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Publications

2020
Xiaolu A. Cambronne and W. Lee Kraus. “Location, Location, Location: Compartmentalization of NAD+ Synthesis and Functions in Mammalian Cells.” Trends in Biochemical Sciences, 45, 10, Pp. 858-873. Publisher's Version Abstract
LocLocLocImageThe 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.
location_location_location_compartmentalization_of_nad_synthesis_and_functions_in_mammalian_cells.pdf
Timothy S. Luongo, Jared M. Eller, Mu-Jie Lu, Marc Niere, Fabio Raith, Caroline Perry, Marc R. Bornstein, Paul Oliphint, Lin Wang, Melanie R. McReynolds, Marie E. Migaud, Joshua D. Rabinowitz, F. Brad Johnson, Kai Johnsson, Mathias Ziegler, Xiaolu A. Cambronne, and Joseph A. Baur. “SLC25A51 is a mammalian mitochondrial NAD+ transporter..” Nature, 558, Pp. 174-179. Publisher's Version Abstract
Mitochondria require nicotinamide adenine dinucleotide (NAD+
) to carry out the
fundamental processes that fuel respiration and mediate cellular energy
transduction. Mitochondrial NAD+ transporters have been identified in yeast and
plants1,2
, but their existence in mammals remains controversial3–5
. Here we
demonstrate that mammalian mitochondria can take up intact NAD+
, and identify
SLC25A51 (also known as MCART1)—an essential6,7 mitochondrial protein of
previously unknown function—as a mammalian mitochondrial NAD+ transporter. Loss
of SLC25A51 decreases mitochondrial—but not whole-cell—NAD+ content, impairs
mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria.
Conversely, overexpression of SLC25A51 or SLC25A52 (a nearly identical paralogue of
SLC25A51) increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast
mitochondria lacking endogenous NAD+ transporters. Together, these findings
identify SLC25A51 as a mammalian transporter capable of importing NAD+ into
mitochondria.
2018
Jared M. Eller, Melissa L. Stewart, Alexandria J. Slepian, Sheila Markwardt, Jack Wiedrick, Michael S. Cohen, Richard H. Goodman, and Xiaolu A. Cambronne. “Flow Cytometry Analysis of Free Intracellular NAD+ Using a Targeted Biosensor..” Current Protocols in Cytometry, 88, 1, Pp. e54. Publisher's Version Abstract
Flow cytometry approaches combined with a genetically encoded targeted flu-
orescent biosensor are used to determine the subcellular compartmental avail-
ability of the oxidized form of nicotinamide adenine dinucleotide (NAD+).
The availability of free NAD+ can affect the activities of NAD+-consuming
enzymes such as sirtuin, PARP/ARTD, and cyclic ADPR-hydrolase family
members. Many methods for measuring the NAD+ available to these enzymes
are limited because they cannot determine free NAD+ as it exists in various
subcellular compartments distinctly from bound NAD+ or NADH. Here, an
approach to express the sensor in mammalian cells, monitor NAD+-dependent
fluorescence intensity changes using flow cytometry approaches, and analyze
data obtained is described. The benefit of flow cytometry approaches with the
NAD+ sensor is the ability to monitor compartmentalized free NAD+ fluctua-
tions simultaneously within many cells, which greatly facilitates analyses and
calibration.
flow_cytometry_analysis_of_free_intracellular_nad_using_a_targeted_biosensor.pdf
Micheal S. Cohen, Melissa L. Stewart, Richard H. Goodman, and Xiaolu A. Cambronne. “Methods for Using a Genetically Encoded Fluorescent Biosensor to Monitor Nuclear NAD+.” Methods in Molecular Biology, 1813. Abstract
Free nicotinamide adenine dinucleotide (NAD + ) serves as substrate for NAD +
-consuming enzymes. As
such, the local concentration of free NAD+ can influence enzymatic activities. Here we describe methods
for using a fluorescent, genetically-encoded sensor to measure subcellular NAD+ concentrations. We also
include a discussion of the limitations and potential applications for the current sensor. Presented in this
chapter are (1) guidelines for calibrating instrumentation and experimental setups using a bead-based
method, (2) instructions for incorporating required controls and properly performing ratiometric mea-
surements in cells, and (3) descriptions of how to evaluate relative and quantitative fluctuations using
appropriate statistical methods for ratio-of-ratio measurements.
methods_for_using_a_genetically_encoded_fluorescent_biosensor_to_monitor_nuclear_nad.pdf
2016
Xiaolu A. Cambronne, Rory K.Morgan, Melissa L. Stewart, DongHo Kim, Amber M. Jones-Brunette, David L. Farrens, Michael S. Cohen, and Richard H. Goodman. “Biosensor reveals multiple sourcesfor mitochondrial NAD+.” Science, 352, 6292, Pp. 1474-1477. Publisher's Version Abstract
Nicotinamide adenine dinucleotide (NAD+) is an essential substrate for sirtuins and
poly(adenosine diphosphate–ribose) polymerases (PARPs), which are NAD+-consuming
enzymes localized in the nucleus, cytosol, and mitochondria. Fluctuations in NAD+
concentrations within these subcellular compartments are thought to regulate the
activity of NAD+-consuming enzymes; however, the challenge in measuring
compartmentalized NAD+ in cells has precluded direct evidence for this type of
regulation. We describe the development of a genetically encoded fluorescent biosensor
for directly monitoring free NAD+ concentrations in subcellular compartments. We
found that the concentrations of free NAD+ in the nucleus, cytoplasm, and mitochondria approximate the Michaelis constants for sirtuins and PARPs in their respective compartments. Systematic depletion of enzymes that catalyze the final step of NAD+ biosynthesis revealed cell-specific mechanisms for maintaining mitochondrial NAD+ concentrations.
biosensor_reveals_multiple_sources_for_mitochondrial_nad.pdf