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ACS Chem Biol. 2016 Sep 16;11(9):2492-8. doi: 10.1021/acschembio.6b00330. Epub 2016 Jul 14.

A Genetically Encoded FRET Sensor for Hypoxia and Prolyl Hydroxylases.

Author information

1
Department of Chemistry, University of California , Riverside, 501 Big Springs Road, Riverside, California 92521, United States.

Abstract

Oxygen is vital for all aerobic life forms. Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-1α by prolyl hydroxylase domain enzymes (PHDs) is an important step for controlling the expression of oxygen-regulated genes in metazoan species, thereby constituting a molecular mechanism for oxygen sensing and response. Herein, we report a genetically encoded dual-emission ratiometric fluorescent sensor, ProCY, which responds to PHD activities in vitro and in live cells. We demonstrated that ProCY could monitor hypoxia in mammalian cells. By targeting this novel genetically encoded biosensor to the cell nucleus and cytosol, we determined that, under normoxic conditions, the HIF-prolyl hydroxylase activity was mainly confined to the cytosol of HEK 293T cells. The results collectively suggest broad applications of ProCY on the evaluation of hypoxia and PHD activities and understanding of pathways for the control of hypoxic responses.

PMID:
27385075
PMCID:
PMC5026574
DOI:
10.1021/acschembio.6b00330
[Indexed for MEDLINE]
Free PMC Article

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