Mezhenska O. Novel protein targets of thiamine and its derivatives in nervous tissue

The thesis is devoted to the identification of nerve tissue proteins that exhibit affinity for thiamine, in particular the thiamine binding protein (ThBР), previously described at the Department of Biochemistry of Vitamins and Coenzymes. Using specific elution and two types of affinity sorbents, binding of thiamine to proteins that exhibit dehydrogenase, such as MDH and GDH, and thiaminphosphatase activity is determined to be specific. The effect of thiamine on malate dehydrogenase and thiamine and its diphosphate on glutamate dehydrogenase activity in vitro and the effect of providing thiamine on these activities in vivo on model of alimentary thiamine deficiency were determined. After affinity chromatography of a commercial MDH preparation using mass spectrometric analysis (MS analysis), MDH isozymes and some minor proteins from the commercial MDH composition capable of binding to thiamine and not previously known to be thiamine dependent were identified. The proteins from nerve tissue extracts that specifically bind thiamine in the affinity sorbent during affinity chromatography and enter to the ThBP preparation after gel filtration, for the first time variants of proteins that may be ThBP components have been identified by MS analysis and Western blot analysis, in particular, the LRP4-Agrin protein complex, which is a component of the nAChR cluster. For the first time, using the molecular docking method, the amino acid residues of both agrin and LRP4 polypeptide chains involved in thiamine binding were determined. Comparative analysis of the binding sites of thiamine and its phosphorous esters with these proteins and with known thiamine-dependent proteins revealed their high similarity and low likelihood of replacement of thiamine with adenosine compounds or acetylcholine, as well as explaining the causes of lower affinity for thiamine phosphate esters compared to thiamine. Bioinformatics tools suggested the possible biological roles of thiamine and its biologically active derivatives when binding to the Agrin-LRP4 complex. The results obtained in these studies are important as they expand the current understanding of cell-molecular mechanisms of vitamin B1 functions realization. This further substantiates the advisability of using thiamine and its pharmacological forms for the prevention and/or treatment of pathologies that are induced or accompanied by thiamine deficiency.