ADP-ribosylation and ADP-riboxanation
ADP-ribosylation involves the attachment of ADP-ribose (ADPr) groups
from nicotinamide adenine dinucleotide (NAD+) to
substrates by enzymes called ADP-ribosyltransferases (ARTs), together
with the release of nicotinamide (NA) (Palazzo et al., 2017). Given
bacterial toxins (i.e., Cholera and Diphtheria toxins) as the first
reported enzymes of this group (Honjo et al., 1968; Cassel & Pfeuffer,
1978), the other ARTs identified afterwards are often classified as
either Cholera toxin-like ART (ARTC) or Diphtheria toxin-like ART (ARTD)
superfamily (Hottiger et al., 2010). Recently, there seems to be a spike
in the number of reports on bacterial virulence factors possessing ART
activities. For instance, Salmonella Typhimurium effector SopF
targets Gln124 of ATP6V0C in host V-ATPase for ADP-ribosylation, thereby
blocking antibacterial autophagy (xenophagy) via disrupting
V-APTase-ATG16L1 association (Xu et al., 2019). In a follow-up study,
the authors found SopF stably associates with ADP-ribosylation factor
(ARF) GTPases and requires these host factors for its activation (Xu et
al., 2022). Another type III effector CetC from Chromobacterium
violaceum catalyzes ADP-ribosylation of ubiquitin to disrupt host
ubiquitin signaling, representing the first of its kind on the threonine
residue (Yan et al., 2020). In the above studies, tandem mass
spectrometry (MS/MS) involving dissociation of modified protein
fragments/peptides plays an indispensable role in ascertaining the
modification in the first place as well as pinpointing the exact
modified sites.
Recently, a novel PTM dubbed as ADP-riboxanation, a derivative of
ADP-ribosylation, was uncovered and catalyzed by bacterial effectors
OspC3 and CopC from S. flexneri and C. violaceum ,
respectively (Li et al., 2021; Peng et al., 2022). Distinct from
ADP-ribosylation, ADP-riboxanation involves an additional deamination
upon the classical modification. In other words, this novel modification
couples two enzymatic reactions through the activity of a single
effector. This unique reaction mechanism was supported by the effector
variants (OspC3D177A and CopCD172E)
capable of catalyzing conventional ADP-ribosylation without the
subsequent deamination. The initial assignment/characterization of this
unconventional modification was greatly facilitated by a suite of MS
tools including multi-stage MS (MS/MS and MS/MS/MS), high-resolution
measurements and stable isotope labeling MS. Unlike canonical arginineNω -ADP-ribosylation (ANT1 by Ceg3 or Rab4a and
Ras by ExoS) (Fu et al., 2022; Bette-Bobillo et al., 1998; Vareechon et
al., 2017), OspC3-catalyzed ADP-riboxanation proceeds withNδ -ADP-ribosylation and subsequent deamination
with the ribosyl-2’-OH of ADPR to remove oneNω , resulting in an oxazolidine ring. Thus far,
both S. flexneri OspC3 and C. violaceum CopC were found to
modify host caspases via ADP-riboxanation. While OspC3 targets
inflammatory caspases (-4 and -11) to block host pyroptosis (Li et al.,
2021), CopC inactivates a broader spectrum of caspases (-3, -7, -8, and
-9) to interfere with host cell death pathways including apoptosis,
necroptosis and pyroptosis (Peng et al., 2022).
Of note, upon elucidation of enzymatic activities of bacterial
effectors, a rate-limiting step of most studies can often be associated
with the discovery of their modified targets. As discussed above,
enzymatic substrates are mostly identified by inference from perturbed
cellular pathways or through conventional genetic and biochemical
assays. Nevertheless, in many cases one could encounter a scenario where
there are no observable phenotypes associated with the effector of
interest. Then we would argue that cellular ADP-ribosylome profiling
(i.e., global analysis of ADP-ribosylated proteome) can be a generic and
unbiased strategy for high-throughput screening of ART substrates. Such
work is facilitated by the development of an engineered macro
domain-containing protein (eAf1521) from Archaeoglobus fulgidus .
This variant of Af1521 exhibits high affinity towards ADP-ribosylated
proteins, thereby allowing efficient enrichment of cellular
ADP-ribosylome prior to MS analyses (Hendriks et al., 2019; Nowak et
al., 2020). Indeed, such a strategy has been applied in the study ofS. Typhimurium SopF (Xu et al., 2019) and L. pneumophilaeffector Ceg3 (Fu et al., 2022; Kubori et al., 2022), though in a more
targeted manner as only those Af1521-enriched proteins with the size of
interest were analyzed by LC-MS. Notably, Ceg3 and Larg1 were found to
target ADP/ATP translocases by reversible ADP-ribosylation in host
mitochondria to temporally regulate their ADP/ATP exchange activity
during L. pneumophila infection. Alternatively, the use of an NAD
derivative, biotin-17-NAD, in in vitro ADP-ribosylation assays
permits pull-down of modified ART substrates prior to MS identification.
By using this strategy, L. pneumophila effector Lart1 was found
to ADP-ribosylate yeast glutamate dehydrogenase 2 (Black et al., 2021).