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The mitochondrial antiviral-signaling protein (MAVS), a core adaptor protein in the retinoic-acid-inducible gene-I-like receptors (RLRs)-MAVS pathway, has been demonstrated to play an important role in antiviral immune response and tumor immunology. Previous studies revealed that ubiquitylation is a key mechanism in the regulation of the RLRs-MAVS axis and immune response. Multiple E3 ubiquitin ligases and deubiquitinating enzymes control MAVS ubiquitylation and changes in MAVS function. In this review, we summarize the biological function of ubiquitylation in MAVS-related signaling and provide new insight into immunotherapy approaches that target MAVS.

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Targeted protein degradation (TPD) has opened the door for drugging transcriptional regulators, yet the number of proteins targeted and E3 ligases utilized remain limited. Here, we highlight UBR5 and propose multiple strategies by which this E3 ligase could be modulated to drive degradation of key transcriptional targets implicated in disease.

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We used the deep learning tool ProteinMPNN to redesign ubiquitin (Ub) as a specific and functionally stimulating/enhancing binder of the Rsp5 E3 ligase. We generated 20 extensively mutated─up to 37 of 76 residues─recombinant Ub variants (UbVs), named R1 to R20, displaying well-folded structures and high thermal stabilities. These UbVs can also form stable complexes with Rsp5, as predicted using AlphaFold2. Three of the UbVs bound to Rsp5 with low micromolar affinity, with R4 and R12 effectively enhancing the Rsp5 activity six folds. AlphaFold2 predicts that R4 and R12 bind to Rsp5's exosite in an identical manner to the Rsp5-Ub template, thereby allosterically activating Rsp5-Ub thioester formation. Thus, we present a virtual solution for rapidly and cost-effectively designing UbVs as functional modulators of Ub-related enzymes.

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The attachment of ubiquitin to a substrate (ubiquitination or ubiquitylation) impacts its lifetime and regulates its function within the cell. Several classes of enzymes oversee the attachment of ubiquitin to the substrate: an E1 activating enzyme that makes ubiquitin chemically susceptible prior to the following stages of conjugation and ligation, respectively mediated by E2 conjugating enzymes (E2s) and E3 ligases (E3s). Around 40 E2s and more than 600 E3s are encoded in the human genome, and their combinatorial and cooperative behaviour dictate the tight specificity necessary for the regulation of thousands of substrates. The removal of ubiquitin is orchestrated by a network of about 100 deubiquitylating enzymes (DUBs). Many cellular processes are tightly controlled by ubiquitylation, which is essential in maintaining cellular homeostasis. Because of the fundamental role(s) of ubiquitylation, there is an interest in better understanding the function and specificity of the ubiquitin machinery. Since 2014, an expanding array of Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) Mass Spectrometry (MS) assays have been developed to systematically characterise the activity of a variety of ubiquitin enzymes in vitro. Here we recapitulate how MALDI-TOF MS aided the in vitro characterization of ubiquitin enzymes and the discovery of new and unexpected of E2s and DUBs functions. Given the versatility of the MALDI-TOF MS approach, we foreseen the use of this technology to further expand our understanding of ubiquitin and ubiquitin-like enzymes.

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Alzheimer's disease (AD) is one of the most common progressive neurodegenerative disorders that cause deterioration of cognitive functions. Recent studies suggested that the accumulation of inflammatory molecules and impaired protein degradation mechanisms might both play a critical role in the progression of AD. Autophagy is a major protein degradation pathway that can be controlled by several HECT-E3 ligases, which then regulates the expression of inflammatory molecules. E3 ubiquitin ligases are known to be upregulated in several neurodegenerative diseases. Here, we studied the expressional change of HECT-E3 ligase using M01 on autophagy and inflammasome pathways in the context of AD pathogenesis. Our results demonstrated that the M01 treatment reversed the working memory deficits in 3xTg-AD mice when examined with the T-maze and reversal learning with the Morris water maze. Additionally, the electrophysiology recordings indicated that M01 treatment enhanced the long-term potentiation in the hippocampus of 3xTg-AD mice. Together with the improved memory performance, the expression levels of the NLRP3 inflammasome protein were decreased. On the other hand, autophagy-related molecules were increased in the hippocampus of 3xTg-AD mice. Furthermore, the protein docking analysis indicated that the binding affinity of M01 to the WWP1 and NEDD4 E3 ligases was the highest among the HECT family members. The western blot analysis also confirmed the decreased expression level of NEDD4 protein in the M01-treated 3xTg-AD mice. Overall, our results demonstrate that the modulation of HECT-E3 ligase expression level can be used as a strategy to treat early memory deficits in AD by decreasing NLRP3 inflammasome molecules and increasing the autophagy pathway.

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To overcome the lack of specificity of cancer therapeutics and thus create a more potent and effective treatment, we developed a novel chimeric protein, IL2-Smurf2. Here, we describe the production of this chimeric IL2-Smurf2 protein and its variants, with inactive or over-active killing components. Using Western blots, we demonstrated the chimeric protein's ability to specifically enter target cells alone. After entering the cells, the protein showed biological activity, causing cell death that was not seen with an inactive variant, and that was shown to be apoptotic. The chimeric protein also proved to be active as an E3 ligase, as demonstrated by testing total ubiquitination levels along with targeted ubiquitination for degradation. Finally, we tested IL2-Smurf2 and its variants in an in vivo mouse model of leukemia and demonstrated its potential as a drug for the targeted treatment of cancer cells. In the course of this work, we established for the first time the feasibility of the use of Smurf2 as a killing component in chimeric targeting proteins. Utilizing the IL2 cytokine to target cells overexpressing IL-2R and Smurf2 to cause protein degradation, we were able to produce a chimeric protein with dual functionality which causes targeted cell death.

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WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) is a member of C2-WW-HECT E3 ligase family. Although it may execute carcinostatic actions in some scenarios, WWP1 functions as an oncoprotein under most circumstances. Here, we comprehensively review reports on regulation of WWP1 and its roles in tumorigenesis. We summarize the WWP1-mediated ubiquitinations of diverse proteins and the signaling pathways they involved, as well as the mechanisms how they affect cancer formation and progression. According to our analysis of database, in combination with previous reports, we come to a conclusion that WWP1 expression is augmented in various cancers. Gene amplification, as well as expression regulation mediated by molecules such as non-coding RNAs, may account for the increased mRNA level of WWP1. Regulation of enzymatic activity is another important facet to upregulate WWP1-mediated ubiquitinations. Based on the published data, we conclude that WWP1 employs interactions between multiple domains to autoinhibit its polyubiquitination activity in a steady state. Association of some substrates can partially release certain autoinhibition-related domains and make WWP1 have a moderate activity of polyubiquitination. Some cancer-related mutations can fully disrupt the inhibitory interactions and make WWP1 hyperactive. High expression level or hyperactivation of WWP1 may abnormally enhance polyubiquitinations of some oncoproteins or tumor suppressors, such as ΔNp63α, PTEN and p27, and ultimately promote cell proliferation, survival, migration and invasion in tumorigenesis. Given the dysregulation and oncogenic functions of WWP1 in some cancer types, it is promising to explore some therapeutic inhibitors to tune down its activity.

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Plant-parasitic nematodes secrete effectors that manipulate plant cell morphology and physiology to achieve host invasion and establish permanent feeding sites. Effectors from the highly expanded SPRYSEC (SPRY domain with a signal peptide for secretion) family in potato cyst nematodes have been implicated in activation and suppression of plant immunity, but the mechanisms underlying these activities remain largely unexplored. To study the host mechanisms used by SPRYSEC effectors, we identified plant targets of GpRbp-1 from the potato cyst nematode Globodera pallida. Here, we show that GpRbp-1 interacts in yeast and in planta with a functional potato homologue of the Homology to E6-AP C-Terminus (HECT)-type ubiquitin E3 ligase UPL3, which is located in the nucleus. Potato lines lacking StUPL3 are not available, but the Arabidopsis mutant upl3-5 displaying a reduced UPL3 expression showed a consistently small but not significant decrease in susceptibility to cyst nematodes. We observed a major impact on the root transcriptome by the lower levels of AtUPL3 in the upl3-5 mutant, but surprisingly only in association with infections by cyst nematodes. To our knowledge, this is the first example that a HECT-type ubiquitin E3 ligase is targeted by a pathogen effector and that a member of this class of proteins specifically regulates gene expression under biotic stress conditions. Together, our data suggest that GpRbp-1 targets a specific component of the plant ubiquitination machinery to manipulate the stress response in host cells.

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The causative agent of Legionnaires' disease, Legionella pneumophila, delivers more than 330 virulent effectors to its host to establish an intracellular membrane-bound organelle called the Legionella containing vacuole. Among the army of Legionella effectors, SidC and its paralog SdcA have been identified as novel bacterial ubiquitin (Ub) E3 ligases. To gain insight into the molecular mechanism of SidC/SdcA as Ub ligases, we determined the crystal structures of a binary complex of the N-terminal catalytic SNL domain of SdcA with its cognate E2 UbcH5C and a ternary complex consisting of the SNL domain of SidC with the Ub-linked E2 UbcH7. These two structures reveal the molecular determinants governing the Ub transfer cascade catalyzed by SidC. Together, our data support a common mechanism in the Ub transfer cascade in which the donor Ub is immobilized with its C-terminal tail locked in an extended conformation, priming the donor Ub for catalysis.

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Ubiquitination, the covalent attachment of ubiquitin to proteins, by E3 ligases of the HECT (homologous to E6AP C terminus) family is critical in controlling diverse physiological pathways. Stringent control of HECT E3 ligase activity and substrate specificity is essential for cellular health, whereas deregulation of HECT E3s plays a prominent role in disease. The cell employs a wide variety of regulatory mechanisms to control HECT E3 activity and substrate specificity. Here, we summarize the current understanding of these regulatory mechanisms that control HECT E3 function. Substrate specificity is generally determined by interactions of adaptor proteins with domains in the N-terminal extensions of HECT E3 ligases. These N-terminal domains have also been found to interact with the HECT domain, resulting in the formation of inhibitory conformations. In addition, catalytic activity of the HECT domain is commonly regulated at the level of E2 recruitment and through HECT E3 oligomerization. The previously mentioned regulatory mechanisms can be controlled through protein-protein interactions, post-translational modifications, the binding of calcium ions, and more. Functional activity is determined not only by substrate recruitment and catalytic activity, but also by the type of ubiquitin polymers catalyzed to the substrate. While this is often determined by the specific HECT member, recent studies demonstrate that HECT E3s can be modulated to alter the type of ubiquitin polymers they catalyze. Insight into these diverse regulatory mechanisms that control HECT E3 activity may open up new avenues for therapeutic strategies aimed at inhibition or enhancement of HECT E3 function in disease-related pathways.

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HECT E3 ubiquitin ligases are responsible for many human disease phenotypes and are promising drug targets; however, screening assays for HECT E3 inhibitors are inherently complex, requiring upstream E1 and E2 enzymes as well as ubiquitin, ATP, and detection reagents. Intermediate ubiquitin thioesters and a complex mixture of polyubiquitin products provide further opportunities for off-target inhibition and increase the complexity of the assay. UbFluor is a novel ubiquitin thioester that bypasses the E1 and E2 enzymes and undergoes direct transthiolation with HECT E3 ligases. The release of fluorophore upon transthiolation allows fluorescence polarization detection of HECT E3 activity. In the presence of inhibitors, HECT E3 activity is ablated, and thus no reaction and no change in FP are observed. This assay has been adapted for high-throughput screening of small molecules against HECT E3 ligases, and its utility has been proven in the discovery of HECT E3 ligase inhibitors. © 2017 by John Wiley & Sons, Inc.

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INTRODUCTION: The labyrinthine zone of the placenta is where exchange of nutrients and waste occurs between maternal and fetal circulations. Proper development of the placental labyrinth is essential for successful growth of the developing fetus and abnormalities in placental development are associated with intrauterine growth restriction (IUGR), preeclampsia and fetal demise. Our previous studies demonstrate that Hectd1 is essential for development of the junctional and labyrinthine zones of the placenta. Here we further characterize labyrinthine zone defects in the Hectd1 mutant placenta. METHODS: The structure of the mutant placenta was compared to wildtype littermates using histological methods. The expression of cell type specific markers was examined by immunohistochemistry and in situ hybridization. RESULTS: Hectd1 is expressed in the labyrinthine zone throughout development and the protein is enriched in syncytiotrophoblast layer type I cells (SynT-I) and Sinusoidal Trophoblast Giant cells (S-TGCs) in the mature placenta. Mutation of Hectd1 results in pale placentas with frequent hemorrhages along with gross abnormalities in the structure of the labyrinthine zone including a smaller overall volume and a poorly elaborated fetal vasculature that contain fewer fetal blood cells. Examination of molecular markers of labyrinthine trophoblast cell types reveals increased Dlx3 positive cells and Syna positive SynT-I cells, along with decreased Hand1 and Ctsq positive sinusoidal trophoblast giant cells (S-TGCs). DISCUSSION: Together these defects indicate that Hectd1 is required for development of the labyrinthine zonethe mouse placenta.

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The placenta plays a critical role in the growth and survival of the fetus. Here we demonstrate that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain E3 ubiquitin ligase, Hectd1, is essential for development of the mouse placenta. Hectd1 is widely expressed during placentation with enrichment in trophoblast giant cells (TGCs) and other trophoblast-derived cell subtypes in the junctional and labyrinth zones of the placenta. Disruption of Hectd1 results in mid-gestation lethality and intrauterine growth restriction (IUGR). Variable defects in the gross structure of the mutant placenta are found including alterations in diameter, thickness and lamination. The number and nuclear size of TGCs is reduced. Examination of subtype specific markers reveals altered TGC development with decreased expression of Placental lactogen-1 and -2 (Pl1 and Pl2) and increased expression of Proliferin (Plf). Reduced numbers of spongiotrophoblasts and glycogen trophoblasts were also found at the junctional zone of the Hectd1 mutant placenta. Finally, there was an increase in immature uterine natural killer (uNK) cells in the maternal decidua of the Hectd1 mutant placenta. Proliferation and apoptosis are differentially altered in the layers of the placenta with an increase in both apoptosis and proliferation in the maternal decidua, a decrease in proliferation and increase in apoptosis in the labyrinth layer and both unchanged in the junctional zone. Together these data demonstrate that Hectd1 is required for development of multiple cell types within the junctional zone of the placenta.

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