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

Cláudio F Costa; Hongli Li; Mohamed A F Hussein; Yi Yang; Celien Lismont; Marc Fransen

doi:10.1007/978-1-0716-3048-8_13

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

Methods Mol Biol. 2023:2643:183-197. doi: 10.1007/978-1-0716-3048-8_13.

Authors

Cláudio F Costa¹, Hongli Li¹, Mohamed A F Hussein^{1

2}, Yi Yang^{3

4}, Celien Lismont¹, Marc Fransen⁵

Affiliations

¹ Department of Cellular and Molecular Medicine, Laboratory of Peroxisome Biology and Intracellular Communication, KU Leuven, Leuven, Belgium.
² Department of Biochemistry, Faculty of Pharmacy, Assiut University, Asyut, Egypt.
³ Synthetic Biology and Biotechnology Laboratory, State Key Laboratory of Bioreactor Engineering, Shanghai Collaborative Innovation Center for Biomanufacturing Technology, East China University of Science and Technology, Shanghai, China.
⁴ State Key Laboratory of Neuroscience, CAS Center for Excellence in Brain Science, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China.
⁵ Department of Cellular and Molecular Medicine, Laboratory of Peroxisome Biology and Intracellular Communication, KU Leuven, Leuven, Belgium. marc.fransen@kuleuven.be.

PMID: 36952186
DOI: 10.1007/978-1-0716-3048-8_13

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 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.

Keywords: Fluorescent biosensors; Live-cell imaging; NAD(H); NADP(H); Peroxisomes; Redox state; SoNar; iNAP.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Luminescent Proteins / metabolism
NAD* / metabolism
NADP / metabolism
Oxidation-Reduction

Substances

NAD
NADP
Luminescent Proteins