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).