ИСТИНА

Prion protein (PrP) is a membrane-bound protein predominantly expressed in neural tissue. Spatial structure of PrP consists of two domains: unstructured N-terminal and C-terminal, which in turn consists of three alpha-helices and two beta-sheets. This protein plays a crucial role in development of a number of amyloid diseases, such as syndrome Kreutzfeldt-Jacobs, fatal familial insomnia, Kuru, scrapie and others. Interaction of the infectious amyloid form of prion with native prion protein causes transformation of the latter and the subsequent accumulation of amyloid fibrils which further leads to the development of neurodegeneration. The aim of our work was to study the effect of prion protein glycation on its ability to form toxic oligomers and fibrils in order to clarify the relationship between carbohydrate metabolism disorders (first of all, occurring in diabetes) on the development of prion amyloidoses. We modified recombinant ovine prion protein by glucose and methylglyoxal. Most effective glycation of lysine and arginine residues occurred in case of methylglyoxal. The level of PrP glycation was measured by nitroblue tetrazolium test. It was found that glycation itself does not cause prion protein aggregation. We have performed titration of prion protein by methylglyoxal in order to monitor the accumulation of modified lysine and arginine residues, as well as the advanced glycation products (AGEs), some of which are capable of fluorescence. As we could see in figures 5 and 6, the modification of amino acid residues begins at sufficiently high concentrations of MGO. We also observed the accumulation of large amount of AGEs at an average concentration of MGO. We found that glycation of prion protein changes its secondary structure. First, it is seen as a reduction of intrinsic tryptophan fluorescence and the shift of fluorescence peak to the right. This could mean that prion protein has changed its conformation so that tryptophan residues are now shielded by new neighbors. Second, we have observed a decrease in ellipticity in 208 nm band of CD spectra, which indicated the decrease in the amount of alpha-helices, which in turn could mean that some alpha-helices have transitioned into other structures: disordered or beta-sheet. We have compared the ability of glycosylated native prion protein to oligomerize. As a result, the modified PrP formed precipitating amorphous aggregates. We were also able to detect large prion protein particles in the supernatant. However, according to the thioflavin T fluorescence these units have not an amyloid, but an amorphous structure. Therefore, prion protein glycation prevents its oligomerization. In order to track the formation of amyloid fibrils, we have preformed a seeding experiment in the presence of modified and non-modified prion protein. As we could see in figure 11, PrP glycation reduces the rate of transition of cellular isoform of PrP into scrapie isoform, thereby preventing the formation of amyloid structures. We have shown that prion protein glycation which can occur under elevated blood levels of glucose and other sugars is capable of reducing the efficiency of amyloid transformation and thus can affect the development of prion amyloidoses.