Formation of β-structures and fibrillar associates by NovoRapid insulin molecules in the presence of Saccharomyces cerevisiae yeast cell walls

Prozorov M.A., Rekstina V.V., Kudryashova I.B., Dragoni O.A., Ziganshin R.H., Kamzolkina O.V., Kalebina T.S.

Lomonosov Moscow State University, Moscow, Russia
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia

The aim of the study was to investigate the ability of NovoRapid insulin preparation to form β-structures and fibrils in the presence and absence of inductors. The formation of β-structures was studied by fluorescence spectroscopy using a specific dye Thioflavin T. The ability of insulin to form fibrils was investigated by electron microscopy. Saccharomyces cerevisiae yeast cell wall (CW) preparations containing Bgl2p, a protein with amyloid properties and CW lacking it were used as inductors. The absence of Bgl2p in the CW was caused by a deletion of the gene encoding it. LC-MS/MS analysis showed that the set of proteins covalently and non-covalently bound to the CW polysaccharide molecules of these yeast is the same and differs only in the absence of Bgl2p in the case of the bgl2Δ strain. It was shown that in the NovoRapid insulin incubation medium, to which wild-type yeast cell walls were added as an inductor, not only the intensity of Thioflavin T fluorescence at a wavelength of 485 nm increases, but also fibrillar structures are detected, reaching a length of several micrometers. The results of this study suggest that Bgl2p can be considered as an inductor of the process of fibril formation in the presence of NovoRapid insulin, while other components of the CW are involved in the induction of the formation of β-structures in the molecules of this insulin.