[Interaction of prostacyclin synthase with cytochromes P450]. / Vzaimodeistvie prostatsiklinsintazy s tsitokhromami P450.

Biosensor experiments on investigation of interaction between prostacyclin synthase (PGIS) and different proteins of the cytochrome P450 monooxygenase systems were perfomed. Interaction of PGIS with microsomal (CYP21A2, CYP2E1) and mitochondrial (CYP27A1, CYP11B1, CYP11B2, CYP11A1) cytochrome P450s was detected. Kinetic and equilibrium parameters of protein complexes formation were determined. Data obtained suggest an essential role of these hemoproteins interaction in regulation of prostacyclin and thromboxane A2 biosynthesis.

[Study specificity of isatin interactions with P450 cytochromes]. / Spetsifichnost' vzaimodeistviia izatina s tsitokhromami R450.

Cytochrome P450-dependent monooxygenase systems exist basically in all living organisms, where they perform various important functions. The coordinated functioning of these systems involves many proteins participating in different protein-protein interactions (PPI). Previously, we have found that the endogenous non-peptide bioregulator isatin (indoledione-2,3), synthesized from indole by means of certain cytochromes P450 (e.g. P450 2E1, P450 2C19, P450 2A6) regulates affinity of some PPI. In this work, an attempt has been undertaken to register a direct interaction of isatin with a set of different proteins related to the functioning of cytochrome P450-dependent monooxygenase: five isoforms of cytochromes P450, two isoforms of cytochrome b5, cytochrome P450 reductase, adrenodoxin, adrenodoxin reductase and ferrochelatase. The study has shown that isatin binds specifically only to cytochromes P450 with high affinity (the equilibrium dissociation constant (Kd) is about 10-8 M).

Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase.

Thromboxane synthase (TBXAS1) catalyzes the isomerization reaction of prostaglandin H2 producing thromboxane A2, the autocrine and paracrine factor in many cell types. A high activity and metastability by these arachidonic acid derivatives suggests the existence of supramolecular structures that are involved in the regulation of the biosynthesis and directed translocation of thromboxane to the receptor. The objective of this study was to identify TBXAS1 protein partners from human liver tissue lysate using a complex approach based on the direct molecular fishing technique, LC-MS/MS protein identification, and protein-protein interaction validation by surface plasmon resonance (SPR). As a result, 12 potential TBXAS1 protein partners were identified, including the components regulating cytoskeleton organization (BBIP1 and ANKMY1), components of the coagulation cascade of human blood (SERPINA1, SERPINA3, APOH, FGA, and FN1), and the enzyme involved in the metabolism of xenobiotics and endogenous bioregulators (CYP2E1). SPR validation on the Biacore 3000 biosensor confirmed the effectiveness of the interaction between CYP2E1 (the enzyme that converts prostaglandin H2 to 12-HHT/thromboxane A2 proantagonist) and TBXAS1 (Kd = (4.3 ± 0.4) × 10-7 M). Importantly, the TBXAS1•CYP2E1 complex formation increases fivefold in the presence of isatin (indole-2,3-dione, a low-molecular nonpeptide endogenous bioregulator, a product of CYP2E1). These results suggest that the interaction between these hemoproteins is important in the regulation of the biosynthesis of eicosanoids.