Serine ADP-ribosylation in Drosophila provides insights into the evolution of reversible ADP-ribosylation signalling

Abstract

In the mammalian DNA damage response, ADP-ribosylation signalling is of crucial importance to mark sites of DNA damage as well as recruit and regulate repairs factors. Specifically, the PARP1:HPF1 complex recognises damaged DNA and catalyses the formation of serine-linked ADP-ribosylation marks (mono-Ser-ADPr), which are extended into ADP-ribose polymers (poly-Ser-ADPr) by PARP1 alone. Poly-Ser-ADPr is reversed by PARG, while the terminal mono-Ser-ADPr is removed by ARH3. Despite its significance and apparent evolutionary conservation, little is known about ADP-ribosylation signalling in non-mammalian Animalia. The presence of HPF1, but absence of ARH3, in some insect genomes, including Drosophila species, raises questions regarding the existence and reversal of serine-ADP-ribosylation in these species. Here we show by quantitative proteomics that Ser-ADPr is the major form of ADP-ribosylation in the DNA damage response of Drosophila melanogaster and is dependent on the dParp1:dHpf1 complex. Moreover, our structural and biochemical investigations uncover the mechanism of mono-Ser-ADPr removal by Drosophila Parg. Collectively, our data reveal PARP:HPF1-mediated Ser-ADPr as a defining feature of the DDR in Animalia. The striking conservation within this kingdom suggests that organisms that carry only a core set of ADP-ribosyl metabolising enzymes, such as Drosophila, are valuable model organisms to study the physiological role of Ser-ADPr signalling.

A site-specific analysis of the ADP-Ribosylome unveils Homogeneous DNA Damage-Induced Serine ADP-Ribosylation across wild-type and BRCA-mutant Breast Cancer cell lines

“…By contrast, among the H 2 O 2 -treated cells, the largest number of ADPr sites were identified in T47D cells (1,133 sites on 576 proteins) and the lowest in MDAMB436 cells (649 sites on 371 proteins). As previously described in HeLa cells as well as Drosophila cell lines 35,36 , serine emerged as a major ADPr target amino acid, with 92.8% of the total identified ADPr acceptor sites localizing to serine residues ( Fig. 3F ).…”

“…We found that a large part of the ADP-ribosylation sites in PARPi sensitive and PARPi resistant breast cancer cells overlap. Furthermore, comparison with previously published reports identifying proteome-wide ADPr target sites after H 2 O 2 treatment 35,36 demonstrates high site- and protein-level overlap with other mammalian and non-mammalian cell types. This supports to the idea of a highly conserved and homogeneous core ADPr signaling network in response to H 2 O 2 induced DNA damage.…”

“…On the protein level, 699 out of 777 proteins ADP-ribosylated in the breast cancer cell lines have been shown to also be ADP-ribosylated in HeLa cells upon H 2 O 2 treatment 35 , additionally demonstrating the homogeneity of the ADPr response across different human cell lines. We also compared the ADPr sites identified in our dataset to those identified in Drosophila S2R+ cells 36 , because about 65% of human disease-causing genes have functional homologues in Drosophila 61 . We used flybase ID to find human orthologues for 219 for the proteins ADP-ribosylated upon H 2 O 2 treatment in Drosophila proteins.…”

“…In the Drosophila ADPr profiling study 36 , 514 ADPr sites on 296 proteins were identified in Drosophila S2R+ cell lines. Flybase IDs of the ADP-ribosylated proteins were matched to orthologous human genes using a list of Drosophila melanogaster- Human Orthologs generated from the flybase API on 22.05.2023 and downloaded on 23.07.2023 here: https://ftp.flybase.net/releases/FB2023_03/precomputed_files/orthologs/.…”

“…We found that H 2 O 2 treatment induced robust and homogeneous ADPr of DNA damage repair proteins in all cell lines, regardless of BRCA1/2 mutation or PARPi sensitivity. Furthermore, ADPr sites and proteins were conserved across different cell lines 35 and organisms 36 . Interestingly, we found reduced PARG expression in PARPi resistant BRCA1 mutant cells, and we found that ADPr on USF1 was significantly upregulated in PARPi sensitive cells compared to PARPi resistant cells.…”