DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on nucleic acids

Abstract

Although ubiquitylation had traditionally been considered limited to proteins, the discovery of non-proteinaceous substrates (e.g. lipopolysaccharides and adenosine diphosphate ribose (ADPr)) challenged this perspective. Our recent study showed that DTX2 E3 ligase efficiently ubiquitylates ADPr. Here, we show that the ADPr ubiquitylation activity is also present in another DELTEX family member, DTX3L, analysed both as an isolated catalytic fragment and the full-length PARP9:DTX3L complex, suggesting that it is a general feature of the DELTEX family. Since structural predictions show that DTX3L possesses single-stranded nucleic acids binding ability and given the fact that nucleic acids have recently emerged as substrates for ADP-ribosylation, we asked whether DELTEX E3s might catalyse ubiquitylation of an ADPr moiety linked to nucleic acids. Indeed, we show that DTX3L and DTX2 are capable of ubiquitylating ADP-ribosylated DNA and RNA synthesized by PARPs, including PARP14. Furthermore, we demonstrate that the Ub-ADPr-nucleic acids conjugate can be reversed by two groups of hydrolases, which remove either the whole adduct (e.g. SARS-CoV-2 Mac1 or PARP14 macrodomain 1) or just the Ub (e.g. SARS-CoV-2 PLpro). Overall, this study reveals ADPr ubiquitylation as a general function of the DELTEX family E3s and presents the evidence of reversible ubiquitylation of ADP-ribosylated nucleic acids.

Ubiquitylation of nucleic acids by DELTEX ubiquitin E3 ligase DTX3L

“…In our previous studies, we showed that the tandem RING-DTC (RD) domains of DELTEX family E3s are capable of ubiquitylating ADPr on the 3’ hydroxyl group of the adenine-proximal ribose (Zhu et al ., 2023; Zhu et al ., 2022). Specifically, the DTC domain first accommodates ADPr molecule to position the 3’ hydroxyl group of ADPr proximal ribose close to the E2∼Ub conjugate bound by the RING domain.…”

“…The DTC domain appears to then utilize its two crucial catalytic residues to deprotonate the 3’ hydroxyl group, thus facilitating ADPr ubiquitylation. The available experimental structures show that the DTC domain uses the same conserved pocket to bind either ADPr or NAD + , and can facilitate Ub transfer to both (Figure 1A) (Ahmed et al , 2020; Chatrin et al ., 2020; Zhu et al ., 2023). Both ADPr and NAD + contain an AMP moiety (Figure EV1), and it is this part that becomes ubiquitylated on the 3’ hydroxyl.…”

“…However, several recent studies changed this view, by demonstrating that Ub can be covalently attached to non-proteinaceous substrates, such as lipopolysaccharides (LPS) (Otten et al , 2021), phosphatidylethanolamine (PE) (Sakamaki et al , 2022), or glucosaccharides (Kelsall et al , 2022). Furthermore, Ub can also be attached on another modification called ADP-ribosylation (ADPr) (Zhu et al , 2023; Zhu et al , 2022). ADP-ribosylation is a chemical modification of proteins and nucleic acids involving the addition of one or more ADPr moieties.…”

“…ADPr is transferred from nicotinamide adenine dinucleotide (NAD + ) onto the targets by ADP-ribosyltransferases (ARTs) including the best-studied PARPs (Groslambert et al, 2021; Pascal, 2018; Suskiewicz et al, 2023b). Hybrid ADPr-Ub modification is efficiently synthesized in vitro by the DELTEX family of E3s on both proteins and nucleic acids substrates (Zhu et al ., 2023; Zhu et al ., 2022), but its physiological relevance is not clear yet.…”

“…DELTEX family in human is composed of five members, namely DTX1, DTX2, DTX3, DTX4 and DTX3L (Takeyama et al, 2003; Wang et al, 2021). Different family members have distinct N-terminal domains, either WWE domains (in DTX1, DTX2 and DTX4), or KH domain(s) (in DTX3 and DTX3L)(Figure EV8) (Zhu et al, 2023). WWE domains in proteins are known to bind poly(ADP-ribose) chains (DaRosa et al , 2015) whereas KH domains are known for binding to single stranded nucleic acids (Nicastro et al , 2015a; Suskiewicz et al , 2023a; Valverde et al , 2008).…”