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A Genetically Encoded FRET Sensor for Hypoxia and Prolyl Hydroxylases.

ABSTRACT: Oxygen is vital for all aerobic life forms. Oxygen-dependent hydroxylation of hypoxia-inducible factor (HIF)-1? 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.

SUBMITTER: Youssef S

PROVIDER: S-EPMC5026574 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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A Genetically Encoded FRET Sensor for Hypoxia and Prolyl Hydroxylases.

Youssef Suzan S Ren Wei W Ai Hui-Wang HW

ACS chemical biology 20160714 9

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 ...[more]

PMID: 27385075

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Project description:Lactate is shuttled between and inside cells, playing metabolic and signaling roles in healthy tissues. Lactate is also a harbinger of altered metabolism and participates in the pathogenesis of inflammation, hypoxia/ischemia, neurodegeneration and cancer. Many tumor cells show high rates of lactate production in the presence of oxygen, a phenomenon known as the Warburg effect, which has diagnostic and possibly therapeutic implications. In this article we introduce Laconic, a genetically-encoded Forster Resonance Energy Transfer (FRET)-based lactate sensor designed on the bacterial transcription factor LldR. Laconic quantified lactate from 1 µM to 10 mM and was not affected by glucose, pyruvate, acetate, betahydroxybutyrate, glutamate, citrate, ?-ketoglutarate, succinate, malate or oxalacetate at concentrations found in mammalian cytosol. Expressed in astrocytes, HEK cells and T98G glioma cells, the sensor allowed dynamic estimation of lactate levels in single cells. Used in combination with a blocker of the monocarboxylate transporter MCT, the sensor was capable of discriminating whether a cell is a net lactate producer or a net lactate consumer. Application of the MCT-block protocol showed that the basal rate of lactate production is 3-5 fold higher in T98G glioma cells than in normal astrocytes. In contrast, the rate of lactate accumulation in response to mitochondrial inhibition with sodium azide was 10 times lower in glioma than in astrocytes, consistent with defective tumor metabolism. A ratio between the rate of lactate production and the rate of azide-induced lactate accumulation, which can be estimated reversibly and in single cells, was identified as a highly sensitive parameter of the Warburg effect, with values of 4.1 ± 0.5 for T98G glioma cells and 0.07 ± 0.007 for astrocytes. In summary, this article describes a genetically-encoded sensor for lactate and its use to measure lactate concentration, lactate flux, and the Warburg effect in single mammalian cells.

Dataset Information

A Genetically Encoded FRET Sensor for Hypoxia and Prolyl Hydroxylases.

Publications

A Genetically Encoded FRET Sensor for Hypoxia and Prolyl Hydroxylases.

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