Identifying poly(ADP-ribosyl)ation targets during genotoxic stress
Poly(ADP-ribosyl)ation, also referred to as PARylation, is a dynamic post-translational modification (PTM) that plays an integral part in signalling pathways. The modification is catalyzed by a specific family of enzymes referred to as ADP-ribosyltransferases with diphtheria toxin homology (ARTD), also known as PARPs. These enzymes transfer the ADP-ribose group from nicotinamide adenine dinucleotide (NAD+) onto acceptor proteins, where they attach to amino acids such as arginine, lysine, glutamic acid, or aspartic acid. The modification is reversibly removed by ADP-ribosylarginine hydrolases (PARGS).
Activation of PARP enzymes entails the rapid synthesis of long, branched poly(ADP-ribose) (PAR) chains from NAD+ and can lead to a transient 10 to 500-fold increase of cellular PAR. These PAR polymers are known to play key roles in the regulation of chromatin structure modulation, DNA repair, transcription and cell death related processes. However, despite PARylation being known for 50 years, surprisingly little is known about the molecular targets of the modification, and which processes these specifically regulate.
Associate Professor Nielsen´s research team in the Department of Proteomics, in collaboration with the Chromosome Stability and Dynamics Group at CPR and international partners, made use of functional proteomics to decipher which proteins become modified with poly(ADP-ribos)ylation during various types of genotoxic stress. Using high-resolution Orbitrap mass spectrometry the researchers were able to identify more than 200 proteins as specific PARP substrates during DNA damage.
Distinct differences in proteins becoming PARylated upon various genotoxic insults were observed. This exemplified by a large number of PARylated proteins being involved in RNA metabolism, positioning for the first time PARylation as an important functional link between the DNA and RNA metabolic processes. High-content imaging revealed that PARylation specifically affects nuclear re-localization of several novel PARP substrates such as THRAP3 and TAF15, demonstrating that the established methodology is able to uncover hitherto unappreciated processes controlled by specific genotoxic stress-induced PARylation.
Title: Proteome-wide Identification of Poly(ADP Ribosyl)ation Targets in Different Genotoxic Stress Responses
Authors: Stephanie Jungmichel, Florian Rosenthal, Matthias Altmeyer, Jiri Lukas, Michael Hottiger and Michael L. Nielsen