Dataset Information

Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents

ABSTRACT: Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a new type of disulfide-bond-activated heterotypic Ub reagents, which allowed to enrich heterotypic Ub-interacting proteins and modifying enzymes simultaneously. The sequential release of readers which are non-covalently bound and writers or erasers which are covalently conjugated by using urea and reductant, respectively, combined with label-free quantitative (LFQ) MS indicated that these innovative heterotypic Ub tools would facilitate future investigations into functional roles played by heterotypic Ub chains.

ORGANISM(S): Saccharomyces Cerevisiae

SUBMITTER: Qingyun Zheng

PROVIDER: PXD045936 | iProX | Thu Sep 28 00:00:00 BST 2023

REPOSITORIES: iProX

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Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents.

Cai Hongyi H Wu Xiangwei X Mao Junxiong J Tong Zebin Z Yan Dingfei D Weng Yicheng Y Zheng Qingyun Q

Bioorganic chemistry 20240209

Heterotypic ubiquitin (Ub) chains have emerged as fundamental components in a wide range of cellular processes. The integrative identification of Ub-interacting proteins (readers) and Ub-modifying enzymes (writers and erasers) that selectively recognize and regulate heterotypic ubiquitination may provide crucial insights into these processes. In this study, we employed the bifunctional molecule-assisted (CAET) strategy to develop a type of disulfide bond-activated heterotypic Ub reagents, which ...[more]

PMID: 38387394

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Project description:Post-translational control by ubiquitin regulates various aspects of cellular biology. This chemical modification on proteins presents itself in numerous iterations, from a single mono-ubiquitination event to chains of poly-ubiquitin. Among the various types of poly-ubiquitin constructed are untethered species that comprise a series of ubiquitin moieties linked to one another, but free from another protein. The current notion is that these unanchored poly-ubiquitin species are deleterious to the cell and are rapidly deconstructed. We recently examined the toxicity and utilization of untethered poly-ubiquitin in an intact organism by using the fruit fly, Drosophila melanogaster. We found that these ubiquitin species are largely innocuous to flies and that free poly-Ub can be controlled by being degraded by the proteasome or being conjugated onto another protein as a single unit. However, whether the fly is mounting an organismal defense against untethered chains was not explored in detail. Here, we conducted RNA-seq analyses to examine at the transcriptome level the impact of unanchored poly-Ub in the fly. We found ~90 transcripts whose expression is altered in the presence of different types of unanchored poly-ubiquitin. The set of genes identified was mostly devoid of ubiquitin-, proteasome- or autophagy-related components. The largest gene ontology category identified, housing ~15 of the altered genes, was proteolysis. The seeming absence of a large and multipronged response to unanchored ubiquitin chains in the fly supports the conclusion that these species need not be toxic in vivo and underscores the need to reexamine the role of free ubiquitin chains in the cell. We designed two types of poly-Ub chains with six Ub molecules in tandem. They cannot be cleaved by DUBs (there are no "GG" motifs linking the Ub molecules). Ub6-GG can be conjugated in toto onto other proteins; Ub6-Stop cannot. We drove ubiquitous expression using sqh-Gal4, and then performed RNA-Seq on adult flies to determine any cellular response to the presence of our unanchored Ub chains.

Project 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.

Dataset Information

Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents

Publications

Sequential release of interacting proteins and Ub-modifying enzymes by disulfide heterotypic ubiquitin reagents.

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