ИСТИНА

Physiological role of thiamine (vitamin B1) is not limited to coenzyme function of its diphosphate (ThDP), but also includes non-coenzyme action of thiamine and derivatives. However, molecular targets and pathways mediating this action are mostly unknown. Recently, proteins of rat synaptosomes bound to the thiamine or thiazolium-modified sepharose have been identified by mass spectrometry. Here, we use bioinformatics resources (DAVID, STRING, PANTHER and TR GeneGO Metacore) to reveal pathways and functional terms enriched in such partial synaptosomal proteomes. Binding of thiamine and derivatives to abundant enzymes in the proteomes, whose function does not depend on coenzyme action of ThDP, was characterized by enzyme kinetics. Response of such enzymes to thiamine was tested in rats. Functional annotation by DAVID revealed that the thiamine and thiazolium binding proteomes are highly enriched with acetylation-related proteins, suggesting dependence of this regulatory post-translational modification on thiamin. A significant part of the proteomes also relates to phosphorylation, which agrees with the known thiamin triphosphate (ThTP)-dependent phosphorylation of synaptosomal proteins. DAVID analysis of functional relationships between the proteome proteins exhibiting similar enrichment score reveals clusters of metabolic, mitochondrial and vesicular proteins. Functional classification by PANTHER indicated the three major functions of the thiamine- and thiazolium-binding proteins: catalytic activity, binding and structural molecule activity. Catalytic proteins are mostly represented by transferases and hydrolases, i.e. those catalyzing the reactions known for thiamine and its phosphates. Binding function is mainly distributed between binding of proteins, Ca2+ and nucleic acids. Proteins from Ca2+-binding group are involved in neurotransmission, suggesting the thiamine regulation of synaptic transmission through calcium-dependent processes. STRING and TR Metacore analysis visualized known interactions between the proteins of the thiamine and thiazolium proteomes (interactomes). The interactions defined clusters of proteins, involved in metabolism and signaling through 14-3-3, Ca2+, glutamate and redox state. Remarkable similarity in the bioinformatics-assigned features of the thiamine and thiazolium proteomes, which differ significantly from the features revealed by us through the same analysis of the published data on another partial brain proteome, points to specificity of the protein interactions with the thiamine/thiazolium part of the affine sorbents. The specificity was further confirmed by influence of the thiamine/thiazolium baits on activities of selected enzymes abundant in the proteomes. Ringer buffer was used in these experiments to imitate conditions of protein binding to affine sorbents. Because Ringer buffer contains many ions potentially having regulatory effects, particularly calcium ions, the study was also done in TRIS buffer. As a result, thiamine compounds were shown to be involved into the Ca2+-, ADP- and NAD(H)-dependent regulation of mitochondrial metabolism related to the 2-oxoglutarate/glutamate transformation node. Activities of these enzymes also responded to i.p. injection of 400 mg thiamine per kg in rats under normal and/or hypoxic conditions. Thus, our bioinformatics and biochemical analysis unravels that thiamine may participate in the calcium-, ADP and NAD(H)-dependent regulation of synaptosomal enzymes, which do not directly depend on the coenzyme action of ThDP, but are metabolically related to the ThDP-dependent dehydrogenases of 2-oxo acids.