ИСТИНА

It is known that a protein molecule must have a native tertiary or quaternary structure for normal functioning. Spontaneous correct folding of a polypeptide chain is a slow and inefficient process. Therefore, there exists a special class of proteins in the cell - molecular chaperones - which increase the speed and efficiency of bio-macromolecules folding and prevents their harmful aggregation. The proper work of the chaperone system is the key to the correct functioning of many cell systems that can be exposed to damaging stress factors. Nowadays there is a number of serious diseases are caused by the accumulation of protein aggregates in the nervous tissue, for example Parkinson's disease or prion diseases. In this case, the cellular system of chaperones receives an alarm signal and begins to interact with the assembled protein formations. However, the result of such interaction between chaperones and a healthy form of protein or its aggregates has not been fully clarified [1]. Thus, the aim of our work was to study the interaction of chaperones with amyloidogenic proteins in normal state and in case of protein glycation taking place in case of diabetes. To work out the methodology we used two model systems: ovine prion protein (PrP) as an agent capable of aggregation and ATP-dependent bacterial chaperonin complex GroEL14/GroES7. We modified prion protein by methylglyoxal in order to show how the disorder of carbohydrate metabolism could affect the protein of interest. It turned out that mainly arginine residues underwent glycation in prion protein. Also, dynamic light scattering (DLS) showed that glycation itself did not lead to the aggregation of PrP. Furthermore, we investigated the effect of different forms of prion protein (native, glycated and also oligomers and fibrils) on the activity of chaperonin complex GroEL14/GroES7. To study changes in chaperonin work we used a marker – a complex glycolytic enzyme glycerol-3-phosphate dehydrogenase. Firstly, we completely denatured GAPDH with the addition of 4 M guanidine hydrochloride. Then we incubated the enzyme with chaperonin complex, which was in turn pre-incubated with PrP, and observed the recovery of the GAPDH activity. Chaperonin-dependent reactivation of GAPDH was blocked by monomeric form of the PrP. This effect was most significant in case of glycated protein. PrP oligomers only slowed down chaperone-dependent reactivation of GAPDH, and the PrP fibrils practically did not affect that process. Since we got an interesting outcome, we expanded our experiments using ovine ATP-dependent cytoplasmic chaperonin TriC. The presented data shows that both eukaryotic chaperonin TriC and bacterial complex GroEL14/GroES7 bind different forms of prion protein with better affinity than denatured GAPDH. This significantly alters the efficiency of amyloid transformation and can affect the development of amyloidosis. 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.