Expanding the use of fluorogenic enzyme reporter substrates to imaging metabolic flux changes: the activity measurement of 5α-steroid reductase in intact mammalian cells.
The study of dynamic properties of metabolic and signaling networks is hindered by the lack of methods for imaging metabolic fluxes in individual intact cells. We describe a novel optical approach for measuring the changes of metabolic fluxes in cells, based on a two-substrate competition between a physiological substrate and a fluorogenic reporter substrate. We have constructed a model cell system for a two-step metabolic pathway involved in the metabolism of testosterone. Potent androgen testosterone is converted by steroid 5α-reductase to DHT (5α-dihydrotestosterone), which is subsequently metabolized to 3α-diol (3α,17β-androstanediol) by the reductase AKR1C2 (aldo-ketoreductase 1C2), for which we have previously developed the fluorogenic reporter substrate Coumberone. Despite the medicinal importance of 5α-reductase, there are presently no probes or methods for the continuous activity readout of this enzyme in cells. We show that the activity of 5α-R1 (5α-reductase type 1) can be measured in COS-1 cells via the changes of DHT flux. Our system enables a measurement of 5α-reductase activity in cells, via either fluorimetry or fluorescence microscopy, with a wide dynamic range of activities, and provides a continuous optical assay for evaluation of small molecule inhibitors for this important enzyme. Furthermore, this paper demonstrates a novel optical approach to measuring metabolic flux changes in living cells and expands the utility of fluorogenic enzyme reporter substrates: optical reporters can measure not only the activity of the target enzyme but also the activity of other enzymes upstream in the pathway, for which there are no probes available.
Rodriguez
Pamela C
PC
Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, USA.
Yee
Dominic J
DJ
Sames
Dalibor
D
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase / Q000032:analysis* / Q000378:metabolism; D043582:5-alpha-Dihydroprogesterone / Q000096:biosynthesis*; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism*; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)
D013741:3-Oxo-5-alpha-Steroid 4-Dehydrogenase; D043582:5-alpha-Dihydroprogesterone; D005456:Fluorescent Dyes; D006913:Hydroxysteroid Dehydrogenases; D013739:Testosterone; C435496:AKR1C2 protein, human; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); C476566:steroid-5alpha-reductase type 1