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

1
Department of Proteomic Research and Mass Spectrometry, Institute of Biomedical Chemistry (IBMC), 10 Pogodinskaya str., 119121 Moscow, Russia
2
Laboratory of Molecular Diagnostics and Biotechnology, Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, 5, bld. 2 V.F. Kuprevich str., 220141 Minsk, Belarus
*
Author to whom correspondence should be addressed.
Biology 2019, 8(2), 49; https://doi.org/10.3390/biology8020049
Received: 24 April 2019 / Revised: 6 June 2019 / Accepted: 18 June 2019 / Published: 20 June 2019
(This article belongs to the Section Biochemistry and Molecular Biology)
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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. View Full-Text
Keywords: prostacyclin synthase; protein–protein interactions; surface plasmon resonance; mass spectrometry; size-exclusion chromatography; subinteractome; direct molecular fishing; isatin prostacyclin synthase; protein–protein interactions; surface plasmon resonance; mass spectrometry; size-exclusion chromatography; subinteractome; direct molecular fishing; isatin
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Ershov, P.V.; Mezentsev, Y.V.; Kopylov, A.T.; Yablokov, E.O.; Svirid, A.V.; Lushchyk, A.Y.; Kaluzhskiy, L.A.; Gilep, A.A.; Usanov, S.A.; Medvedev, A.E.; Ivanov, A.S. Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome. Biology 2019, 8, 49.

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