Deciphering the molecular mechanisms governing PARP14 ADP-ribosylation activity and its interplay with ubiquitylation signalling

Intricate networks of post-translational modifications (PTMs) shape how cells respond to environmental threats. Adenosine diphosphate (ADP)-ribosylation (ADPr) has emerged as a pivotal PTM elicited by DNA damage or host immune responses to combat viruses, including SARS-CoV-2. This protective ADPr signature can be regulated by PARP14, a versatile enzyme that can both install and remove ADPr. While the connection between ADPr signalling and these damaging events is clear, the physiological targets and functions of PARP14-mediated ADPr remain ill-defined. Recently, a cryptic link between PARP14 ADPr and ubiquitylation signalling has arisen, adding complexity to PARP14-regulated processes. This ambitious research program 'UbADPrLINK' aims to dissect the mechanistic underpinnings of PARP14 ADPr and its link with ubiquitylation using an integrative approach, merging proteomics, structural, biochemical methods. UbADPrLINK will focus on four areas. First, we will precisely map and characterise cellular PARP14-catalysed ADPr sites on important targets by reconstituting ADPr in vitro. Concurrently, we will study the structure-function relationships of PARP14 and explore the roles of uncharacterised PARP14 domains and auto-modification in regulating its activity. Next, we will attempt to decipher the interplay between PARP14 and the PARP9-DTX3L complex that is known to regulate PARP14 activity using unclear mechanisms. We will structurally characterise the PARP14-PARP9-DTX3L complex and assess its potential to simultaneously modify targets with ADPr and ubiquitin. Finally, we will launch a drug discovery campaign to discover cyclic peptides capable of modulating SARS-CoV-2 by targeting PARP14 and SARS-CoV-2 domains involved in ADPr during infection. By seeking to unravel the molecular details of PARP14-catalysed ADPr, UbADPrLINK has the potential to advance our understanding of ADPr signalling beyond the state-of-the-art and may contribute to the development of new therapies.