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Antioxid Redox Signal. 2019 Aug 28. doi: 10.1089/ars.2019.7799. [Epub ahead of print]

Subcellular characterization of NAD+ biosynthesis in metastatic melanoma by using organelle-specific biosensors.

Author information

1
University of Turin, Medical Science, Turin, IT 10126, Turin, Italy; federica.gaudino@unito.it.
2
University of Turin, Medical Sciences, Turin, Italy; ilaria.manfredonia@edu.unito.it.
3
University of Turin, Medical Sciences, Turin, Italy; antonella.manago@unito.it.
4
University of Turin, Medical Sciences, Turin, Italy; valentina.audrito@unito.it.
5
Università Politecnica delle Marche, 9294, Ancona, Marche, Italy; n.raffaelli@staff.univpm.it.
6
University of Turin, Medical Sciences, Turin, Italy; tiziana.vaisitti@unito.it.
7
University of Turin, Medical Sciences, Turin, Italy; silvia.deaglio@unito.it.

Abstract

AIM:

NAD+ plays central roles in a wide array of normal and pathological conditions. Inhibition of NAD+ biosynthesis can be exploited therapeutically in cancer, including melanoma. To obtain quantitation of NAD+ levels in live cells and to address the issue of the compartmentalization of NAD+ biosynthesis, we exploited a recently described, genetically-encoded NAD+ biosensor (LigA-cpVENUS), which was targeted to the cytosol, mitochondria and nuclei of BRAF-V600E A375 melanoma cells, a model of metastatic melanoma (MM).

RESULTS:

FK866, a specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), the main NAD+-producing enzyme in MM cells, was used to monitor NAD+ depletion kinetics at the subcellular level in biosensor-transduced A375 cells. In addition, we treated FK866-blocked A375 cells with NAD+ precursors, including nicotinamide, nicotinic acid, nicotinamide riboside and quinolinic acid, highlighting an organelle-specific capacity of each substrate to rescue from NAMPT block. Expression patterns of NAD+ biosynthetic enzymes were then biochemically studied in isolated organelles, revealing expression of NAMPT in all three cellular compartments, while NAPRT was predominantly cytosolic and mitochondrial, and NRK mitochondrial and nuclear. In keeping with biosensor data, QPRT was expressed at extremely low levels. Innovation & Conclusions: Throughout this work, we validated the use of genetically encoded NAD+ biosensors to characterize subcellular distribution of NAD+ production routes in MM. The chance of real time monitoring of NAD+ fluctuations after chemical perturbations, together with a deeper comprehension of the cofactor biosynthesis compartmentalization, strengthens the foundation for a targeted strategy of NAD+ pool manipulation in cancer and metabolic diseases.

PMID:
31456414
DOI:
10.1089/ars.2019.7799

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