Enzyme–substrate reporters for evaluation of substrate specificity of HIF prolyl hydroxylase isoforms
| Autor: | N. A. Smirnova, Vladimir I. Tishkov, Irina G. Gazaryan, Sergey Nikulin, Andrey A. Poloznikov, A. Yu. Khristichenko, A. I. Osipyants, A. A. Zakhariants, T. A. Chubar, D. M. Hushpulian |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Gene isoform Biology Biochemistry Isozyme Hypoxia-Inducible Factor-Proline Dioxygenases Substrate Specificity Hydroxylation 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Genes Reporter Cell Line Tumor RNA Ribosomal 18S Humans RNA Messenger Structural motif chemistry.chemical_classification Expression vector Substrate (chemistry) General Medicine Recombinant Proteins Isoenzymes Kinetics 030104 developmental biology Enzyme chemistry 030217 neurology & neurosurgery |
| Zdroj: | Biochemistry (Moscow). 82:1207-1214 |
| ISSN: | 1608-3040 0006-2979 |
| DOI: | 10.1134/s0006297917100145 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|