Chronic depletion of subcellular NAD pools reveals their interconnectivity and a buffering function of mitochondria

Abstract

The coenzyme NAD is consumed by signaling enzymes including 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, or peroxisomes. Irrespective of the compartment targeted, total cellular NAD concentrations declined by ~40%. Isotope-tracer flux measurements and mathematical modeling 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.

Welcome to the Family: Identification of the NAD+ Transporter of Animal Mitochondria as Member of the Solute Carrier Family SLC25

“…To date, the cytosolic, nuclear, and mitochondrial NAD + pools have been best described. Using the PARAPLAY assay, which is based on expression of the catalytic domain of poly-ADP-ribosyltransferase 1 (PARP1) in various subcellular compartments [28], pools of NAD + in peroxisomes, ER, and Golgi complex [29,30] were demonstrated. It was shown that NAD + is transported into peroxisomes using the mitochondrial type carrier SLC25A17 [13].…”

Accounting for NAD Concentrations in Genome-Scale Metabolic Models Captures Important Metabolic Alterations in NAD-Depleted Systems

“…The free NAD concentration has been measured to be approximately 230 μM in the mitochondria and 110 μM in the cytosol of parental HEK293 cells [52], whereas the 293mitoPARP cell line displays an up to 40-50% decrease in cellular NAD [41] and a severe depletion of NAD in mitochondria with only 10-20% of the mitochondrial NAD remaining [41] compared to parental HEK293 cells. The free NAD concentration in 293mitoPARP was thus decreased to approximately 66 μM in the cytosol and 23 μM in the mitochondria [41]. To model the changes related to free NAD concentrations, we retrieved 𝐾 𝑚 values from the Brenda [49] enzyme database and adjusted the flux boundaries (cf.…”

“…A cell line expressing PARP1 targeted to mitochondria has been used [40][41][42][43]. Proteins and peptides were quantified by VanLinden et al [41] via an MS/MS-based approach using tandem mass tags [44].…”

“…A cell line expressing PARP1 targeted to mitochondria has been used [40][41][42][43]. Proteins and peptides were quantified by VanLinden et al [41] via an MS/MS-based approach using tandem mass tags [44]. All analyses of cell lines were performed in triplicates, with abundance ratios calculated for each replicate by comparing its measurement to a reference sample, resulting in a dataset containing 6392 proteins.…”

“…The scaling factor allows us to correct for the case where the models do not assume full cofactor saturation of all enzymes by normalizing to the reaction flux under condition C 0 , which is 0.11 mM in the cytosol and 0.23 mM in the mitochondria (see Table 1) [40,41,52,53]. NADP(H) was treated identically to NAD(H); i.e., the same concentrations were used for NADP(H)-dependent reactions.…”