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Cryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylation

ABSTRACT: Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1) and other gluconeogenic enzymes. “GID” is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core and supramolecular module along with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex targeting Fbp1. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Mario Oroshi

LAB HEAD: Matthias Mann

PROVIDER: PXD031713 | Pride | 2022-06-01

REPOSITORIES: Pride

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Cryo-EM structures of Gid12-bound GID E3 reveal steric blockade as a mechanism inhibiting substrate ubiquitylation

Project description:Protein degradation, a major eukaryotic response to cellular signals, is subject to numerous layers of regulation. In yeast, the evolutionarily conserved GID E3 ligase mediates glucose-induced degradation of fructose-1,6-bisphosphatase (Fbp1) and other gluconeogenic enzymes. “GID” is a collection of E3 ligase complexes; a core scaffold, RING-type catalytic core and supramolecular module along with interchangeable substrate receptors select targets for ubiquitylation. However, knowledge of additional cellular factors directly regulating GID-type E3s remains rudimentary. Here, we structurally and biochemically characterize Gid12 as a modulator of the GID E3 ligase complex targeting Fbp1. Our collection of cryo-EM reconstructions shows that Gid12 forms an extensive interface sealing the substrate receptor Gid4 onto the scaffold, and remodeling the degron binding site. Gid12 also sterically blocks a recruited Fbp1 from the ubiquitylation active sites. Our analysis of the role of Gid12 establishes principles that may more generally underlie E3 ligase regulation.

2022-05-29 | PXD028579 | Pride

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

Cryo-EM visualizes substrate channeling and E2 multisite phosphorylation configuring ubiquitin ligation by GID/CTLH E3

Project description:Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including for RING-family GID/CTLH E3 ubiquitin ligases and their dedicated E2, Ubc8/UBE2H (yeast/human nomenclature). GID/CTLH-Ubc8/UBE2H-mediated ubiquitylation regulates biological processes ranging from yeast metabolic signaling to human development. Here, cryo-EM, biochemistry, and cell biology reveal exquisitely specific GID/CTLH-Ubc8/UBE2H pairing through an unconventional catalytic assembly and auxiliary interactions 70-100 Å away, mediated by E2 phosphorylation. Rather than dynamic polyelectrostatic interactions reported for other ubiquitylation complexes, multiple Ubc8/UBE2H phosphorylation sites within acidic CK2-targeted sequences specifically anchor the E2 C-termini to E3 basic patches. Positions of phospho-dependent interactions relative to the catalytic domains correlate across evolution. Overall, our data illustrate phosphorylation-dependent multivalency establishes a specific E3-E2 partnership, is antagonistic with dephosphorylation, immobilizes the catalytic centers within a flexing GID E3-substrate assembly, and facilitates substrate collision with ubiquitylation active sites.

2023-12-18 | PXD043646 | Pride

1A 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 environmentalconditions. Ubiquitin E3 ligases enablethis response, by facilitatingrapid andreversible changes to protein stability, localization, or interaction partners. In S. cerevisiae, the GID E3 ligase regulatesthe switch from gluconeogenic to glycolytic conditions throughinduction and incorporation of the substrate receptorsubunitGid4, whichpromotes 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,andregulates the ART-Rsp5 pathway. Art2 levels are elevated upon GID10deletion, a crystal structure shows the basis for Gid10-Art2 interactions, andGid10 directs a GID E3 ligase complex to ubiquitinate Art2. We also findthat the GID E3 ligase affects the flux of plasma membrane nutrient transportersduring heat stress. The data revealGID as a system of E3 ligases with metabolic regulatory functions outside of glycolysis and gluconeogenesis, controlled by distinct stress-specific substrate receptors.

2022-03-02 | PXD030902 | Pride

Genome-wide screening of Mycobacterium tuberculosis-infected macrophages identified the GID/CTLH complex as a determinant of intracellular bacterial growth

Project description:The eukaryotic GID/CTLH complex is a highly conserved E3 ubiquitin ligase involved in a broad range of biological processes. However, a role of this complex in host antimicrobial defenses has not been described. We exploited Mycobacterium tuberculosis (Mtb) induced cytotoxicity in macrophages in a FACS based CRISPR genetic screen to identify host determinants of intracellular Mtb growth restriction. Our screen identified 5 (GID8, YPEL5, WDR26, UBE2H, MAEA) of the 10 predicted members of the GID/CTLH complex as determinants of intracellular growth of both Mtb and Salmonella serovar Typhimurium. We show that the antimicrobial properties of the GID/CTLH complex knockdown macrophages are mediated by enhanced GABAergic signaling, activated AMPK, increased autophagic flux and resistance to cell death. Meanwhile, Mtb isolated from GID/CTLH knockdown macrophages are nutritionally starved and oxidatively stressed. Our study identifies the GID/CTLH complex activity as broadly suppressive of host antimicrobial responses against intracellular bacterial infections.

2024-05-08 | GSE267063 | GEO

Doa10/MARCH6 architecture interconnects E3 ligase activity with lipid-binding transmembrane channel to regulate SQLE

Project description:Transmembrane E3 ligases play crucial roles in homeostasis. Much protein and organelle quality control, and metabolic regulation, are determined by ER-resident MARCH6 E3 ligases, including Doa10 in yeast. Here, we present Doa10/MARCH6 structural analysis by cryo-EM and AlphaFold predictions, and a structure-based mutagenesis campaign. The majority of MARCH6/Doa10 adopts a unique circular structure within the membrane. This channel is established by a lipid-binding scaffold, gated by a flexible helical bundle. The ubiquitylation active site is positioned over the channel by connections between the cytosolic E3 ligase RING domain and the membrane-spanning scaffold and gate. Assaying 95 MARCH6 variants for effects on stability of the well-characterized substrate SQLE, which regulates cholesterol levels, revealed crucial roles of the gated channel and RING domain consistent with AlphaFold-models of substrate-engaged and ubiquitylation complexes. SQLE degradation further depends on connections between the channel and RING domain, and lipid binding sites, revealing how interconnected MARCH6/Doa10 elements could orchestrate metabolic signals, substrate binding, and E3 ligase activity.

2024-01-26 | PXD047499 | Pride

CUL4B ChIP-sequencing in non-treated MDA-MB-231 cells

Project description:Cullin 4B (CUL4B) is a scaffold protein of the CUL4B-Ring E3 ligase (CRL4B) complex. Here, we found that CRL4B interacted with transcriptional corepressor complexes. Our results supporting CUL4B as a potential target of cancer therapy.

2021-03-25 | GSE124611 | GEO

Dzip3 regulates developmental genes in mouse embryonic stem cells by reorganizing 3D chromatin conformation [ChIP-seq]

Project description:In mouse embryonic stem cells (mES cells), ubiquitylation of histone H2A lysine 119 represses a large number of developmental genes and maintains mES cell pluripotency. It has been suggested that a number of H2A ubiquitin ligases as well as deubiquitylases and related peptide fragments contribute to a delicate balance between self-renewal and multi-lineage differentiation in mES cells. Here, we tested whether known H2A ubiquitin ligases and deubiquitylases are involved in mES cell regulation and discovered that Dzip3, the E3 ligase of H2AK119, represses differentiation-inducible genes as well as Ring1B. The two sets of target genes partially overlapped but hadãdifferent spectra. We found that Dzip3 represses gene expression by orchestrating changes in 3D organization in addition to regulating ubiquitylation of H2A. Our results shed light on the epigenetic mechanism of transcriptional regulation, which depends on 3D chromatin reorganization to regulate mES cell differentiation. Examination of 3 different histone modifications in 1 cell type.

2015-12-08 | E-GEOD-71883 | biostudies-arrayexpress

Sumoylation at Chromatin Governs Coordinated Repression of a Transcriptionnal Program Essential for Cell Growth and Proliferation

Project description:mRNA profiling of WI38 wild-type or overexpressiong the SUMO E3 ligase PIASy in human primary fibroblasts 24h post-infection. The goal of this study is to analyse transcriptional changes in cells over-expressing the SUMO E3 ligase PIASy and to compare them with ChIPseq data for several histones marks and proteins of the SUMO machinery including PIASy

2013-11-25 | E-MEXP-3776 | biostudies-arrayexpress

DIA-based systems biology approach unveils novel E3-dependent responses to a metabolic shift

Project description:Yeast Saccharomyces cerevisiae is a powerful model system for systems-wide biology screens and large-scale proteomics methods. The proteomics community has achieved nearly complete coverage for this organism owing to advances in mass spectrometry. However, it remains challenging to scale the technology for rapid and high-throughput analysis of the yeast proteome to investigate biological pathways on a global scale. Here we describe a systems biology approach employing plate-based sample preparation and rapid, single-run data independent mass spectrometry analysis (DIA). Our approach is straightforward, easy to implement and enables quantitative profiling and comparisons of hundreds of largely covered yeast proteomes in only a few days. We evaluate its capability by characterizing changes in the yeast proteome in response to a variety of stresses commonly used in yeast research, identify distinct responses to each stress, and provide a comprehensive resource of these responses. Using our facile, rapid and robust methodology, we observe many previously characterized stress responses, including carbon source dependent regulation of the GID E3 ligase, an important regulator of cellular metabolism during the switch between gluconeogenic and glycolytic growth conditions. Furthermore, we applied our methodology to search for new regulatory targets of the GID ligase during a metabolic switch. We are able to pinpoint effects of a single deletion or point mutation in the GID complex on the global proteome, and thereby identify and validate novel targets of the GID E3 ligase. Moreover, our approach allowed the identification of targets from multiple cellular pathways that display distinct patterns of regulation.

2020-11-26 | PXD021559 | Pride

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