Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors.

Skraban-Deardorff intellectual disability syndrome-associated mutations in WDR26 impair CTLH E3 complex assembly.

Patients with Skraban-Deardorff syndrome (SKDEAS), a neurodevelopmental syndrome associated with a spectrum of developmental and intellectual delays and disabilities, harbor diverse mutations in WDR26, encoding a subunit of the multiprotein CTLH E3 ubiquitin ligase complex. Structural studies revealed that homodimers of WDR26 bridge two core-CTLH E3 complexes to generate giant, hollow oval-shaped supramolecular CTLH E3 assemblies. Additionally, WDR26 mediates CTLH E3 complex binding to subunit YPEL5 and functions as substrate receptor for the transcriptional repressor HBP1.

Multisite phosphorylation dictates selective E2-E3 pairing as revealed by Ubc8/UBE2H-GID/CTLH assemblies.

Ubiquitylation is catalyzed by coordinated actions of E3 and E2 enzymes. Molecular principles governing many important E3-E2 partnerships remain unknown, including those 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.

Catalysis of non-canonical protein ubiquitylation by the ARIH1 ubiquitin ligase.

Protein ubiquitylation typically involves isopeptide bond formation between the C-terminus of ubiquitin to the side-chain amino group on Lys residues. However, several ubiquitin ligases (E3s) have recently been identified that ubiquitylate proteins on non-Lys residues. For instance, HOIL-1 belongs to the RING-in-between RING (RBR) class of E3s and has an established role in Ser ubiquitylation.

Modular UBE2H-CTLH E2-E3 complexes regulate erythroid maturation.

The development of haematopoietic stem cells into mature erythrocytes - erythropoiesis - is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood.

The ubiquitin ligation machinery in the defense against bacterial pathogens.

The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which-inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes-become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling.

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

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, which resembles an organometallic supramolecular chelate. The Chelator-GID assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites.

Integrative proteomics reveals principles of dynamic phosphosignaling networks in human erythropoiesis.

Human erythropoiesis is an exquisitely controlled multistep developmental process, and its dysregulation leads to numerous human diseases. Transcriptome and epigenome studies provided insights into system-wide regulation, but we currently lack a global mechanistic view on the dynamics of proteome and post-translational regulation coordinating erythroid maturation. We established a mass spectrometry (MS)-based proteomics workflow to quantify and dynamically track 7,400 proteins and 27,000 phosphorylation sites of five distinct maturation stages of in vitro reconstituted erythropoiesis of CD34 HSPCs.

FBXO11-mediated proteolysis of BAHD1 relieves PRC2-dependent transcriptional repression in erythropoiesis.

The histone mark H3K27me3 and its reader/writer polycomb repressive complex 2 (PRC2) mediate widespread transcriptional repression in stem and progenitor cells. Mechanisms that regulate this activity are critical for hematopoietic development but are poorly understood. Here we show that the E3 ubiquitin ligase F-box only protein 11 (FBXO11) relieves PRC2-mediated repression during erythroid maturation by targeting its newly identified substrate bromo adjacent homology domain-containing 1 (BAHD1), an H3K27me3 reader that recruits transcriptional corepressors.

Interconversion between Anticipatory and Active GID E3 Ubiquitin Ligase Conformations via Metabolically Driven Substrate Receptor Assembly.

Cells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, in Saccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase GID recruiting N termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryoelectron microscopy define molecular underpinnings of glucose-induced degradation.

ARIH2 Is a Vif-Dependent Regulator of CUL5-Mediated APOBEC3G Degradation in HIV Infection.

The Cullin-RING E3 ligase (CRL) family is commonly hijacked by pathogens to redirect the host ubiquitin proteasome machinery to specific targets. During HIV infection, CRL5 is hijacked by HIV Vif to target viral restriction factors of the APOBEC3 family for ubiquitination and degradation. Here, using a quantitative proteomics approach, we identify the E3 ligase ARIH2 as a regulator of CRL5-mediated APOBEC3 degradation.

Coupled monoubiquitylation of the co-E3 ligase DCNL1 by Ariadne-RBR E3 ubiquitin ligases promotes cullin-RING ligase complex remodeling.

Cullin-RING E3 ubiquitin ligases (CRLs) are large and diverse multisubunit protein complexes that contribute to about one-fifth of ubiquitin-dependent protein turnover in cells. CRLs are activated by the attachment of the ubiquitin-like protein neural precursor cell expressed, developmentally down-regulated 8 (NEDD8) to the cullin subunits. This cullin neddylation is essential for a plethora of CRL-regulated cellular processes and is vital for life.

Blocking an N-terminal acetylation-dependent protein interaction inhibits an E3 ligase.

N-terminal acetylation is an abundant modification influencing protein functions. Because ∼80% of mammalian cytosolic proteins are N-terminally acetylated, this modification is potentially an untapped target for chemical control of their functions. Structural studies have revealed that, like lysine acetylation, N-terminal acetylation converts a positively charged amine into a hydrophobic handle that mediates protein interactions; hence, this modification may be a druggable target.

Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3.

Ubiquitin signalling is a fundamental eukaryotic regulatory system, controlling diverse cellular functions. A cascade of E1, E2, and E3 enzymes is required for assembly of distinct signals, whereas an array of deubiquitinases and ubiquitin-binding modules edit, remove, and translate the signals. In the centre of this cascade sits the E2-conjugating enzyme, relaying activated ubiquitin from the E1 activating enzyme to the substrate, usually via an E3 ubiquitin ligase.

Two Distinct Types of E3 Ligases Work in Unison to Regulate Substrate Ubiquitylation.

Hundreds of human cullin-RING E3 ligases (CRLs) modify thousands of proteins with ubiquitin (UB) to achieve vast regulation. Current dogma posits that CRLs first catalyze UB transfer from an E2 to their client substrates and subsequent polyubiquitylation from various linkage-specific E2s. We report an alternative E3-E3 tagging cascade: many cellular NEDD8-modified CRLs associate with a mechanistically distinct thioester-forming RBR-type E3, ARIH1, and rely on ARIH1 to directly add the first UB and, in some cases, multiple additional individual monoubiquitin modifications onto CRL client substrates.

The Fanconi Anemia DNA Repair Pathway Is Regulated by an Interaction between Ubiquitin and the E2-like Fold Domain of FANCL.

The Fanconi Anemia (FA) DNA repair pathway is essential for the recognition and repair of DNA interstrand crosslinks (ICL). Inefficient repair of these ICL can lead to leukemia and bone marrow failure. A critical step in the pathway is the monoubiquitination of FANCD2 by the RING E3 ligase FANCL.

TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes.

RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes.

Structure of HHARI, a RING-IBR-RING ubiquitin ligase: autoinhibition of an Ariadne-family E3 and insights into ligation mechanism.

A distinct mechanism for ubiquitin (Ub) ligation has recently been proposed for the RING1-IBR-RING2 (RBR) family of E3s: an N-terminal RING1 domain recruits a thioester-linked intermediate complex between Ub and the E2 UbcH7, and a structurally distinct C-terminal RING2 domain displays a catalytic cysteine required for Ub ligation. To obtain insights into RBR E3s, we determined the crystal structure of the human homolog of Ariadne (HHARI), which reveals the individual RING1, IBR, and RING2 domains embedded in superdomains involving sequences specific to the Ariadne RBR subfamily. The central IBR is flanked on one side by RING1, which is exposed and binds UbcH7.

C. elegans ring finger protein RNF-113 is involved in interstrand DNA crosslink repair and interacts with a RAD51C homolog.

The Fanconi anemia (FA) pathway recognizes interstrand DNA crosslinks (ICLs) and contributes to their conversion into double-strand DNA breaks, which can be repaired by homologous recombination. Seven orthologs of the 15 proteins associated with Fanconi anemia are functionally conserved in the model organism C. elegans.

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells.

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway.

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair.

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage.