E2-Ub-R74G strategy reveals E2-specific ubiquitin conjugation profiles in live cells
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ABSTRACT:
ORGANISM(S): Homo Sapiens
SUBMITTER: 
Hang Yin
PROVIDER: PXD058017 | iProX | Wed Dec 11 00:00:00 GMT 2024
REPOSITORIES: iProX
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Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles Ube2m and Ube2f are E2 enzymes that direct protein modification by NEDD8. Here we explore the specific functions of Ube2f and Ube2m by comparing gene expression profiles following knockdown of their function in NIH 3T3 cells. We used microarrays to detail the global programme of gene expression changes following knockdown of Ube2m and Ube2f in NIH 3T3 cells. NIH 3T3 cells were transduced with retroviral constructs containing shRNA directed against Ube2m or Ube2f. Three replicates of each condition were analyzed.
2009-03-23 | E-GEOD-14088 | biostudies-arrayexpress
Project description:Ubiquitin and ubiquitin-like proteins (UBLs) are directed to targets by cascades of E1, E2, and E3 enzymes. The largest ubiquitin E3 subclass consists of cullin-RING ligases (CRLs), which contain one each of several cullins (CUL1, -2, -3, -4, or -5) and RING proteins (RBX1 or -2). CRLs are activated by ligation of the UBL NEDD8 to a conserved cullin lysine. How is cullin NEDD8ylation specificity established? Here we report that, like UBE2M (also known as UBC12), the previously uncharacterized E2 UBE2F is a NEDD8-conjugating enzyme in vitro and in vivo. Biochemical and structural analyses indicate how plasticity of hydrophobic E1-E2 interactions and E1 conformational flexibility allow one E1 to charge multiple E2s. The E2s have distinct functions, with UBE2M/RBX1 and UBE2F/RBX2 displaying different target cullin specificities. Together, these studies reveal the molecular basis for and functional importance of hierarchical expansion of the NEDD8 conjugation system in establishing selective CRL activation. Mol Cell 33:483-495, 2009. Keywords: Comparison of gene expression profiles
2009-03-24 | GSE14088 | GEO
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.
2025-06-20 | PXD058087 | Pride
Project description:Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a simple, proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2~Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich the bona fide substrates and potential new substrates of the ligase using a one-step Streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase Rad18. Rad18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single Ub-POD experiment. We further discovered an unintended but important application of Ub-POD, where we were able to pin down the cellular localization of Rad18- mediated ubiquitination to the damaged DNA nuclear puncta through Streptavidin immunofluorescence. We also applied our method to TRAF6, another RING ubiquitin ligase involved in TNF signaling, successfully identifying known and potentially new substrates. Finally, we adapted our method to the U-box type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.
2024-11-26 | PXD045521 | Pride
Project description:E6AP/UBE3A, the founding member of HECT-type (Homologous to E6AP C-terminus) E3 ligases, is implicated in human papillomaviruses (HPV)-mediated cervical cancer and neurodevelopmental disorders like Angelman and Dup15q syndromes. To elucidate the function of E3 ligases, knowledge about their substrates is crucial. Their identification, however, is challenging due to complex cross-reactivities between E2, E3 and their substrates. To address this, we developed a novel ubiquitin-based photo labeling approach for HECT-type ligases. Our method modifies ubiquitin with a diazirine moiety and attaches it to E6AP's active site using the ubiquitination cascade and a Cys to Lys mutation. The resulting E6AP-Ub conjugate enables substrate trapping via photoaffinity labeling and proteomic profiling. We validated this approach through literature comparison and identified Ub C-terminal hydrolase 7 (Uchl5/Uch37) as a new, proteasome-independent ubiquitination target of E6AP. This method expands the toolbox for substrate identification, potentially providing deeper insights into E6AP-related cellular processes and other HECT-type E3 ligases.
2025-01-27 | PXD055649 | Pride
Project description:NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation both in vitro and in cells. Furthermore, we identified SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in engineered SENP8-deficient cells. Importantly, loss of SENP8 function contributes to reduced CRL activity, accumulation of CRL substrates, and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels and CRL-dependent proteostasis, implicating this protease as a potential therapeutic target for inhibiting CRL activity.
2017-04-13 | MSV000080917 | MassIVE
Project description:Ubiquitination is the key post-translational modification in eukaryotic cells that governs protein degradation, localisation, and activity. This process results in the tagging of ubiquitin to protein substrates by a concerted enzyme cascade of E1, E2 and E3 enzymes. The largest superfamily of these proteins includes the Cullin-Ring-Ligase (CRL) complexes of E3 ligases. Plasmodium falciparum, the causative agent of the most severe form of malaria in humans, encodes the critical proteins required for ubiquitination, but we do not yet understand the function of this pathway. Here the P. falciparum CRL E3 ligases were characterised to reveal an essential but minimal repertoire controlled by two Cullin scaffolds. A PfCullin1-linked CRL complex was identified as being required for parasite inner-membrane biogenesis and DNA replication. This complex recruits only one substrate receptor, compared to the ~70 receptors used interchangeably by human cells. A second CRL complex functioning through a PfCullin4 scaffold was identified that utilised a previously unidentified adaptor protein and receptors to support DNA replication. These results show that the P. falciparum E3 ubiquitin ligases play an essential role in the biology of this important infectious agent.
2025-07-11 | PXD063957 | Pride
Project description:Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, "cullin-RING" and "RBR", are individually found in several hundred E3 ligases in humans, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry, and cellular assays elucidate a 1.8 MDa hexameric CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique amongst RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects from deneddylation. Mono-ubiquitylation of TP53 relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.
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Project description:Ubiquitin ligation is typically executed by hallmark E3 catalytic domains. Two such domains, "cullin-RING" and "RBR", are individually found in several hundred E3 ligases in humans, and collaborate with E2 enzymes to catalyze ubiquitylation. However, the vertebrate-specific CUL9 complex with RBX1 (also called ROC1), of interest due to its tumor suppressive interaction with TP53, uniquely encompasses both cullin-RING and RBR domains. Here, cryo-EM, biochemistry, and cellular assays elucidate a 1.8 MDa hexameric CUL9-RBX1 assembly. Within one dimeric subcomplex, an E2-bound RBR domain is activated by neddylation of its own cullin domain and positioning from the adjacent CUL9-RBX1 in trans. Our data show CUL9 as unique amongst RBX1-bound cullins in dependence on the metazoan-specific UBE2F neddylation enzyme, while the RBR domain protects it from deneddylation. Substrate ubiquitylation relies on both CUL9's neddylated cullin and RBR domains achieving self-assembled and chimeric cullin-RING/RBR E3 ligase activity.
2024-04-09 | PXD047229 | Pride
Project description:Attachment of the ubiquitin (UB) peptide to proteins via the E1-E2-E3 enzymatic machinery regulates diverse biological pathways, yet identification of the substrates of E3 UB ligases remains a challenge. We overcame this challenge by constructing an “orthogonal UB transfer (OUT) cascade with yeast E3 Rsp5 to enable the exclusive delivery of an engineered UB (xUB) to Rsp5 and its substrate proteins. The OUT screen uncovered new Rsp5 substrates in yeast, such as Pal1 and Pal2 that are partners of endocytic protein Ede1, and chaperones Hsp70-Ssb, Hsp82, and Hsp104 that counteract protein misfolding and control self-perpetuating amyloid aggregates (prions), resembling those involved in human amyloid diseases. We showed that prion formation and effect of Hsp104 on prion propagation are modulated by Rsp5. Overall, our work demonstrates the capacity of OUT to deconvolute the complex E3-substrate relationships in crucial biological processes such as endocytosis and protein assembly disorders through protein ubiquitination.
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