Establishment of a Mass-Spectrometry-Based Method for the Identification of the In Vivo Whole Blood and Plasma ADP-Ribosylomes.

Blood and plasma proteins are heavily investigated as biomarkers for different diseases. However, the post-translational modification states of these proteins are rarely analyzed since blood contains many enzymes that rapidly remove these modifications after sampling. In contrast to the well-described role of protein ADP-ribosylation in cells and organs, its role in blood remains mostly uncharacterized. Here, we discovered that plasma phosphodiesterases and/or ADP-ribosylhydrolases rapidly demodify in vitro ADP-ribosylated proteins. Thus, to identify the in vivo whole blood and plasma ADP-ribosylomes, we established a mass-spectrometry-based workflow that was applied to blood samples collected from LPS-treated pigs (Sus scrofa domesticus), which serves as a model for human systemic inflammatory response syndrome. These analyses identified 60 ADP-ribosylated proteins, 17 of which were ADP-ribosylated plasma proteins. This new protocol provides an important step forward for the rapidly developing field of ADP-ribosylation and defines the blood and plasma ADP-ribosylomes under both healthy and disease conditions.

Mitochondrial NAD+ Controls Nuclear ARTD1-Induced ADP-Ribosylation.

In addition to its role as an electron transporter, mitochondrial nicotinamide adenine dinucleotide (NAD+) is an important co-factor for enzymatic reactions, including ADP-ribosylation. Although mitochondria harbor the most intra-cellular NAD+, mitochondrial ADP-ribosylation remains poorly understood. Here we provide evidence for mitochondrial ADP-ribosylation, which was identified using various methodologies including immunofluorescence, western blot, and mass spectrometry. We show that mitochondrial ADP-ribosylation reversibly increases in response to respiratory chain inhibition. Conversely, H2O2-induced oxidative stress reciprocally induces nuclear and reduces mitochondrial ADP-ribosylation. Elevated mitochondrial ADP-ribosylation, in turn, dampens H2O2-triggered nuclear ADP-ribosylation and increases MMS-induced ARTD1 chromatin retention. Interestingly, co-treatment of cells with the mitochondrial uncoupler FCCP decreases PARP inhibitor efficacy. Together, our results suggest that mitochondrial ADP-ribosylation is a dynamic cellular process that impacts nuclear ADP-ribosylation and provide evidence for a NAD+-mediated mitochondrial-nuclear crosstalk.