Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms
- 作者: Osipyants A.1, Smirnova N.1, Khristichenko A.1, Hushpulian D.1, Nikulin S.1, Chubar T.2, Zakhariants A.3, Tishkov V.2,3, Gazaryan I.1,2, Poloznikov A.1
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隶属关系:
- Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
- Lomonosov Moscow State University, Chemistry Faculty
- Innovations and High Technologies MSU Ltd.
- 期: 卷 82, 编号 10 (2017)
- 页面: 1207-1214
- 栏目: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151491
- DOI: https://doi.org/10.1134/S0006297917100145
- ID: 151491
如何引用文章
详细
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.
作者简介
A. Osipyants
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
N. Smirnova
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
A. Khristichenko
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
D. Hushpulian
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
S. Nikulin
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
T. Chubar
Lomonosov Moscow State University, Chemistry Faculty
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 119991
A. Zakhariants
Innovations and High Technologies MSU Ltd.
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 109451
V. Tishkov
Lomonosov Moscow State University, Chemistry Faculty; Innovations and High Technologies MSU Ltd.
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 119991; Moscow, 109451
I. Gazaryan
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology; Lomonosov Moscow State University, Chemistry Faculty
编辑信件的主要联系方式.
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997; Moscow, 119991
A. Poloznikov
Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology
Email: igazaryan@gmail.com
俄罗斯联邦, Moscow, 117997
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