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

• Published in: Biology (Basel, Switzerland) Switzerland), 2019-06, Vol.8 (2), p.49
• Main Authors: Ershov, Pavel V, Mezentsev, Yuri V, Kopylov, Arthur T, Yablokov, Evgeniy O, Svirid, Andrey V, Lushchyk, Aliaksandr Ya, Kaluzhskiy, Leonid A, Gilep, Andrei A, Usanov, Sergey A, Medvedev, Alexey E, Ivanov, Alexis S
• Format: Article
• Language: English
• Subjects: Acids; Affinity; Aldo-keto reductase; Aqueous solutions; Biosensors; Chromatography; Cytochrome P450; direct molecular fishing; Drug metabolism; E coli; Experiments; Glutaredoxin; Glutathione transferase; Glycerol; GSTM1 protein; Heme; Histidine; isatin; Isomerization; Lysates; mass spectrometry; Mass spectroscopy; Oxidative metabolism; Oxidative stress; Platelet aggregation; Potassium; Prostacyclin; Prostacyclin synthase; Prostaglandin H2; Protein expression; Protein interaction; Proteins; protein–protein interactions; size-exclusion chromatography; subinteractome; Surface plasmon resonance; Synthetic peptides; Uterus
• ISSN: 2079-7737; 2079-7737
• DOI: 10.3390/biology8020049

Prostacyclin synthase (PTGIS; EC 5.3.99.4) catalyzes isomerization of prostaglandin H 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.