Abstract
Nicotinamide adenine dinucleotide (NAD+) is a coenzyme for many NAD+-consuming proteins with diverse biological functions. Oscillations in NAD+ levels may influence several cellular signaling pathways. NAD+ synthesis via Preiss-Handler route (salvage reactions) has been extensively reported. However, the contribution of L-tryptophan/kynurenine catabolism in de novo NAD+ synthesis is poorly understood. Using L-[14C]-tryptophan tracing in four liver cancer cell lines and siRNA-mediated silencing of arylformamidase (AFMID), a key enzyme involved in L-tryptophan degradation, we demonstrate the contribution of L-tryptophan catabolism in de novo synthesis of NAD+ pools. NAD+ modulation is therefore important in maintaining cellular homeostasis and appropriate cellular functions according to nutrients availability.
Keywords: Kynurenine pathway, De novo NAD+ synthesis, [14C]-Tryptophan metabolic tracing, Arylformamidase (AFMID), Liver cancer
Materials and Reagents
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Procedure
Notes
The experiment has been carried out using the 4 different liver tumor cell lines for at least 4 times and this blot is representative of these 4 different experiments. Consistent incorporation of label has been observed. Fiji software (www.fiji.sc/Fiji) was used to quantify the pixilation of different bands in the image and the relative band intensities were calibrated in respect to the siCtr samples. Data interpretation should be done based on the intensity of the bands at the right size, greater is the band intensity, and greater is the NAD+ content.
Recipes
Acknowledgments
This protocol was adapted as previously reported (Merdanovic et al., 2005). N.D is a recipient of the Spanish Ramón y Cajal fellowship. This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2010-18518), the Association for International Cancer Research AICR-UK (11-0242), CNIO (BC1104-08) and the European Foundation for the Study of Diabetes (EFSD).
References
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