A ribose-functionalized NAD+ with unexpected high activity and selectivity for protein poly-ADP-ribosylation

Abstract

Nicotinamide adenine dinucleotide (NAD+)-dependent ADP-ribosylation plays important roles in physiology and pathophysiology. It has been challenging to study this key type of enzymatic post-translational modification in particular for protein poly-ADP-ribosylation (PARylation). Here we explore chemical and chemoenzymatic synthesis of NAD+ analogues with ribose functionalized by terminal alkyne and azido groups. Our results demonstrate that azido substitution at 3′-OH of nicotinamide riboside enables enzymatic synthesis of an NAD+ analogue with high efficiency and yields. Notably, the generated 3′-azido NAD+ exhibits unexpected high activity and specificity for protein PARylation catalyzed by human poly-ADP-ribose polymerase 1 (PARP1) and PARP2. And its derived poly-ADP-ribose polymers show increased resistance to human poly(ADP-ribose) glycohydrolase-mediated degradation. These unique properties lead to enhanced labeling of protein PARylation by 3′-azido NAD+ in the cellular contexts and facilitate direct visualization and labeling of mitochondrial protein PARylation. The 3′-azido NAD+ provides an important tool for studying cellular PARylation.

Uncovering the Invisible: Mono-ADP-ribosylation Moved into the Spotlight

“…Additional targeting of recombinant ARH3 to the mitochondrial matrix could reverse the artificially induced PAR, demonstrating that the conditions present in mitochondria support the full life cycle of the modification [187,188]. Furthermore, permeabilization of cells and subsequent incubation with 3 -azido_NAD + allowed detecting mitochondrial-localized PAR chains by confocal microscopy [171]. However, a recent analysis using ARTD1 knockout cells revealed that the observed mitochondrial ADP-ribosylation is most likely not dependent on ARTD1 [155].…”

“…To further facilitate the incorporation of both modified metabolites into ADP-ribosylation, ARTs were genetically modified to favor NAD + analogues over natural NAD + [ 167 ]. In addition to these approaches that involve click-it chemistry, another study investigated in the synthesis of an NR analog that can be taken up by cells and is locally converted to 3′-azido_NAD + by the nicotinamide riboside kinase 1 and NMNAT1 [ 171 ]. The authors describe 3′-azido_NAD + to exhibit a high activity and specificity to ARTD1 and ATD2-catalyzed protein ADP-ribosylation and the resulting PAR chains to show a certain degree of resistance towards PARG-mediated degradation.…”

New Perspectives for Resistance to PARP Inhibitors in Triple-Negative Breast Cancer

“…The PARP family comprises 17 nucleoproteins with four domains of interest: a DNA-binding domain, a caspase-cleaved domain, an auto-modification domain, and a catalytic domain (43). When PARP detects DNA double stand breaks, it initiates a polymeric adenosine diphosphate ribose (polyADP-ribose or PAR) chain with NAD + as the substrate (44). The highly conserved PAR polymer reaches as long as 200 nucleotides and transfers one unit to target proteins, whereas PARP1, PARP2, PARP3, and PARP5a characteristically add repeated ADP-ribose units (45).…”

NAD Analogs in Aid of Chemical Biology and Medicinal Chemistry

“…Recently, another group has for the first time explored the modifications of nicotinamide riboside for protein PARylation studies [54]. To that end, Zhang et al reported a set of three NAD + analogs modified with either an alkyne or azide group at the 3′-position of nicotinamide riboside (Figure 3, 21 – 23 ) and their 2′-counterparts (not shown), which are accessible by either chemical or chemoenzymatic methods.…”