Catalytic mechanism of an Actin-dependent adenylyltransferase involved in ubiquitin signaling
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Jiaqi Fu
PROVIDER: PXD062291 | iProX | Thu Mar 27 00:00:00 GMT 2025
REPOSITORIES: iProX
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Project description:Ubiquitination is a crucial posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila, the bacterium responsible for Legionnaires’ disease. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. In this study, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on the phosphoribosyl-Ub (PR-Ub) conjugated to host targets by Sde. Remarkably, the Ub moieties within the poly-Ub chains are either modified with a phosphoribosyl group by Sde and other PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated PR-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors, such as p62, and therefore exclude host autophagy adaptors from the LCV. Our findings shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.
2024-09-11 | PXD045494 | Pride
Project description:Ubiquitination is a crucial posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila, the bacterium responsible for Legionnaires’ disease. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. In this study, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on the phosphoribosyl-Ub (PR-Ub) conjugated to host targets by Sde. Remarkably, the Ub moieties within the poly-Ub chains are either modified with a phosphoribosyl group by Sde and other PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated PR-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors, such as p62, and therefore exclude host autophagy adaptors from the LCV. Our findings shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.
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Project description:We utilized a large-scale proteomic profiling strategy to quantitatively measure ADP-riboxanation (and classical ADP-ribosylation) levels of host proteins upon introduction of bacterial effectors. Notably, we identified the eukaryotic translation initiation factor 3 (eIF3) complex as a novel target of the OspC family proteins. ADP-riboxanation of eIF3 resulted in inhibition of bulk protein synthesis and SG assembly in infected host cells, which promoted intracellular bacterial proliferation.
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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.
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Project description:Vibrio vulnificus is an foodborne pathogen that can cause gastroenteritis and septicemia in humans. V. vulnificus secretes a multifunctional autoprocessing repeats-in-toxin (MARTX) toxin as an essential virulence factor to cause disease. MARTX toxins are pore-forming toxins that translocate multiple functionally independent effector domains into a target cell. MARTX toxins of V. vulnificus can contain anywhere from 3 to 5 of the 10 identified effector domains and strains with different effector repertories having varying virulence potential. The goal of this study was to compare how different effector combinations from an F-type MARTX toxin differentially remodel the transcriptional response of human intestinal epithelial cells (IECs). F-type MARTX toxins contain five effector domains – the actin crosslinking domain (ACD), two copies the makes caterpillar floppy-like domain (MCF), and alpha-beta hydrolase (ABH) domain, and the Ras/Rap1 specific endopeptidase (RRSP). Cultured human IECs were treated with V. vulnificus or strains modified to secrete a toxin with only ACD, ACD with MCF-ABH, ACD with RRSP, or no active effectors. We demonstrate that when no active effectors are present, the bacterium induces minimal changes in the transcriptional profile of IECs. However, the strains containing different effector combinations each uniquely remodeled the transcriptional profile of IECs. These data provide insight into how V. vulnificus strains with varying effector combinations can differentially regulate the host cell response to cause disease.
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Project description:These files contain the raw spectra for intact protein and trypsin digests of untreated (unmodified) or treated (pR-Ub) tetra ubiquitin.
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