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

doi:10.1016/j.tibs.2020.05.010

Trends in Biochemical Sciences

Volume 45, Issue 10, October 2020, Pages 858-873

https://doi.org/10.1016/j.tibs.2020.05.010 Get rights and content

Highlights

NAD⁺ serves an essential role as an electron acceptor (via hydride transfer) in central carbon metabolism. In the absence of intracellular NAD⁺, cells cannot produce ATP. However, even moderate diminishments in NAD⁺ levels can limit the signaling activity of NAD⁺-consuming enzymes.
NAD⁺ concentrations differ in different parts of the cell, and there are distinct subcellular requirements for NAD⁺.
Dynamic modulation of subcellular, and possibly extracellular, NAD⁺ concentrations represent an emerging mechanism for regulating specific NAD⁺-dependent pathways.
Compartmentalization of NAD⁺ helps to time responses, communicate cellular status, and protect crucial NAD⁺ pools.
Genetically encoded sensors represent promising approaches for additional development to generate a molecular toolbox for measuring and studying fluctuations in levels of compartmentalized NAD⁺.

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.

Section snippets

Subcellular Compartmentalization of NAD⁺ Synthesis, Catabolism, and Function

Oxidized nicotinamide adenine dinucleotide (NAD⁺) is a metabolic cofactor that plays essential roles in all domains of life. In over a century of research on NAD⁺, great strides have been made in understanding the synthesis, degradation, and biological functions of NAD⁺ (Box 1 and Table 1, Table 2). Recent interest in the biology of NAD⁺ has been driven by a desire to modulate its metabolic and signaling pathways to counteract illnesses stemming from metabolic disorders, tumorigenesis,

Non-Redundant Roles of NAD⁺ in Different Cellular Compartments

The concept that NAD⁺ in different parts of the cell has non-overlapping roles is illustrated by mouse genetic models targeting individual nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) enzymes. Many species, including humans, depend on more than one NMNAT enzyme to synthesize NAD⁺ [10., 11., 12., 13., 14., 15.] (Table 3). Mammalian NMNAT enzymes have varying expression levels, activities, and subcellular localizations across different tissues [16., 17., 18.]. NMNAT-1 is nuclear,

NAD⁺ Compartmentalization for Regulation

NAD⁺ compartmentalization plays an important role in regulating biological outcomes, as illustrated in the following examples.

When NAD⁺ Compartmentalization Goes Awry

Given the importance of NAD⁺ compartmentalization in key biological systems such as those noted previously, non-physiological alterations in NAD⁺ compartmentalization are expected to be a driver of disease. Although direct evidence of a role for NAD⁺ compartmentalization in NAD⁺-related pathologies (e.g., cancer, diseases of aging) is limited, recent studies are beginning to hint at its importance. For example, the same NMNAT-1/NMNAT-2-driven NAD⁺ compartmentalization that regulates

On the Outside Looking In: An Extracellular Compartment for NAD⁺?

In this review we have focused on the synthesis and functions of NAD⁺ in intracellular compartments. NAD⁺, however, is also present in the extracellular milieu as extracellular NAD⁺ (eNAD⁺) [68], which may be considered as another compartment for NAD⁺.

Methods to Study NAD⁺ Compartmentalization

The interior of any cell is not a uniform milieu: cells are organized with membrane-bound organelles, macromolecular scaffolding and tracks, and subcompartments with distinct viscosities. The complexities of NAD⁺ compartmentalization – including being able to distinguish free intracellular fractions from total NAD⁺ levels – present challenges for monitoring and determining NAD⁺ concentrations in real time and in vivo. An ideal method would dynamically monitor free NAD⁺ in an organism with

Concluding Remarks

NAD⁺ is a crucial metabolite and signaling molecule whose detailed functions and biology are incompletely characterized, at best. We are only beginning to elucidate how the temporal and spatial compartmentalization of NAD⁺ contributes to its numerous biological roles. The emerging view is that NAD⁺ concentrations are partitioned and dynamically modulated by NAD⁺ synthesis within the cell. Determining the extent of these mechanisms in vivo and their impact on signaling pathways will be a rich

Acknowledgments

We thank A. Jones, K. Ryu, S. Challa, J. Eller, and M. Stokes for critical comments and suggestions on this work. NAD⁺-related research in our laboratories is supported by the National Institutes of Health (NIH)/National Institute of General Medical Sciences (NIGMS) grant DP2GM126897 (to X.A.C.), and by the NIH National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant R01 DK069710 and funds from the Cecil H. and Ida Green Center for Reproductive Biology Sciences Endowment

Disclaimer Statement

X.A.C. is an inventor on US patent 10 392 649 covering the NAD⁺ cpVenus-based sensor described herein. W.L.K. is a founder and consultant for Ribon Therapeutics. He is also an inventor on US patent 9 599 606 covering a set of ADP-ribose detection reagents which have been licensed to and are sold by EMD Millipore.

Glossary

Adipogenesis: the developmental process by which precursor cells develop into adipocytes (fat cells).
Compartmentalization: separated into isolated or distinct subcellular locales.
Ectoenzyme: an enzyme that is located on, and has catalytic activity at, the surface of a cell, directed towards the exterior of the cell.
Free metabolite: the fraction of a metabolite that is not currently bound to or associated with protein. Typically, this can be considered as the amount of available metabolite.
Multiplexing

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Trends in Biochemical Sciences

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

Highlights

Section snippets

Subcellular Compartmentalization of NAD+ Synthesis, Catabolism, and Function

Non-Redundant Roles of NAD+ in Different Cellular Compartments

NAD+ Compartmentalization for Regulation

When NAD+ Compartmentalization Goes Awry

On the Outside Looking In: An Extracellular Compartment for NAD+?

Methods to Study NAD+ Compartmentalization

Concluding Remarks

Acknowledgments

Disclaimer Statement

Glossary

Cell Metab.

Cell Metab.

Gene

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Cell Biol.

Exp. Neurol.

Exp. Neurol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Trends Pharmacol. Sci.

Cell Metab.

Trends Cell Biol.

Biochem. Biophys. Res. Commun.

Comput. Struct. Biotechnol. J.

Biochim. Biophys. Acta Gen. Subj.

Cell Metab.

Semisynthetic biosensors for mapping cellular concentrations of nicotinamide adenine dinucleotides

eLife

Biosensor reveals multiple sources for mitochondrial NAD+

Science

Metabolic regulation of transcription through compartmentalized NAD+ biosynthesis

Science

The secret life of NAD+: an old metabolite controlling new metabolic signaling pathways

Endocr. Rev.

Visualization of subcellular NAD pools and intra-organellar protein localization by poly-ADP-ribose formation

Cell. Mol. Life Sci.

The redox state of free nicotinamide-adenine dinucleotide in the cytoplasm and mitochondria of rat liver

Biochem. J.

Regulation of corepressor function by nuclear NADH

Science

Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase

J. Biol. Chem.

Insight into molecular and functional properties of NMNAT-3 reveals new hints of NAD homeostasis within human mitochondria

PLoS ONE

Identification of evolutionary and kinetic drivers of NAD-dependent signaling

Proc. Natl. Acad. Sci. U. S. A.

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis

Biochemistry

Simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues

PLoS ONE

Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues

PLoS ONE

Nmnat3 is dispensable in mitochondrial NAD Level maintenance in vivo

PLoS ONE

Reducing expression of NAD+ synthesizing enzyme NMNAT-1 does not affect the rate of Wallerian degeneration

FEBS J.

Nicotinamide mononucleotide adenylyltransferase 2 (Nmnat2) regulates axon integrity in the mouse embryo

PLoS ONE

Endogenous Nmnat2 is an essential survival factor for maintenance of healthy axons

PLoS Biol.

Rescue of peripheral and CNS axon defects in mice lacking NMNAT-2

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

Subcellular Compartmentalization of NAD⁺ Synthesis, Catabolism, and Function

Non-Redundant Roles of NAD⁺ in Different Cellular Compartments

NAD⁺ Compartmentalization for Regulation

When NAD⁺ Compartmentalization Goes Awry

On the Outside Looking In: An Extracellular Compartment for NAD⁺?

Methods to Study NAD⁺ Compartmentalization

Biosensor reveals multiple sources for mitochondrial NAD⁺

Metabolic regulation of transcription through compartmentalized NAD⁺ biosynthesis

The secret life of NAD⁺: an old metabolite controlling new metabolic signaling pathways

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD⁺ biosynthesis

Reducing expression of NAD⁺ synthesizing enzyme NMNAT-1 does not affect the rate of Wallerian degeneration