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Chemical Proteomics Approach for Profiling the NAD Interactome
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ABSTRACT: Nicotinamide adenine dinucleotide (NAD+) is a multifunctional molecule. Beyond redox metabolism, NAD+ has an
equally important function as a substrate for post-translational modification enzymes, the largest family being the poly-ADP−ribose
polymerases (PARPs, 17 family members in humans). The recent surprising discoveries of noncanonical NAD (NAD+/NADH)-
binding proteins suggests that the NAD interactome is likely larger than previously thought; yet, broadly useful chemical tools for
profiling and discovering NAD-binding proteins do not exist. Here, we describe the design, synthesis, and validation of clickable,
photoaffinity labeling (PAL) probes, 2- and 6-ad-BAD, for interrogating the NAD interactome. We found that 2-ad-BAD efficiently
labels PARPs in a UV-dependent manner. Chemical proteomics experiments with 2- and 6-ad-BAD identified known and unknown
NAD+/NADH-binding proteins. Together, our study shows the utility of 2- and 6-ad-BAD as clickable PAL NAD probes.
analogues are not cleaved by NAD+ consumers.13,14 BAD,
icotinamide adenine dinucleotide (NAD+) wears differ-
Nent hats in the cell: on the one hand, it serves as a but not carba-NAD+, binds to PARP-1 and inhibits its
enzymatic activity.16 Additionally, BAD inhibits NAD+/
coenzyme for oxidoreductases in metabolism, and on the
NADH-binding enzymes.13 Therefore, we focused our design
other, it is a substrate for signaling enzymes that mediate post-
translational modifications.1 Unlike oxidoreductases, which
efforts based on BAD.
To convert BAD into a clickable PAL probe for NAD+
consumers and other NAD-binding proteins, we sought
positions on BAD that could be modified with a photoreactive
group and a clickable tag without perturbing BAD’s
interactions with its targets. We focused on the adenine ring
of BAD, because our previous studies on orthogonal NAD+
analogues for engineered PARPs showed that substitutions on
the nicotinamide ring were not tolerated by wild-type
PARPs.17,18 On the adenine ring of BAD, there are three
possible positions that could be modified: N-6, C-2, and C-8.
We scrutinized the crystal structure of BAD bound to PARP-1
(PDB: 6bhv).16 In this structure, the C-2 position of the
adenine ring of BAD is solvent-exposed, whereas the N-6
position is partially solvent-exposed (Figure 1a). In contrast,
the C-8 position is buried in the NAD+-binding pocket. We
therefore designed and synthesized modified BAD analogues
with a “minimalist” linker19 containing a diazirine and a
terminal alkyne at the C-2 or N-6 positions of the adenine ring
(2-ad-BAD, 1, and 6-ad-BAD, 2) (Figure 1b and Schemes S1−
mediate the reversible two-electron reduction of NAD+ to
NADH, the enzymes that use NAD+ as a substrate cleave the
nicotinamide glycosidic bond of NAD+ leading to the
consumption of NAD+. The most prominent NAD+ consumers
in human cells are poly-ADP−ribose polymerases (PARP-1−
17) and sirtuins (SIRT1−7).2 Intriguingly, recent studies show
that noncanonical NAD+ consumers exist (e.g., SARM1,3−6
DTX3L7), which have NAD+-binding sites that are quite
distinct from the conserved structural motifs found in
canonical NAD+ consumers. Additionally, NADH can act as
an allosteric modulator of proteins (e.g., NAD-dependent
isocitrate dehydrogenase).8 Hence, the NAD (NAD+/NADH)
interactome is likely much more diverse than previously
anticipated based solely on analysis of protein sequences.
We sought an unbiased strategy to profile NAD-binding
proteins. Chemical proteomics using photoaffinity labeling
(PAL) is a powerful approach for unbiased profiling of
proteome-wide small-molecule−protein interactions.9 Small-
molecule probes for PAL are bifunctional: they contain a
photoreactive moiety as well as a “clickable” tag (e.g., alkyne).
While several photoreactive groups have been used for PAL,
diazirines are popular because of their compact structure and
excellent photo-cross-linking properties.10 Although clickable
PAL probes have been developed for nucleotides such as S-
adenosyl methionine (SAH),11 and, more recently, adenosine
triphosphate (ATP),12 a clickable PAL NAD probe has
heretofore not been described.
We first evaluated our clickable PAL NAD probes on PARP
enzymatic activity. PARPs catalyze the transfer of the ADP−
ribose (ADPr) moiety of NAD+ to target proteins in a process
known as ADP-ribosylation. In humans, PARPs fall into two
major subgroups: (i) PARPs that catalyze poly-ADP-
Received: February 2, 2021
Published: April 29, 2021
We reasoned that a clickable PAL NAD probe should
contain an enzymatically stable nicotinamide glycosidic bond.
There are several NAD+ analogues that fit this criterion:
benzamide adenine dinucleotide (BAD),13 carba-NAD+,14 and
4-thioribose NAD+ (S-NAD+).15 Importantly, these NAD+
J. Am. Chem. Soc. 2021, 143, 6787−6791
© 2021 American Chemical Society
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