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.

Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome

“…The PTGIS protein was covalently immobilized on the activated CNBr-sepharose 4B via amino groups of the protein. A similar protocol has already been used to immobilize recombinant thromboxane synthase [16] and some recombinant proteins encoded by genes on Chromosome 18 [12,14,25] on this sorbent. The specificity of direct molecular fishing was analyzed by SPR.…”

“…Recently, we found that the non-peptide endogenous bioregulator isatin (Mw = 147 Da) bound some cytochrome P450s isoenzymes and influenced the affinity of protein–protein interactions [16,53,54]. Therefore, to continue this investigation, SPR validation of the isatin binding (injected as analyte) with PTGIS protein immobilized on the optical chip was performed.…”

“…CNBr-activated Sepharose 4B was used for covalent PTGIS coupling via amino groups of the protein according to the protocol described in [16]. Briefly, 200 μL of immobilization buffer containing 500 mM NaCl, 100 mM NaHCO 3 (pH 8.3) and PTGIS protein were added to about 200 µL suspension of CNBr—activated Sepharose 4B (100 mg of dry sorbent).…”

“…We have previously demonstrated the applicability of different approaches to direct molecular fishing [12,13,14] for isolation and identification of previously unknown and real protein partners for the bait proteins from tissue lysates. These approaches include a combination of paramagnetic nanoparticle technology, affinity chromatography, LC/MS-MS and surface plasmon resonance (SPR) optical biosensors [12,15,16,17]. The present study is aimed at the isolation and identification of new protein partners of prostacyclin synthase using direct molecular fishing combined with size exclusion chromatography (SEC).…”