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GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme.

ABSTRACT: How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GID^SR4, which resembles an organometallic supramolecular chelate. The Chelator-GID^SR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

SUBMITTER: Sherpa D

PROVIDER: S-EPMC8189437 | biostudies-literature |

REPOSITORIES: biostudies-literature

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GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

Project description:How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex, mutation of which is Glucose-Induced Degradation deficient, we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryo EM structures show that to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two otherwise functional GID E3s assemble into a 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons and positions Fbp1 so that its protomers are simultaneously ubiquitylated at lysines near its allosteric and substrate binding sites. Significantly, key structural and biochemical features - including capacity for supramolecular assembly - are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical and cell biological data, we propose that higher-order E3 ligase assembly generally underlies multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

2021-04-28 | PXD024462 | Pride

The yeast GID complex, a novel ubiquitin ligase (E3) involved in the regulation of carbohydrate metabolism.

Project description:Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.

| S-EPMC2488282 | biostudies-literature

Structural Insights into the Ubiquitylation Strategy of the Oligomeric CRL2FEM1B E3 Ubiquitin Ligase

Project description:Cullin-RING E3 ubiquitin ligase (CRL) family members play critical roles in numerous biological processes and diseases including cancer and Alzheimer's disease. Oligomerization of CRLs has been reported to be crucial for the regulation of their activities. However, the structural basis for its regulation and mechanism of its oligomerization are not fully known. Here, we present cryo-EM structures of oligomeric CRL2FEM1B in its unneddylated state, neddylated state in complex with BEX2 as well as neddylated state in complex with FNIP1/FLCN. These structures reveal that asymmetric dimerization of N8-CRL2FEM1B is critical for the ubiquitylation of BEX2 while FNIP1/FLCN is ubiquitylated by monomeric CRL2FEM1B. Our data present an example of the asymmetric homo-dimerization of CRL. Taken together, this study sheds light on the ubiquitylation strategy of oligomeric CRL2FEM1B according to substrates with different scales.

| S-SCDT-10_1038-S44318-024-00047-Y | biostudies-other

The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation.

Project description:In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.

| S-EPMC6037477 | biostudies-literature

A human ubiquitin conjugating enzyme (E2)-HECT E3 ligase structure-function screen.

Project description:Here we describe a systematic structure-function analysis of the human ubiquitin (Ub) E2 conjugating proteins, consisting of the determination of 15 new high-resolution three-dimensional structures of E2 catalytic domains, and autoubiquitylation assays for 26 Ub-loading E2s screened against a panel of nine different HECT (homologous to E6-AP carboxyl terminus) E3 ligase domains. Integration of our structural and biochemical data revealed several E2 surface properties associated with Ub chain building activity; (1) net positive or neutral E2 charge, (2) an "acidic trough" located near the catalytic Cys, surrounded by an extensive basic region, and (3) similarity to the previously described HECT binding signature in UBE2L3 (UbcH7). Mass spectrometry was used to characterize the autoubiquitylation products of a number of functional E2-HECT pairs, and demonstrated that HECT domains from different subfamilies catalyze the formation of very different types of Ub chains, largely independent of the E2 in the reaction. Our data set represents the first comprehensive analysis of E2-HECT E3 interactions, and thus provides a framework for better understanding the molecular mechanisms of ubiquitylation.

| S-EPMC3412965 | biostudies-literature

Structural insights into the ubiquitylation strategy of the oligomeric CRL2<sup>FEM1B</sup> E3 ubiquitin ligase.

| S-EPMC10943247 | biostudies-literature

A GID E3 ligase assembly ubiquitinates an Rsp5 E3 adaptor and regulates plasma membrane transporters

Project description:Cells rapidly remodel their proteomes to align their cellular metabolism to environmental conditions. Ubiquitin E3 ligases enable this response, by facilitating rapid and reversible changes to protein stability, localization, or interaction partners. In S. cerevisiae, the GID E3 ligase regulates the switch from gluconeogenic to glycolytic conditions through induction and incorporation of the substrate receptor subunit Gid4, which promotes the degradation of gluconeogenic enzymes. Here, we show an alternative substrate receptor, Gid10, which is induced in response to changes in temperature, osmolarity and nutrient availability, regulates the ART-Rsp5 ubiquitin ligase pathway, a component of plasma membrane quality control. Proteomic studies reveal that the levels of the adaptor protein Art2 are elevated upon GID10 deletion. A crystal structure shows the basis for Gid10-Art2 interactions, and we demonstrate that Gid10 directs a GID E3 ligase complex to ubiquitinate Art2. Our data suggest that the GID E3 ligase affects Art2-dependent amino acid transport. This study reveals GID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress-specific substrate receptors.

| S-SCDT-EMBOR-2021-53835V1 | biostudies-other

The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

Project description:The tumor suppressor CYLD is a deubiquitinating enzyme that suppresses polyubiquitin-dependent signaling pathways, including the proinflammatory and cell growth-promoting NF-?B pathway. Missense mutations in the CYLD gene are present in individuals with syndromes such as multiple familial trichoepithelioma (MFT), but the pathogenic roles of these mutations remain unclear. Recent studies have shown that CYLD interacts with a RING finger domain protein, mind bomb homologue 2 (MIB2), in the regulation of NOTCH signaling. However, whether MIB2 is an E3 ubiquitin ligase that acts on CYLD is unknown. Here, using the cell-free-based AlphaScreen and pulldown assays to detect protein-protein interactions, along with immunofluorescence assays and murine Mib2 knockout cells and animals, we demonstrate that MIB2 promotes proteasomal degradation of CYLD and enhances NF-?B signaling. Of note, arthritic inflammation was suppressed in Mib2-deficient mice. We further observed that the ankyrin repeat in MIB2 interacts with the third CAP domain in CYLD and that MIB2 catalyzes Lys-48-linked polyubiquitination of CYLD at Lys-338 and Lys-530. MIB2-dependent CYLD degradation activated NF-?B signaling via tumor necrosis factor alpha (TNF?) stimulation and the linear ubiquitination assembly complex (LUBAC). Mib2-knockout mice had reduced serum interleukin-6 (IL-6) and exhibited suppressed inflammatory responses in the K/BxN serum-transfer arthritis model. Interestingly, MIB2 significantly enhanced the degradation of a CYLDP904L variant identified in an individual with MFT, although the molecular pathogenesis of the disease was not clarified here. Together, these results suggest that MIB2 enhances NF-?B signaling in inflammation by promoting the ubiquitin-dependent degradation of CYLD.

| S-EPMC6755803 | biostudies-literature

Structural Diversity of Ubiquitin E3 Ligase

Project description:The post-translational modification of proteins regulates many biological processes. Their dysfunction relates to diseases. Ubiquitination is one of the post-translational modifications that target lysine residue and regulate many cellular processes. Three enzymes are required for achieving the ubiquitination reaction: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). E3s play a pivotal role in selecting substrates. Many structural studies have been conducted to reveal the molecular mechanism of the ubiquitination reaction. Recently, the structure of PCAF_N, a newly categorized E3 ligase, was reported. We present a review of the recent progress toward the structural understanding of E3 ligases.

| S-EPMC8586995 | biostudies-literature

Ubiquitin E3 ligase FIEL1 regulates fibrotic lung injury through SUMO-E3 ligase PIAS4.

Project description:The E3 small ubiquitin-like modifier (SUMO) protein ligase protein inhibitor of activated STAT 4 (PIAS4) is a pivotal protein in regulating the TGF? pathway. In this study, we discovered a new protein isoform encoded by KIAA0317, termed fibrosis-inducing E3 ligase 1 (FIEL1), which potently stimulates the TGF? signaling pathway through the site-specific ubiquitination of PIAS4. FIEL1 targets PIAS4 using a double locking mechanism that is facilitated by the kinases PKC? and GSK3?. Specifically, PKC? phosphorylation of PIAS4 and GSK3? phosphorylation of FIEL1 are both essential for the degradation of PIAS4. FIEL1 protein is highly expressed in lung tissues from patients with idiopathic pulmonary fibrosis (IPF), whereas PIAS4 protein levels are significantly reduced. FIEL1 overexpression significantly increases fibrosis in a bleomycin murine model, whereas FIEL1 knockdown attenuates fibrotic conditions. Further, we developed a first-in-class small molecule inhibitor toward FIEL1 that is highly effective in ameliorating fibrosis in mice. This study provides a basis for IPF therapeutic intervention by modulating PIAS4 protein abundance.

| S-EPMC4886359 | biostudies-literature

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