Preserving ester-linked modifications reveals glutamate and aspartate mono-ADP-ribosylation by PARP1 and its reversal by PARG

• Published in: Nature communications Vol. 15; no. 1; p. 4239
• Main Authors: Longarini, Edoardo José, Matić, Ivan
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 18.05.2024; Nature Publishing Group; Nature Portfolio
• Subjects: 13; 13/1; 14; 14/63; 631/45/173; 631/45/475; 631/92/458/2389; 82/29; 82/58; 96; 96/35; Adenosine diphosphate; ADP; ADP-Ribosylation; Antibodies; Aspartic Acid - metabolism; Damage; Deoxyribonucleic acid; DNA; DNA Damage; Esters - chemistry; Esters - metabolism; Glutamic Acid - metabolism; Glycoside Hydrolases - metabolism; HEK293 Cells; Humanities and Social Sciences; Humans; Lability; Mass spectrometry; Mass spectroscopy; multidisciplinary; Poly (ADP-Ribose) Polymerase-1 - metabolism; Poly(ADP-ribose) polymerase; Post-translation; Protein Processing, Post-Translational; Ribose; Science; Science (multidisciplinary); Serine; Signal Transduction; Threonine; Ubiquitination
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-48314-0

Ester-linked post-translational modifications, including serine and threonine ubiquitination, have gained recognition as important cellular signals. However, their detection remains a significant challenge due to the chemical lability of the ester bond. This is the case even for long-known modifications, such as ADP-ribosylation on aspartate and glutamate, whose role in PARP1 signaling has recently been questioned. Here, we present easily implementable methods for preserving ester-linked modifications. When combined with a specific and sensitive modular antibody and mass spectrometry, these approaches reveal DNA damage-induced aspartate/glutamate mono-ADP-ribosylation. This previously elusive signal represents an initial wave of PARP1 signaling, contrasting with the more enduring nature of serine mono-ADP-ribosylation. Unexpectedly, we show that the poly-ADP-ribose hydrolase PARG is capable of reversing ester-linked mono-ADP-ribosylation in cells. Our methodology enables broad investigations of various ADP-ribosylation writers and, as illustrated here for noncanonical ubiquitination, it paves the way for exploring other emerging ester-linked modifications. Ester-linked modifications are common but difficult to detect. Here, the authors present methods based on ester preservation and a sensitive antibody to reveal DNA damage-induced mono-ADP-ribosylation on aspartate and glutamate. This signal, part of the first wave of PARP1 signaling, is removed by PARG.