Abstract
Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H2 to prostacyclin, a potent vasodilator and inhibitor of platelet aggregation. At present, limited data exist on functional coupling and possible ways of regulating PTGIS due to insufficient information about protein–protein interactions in which this crucial enzyme is involved. The aim of this study is to isolate protein partners for PTGIS from rat tissue lysates. Using CNBr-activated Sepharose 4B with covalently immobilized PTGIS as an affinity sorbent, we confidently identified 58 unique proteins by mass spectrometry (LC-MS/MS). The participation of these proteins in lysate complex formation was characterized by SEC lysate profiling. Several potential members of the PTGIS subinteractome have been validated by surface plasmon resonance (SPR) analysis. SPR revealed that PTGIS interacted with full-length cytochrome P450 2J2 and glutathione S-transferase (GST). In addition, PTGIS was shown to bind synthetic peptides corresponding to sequences of for GSTA1, GSTM1, aldo-keto reductase (AKR1A1), glutaredoxin 3 (GLRX3) and histidine triad nucleotide binding protein 2 (HINT2). Prostacyclin synthase could potentially be involved in functional interactions with identified novel protein partners participating in iron and heme metabolism, oxidative stress, xenobiotic and drugs metabolism, glutathione and prostaglandin metabolism. The possible biological role of the recognized interaction is discussed in the context of PTGIS functioning.
Highlights
Prostacyclin synthase (PTGIS; EC 5.3.99.4), known as prostaglandin I2 synthase, catalyzes the reaction of prostaglandin H2 isomerization to prostacyclin
We optimized the lysate sample preparation to increase the efficiency of isolation of protein partners from the lysate by carrying out the preliminary dissociation of protein complexes by a short-term acid treatment followed by NaOH neutralization to restore the initial pH value of the samples [24]
The addition of exogenous PTGIS to the acid-dissociated and neutralized lysate sample resulted in almost 50% reduction in the binding levels of protein material to PTGIS immobilized on the chip as compared to the control lysate
Summary
Prostacyclin synthase (PTGIS; EC 5.3.99.4), known as prostaglandin I2 synthase, catalyzes the reaction of prostaglandin H2 isomerization to prostacyclin (or prostaglandin I2 , PGI2 ). PTGIS is characterized by tissue specificity; according to the GeneCards database [https://www.genecards.org], it is mainly determined in tonsil, lung, heart, urinary bladder, uterus, testis. PTGIS co-localizes with cyclooxygenase in the endoplasmic reticulum, plasma membrane and nuclear membrane [1]. An imbalance between prostacyclin and its physiological antagonist thromboxane A2 contributes to the development of myocardial infarction, stroke, and atherosclerosis [2,3,4,5]. Certain evidence exists that PTGIS and thromboxane synthase (TBXAS1) may be involved in carcinogenesis [6,7]
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