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

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.

A new insight into subinteractomes of functional antagonists: Thromboxane (CYP5A1) and prostacyclin (CYP8A1) synthases

“…In previous publications, it was shown that both thromboxane and prostacyclin synthases had formed stable molecular complexes with a number of protein partners (Ershov et al, 2019a; Gnedenko et al, 2019; Svirid et al, 2017). Taken together, the SPR‐validated PPIs are shown schematically in Figure 1.…”

“…At the same time, CYP8A1 formed molecular complexes with other microsomal (CYP21A2 and CYP2J2) and mitochondrial (CYP11A1 and CYP11B1) cytochrome P450s. Negative results of direct complex formation between CYP5A1 and CYP8A1 as well as between these synthases and cytochrome P450's redox partners (CYB5A, CPR, and Adx), which are strong binders of different CYP isoenzymes, were obtained (Ershov et al, 2019a; Yablokov et al, 2019). It is quite obvious that the CPR protein, isolated from the liver tissue, using CYP5A1 as a bait, could be a protein partner of co‐isolated CYP2D10 (Table 1).…”

“…Taking into account the high capacity of GAPDH to forming of hetero‐complexes with different proteins (E Silva et al, 2019), it could have been isolated as a part of a multiprotein complex сontaning certain direct partners of CYP8A1. The SPR‐validated PPIs of СYP8A1 with several full‐length cytochrome P450s as well as peptide–protein interactions with AKR1A1, GSTA1, GSTM1, GLRX3, and HINT2 from different functional groups (Figure 1) indicate that prostacyclin synthase can be a multifunctional protein (Ershov et al, 2019a).…”

Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

“…Содержание общего белка в образцах тканевого лизата определяли по методу Бредфорда. Диссоциацию белковых комплексов тканевого лизата выполняли согласно протоколу [3].…”

“…Было выявлено, что почти три четверти всех идентифицированных во фракциях белков находятся в виде гомо-и гетеродимерных комплексов и белковых комплексов более высокого порядка. Также в работах [2,3] был продемонстрирован оригинальный способ диссоциации интактных белковых комплексов лизата, заключающийся в кратковременной кислотной обработке лизата с последующей нейтрализацией, для увеличения чувствительности и специфичности прямого молекулярного фишинга белковых партнеров целевого белка. Вместе с тем, остается пока неизвестным, насколько отличается обычный SEC профиль белков/белковых комплексов лизата от профиля ре-ассоциированных белков обработанного лизата.…”