PXD058087-1

PXD058087 is an original dataset announced via ProteomeXchange.

Title	Identification of RNF114 as ADPrUb reader through chemical synthesis of non-hydrolysable Ubiquitylated ADPribose
Description	Ubiquitination is a much-studied post-translational modification (PTM) playing crucial roles in regulating cellular processes across eukaryotes. Canonical ubiquitination relies on a cascade of E1-, E2-, and E3 enzymes which conjugate the Gly76 C-terminus of ubiquitin (Ub) to the lysine side chains in proteins creating a stable isopeptide bond(1). However, it became evident that Ub can also be conjugated to non-lysine residues in proteins to form a thioester bond with cysteine(2, 3) and an oxyester bond with serine or threonine(4-6). Beyond protein modification, recent discoveries reveal ubiquitination on several non-protein substrates, such as lipopolysaccharide(7), oligosaccharide(8), phosphatidylethanolamine(9), nucleic acids(10, 11), as well as on another PTM, ADP-ribose (ADPr) (12, 13). ADP-ribosylation is an ancient modification which is conserved across all kingdoms of life. This modification relies on ADP-ribosyltransferase enzymes which utilise NAD+ and transfer ADP-ribose onto proteins and nucleic acids(14, 15). Crosstalk between ubiquitination and ADP-ribosylation occurs in mammals where for instance poly-ADP-ribose (PAR) chains direct subsequent ubiquitination events in DNA damage responses(16) and ADPr-dependent ubiquitination in immunity responses (17). Such crosstalk is also cleverly utilised by bacteria, which themselves lack Ub, to disrupt host Ub signaling by modifying it with ADPr, as shown by Legionella and Chromobacterium(18, 19). These are prime examples where the already complex ubiquitination code is made even more diverse upon crosstalk with ADPr. In humans, the family of Deltex RING E3 Ub ligases (DTX1, DTX2, DTX3, DTX3L, DTX4) catalyse Ub conjugation to the 3’-hydroxyl group of ADPr, which results in a hybrid ADPr-3’-Ub modification (Fig. 1A)(12, 20). It was first reported that the heterodimeric complex of DTX3L/PARP9 conjugates ADPr to the Gly76 residue of Ub(21). This unique reaction is catalysed by all members of Deltex E3 ligases through their conserved RING and DTC domains, which interacts with Ub-loaded E2 (E2~Ub) and NAD+/ADPr, respectively(20). The DTC domains of Deltex E3 ligases bind to NAD+ and ADPr, and can recruit ADP-ribosylated proteins for ubiquitination(22). However, instead of ADP-ribosylation of Ub which occurs on the C1” of nicotinamide ribose, Deltex E3 ligases catalyse the ubiquitination of ADPr on the 3’-hydroxyl of adenosine ribose (Fig 1A) (12). To support these reactions, the DTC domains of Deltex E3 ligases harbour a conserved Glu-His diade to deprotonate the 3’-hydroxyl of the proximal adenosine ribose in ADPr to attack the thioester E2~Ub conjugate, resulting in a covalent ester bond between Ub Gly76 and the ADPr 3’-hydroxyl group(12). This allows formation of dual hybrid modification composed of ubiquitin and ADPr (ADPr-3’-Ub) either on protein and nucleic acids substrates(12, 13). However, the biological function of such Deltex-catalysed hybrid ADPr-3’-Ub-substrates remains unknown. In this work, we wondered if there are modules involved in reading this new expansion of the ubiquitin code, which would establish a first indication regarding the biological consequence and the underlying molecular mechanisms of the hybrid ADPr-3’-Ub. One of the major challenges in studying the ADPr-3’-Ub modification is the inherent lability of the ester bond. Here we synthesised a non-hydrolysable tool that mimics the formed ubiquitinated ADP-ribose (further annotated as ADPr3’-Ub) to perform an unbiased pulldown from cell lysate and start exploring its interactome. We identified RNF114 as one of the top binders for ADPr3’-Ub. RNF114 was previously suggested to bind mono-ADP-ribose (MAR) and poly-ADP-ribose (PAR) through its Zinc finger (ZnF) domains (23, 24). Here, we discovered that RNF114 mediate low micromolar binding to ADPr3’-Ub through its tandem ZnF2+ZnF3 and Ub-interacting motif (UIM) domains. We further showed that RNF114 can engage ADPr3’-Ub and elongate it with K11-linked polyubiquitin chains forming a novel mixed Ub chain linkage product. Finally, the tandem ZnF2+ZnF3+UIM domains of RNF114 are indispensable for its recruitment to the sites of DNA damage in cells, which suggest RNF114 as a reader of ADPr-3’-Ub signal during response to DNA damage.
HostingRepository	PRIDE
AnnounceDate	2025-06-20
AnnouncementXML	Submission_2025-06-20_00:54:09.920.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Rayman Tjokrodirijo
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	acetylated residue; monohydroxylated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Exploris 480

Revision	Datetime	Status	ChangeLog Entry
0	2024-11-20 05:16:47	ID requested
⏵ 1	2025-06-20 00:54:10	announced

Dataset with its publication pending

submitter keyword: RNF114, chemoproteomics, ADP-ribosylation, ubiquitin

Peter A. van Veelen
contact affiliation	Head Proteomics Group, Leiden University Medical Center, Center for Proteomics and Metabolomics, PO Box 9600, Postal zone P1-Q, 2300 RC Leiden, The Netherlands
contact email	P.A.van_Veelen@lumc.nl
lab head
Rayman Tjokrodirijo
contact affiliation	Leiden University Medical Center, Proteomics group
contact email	R.T.N.Tjokrodirijo@lumc.nl
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD058087
     1. Label: PRIDE project
     2. Name: Identification of RNF114 as ADPrUb reader through chemical synthesis of non-hydrolysable Ubiquitylated ADPribose