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Noncanonical assembly, neddylation, and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD047326 | Pride | 2024-04-23

REPOSITORIES: Pride

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Noncanonical assembly, neddylation, and chimeric cullin-RING/RBR ubiquitylation by the 1.8 MDa CUL9 E3 ligase complex

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

Transcriptome data of Foxp3-Cre, Rbx1&Sag-deficient Treg cells from mice

Project description:Neddylation is necessary for activation of Cullin-RING Ligases (CRLs) which degrade various immune regulatory proteins. Our recent study showed that while depletion of neddylation E2-E3 pair Ube2f-Sag in regulatory T cells (Treg cells) had no obvious phenotype, the same depletion of either Ube2m or Rbx1 caused inflammation disorders with different severity. Whether these E2s or E3s compensate each other in functional regulations of Treg cells is, however, previously unknown. In this report, we generated Foxp3Cre;Ube2mfl/fl;Ube2ffl/fl or Foxp3Cre;Rbx1fl/fl;Sagfl/fl double null mice by simultaneously deletion of both neddylation E2s or E3s in Treg cells, respectively. Remarkably, Ube2m&Ube2f double null mice developed much severe autoimmune phenotypes than Ube2m-null mice, indicating Ube2m significantly compensates Ube2f in Treg cells. The minor worsen autoimmune phenotypes seen at very early stage in Rbx1&Sag double null than Rbx1-null mice, is likely due to already severe phenotypes of the later, indicating a minor compensation of Rbx1 for Sag. The RNA profiling-based analyses revealed that up- and down-regulations of few signaling pathways in Treg cells are associated with the severity of autoimmune phenotypes. Finally, severer inflammation phenotypes seen in mice with double E3-null than with double E2-null Treg cells indicate a neddylation-independent mechanism of two E3s, also known to serve as the RING component of CRLs in regulation of Treg cell fitness.

2023-08-12 | GSE237498 | GEO

Transcriptome data of Foxp3-Cre, Ube2m&Ube2f-deficient Treg cells from mice

2023-08-12 | GSE237497 | GEO

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

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

E2-RING expansion of the NEDD8 cascade confers specificity to cullin modification

2009-03-24 | GSE14088 | GEO

Transcriptome data of wt, Rbx1- or Ube2m- deficient Treg cells from mice

Project description:Cullin-RING Ligases (CRLs), upon activation by neddylation, play the key roles in regulating many biological processes. However, how CRL-neddylation regulates the function of Treg cells remains elusive. Here we show that mice with Treg-specific deletion of Rbx1, a RING component of CRLs required for its activity, developed an early-onset fetal inflammatory disorders and death at day ~25 after birth with disrupted homeostasis and impaired suppressive functions of Treg cells. Specifically, Rbx1 is essential for maintenance of the effector subpopulations in Treg cells, and regulates several inflammatory pathways. Similar phenotypes were seen in mice with deletion of Ube2m, a neddylation E2, in Treg cells, but with much lesser severity. Interestingly, Treg-specific deletion of Rbx2/Sag or Ube2f, the family member of Rbx1 or Ube2m, respectively, had no obvious phenotype. Thus, the Ube2m-Rbx1 axis is required for the maintenance of homeostasis and functions of Treg cells; and Rbx1 has Ube2m-independent roles in the fitness of Treg cells, suggesting a layer of complexity in neddylation activation of CRLs. We use the tranpritome dara to show the mechanism of the function of Rbx1 and Ube2m in Treg cells, also research the relationship of Rbx1 and Ube2m in Treg cells.

2021-12-28 | GSE192664 | GEO

NEDDylated-Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors (RNA-Seq)

Project description:DNA topoisomerase 1 (Top1) inhibitors are mainstays of anticancer therapy. These drugs trap Top1 on DNA, stabilizing the Top1-cleavage complex (Top1-cc). The accumulation of Top1-ccs perturbs DNA replication fork progression, leading to DNA breaks and cell death. By analyzing the genomic occupancy and activity of Top1, we show that cells adapt to treatment with multiple doses of Top1 inhibitor by promoting the degradation of Top1-ccs, allowing cells to better tolerate subsequent doses of Top1 inhibitor. The E3-RING Cullin 3 Ligase in complex with the BTBD1 and BTBD2 adaptor proteins promote Top1-cc ubiquitination and subsequent proteasomal degradation. NEDDylation of Cullin 3 activates this pathway and inhibition of protein NEDDylation or depletion of Cullin 3 sensitizes cancer cells to Top1 inhibitors. Collectively, our data identify a novel NEDD8-Cullin 3 pathway involved in the adaptive response to Top1 inhibitors, which can be targeted to improve the efficacy of Top1 drugs in cancer therapy.

2022-11-04 | GSE212469 | GEO

NEDDylated-Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors (ChIP-Seq)

2022-11-04 | GSE212468 | GEO

SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin- 1 RING ubiquitin ligase function

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

Discovery of a small molecule inhibitor of the UBE2F-CRL5 axis that induces apoptosis and G2/M arrest and confers radiosensitization

Project description:Protein neddylation modification is catalyzed by a neddylation activating enzyme (NAE, E1), an E2 conjugating enzyme and an E3 ligase. In various types of human cancers, the neddylation pathway was abnormally activated. Our previous study validated that neddylation E2 UBE2F is a promising lung cancer target. However, although NAE inhibitor MLN4924/pevonedistat is currently in few clinical trials for anticancer application, no small molecule was reported that targets UBE2F. Here, we report, for the first time, the discovery, via structure-based virtual screen and chemical optimization, of such a small molecule, designated as HA-9104. HA-9104 binds to UBE2F, reduces its protein levels, and consequently inhibits cullin-5 neddylation to inactivate CRL5 (cullin-RING ligase-5) ligase, leading to accumulation of CRL5 substrate, NOXA, to induce apoptosis. Moreover, HA-9104 appears to form the DNA adduct via its 7-azaindole group to induce DNA damage and G2/M arrest. Biologically, HA-9104 effectively suppresses the growth and survival of lung cancer cells and confers radiosensitization in both in vitro cell culture and in vivo xenograft tumor models. Taken together, our study discovered a small molecule HA-9104 that targets the UBE2F-CRL5 axis with anticancer activity alone or in combination with radiation.

2022-10-19 | PXD036191 | Pride

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