[Back to Issue 10 ToC] [Back to Journal Contents] [Back to Biochemistry (Moscow) Home page]
[View Full Article] [Download Reprint (PDF)]

Enzyme–Substrate Reporters for Evaluation of Substrate Specificity of HIF Prolyl Hydroxylase Isoforms

A. I. Osipyants1, N. A. Smirnova1, A. Yu. Khristichenko1, D. M. Hushpulian1, S. V. Nikulin1, T. A. Chubar2, A. A. Zakhariants3, V. I. Tishkov2,3, I. G. Gazaryan1,2*, and A. A. Poloznikov1

1Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology, Healthcare Ministry of Russia, 117997 Moscow, Russia; E-mail: igazaryan@gmail.com

2Lomonosov Moscow State University, Chemistry Faculty, 119991 Moscow, Russia

3Innovations and High Technologies MSU Ltd., 109451 Moscow, Russia

* To whom correspondence should be addressed.

Received August 26, 2017; Revision received September 8, 2017
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 isoform 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.
KEY WORDS: activation, luciferase fusion protein, enzyme inhibitors, branched oxyquinolines, ciclopirox, real-time PCR

DOI: 10.1134/S0006297917100145