Location, Location, Location: Compartmentalization of NAD+ Synthesis and Functions in Mammalian Cells

Abstract

The numerous biological roles of NAD⁺ are organized and coordinated via its compartmentalization within cells. The spatial and temporal partitioning of this intermediary metabolite is intrinsic to understanding the impact of NAD⁺ on cellular signaling and metabolism. We review evidence supporting the compartmentalization of steady-state NAD⁺ levels in cells, as well as how the modulation of NAD⁺ synthesis dynamically regulates signaling by controlling subcellular NAD⁺ concentrations. We further discuss potential benefits to the cell of compartmentalizing NAD⁺, and methods for measuring subcellular NAD⁺ levels.

Keywords: CD38; nicotinamide mononucleotide adenylyltransferase (NMNAT); nicotinamide phosphoribosyltransferase (NAMPT); poly(ADP-ribose) polymerase (PARP); sirtuin.

Figure 1.. NAD⁺ Compartmentalization for Regulation.

The levels of free NAD⁺ are tightly controlled both temporally and spatially to precisely modulate signaling in cells. (A) Temporal fluctuations in NAD⁺ concentrations regulate circadian periodicity rhythms. (B) Spatial partitioning of NAD⁺ biosynthetic pathways permits signal-dependent initiation of adipogenic differentiation. (C) NAD⁺ compartmentalization may protect and prioritize support for particularly crucial cellular processes (e.g., in the mitochondria) in response to stress when other subcellular stores are depleted. Abbreviation: Ac, acylation.

Figure 2.. Specificity and Resolution when Measuring Intracellular NAD⁺.

Schematic representations of the specificity and resolution of various methods for measuring intracellular NAD⁺. (A) Methods are broadly ordered by their ability to both directly and specifically measure the free fraction of intracellular NAD⁺. Consideration of the entire methodology is taken into account. For example, analytical methods, such as LC-MS/MS, HPLC, and NMR, directly and very specifically measure intracellular NAD⁺; however, the preparation of biological samples for analysis may conflate free and bound fractions. (B) Methods are organized according to their ability to obtain dynamic measurements from the same locale, as well as by their spatial resolution. Abbreviations: LC, liquid chromatography; MRI, magnetic resonance imaging; MS, mass spectrometry; NMR, nuclear magnetic resonance.

Figure I.. Chemical Structure and Biosynthesis of NAD⁺.

(A) Chemical structure of oxidized NAD⁺. NAD⁺ comprises two nucleotides, one containing an adenine nucleobase and the other containing nicotinamide, joined through their phosphate groups. The location of various chemical moieties contained within NAD⁺ are indicated: ADP-ribose (ADPR), nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). (B) NAD⁺ is synthesized de novo in a pathway leading from tryptophan (blue), as well as in ‘salvage’ pathways: (i) the nicotinic acid (NA) (‘Preiss–Handler’) salvage pathway (yellow), (ii) the nicotinamide mononucleotide (NMN) salvage pathway (green), and (iii) the nicotinamide riboside (NR) salvage pathway (green). The abbreviations for the enzymes (gray text) and the intermediates (black plain text) are defined in Table 1.