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Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms

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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.

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Abbreviations

αKG:

alpha-ketoglutarate

DMSO:

dimethylsulf-oxide

HIF:

hypoxia-inducible factor

ODD:

oxygen-dependent degradation

PHD:

prolyl hydroxylase domain

VHL:

von Hippel–Lindau protein

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Authors and Affiliations

  1. Rogachev National Medical Research Center for Pediatric Hematology, Oncology and Immunology, Healthcare Ministry of Russia, 117997, Moscow, Russia

    A. I. Osipyants, N. A. Smirnova, A. Yu. Khristichenko, D. M. Hushpulian, S. V. Nikulin, I. G. Gazaryan & A. A. Poloznikov

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

    T. A. Chubar, V. I. Tishkov & I. G. Gazaryan

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

    A. A. Zakhariants & V. I. Tishkov

Authors
  1. A. I. Osipyants
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  2. N. A. Smirnova
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  3. A. Yu. Khristichenko
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  4. D. M. Hushpulian
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  5. S. V. Nikulin
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  6. T. A. Chubar
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  7. A. A. Zakhariants
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  8. V. I. Tishkov
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  9. I. G. Gazaryan
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  10. A. A. Poloznikov
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Corresponding author

Correspondence to I. G. Gazaryan.

Additional information

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM17-394, September 18, 2017.

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Osipyants, A.I., Smirnova, N.A., Khristichenko, A.Y. et al. Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms. Biochemistry Moscow 82, 1207–1214 (2017). https://doi.org/10.1134/S0006297917100145

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  • DOI: https://doi.org/10.1134/S0006297917100145

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