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 TBXAS1CYP2E1 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.

Mechanism of the Affinity-Enhancing Effect of Isatin on Human Ferrochelatase and Adrenodoxin Reductase Complex Formation: Implication for Protein Interactome Regulation

“…This novel regulatory role of isatin has been originally demonstrated by us for the interaction of two pairs of proteins: (1) ferrochelatase (FECH) and adrenodoxin reductase (ADR) [8], (2) thromboxane synthase (TBXAS1) and cytochrome P450 2E1 (CYP2E1) [9]. It was particularly interesting that individual proteins of each investigated pair of the interacting proteins demonstrated low (if any) isatin binding capacity, but the addition of isatin caused a several-fold increase in the affinity of their interaction [8,9].…”

“…However, besides direct action of isatin on its particular targets, characterized by classical binding constants and altered biological functioning of individual proteins [6,7] it can also act as a bidirectional regulator of protein-protein interactions (PPI) increasing or decreasing affinity of interacting protein partners [1,8]. This novel regulatory role of isatin has been originally demonstrated by us for the interaction of two pairs of proteins: (1) ferrochelatase (FECH) and adrenodoxin reductase (ADR) [8], (2) thromboxane synthase (TBXAS1) and cytochrome P450 2E1 (CYP2E1) [9]. It was particularly interesting that individual proteins of each investigated pair of the interacting proteins demonstrated low (if any) isatin binding capacity, but the addition of isatin caused a several-fold increase in the affinity of their interaction [8,9].…”

The Multienzyme Complex Nature of Dehydroepiandrosterone Sulfate Biosynthesis

“…SULTs were covalently immobilized onto CNBr-Sepharose 4B (Cytiva, Marlborough, MA, USA). Sorbent preparation, the immobilization of a target protein, and the isolation of its protein partners from tissue lysate samples were described in our previous work [78]. HBS-P (10 mM HEPES, 150 mM NaCl, and 0.005% Tween-20, pH 7.4) was used as a working buffer for performing the AP-MS analysis of proteins.…”

Bielectrode Strategy for Determination of CYP2E1 Catalytic Activity: Electrodes with Bactosomes and Voltammetric Determination of 6-Hydroxychlorzoxazone

“…The maximal rate of the 6-hydroxylation of chlorzoxazone (Vmax) is 9.3 min −1 and the Michaelis constant (KM) is 75 µM, according to the product information sheet. Recombinant human CYP2E1 with a protein concentration of 72 µM in 50 mM potassium phosphate buffer, pH 7.4, containing 400 mM NaCl, 1 mM EDTA, and 20% glycerol, was obtained in accordance with the previously described procedure [48]. Microsomal human cytochrome b5 (cyt b5) with a protein concentration of 158 µM in 400 mM potassium Conventional methods for assessing CYP2E1's 6-hydroxylase activity towards chlorzoxazone in microsomal or reconstituted systems rely on high-performance liquid chromatography with UV detection [42,44] or mass spectrometry analysis of the resulting metabolite.…”