Assessment of the Peroxisomal Redox State in Living Cells Using NADPH- and NAD+/NADH-Specific Fluorescent Protein Sensors

Abstract

The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular 2 resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD + /NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.

Functional Analysis of GSTK1 in Peroxisomal Redox Homeostasis in HEK-293 Cells

“…Given that (i) the overarching aim of this study was to examine the role of GSTK1 in peroxisomal glutathione redox homeostasis, (ii) organellar glutathione pools rely entirely on cytosolic glutathione import [ 63 ], and (iii) changes in (local) glutathione metabolism may have a direct impact on the levels of other redox metabolites [ 7 , 43 , 44 , 45 ], we first evaluated if GSTK1 inactivation affected the overall peroxisomal and cytosolic GSSG/GSH, NAD + /NADH, NADPH, and H 2 O 2 levels under basal conditions. To monitor possible changes in these metabolites, we used compartment-specific forms of the ratiometric fluorescent redox sensors roGFP2 [ 64 ], SoNar [ 65 ], iNAP1 [ 65 ], and roGFP2-Orp1 [ 64 ], respectively. The typical subcellular distribution patterns for each of these reporter proteins are depicted in Figure S2 .…”