Transcriptome (RNASeq) data of engineered human 293 cell with compromised subcellular NAD availability
The coenzyme NAD is consumed by signalling enzymes such as poly-ADP-ribose-polymerases (PARPs) and sirtuins. Understanding the mechanisms of aging-associated NAD decline and how cells cope with decreased NAD concentrations requires model systems reflecting chronic NAD deficiency. To evoke compartment-specific over-consumption of NAD, we have engineered cell lines expressing PARP activity in mitochondria, the cytosol, endoplasmic reticulum, and peroxisomes. Additionally, we have engineered cell lines that lack a functional gene for the human mitochondrial transporter SLC25A51 and the NAD biosynthesis enzyme NMNAT3. Isotope-tracer flux measurements and mathematical modelling showed that the lowered NAD concentration limits total NAD consumption kinetically. Moreover, NAD biosynthesis rate and capacity remained unchanged, thereby also precluding an increase of total NAD turnover. The chronic NAD deficiency was surprisingly well tolerated unless the mitochondria were targeted. Oxidative phosphorylation and glycolysis were little affected by NAD over-consumption in the other compartments. Likewise, peroxisomal NAD over-consumption was balanced by mitochondrial NAD decrease to maintain beta-oxidation of very long chain fatty acids in peroxisomes. We propose that subcellular NAD pools are interconnected, with mitochondria acting as a rheostat to facilitate NAD-dependent processes in organelles with excessive consumption. Overall design: Human 293 [HEK293] and HeLa S3 cells were stably transfected with vectors encoding fusion proteins composed of enhanced green fluorescent protein (EGFP) and the catalytic domain of human poly-ADP-ribose polymerase 1 (PARP1cd) targeted to the mitochondria, endoplasmic reticulum, cytosol, and peroxisomes under the control of the CMV promoter Human 293 [HEK293] cells were subjected to CRISPR-Cas9-mediated genome editing targeted against SLC25A51 encoding the human mitochondrial NAD transporter Human 293 [HEK293] cells were stably transfected with a vector encoding C-terminally FLAG-tagged SLC25A51 under control of the CMV promoter Human 293 [HEK293] cells were subjected to CRISPR-Cas9-mediated genome editing targeted against NMNAT3 encoding human nicotinamide mononucleotide adenylytransferase 3