrP and then measured the fluorescence emission spectra of ThT of this samples. Eventually we found out that chaperonin stimulated amyloidogenic transformation of not only already formed oligomers and fibrils but also of monomeric prion protein. The same results were also obtained by the dynamic light scattering. We investigated the possibility of blocking the main function of the chaperonin GroEL14 – its ability to reactivate different proteins - by different forms of prion protein. To indicate changes in the work of chaperonin we used a marker – a complex glycolytic enzyme glycerol-3-phosphate dehydrogenase. Firstly, we completely denatured GAPD with the addition of 4 M guanidine hydrochloride. Then we incubated denatured GAPD with chaperonin complex and observed the recovery of the enzyme activity. When chaperonin complex GroEL14/GroES7 was co-incubated with one of the prion protein forms and with denatured GAPD, the functional activity of the chaperonine complex was reduced to almost complete loss of its activity The similarity of bacterial chaperonin to some mammalian chaperonin complexes (i.e. cytoplasmic chaperonin TriC), the key role of GAPDH in glycolysis and the presence of different forms of amyloid-like proteins in the neural tissue allows to suggest the involvement of the described mechanism into development of amyloid neurodegenerative diseases. 1. Stroylova Y.Y., Kiselev G.G., Schmalhausen E.V., Muronetz V.I. (2014) Prions and chaperones: Friends or foes?, Biochemistry (Moscow), 79 (8), 761-775.