Direct Molecular Fishing of New Protein Partners for Human Thromboxane Synthase

Abstract

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.

Keywords: Thromboxane synthase (CYP5A1, TBXAS1); cytochrome P450; direct molecular fishing; isatin; protein partners; surface plasmon resonance.

Fig. 1

Schematic representation of TXA2 biosynthesis supplemented by our experimental results. PLC – phospholipase C, PLA2 – phospholipase A₂, COX – cyclooxygenase. Biosynthesis of TXA₂ begins with the release of arachidonic acid from membrane phospholipids assisted by PLA2. Then, COX catalyzes the transformation of arachidonic acid into prostaglandin H2, which is then metabolized by TBXAS1 to form TXA₂, 12-HHT, and MDA. At the same time, prostaglandin H2 is transformed by CYP2E1 to 12-HHT and MDA. TXA₂ binding to TXAR causes signal transmission via the inositol phosphate pathway with PLC activation and mobilization of intracellular Ca²⁺, which has a stimulating effect on PLA2. Further, 12-HHT is metabolized by 15-hydroxyprostaglandin dehydrogenase to form 12-keto-HHT, which has a partial antagonistic effect on TXAR. TBXAS1 also presumably interacts with BBIP1, which is a component of the protein transport complex of cilia (BBSome). BBIP1 can influence the stability of the microtubulin cytoskeleton, indirectly inhibiting HDAC6 (microtubule deacetylase).

Fig. 2

Typical sensorgrams of the interaction between various concentrations of CYP2E1 and TBXAS1 immobilized on a CM5 optical chip

Fig. 3

Typical sensorgrams of the interaction between various concentrations of CYP11B2 and TBXAS1 immobilized on a CM5 optical chip

Fig. 4

Diagram representation of equilibrium dissociation constant (K_d) values of the TBXAS1•CYP2E1 and TBXAS•CYP11B2 complexes in the absence and presence of 100 μM of isatin; M ± m, n = 3