Identifying a Ubiquitinated Adaptor Protein by a Viral E3 Ligase Through Co-immunoprecipitation.

Co-immunoprecipitation is a technique widely utilized to isolate protein complexes and study protein-protein interactions. Ubiquitinated proteins could be identified by combining co-immunoprecipitation with SDS-PAGE followed by immunoblotting. In this chapter, we use Herpes Simplex Virus 1 immediate-early protein ICP0-mediated polyubiquitination of p50 as an example to describe the method to identify a ubiquitinated adaptor protein by a viral E3 ligase by co-immunoprecipitation.

Ubiquitination regulates autophagy in cancer: simple modifications, promising targets.

Autophagy is an important lysosomal degradation process that digests and recycles bio-molecules, protein or lipid aggregates, organelles, and invaded pathogens. Autophagy plays crucial roles in regulation of metabolic and oxidative stress and multiple pathological processes. In cancer, the role of autophagy is dual and paradoxical. Ubiquitination has been identified as a key regulator of autophagy that can influence various steps in the autophagic process, with autophagy-related proteins being targeted for ubiquitination, thus impacting cancer progression and the effectiveness of therapeutic interventions. This review will concentrate on mechanisms underlying autophagy, ubiquitination, and their interactions in cancer, as well as explore the use of drugs that target the ubiquitin-proteasome system (UPS) and ubiquitination process in autophagy as part of cancer therapy.

MpPUB9, a U-box E3 ubiquitin ligase, acts as a positive regulator by promoting the turnover of MpEXO70.1 under high salinity in Marchantia polymorpha.

Marchantia polymorpha, occupying a basal position in the monophyletic assemblage of land plants, displays a notable expansion of plant U-box (PUB) proteins compared with those in animals. We elucidated the roles of MpPUB9 in regulating salt stress tolerance in M. polymorpha. MpPUB9 expression was rapidly induced by high salinity and dehydration. MpPUB9 possessed an intact U-box domain in the N-terminus. MpPUB9-Citrine localized to punctate structures and was peripherally associated with microsomal membranes. Phenotypic analyses demonstrate that the hyponastic and epinastic thallus growth phenotypes, which were induced by the overexpression and suppression of MpPUB9, may provoke salt stress-resistant and -susceptible phenotypes, respectively. MpPUB9 was also found to directly interact with the exocyst protein MpEXO70.1, leading to its ubiquitination. Under high-salinity conditions, though the stability of MpPUB9 was dramatically increased, MpEXO70.1 showed slightly faster turnover rates. Transcriptome analyses showed that salt treatment and the overexpression of MpPUB9 co-upregulated the genes related to the modulation of H2O2 and cell wall organization. Overall, our results suggest that MpPUB9 plays a crucial role in the positive regulation of salt stress tolerance, resulting from its interaction with MpEXO70.1 and modulating turnover of the protein under high-salt conditions via the coordination of UPS with autophagy.

Tsg101 UEV Interaction with Nedd4 HECT Relieves E3 Ligase Auto-Inhibition, Promoting HIV-1 Assembly and CA-SP1 Maturation Cleavage.

Tsg101, a component of the endosomal sorting complex required for transport (ESCRT), is responsible for recognition of events requiring the machinery, as signaled by cargo tagging with ubiquitin (Ub), and for recruitment of downstream acting subunits to the site. Although much is known about the latter function, little is known about its role in the earlier event. The N-terminal domain of Tsg101 is a structural homologue of Ub conjugases (E2 enzymes) and the protein associates with Ub ligases (E3 enzymes) that regulate several cellular processes including virus budding. A pocket in the domain recognizes a motif, PT/SAP, that permits its recruitment. PT/SAP disruption makes budding dependent on Nedd4L E3 ligases. Using HIV-1 encoding a PT/SAP mutation that makes budding Nedd4L-dependent, we identified as critical for rescue the residues in the catalytic (HECT) domain of the E3 enzyme that lie in proximity to sites in Tsg101 that bind Ub non-covalently. Mutation of these residues impaired rescue by Nedd4L but the same mutations had no apparent effect in the context of a Nedd4 isomer, Nedd4-2s, whose N-terminal (C2) domain is naturally truncated, precluding C2-HECT auto-inhibition. Surprisingly, like small molecules that disrupt Tsg101 Ub-binding, small molecules that interfered with Nedd4 substrate recognition arrested budding at an early stage, supporting the conclusion that Tsg101-Ub-Nedd4 interaction promotes enzyme activation and regulates Nedd4 signaling for viral egress. Tsg101 regulation of E3 ligases may underlie its broad ability to function as an effector in various cellular activities, including viral particle assembly and budding.

Rift Valley fever virus coordinates the assembly of a programmable E3 ligase to promote viral replication.

Viruses encode strategies to degrade cellular proteins to promote infection and pathogenesis. Here, we revealed that the non-structural protein NSs of Rift Valley fever virus forms a filamentous E3 ligase to trigger efficient degradation of targeted proteins. Reconstitution in vitro and cryoelectron microscopy analysis with the 2.9-Å resolution revealed that NSs forms right-handed helical fibrils. The NSs filamentous oligomers associate with the cellular FBXO3 to form a remodeled E3 ligase. The NSs-FBXO3 E3 ligase targets the cellular TFIIH complex through the NSs-P62 interaction, leading to ubiquitination and proteasome-dependent degradation of the TFIIH complex. NSs-FBXO3-triggered TFIIH complex degradation resulted in robust inhibition of antiviral immunity and promoted viral pathogenesis in vivo. Furthermore, it is demonstrated that NSs can be programmed to target additional proteins for proteasome-dependent degradation, serving as a versatile targeted protein degrader. These results showed that a virulence factor forms a filamentous and programmable degradation machinery to induce organized degradation of cellular proteins to promote viral infection.

Mechanism of phospho-Ubls' specificity and conformational changes that regulate Parkin activity.

PINK1 and Parkin mutations lead to the early onset of Parkinson's disease. PINK1-mediated phosphorylation of ubiquitin (Ub), ubiquitin-like protein (NEDD8), and ubiquitin-like (Ubl) domain of Parkin activate autoinhibited Parkin E3 ligase. The mechanism of various phospho-Ubls' specificity and conformational changes leading to Parkin activation remain elusive. Herein, we show that compared to Ub, NEDD8 is a more robust binder and activator of Parkin. Structures and biophysical/biochemical data reveal specific recognition and underlying mechanisms of pUb/pNEDD8 and pUbl domain binding to the RING1 and RING0 domains, respectively. Also, pUb/pNEDD8 binding in the RING1 pocket promotes allosteric conformational changes in Parkin's catalytic domain (RING2), leading to Parkin activation. Furthermore, Parkinson's disease mutation K211N in the RING0 domain was believed to perturb Parkin activation due to loss of pUb binding. However, our data reveal allosteric conformational changes due to N211 that lock RING2 with RING0 to inhibit Parkin activity without disrupting pNEDD8/pUb binding.

UBR-5 and UBE2D mediate timely exit from stem fate via destabilization of poly(A)-binding protein PABP-2 in cell state transition.

UBR5 E3 ligase has been associated with cancer susceptibility and neuronal integrity, with functions in chromatin regulation and proteostasis. However, the functions of ubr5 within animals remain unclear due to lethality in both mammals and flies when disrupted. Using Caenorhabditis elegans, we show that UBR-5 E3 ligase is required for timely exit of stem fate and complete transition into multiple cell type descendants in an ectodermal blast lineage. Animals lacking intact UBR-5 function simultaneously exhibit both stem fate and differentiated fate in the same descendant cells. A functional screen of UBR-5 physical interactors allowed us to identify the UBE2D2/3 E2 conjugase LET-70 working with UBR-5 to exit stem fate. Strikingly, we revealed that another UBR-5 physical interactor, namely the nuclear poly(A)-binding protein PABPN1 ortholog PABP-2, worked antagonistically to UBR-5 and LET-70. Lowering pabp-2 levels restored normal transition of cell state out of stemness and promoted normal cell fusion when either ubr-5 or let-70 UBE2D function was compromised. The UBR-5-LET-70 and PABP-2 switch works independently of the stem pool size determined by pluripotency factors like lin-28. UBR-5 limits PABP-2 protein and reverses the PABP-2-dependent gene expression program including developmental, proteostasis, and innate immunity genes. Loss of ubr-5 rescues the developmental stall when pabp-2 is compromised. Disruption of ubr-5 elevates PABP-2 levels and prolongs expression of ectodermal and muscle stem markers at the transition to adulthood. Additionally, ubr-5 mutants exhibit an extended period of motility during aging and suppress pabp-2-dependent early onset of immobility.

Outer mitochondrial membrane E3 Ub ligase MARCH5 controls de novo peroxisome biogenesis.

We report that the outer mitochondrial membrane (OMM)-associated E3 Ub ligase MARCH5 is vital for generating mitochondria-derived pre-peroxisomes. In human immortalized cells, MARCH5 knockout leads to the accumulation of immature peroxisomes, reduced fatty-acid-induced peroxisomal biogenesis, and abnormal peroxisome biogenesis in MARCH5/Pex14 and MARCH5/Pex3 dko cells. Upon fatty-acid-induced peroxisomal biogenesis, MARCH5 redistributes to peroxisomes, and ubiquitination activity-deficient mutants of MARCH5 accumulate on peroxisomes containing high levels of the OMM protein Tom20 (mitochondria-derived pre-peroxisomes). Similarly, depletion of peroxisome biogenesis factor Pex14 leads to the accumulation of MARCH5- and Tom20-positive pre-peroxisomes, whereas no peroxisomes are detected in MARCH5/Pex14 dko cells. Inconsistent with MARCH5 merely acting as a quality factor, mitochondrial decline is not evident in tested models. Furthermore, reduced expression of peroxisomal proteins is detected in MARCH5-/- cells, whereas some of these proteins are stabilized in peroxisome biogenesis deficiency models lacking MARCH5 expression. Thus, MARCH5 is central for mitochondria-dependent peroxisome biogenesis.

Fragment-based approaches to discover ligands for tumor-specific E3 ligases.

INTRODUCTION: Targeted protein degradation (TPD) has emerged as an innovative therapeutic strategy through selective degradation of specific proteins by harnessing the cellular ubiquitin-proteasome system (UPS), which involves over 600 E3 ubiquitin ligases. Recent proteome profiling reported tumor-specific E3 ligases in human. Development of those tumor-specific E3 ligase ligands would provide a solution for tumor-specific TPD for effective cancer treatment. AREAS COVERED: This review provides a comprehensive list of E3 ligases found only in specific types of tumor from public databases and highlights examples of their ligands discovered through fragment-based approaches. It details their discovery process and potential applications for precise TPD and effective cancer treatments. EXPERT OPINION: Current TPD strategies using proteolysis-targeting chimeras (PROTACs) primarily utilize general E3 ligases, such as CRBN and VHL. Since these E3 ligases demonstrate effective protein degradation activity in most human cell types, CRBN and VHL-based PROTACs can exhibit undesired TPD in off-target tissues, which often leads to the side effects. Therefore, developing tumor-specific E3 ligase ligands can be crucial for effective cancer treatments. Fragment-based ligand discovery (FBLD) approaches would accelerate the identification of these tumor-specific E3 ligase ligands and associated PROTACs, thereby advancing the field of targeted cancer therapies.

Dysfunction of Telomeric Cdc13-Stn1-Ten1 Simultaneously Activates DNA Damage and Spindle Checkpoints.

Telomeres, the ends of eukaryotic linear chromosomes, are composed of repeated DNA sequences and specialized proteins, with the conserved telomeric Cdc13/CTC1-Stn1-Ten1 (CST) complex providing chromosome stability via telomere end protection and the regulation of telomerase accessibility. In this study, SIZ1, coding for a SUMO E3 ligase, and TOP2 (a SUMO target for Siz1 and Siz2) were isolated as extragenic suppressors of Saccharomyces cerevisiae CST temperature-sensitive mutants. ten1-sz, stn1-sz and cdc13-sz mutants were isolated next due to being sensitive to intracellular Siz1 dosage. In parallel, strong negative genetic interactions between mutants of CST and septins were identified, with septins being noticeably sumoylated through the action of Siz1. The temperature-sensitive arrest in these new mutants of CST was dependent on the G2/M Mad2-mediated and Bub2-mediated spindle checkpoints as well as on the G2/M Mec1-mediated DNA damage checkpoint. Our data suggest the existence of yet unknown functions of the telomeric Cdc13-Stn1-Ten1 complex associated with mitotic spindle positioning and/or assembly that could be further elucidated by studying these new ten1-sz, stn1-sz and cdc13-sz mutants.

MetaDegron: multimodal feature-integrated protein language model for predicting E3 ligase targeted degrons.

Protein degradation through the ubiquitin proteasome system at the spatial and temporal regulation is essential for many cellular processes. E3 ligases and degradation signals (degrons), the sequences they recognize in the target proteins, are key parts of the ubiquitin-mediated proteolysis, and their interactions determine the degradation specificity and maintain cellular homeostasis. To date, only a limited number of targeted degron instances have been identified, and their properties are not yet fully characterized. To tackle on this challenge, here we develop a novel deep-learning framework, namely MetaDegron, for predicting E3 ligase targeted degron by integrating the protein language model and comprehensive featurization strategies. Through extensive evaluations using benchmark datasets and comparison with existing method, such as Degpred, we demonstrate the superior performance of MetaDegron. Among functional features, MetaDegron allows batch prediction of targeted degrons of 21 E3 ligases, and provides functional annotations and visualization of multiple degron-related structural and physicochemical features. MetaDegron is freely available at http://modinfor.com/MetaDegron/. We anticipate that MetaDegron will serve as a useful tool for the clinical and translational community to elucidate the mechanisms of regulation of protein homeostasis, cancer research, and drug development.

The Delayed Turnover of Proteasome Processing of Myocilin upon Dexamethasone Stimulation Introduces the Profiling of Trabecular Meshwork Cells' Ubiquitylome.

Glaucoma is chronic optic neuropathy whose pathogenesis has been associated with the altered metabolism of Trabecular Meshwork Cells, which is a cell type involved in the synthesis and remodeling of the trabecular meshwork, the main drainage pathway of the aqueous humor. Starting from previous findings supporting altered ubiquitin signaling, in this study, we investigated the ubiquitin-mediated turnover of myocilin (MYOC/TIGR gene), which is a glycoprotein with a recognized role in glaucoma pathogenesis, in a human Trabecular Meshwork strain cultivated in vitro in the presence of dexamethasone. This is a validated experimental model of steroid-induced glaucoma, and myocilin upregulation by glucocorticoids is a phenotypic marker of Trabecular Meshwork strains. Western blotting and native-gel electrophoresis first uncovered that, in the presence of dexamethasone, myocilin turnover by proteasome particles was slower than in the absence of the drug. Thereafter, co-immunoprecipitation, RT-PCR and gene-silencing studies identified STUB1/CHIP as a candidate E3-ligase of myocilin. In this regard, dexamethasone treatment was found to downregulate STUB1/CHIP levels by likely promoting its proteasome-mediated turnover. Hence, to strengthen the working hypothesis about global alterations of ubiquitin-signaling, the first profiling of TMCs ubiquitylome, in the presence and absence of dexamethasone, was here undertaken by diGLY proteomics. Application of this workflow effectively highlighted a robust dysregulation of key pathways (e.g., phospholipid signaling, ß-catenin, cell cycle regulation) in dexamethasone-treated Trabecular Meshwork Cells, providing an ubiquitin-centered perspective around the effect of glucocorticoids on metabolism and glaucoma pathogenesis.

Antagonistic regulation of plant NLR-mediated autoimmunity by E3 ligase pairs.

KEY MESSAGE: Two plant U-box E3 ligases, PUB5 and PUB44, antagonistically regulate the NLR receptor SUMM2-mediated autoimmunity in Arabidopsis, indicating a new regulatory mechanism for fine-tuning plant immunity.

Geranylgeranylated SCFFBXO10 regulates selective outer mitochondrial membrane proteostasis and function.

Compartment-specific cellular membrane protein turnover is not well understood. We show that FBXO10, the interchangeable component of the cullin-RING-ligase 1 complex, undergoes lipid modification with geranylgeranyl isoprenoid at cysteine953, facilitating its dynamic trafficking to the outer mitochondrial membrane (OMM). FBXO10 polypeptide lacks a canonical mitochondrial targeting sequence (MTS); instead, its geranylgeranylation at C953 and interaction with two cytosolic factors, cytosolic factor-like δ subunit of type 6 phosphodiesterase (PDE6δ; a prenyl-group-binding protein) and heat shock protein 90 (HSP90; a chaperone), orchestrate specific OMM targeting of prenyl-FBXO10. The FBXO10(C953S) mutant redistributes away from the OMM, impairs mitochondrial ATP production and membrane potential, and increases fragmentation. Phosphoglycerate mutase-5 (PGAM5) was identified as a potential substrate of FBXO10 at the OMM using comparative quantitative proteomics of enriched mitochondria. FBXO10 loss or expression of prenylation-deficient FBXO10(C953S) inhibited PGAM5 degradation, disrupted mitochondrial homeostasis, and impaired myogenic differentiation of human induced pluripotent stem cells (iPSCs) and murine myoblasts. Our studies identify a mechanism for FBXO10-mediated regulation of selective mitochondrial proteostasis potentially amenable to therapeutic intervention.

RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation.

As a RIG-I-like receptor, MDA5 plays a critical role in antiviral innate immunity by acting as a cytoplasmic double-stranded RNA sensor capable of initiating type I interferon pathways. Here, we show that RNF144B specifically interacts with MDA5 and promotes K27/K33-linked polyubiquitination of MDA5 at lysine 23 and lysine 43, which promotes autophagic degradation of MDA5 by p62. Rnf144b deficiency greatly promotes IFN production and inhibits EMCV replication in vivo. Importantly, Rnf144b-/- mice has a significantly higher overall survival rate than wild-type mice upon EMCV infection. Collectively, our results identify RNF144B as a negative regulator of innate antiviral response by targeting CARDs of MDA5 and mediating autophagic degradation of MDA5.

Unveiling the hidden interactome of CRBN molecular glues with chemoproteomics.

Targeted protein degradation and induced proximity refer to strategies that leverage the recruitment of proteins to facilitate their modification, regulation or degradation. As prospective design of glues remains challenging, unbiased discovery methods are needed to unveil hidden chemical targets. Here we establish a high throughput affinity purification mass spectrometry workflow in cell lysates for the unbiased identification of molecular glue targets. By mapping the targets of 20 CRBN-binding molecular glues, we identify 298 protein targets and demonstrate the utility of enrichment methods for identifying novel targets overlooked using established methods. We use a computational workflow to estimate target confidence and perform a biochemical screen to identify a lead compound for the new non-ZF target PPIL4. Our study provides a comprehensive inventory of targets chemically recruited to CRBN and delivers a robust and scalable workflow for identifying new drug-induced protein interactions in cell lysates.

SARS-CoV-2 ORF10 hijacking ubiquitination machinery reveals potential unique drug targeting sites.

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis. CUL2ZYG11B known as the substrate receptor of cullin-2 RING E3 ligase, is bound by SARS-CoV-2 ORF10 to increase its E3 ligase activity, leading to degradation of IFT46, a protein component of the intraflagellar transport (IFT) complex B. This results in dysfunctional cilia, which explains certain symptoms that are specific to COVID-19. However, the precise molecular mechanism of how ORF10 recognizes CUL2ZYG11B remains unknown. Here, we determined the crystal structure of CUL2ZYG11B complexed with the N-terminal extension (NTE) of SARS-CoV-2 ORF10 (2.9 Å). The structure reveals that the ORF10 N-terminal heptapeptide (NTH) mimics the Gly/N-degron to bind CUL2ZYG11B. Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2ZYG11B both in ITC assay and in vivo cells. In addition, we prove that enhancement of CUL2ZYG11B activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2ZYG11B. Finally, we used a Global Protein Stability system to show that the NTH of ORF10 mimics the Gly/N-degron motif, thereby binding competitively to CUL2ZYG11B and inhibiting the degradation of target substrates bearing the Gly/N-degron motif. Overall, this study sheds light on how SARS-CoV-2 ORF10 exploits the ubiquitination machinery for proteasomal degradation, and offers valuable insights for optimizing PROTAC-based drug design based on NTH CUL2ZYG11B interaction, while pinpointing a promising target for the development of treatments for COVID-19.

E3 Ubiquitin Ligases and Their Therapeutic Applications in Cancers: Narrative Review.

E3 ubiquitin ligases are a class of enzymes, essential for maintaining the equilibrium of cells by binding ubiquitin molecules to substrates to mark them for destruction. Since many cancer-related proteins, including both oncogenic and tumor-suppressive ones, are controlled by the ubiquitin-proteasome system, E3 ligases have drawn a great deal of interest as potential targets for the creation of anti-cancer drugs. This is because E3 ligases function as modules that select the substrates that are intended for degradation, giving them the ability to particularly affect proteins that are connected to cancer. Their molecular properties and the ways in which they work serve as the basis for these distinctions. Investment in the creation of bioactive substances that can target E3 ligases is essential given the crucial roles they play in cancer. These substances have the potential to be powerful cancer-fighting tools. In this review, we explore the crucial roles that E3 ligases play in the biology of cancer. We also examine the current bioactive substances that have been created to target different E3 ligases, emphasizing their potential as candidates for treating the cancers.

Additional feedforward mechanism of Parkin activation via binding of phospho-UBL and RING0 in trans.

Loss-of-function Parkin mutations lead to early-onset of Parkinson's disease. Parkin is an auto-inhibited ubiquitin E3 ligase activated by dual phosphorylation of its ubiquitin-like (Ubl) domain and ubiquitin by the PINK1 kinase. Herein, we demonstrate a competitive binding of the phospho-Ubl and RING2 domains towards the RING0 domain, which regulates Parkin activity. We show that phosphorylated Parkin can complex with native Parkin, leading to the activation of autoinhibited native Parkin in trans. Furthermore, we show that the activator element (ACT) of Parkin is required to maintain the enzyme kinetics, and the removal of ACT slows the enzyme catalysis. We also demonstrate that ACT can activate Parkin in trans but less efficiently than when present in the cis molecule. Furthermore, the crystal structure reveals a donor ubiquitin binding pocket in the linker connecting REP and RING2, which plays a crucial role in Parkin activity.

Blockade of forkhead box protein O1 signaling alleviates primary sclerosing cholangitis-induced sarcopenia in mice model.

AIMS: Primary sclerosing cholangitis (PSC) is a cholestatic liver disease that affects the hepatic bile ducts, leading to hepatic inflammation and fibrosis. PSC can also impact skeletal muscle through the muscle-liver axis, resulting in sarcopenia, a complication characterized by a generalized loss of muscle mass and strength. The underlying mechanisms and therapy of PSC-induced sarcopenia are not well understood, but one potential regulator is the transcription factor forkhead box protein O1 (FOXO1), which is involved in the ubiquitin proteasome system. Thus, the aim of this study is to assess the pharmacological potential of FOXO1 inhibition for treating PSC-induced sarcopenia. MATERIALS AND METHODS: To establish diet-induced PSC model, we provided mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 4 weeks. Mice were intramuscularly injected with AS1842856 (AS), a FOXO1 inhibitor, at a dose of 3.5 mg/kg twice a week for last two weeks. C2C12 myotubes with cholic acid (CA) or deoxycholic acid (DCA) were treated with AS. KEY FINDINGS: We observed a decrease in muscle size and performance in DDC-fed mice with upregulated expression of FOXO1 and E3 ligases such as ATROGIN1 and MuRF1. We found that myotube diameter and MyHC protein level were decreased by CA or DCA in C2C12 myotubes, but treatment of AS reversed these reductions. We observed that intramuscular injection of AS effectively mitigates DDC diet-induced sarcopenia in a rodent PSC model. SIGNIFICANCE: Our study suggests that a FOXO1 inhibitor could be a potential leading therapeutic drug for relieving PSC-induced sarcopenia.