Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms
- Authors: Osipyants A.I.1, Smirnova N.A.1, Khristichenko A.Y.1, Hushpulian D.M.1, Nikulin S.V.1, Chubar T.A.2, Zakhariants A.A.3, Tishkov V.I.2,3, Gazaryan I.G.1,2, Poloznikov A.A.1
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Affiliations:
- Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
- Lomonosov Moscow State University, Chemistry Faculty
- Innovations and High Technologies MSU Ltd.
- Issue: Vol 82, No 10 (2017)
- Pages: 1207-1214
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151491
- DOI: https://doi.org/10.1134/S0006297917100145
- ID: 151491
Cite item
Abstract
An organism naturally responds to hypoxia via stabilization of hypoxia-inducible factor (HIF). There are three isoforms of HIFα subunits whose stability is regulated by three isozymes of HIF prolyl hydroxylase (PHD1-3). Despite intense studies on recombinant enzyme isoforms using homogeneous activity assay, there is no consensus on the PHD iso-form preference for the HIF isoform as a substrate. This work provides a new approach to the problem of substrate specificity using cell-based reporters expressing the enzyme and luciferase-labeled substrate pair encoded in the same expression vector. The cell is used as a microbioreactor for running the reaction between the overexpressed enzyme and substrate. Using this novel approach, no PHD3 activity toward HIF3 was demonstrated, indirectly pointing to the hydroxylation of the second proline in 564PYIP567 (HIF1) catalyzed by this isozyme. The use of “paired” enzyme–substrate reporters to evaluate the potency of “branched tail” oxyquinoline inhibitors of HIF PHD allows higher precision in revealing the optimal structural motif for each enzyme isoform.
About the authors
A. I. Osipyants
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997
N. A. Smirnova
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997
A. Yu. Khristichenko
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997
D. M. Hushpulian
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997
S. V. Nikulin
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997
T. A. Chubar
Lomonosov Moscow State University, Chemistry Faculty
Email: igazaryan@gmail.com
Russian Federation, Moscow, 119991
A. A. Zakhariants
Innovations and High Technologies MSU Ltd.
Email: igazaryan@gmail.com
Russian Federation, Moscow, 109451
V. I. Tishkov
Lomonosov Moscow State University, Chemistry Faculty; Innovations and High Technologies MSU Ltd.
Email: igazaryan@gmail.com
Russian Federation, Moscow, 119991; Moscow, 109451
I. G. Gazaryan
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology; Lomonosov Moscow State University, Chemistry Faculty
Author for correspondence.
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997; Moscow, 119991
A. A. Poloznikov
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
Russian Federation, Moscow, 117997