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The unfolding dynamics of ubiquitin were studied using a combination of x-ray solution scattering (XSS) and molecular dynamics (MD) simulations. The kinetic analysis of the XSS ubiquitin signals showed that the protein unfolds through a two-state process, independent of the presence of destabilizing salts. In order to characterize the ensemble of unfolded states in atomic detail, the experimental XSS results were used as a constraint in the MD simulations through the incorporation of x-ray scattering derived potential to drive the folded ubiquitin structure toward sampling unfolded states consistent with the XSS signals. We detail how biased MD simulations provide insight into unfolded states that are otherwise difficult to resolve and underscore how experimental XSS data can be combined with MD to efficiently sample structures away from the native state. Our results indicate that ubiquitin samples unfolded in states with a high degree of loss in secondary structure yet without a collapse to a molten globule or fully solvated extended chain. Finally, we propose how using biased-MD can significantly decrease the computational time and resources required to sample experimentally relevant nonequilibrium states. [ABSTRACT FROM AUTHOR]

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields. [ABSTRACT FROM AUTHOR]

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.

Doa10 (MARCHF6 in metazoans) is a large polytopic membrane-embedded E3 ubiquitin ligase in the endoplasmic reticulum (ER) that plays an important role in quality control of cytosolic and ER proteins. Although Doa10 is highly conserved across eukaryotes, it is not understood how Doa10 recognizes its substrates. Here, we define the substrate recognition mechanism of Doa10 by structural and functional analyses on Saccharomyces cerevisiae Doa10 and its model substrates. Cryo-EM analysis shows that Doa10 has unusual architecture with a large lipid-filled central cavity, and its conserved middle domain forms an additional water-filled lateral tunnel open to the cytosol. Our biochemical data and molecular dynamics simulations suggest that the entrance of the substrates degron peptide into the lateral tunnel is required for efficient polyubiquitination. The N- and C-terminal membrane domains of Doa10 seem to form fence-like features to restrict polyubiquitination to those proteins that can access the central cavity and lateral tunnel. Our study reveals how extended hydrophobic sequences at the termini of substrate proteins are recognized by Doa10 as a signal for quality control.

Macroautophagy/autophagy receptors target their substrates to phagophores for subsequent sequestration within autophagosomes. During phagophore membrane expansion in mammalian cells, autophagy receptors simultaneously interact with the ubiquitinated substrates and the LC3/GABARAP proteins on the expanding membrane. In this punctum, we summarize and discuss our recent research progress on synthetic autophagy receptors (AceTACs). The series of AceTACs were designed by engineering the essential interacting domains and motifs of SQSTM1/p62 (sequestosome 1), a major mammalian autophagy receptor. Particularly, we replaced the ubiquitin-associated domain of SQSTM1 with a target-specific antibody, redirecting the bifunctional interactions of wild-type SQSTM1 and directing the degradation target into the autophagy process. We successfully demonstrated the targeted degradation of aggregation-prone proteins using the AceTAC degraders. Moreover, we presented a model system with a guideline to induce targeted degradation of organelles through the autophagy machinery.

The intracellular bacterial pathogen Legionella pneumophila (L.p.) manipulates eukaryotic host ubiquitination machinery to form its replicative vacuole. While nearly 10% of L.p.'s ∼330 secreted effector proteins are ubiquitin ligases or deubiquitinases, a comprehensive measure of temporally resolved changes in the endogenous host ubiquitinome during infection has not been undertaken. To elucidate how L.p. hijacks host cell ubiquitin signaling, we generated a proteome-wide analysis of changes in protein ubiquitination during infection. We discover that L.p. infection increases ubiquitination of host regulators of subcellular trafficking and membrane dynamics, most notably ∼40% of mammalian Ras superfamily small GTPases. We determine that these small GTPases undergo nondegradative ubiquitination at the Legionella-containing vacuole (LCV) membrane. Finally, we find that the bacterial effectors SidC/SdcA play a central role in cross-family small GTPase ubiquitination, and that these effectors function upstream of SidE family ligases in the polyubiquitination and retention of GTPases in the LCV membrane. This work highlights the extensive reconfiguration of host ubiquitin signaling by bacterial effectors during infection and establishes simultaneous ubiquitination of small GTPases across the Ras superfamily as a novel consequence of L.p. infection. Our findings position L.p. as a tool to better understand how small GTPases can be regulated by ubiquitination in uninfected contexts.

Cells entrust ribosomes with the critical task of identifying problematic mRNAs and facilitating their degradation. Ribosomes must communicate when they encounter and stall on an aberrant mRNA, lest they expose the cell to toxic and disease-causing proteins, or they jeopardize ribosome homeostasis and cellular translation. In recent years, ribosomal ubiquitination has emerged as a central signaling step in this process, and proteomic studies across labs and experimental systems show a myriad of ubiquitination sites throughout the ribosome. Work from many labs zeroed in on ubiquitination in one region of the small ribosomal subunit as being functionally significant, with the balance and exact ubiquitination sites determined by stall type, E3 ubiquitin ligases, and deubiquitinases. This review discusses the current literature surrounding ribosomal ubiquitination during translational stress and considers its role in committing translational complexes to decay.

Stress response pathways detect and alleviate adverse conditions to safeguard cell and tissue homeostasis, yet their prolonged activation induces apoptosis and disrupts organismal health1-3. How stress responses are turned off at the right time and place remains poorly understood. Here we report a ubiquitin-dependent mechanism that silences the cellular response to mitochondrial protein import stress. Crucial to this process is the silencing factor of the integrated stress response (SIFI), a large E3 ligase complex mutated in ataxia and in early-onset dementia that degrades both unimported mitochondrial precursors and stress response components. By recognizing bifunctional substrate motifs that equally encode protein localization and stability, the SIFI complex turns off a general stress response after a specific stress event has been resolved. Pharmacological stress response silencing sustains cell survival even if stress resolution failed, which underscores the importance of signal termination and provides a roadmap for treating neurodegenerative diseases caused by mitochondrial import defects.

Protein homeostasis is a tightly conserved process that is regulated through the ubiquitin proteasome system (UPS) in a ubiquitin-independent or ubiquitin-dependent manner. Over the past two decades, the proteasome has become an excellent therapeutic target through inhibition of the catalytic core particle, inhibition of subunits responsible for recognizing and binding ubiquitinated proteins, and more recently, through targeted protein degradation using proteolysis targeting chimeras (PROTACs). The majority of the developed inhibitors of the proteasome's core particle rely on gaining selectivity through binding interactions within the unprimed substrate channel. Although this has allowed for selective inhibitors and chemical probes to be generated for the different proteasome isoforms, much remains unknown about the interactions that could be harnessed within the primed substrate channel to increase potency or selectivity. Herein, we discuss small molecules that interact with the primed substrate pocket and how their differences may give rise to altered activity. Taking advantage of additional interactions with the primed substrate pocket of the proteasome could allow for the generation of improved chemical tools for perturbing or monitoring proteasome 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. [Display omitted] • pNEDD8/NEDD8 shows a higher affinity and activation of Parkin compared to pUb/Ub • Mechanism of specific binding of pUb/pNEDD8/pUbl to Parkin is revealed • Mechanism of pUb/pNEDD8 mediated Parkin activation is revealed • Mechanism of pathogenic mutation K211N mediated loss of Parkin activity is identified Lenka et al. determined the Parkin-pNEDD8 complex structure that reveals the specific recognition of Parkin activators on its two similar basic patches despite the similarities of various ubiquitin-like domains/proteins. They also explain the mechanism behind the reduced Parkin activity of the disease mutant K211N, which is of interest for drug discovery. [ABSTRACT FROM AUTHOR]

Summary The endoplasmic reticulum‐associated degradation (ERAD) system eliminates misfolded and short‐lived proteins to maintain physiological homeostasis in the cell. We have previously reported that ERAD is involved in salt tolerance in Arabidopsis. Given the central role of the phytohormone abscisic acid (ABA) in plant stress responses, we sought to identify potential intersections between the ABA and the ERAD pathways in plant stress response. By screening for the ABA response of a wide array of ERAD mutants, we isolated a gain‐of‐function mutant, doa10a‐1, which conferred ABA hypersensitivity to seedlings. Genetic and biochemical assays showed that DOA10A is a functional E3 ubiquitin ligase which, by acting in concert with specific E2 enzymes, mediates mono‐ubiquitination of the ABA receptor, followed by their relocalization to the plasma membrane. This in turn leads to enhanced ABA perception. In summary, we report here the identification of a novel RING‐type E3 ligase, DOA10A, which regulates ABA perception by affecting the localization and the activity of ABA receptors through their mono‐ubiquitination. [ABSTRACT FROM AUTHOR]

The ubiquitin receptors RPN10 and RPN13 harbor multiple activities including ubiquitin binding; however, solid evidence connecting a particular activity to specific in vivo functions is scarce. Through complementation, the ubiquitin-binding site-truncated Arabidopsis RPN10 (N215) rescued the growth defects of rpn10-2, supporting the idea that the ubiquitin-binding ability of RPN10 is dispensable and N215, which harbors a vWA domain, is fully functional. Instead, a structural role played by RPN10 in the 26S proteasomes is likely vital in vivo. A site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could partially rescue the rpn10-2 growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with rpn10-2 26S proteasomes showed that the loss of RPN10 reduced the abundance of double-capped proteasomes, induced the integration of specific subunit paralogues, and increased the association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. Extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and UCH2-binding sites in the RPN13 C-terminus (246–254). Interestingly, an analysis of homozygous rpn10-2 segregation in a rpn13-1 background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13–RPN2 association in vivo. [ABSTRACT FROM AUTHOR]

Solution nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing protein–protein interactions. NMR-monitored titration proves to be effective in determining dissociation constants, particularly for Kd values in the micromolar to millimolar range. In conventional NMR titrations, planning for optimal titration conditions requires a prior estimate of Kd. In addition, a highly concentrated ligand stock solution is often required, posing challenges when the ligand exhibits limited solubility and stability at elevated concentrations. To overcome these constraints, we propose a simulation-guided "swapping" protocol for NMR titrations. Guided by simulations of the binding curves, two samples, one with zero and the other with maximum ligand concentration, but both containing identical protein concentrations, initiate the titration. Using a "swapping" strategy, intermediate ligand concentrations in between those of the two initial samples are generated without the need of a concentrated ligand stock, while maintaining constant protein concentration. More importantly, this protocol facilitates estimation of Kd by early titration points and allows on-the-fly optimization of the titration points. The proposed approach enhances the efficiency of NMR titrations and provides a straightforward means to optimize the experimental conditions for the titrations. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed to explore the potential of glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) as biomarkers for diagnosis and prognosis in mild and severe TBI cases, including TBI-related deaths. Methods: This prospective cohort study includes 40 cases each of mild, severe, fatal TBI cases, and 40 healthy controls. Serum samples were collected from live patients at 8 and 20 h post injury for UCH-L1 and GFAP respectively, and from deceased patients within 6 h of death. Results: Elevated levels of both GFAP and UCH-L1 were observed in patients with severe and fatal TBI cases. These biomarkers exhibited promising potential for predicting various Glasgow Outcome Scale Extended (GOSE) categories. Combining GFAP and UCH-L1 yielded higher predictive accuracy both for diagnosis and prognosis in TBI cases. The study additionally established specific cut-off levels for GFAP and UCH-L1 stratified according to the severity and prognosis. Conclusion: GFAP and UCH-L1 individually demonstrated moderate to good discrimination capacity in predicting TBI severity and functional outcomes. However, combining these biomarkers is recommended for improved diagnostic and prognostic utility. This precision tool can enhance patient care, enabling tailored treatment plans, ultimately reducing morbidity and mortality rates in TBI cases. [ABSTRACT FROM AUTHOR]

The ubiquitin‐conjugating E2 enzymes play a central role in ubiquitin transfer. Disruptions to the ubiquitin system are implicated in multiple diseases, and as a result, molecules that modulate the activity of the ubiquitin system are of interest. E2 enzyme function relies on interactions with partner proteins, and the disruption of these is an effective way to modulate activity. Here, we report the discovery of ubiquitin variants (UbVs) that inhibit the E2 enzyme, Ube2d2 (UbcH5b). The six UbVs identified inhibit ubiquitin chain building, and the structural and biophysical characterisation of two of these demonstrate they bind to Ube2d2 with low micromolar affinity and high specificity. Both characterised UbVs bind at a site that overlaps with E1 binding, while the more inhibitory UbV has an additional binding site that blocks a critical non‐covalent ubiquitin‐binding site on the E2 enzyme. The discovery of novel protein‐based ubiquitin derivatives that inhibit protein–protein interactions is an important step towards discovering small molecules that inhibit the activity of E2 enzymes. Furthermore, the specificity of the UbVs within the Ube2d family suggests that it may be possible to develop tools to selectively inhibit highly related E2 enzymes. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation has emerged as a crucial mechanism driving the formation of membraneless biomolecular condensates, which play important roles in numerous cellular processes. These condensates, found both in the nucleus and cytoplasm, are formed through multivalent, low-affinity interactions between various molecules. P62-containing condensates serve, among other functions, as proteolytic hubs for the ubiquitin–proteasome system. In this study, we investigated the dynamic interplay between nuclear p62 condensates and promyelocytic nuclear bodies (PML-NBs). We show that p62 condensates stabilize PML-NBs under both basal conditions and following exposure to arsenic trioxide which stimulates their degradation. We further show that this effect on the stability of PML-NBs is due to sequestration of their ubiquitin E3 ligase RNF4 in the p62 condensates with subsequent rapid degradation of the ligase. The sequestration of the ligase is made possible by association between the proline-rich domain of the PML protein and the PB1 domain of p62, which results in the formation of a PML-NB shell around the p62 condensates. Importantly, these hybrid structures do not undergo fusion and mixing of their contents which leaves unsolved the mechanism of sequestration of RNF4 in the condensates. These findings suggest an additional possible mechanism of PML-NB as a tumor suppressor which is mediated via interactions between different biomolecular condensates. [ABSTRACT FROM AUTHOR]

Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein–protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions. [ABSTRACT FROM AUTHOR]

Deubiquitinating enzymes (DUBs) prevent ubiquitination by eliminating ubiquitin from their substrates. Deubiquitinating enzymes have important roles in a number of cell biology subfields that are highly relevant to diseases like neurodegeneration, cancer, autoimmune disorders, and long-term inflammation. Deubiquitinating enzymes feature a well-defined active site and, for the most part, catalytic cysteine, which makes them appealing targets for small-molecule drug development. Ubiquitin-specific protease 46 (USP46) is a member of the ubiquitin-specific protease family, the largest subfamily of DUBs. Over the past 10 years, some studies have steadily demonstrated the significance of USP46 in several biological processes, although it was identified later and early research progress was modest. Specifically, in the last few years, the carcinogenic properties of USP46 have become more apparent. In the current review, we provide a comprehensive overview of the current knowledge about USP46 including its characteristics, structure, inhibitors, function in diseases, especially in the nervous system, and the correlation of USP46 with cancers. [ABSTRACT FROM AUTHOR]

PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2's U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3BD2. Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases. KLHDC2 is a promising E3 ligase for targeted protein degradation (TPD). In this study, the authors demonstrate that heterobifunctional degraders induce cooperative ternary complexes with KLHDC2 and BRD3. They highlight exit vector, neo-substrate, E3 ligase selectivity, and prodrug choice can effectively leverage C-degron E3s for TPD. [ABSTRACT FROM AUTHOR]

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Efficient interbacterial competitions and diverse defensive strategies employed by various bacteria play a crucial role in acquiring a hold within a dense microbial community. The gut symbiont Bacteroides fragilis secretes an antimicrobial ubiquitin homologue (BfUbb) that targets an essential periplasmic PPIase to drive intraspecies bacterial competition. However, the mechanisms by which BfUbb enters the periplasm and its potential for interspecies antagonism remain poorly understood. Here, we employ transposon mutagenesis and identify a highly conserved TonB-dependent transporter SusCD (designated as ButCD) in B. fragilis as the BfUbb transporter. As a putative protein-related nutrient utilization system, ButCD is widely distributed across diverse Bacteroides species with varying sequence similarity, resulting in distinct import efficiency of Bacteroides ubiquitin homologues (BUbb) and thereby determining the species-specific toxicity of BUbb. Cryo-EM structural and functional investigations of the BfUbb–ButCD complex uncover distinctive structural features of ButC that are crucial for its targeting by BfUbb. Animal studies further demonstrate the specific and efficient elimination of enterotoxigenic B. fragilis (ETBF) in the murine gut by BfUbb, suggesting its potential as a therapeutic against ETBF-associated inflammatory bowel disease and colorectal cancer. Our findings provide a comprehensive elucidation of the species-specific toxicity exhibited by BUbb and explore its potential applications. The gut symbiont Bacteroides fragilis secretes an antimicrobial ubiquitin homologue (BfUbb) that drives intraspecies bacterial competition. Here, the authors identify a highly conserved TonB-dependent transporter SusCD in B. fragilis as the BfUbb transporter and show species-specific antagonism of BfUbb and the BfUbb bound SusCD complex structure, revealing potential therapeutic applications of BfUbb against enterotoxigenic B. fragilis-related diseases. [ABSTRACT FROM AUTHOR]

The recent discovery of non-proteinaceous ubiquitylation substrates broadened our understanding of this modification beyond conventional protein targets. However, the existence of additional types of substrates remains elusive. Here, we present evidence that nucleic acids can also be directly ubiquitylated via ester bond formation. DTX3L, a member of the DELTEX family E3 ubiquitin ligases, ubiquitylates DNA and RNA in vitro and that this activity is shared with DTX3, but not with the other DELTEX family members DTX1, DTX2 and DTX4. DTX3L shows preference for the 3′-terminal adenosine over other nucleotides. In addition, we demonstrate that ubiquitylation of nucleic acids is reversible by DUBs such as USP2, JOSD1 and SARS-CoV-2 PLpro. Overall, our study proposes reversible ubiquitylation of nucleic acids in vitro and discusses its potential functional implications. Synopsis: Nucleic acids are a novel type of ubiquitylation substrate. Nucleic acids are ubiquitylated by DELTEX ubiquitin E3 ligase DTX3L, targeting the 3′-terminal adenosine nucleotides at their 3′ hydroxyl groups. KH domain-containing DELTEX E3 ligase DTX3 and DTX3L possess nucleic acids ubiquitylation activity while WWE domain-containing DELTEX E3 ligase DTX1, DTX2 as well as DTX4 do not. Ubiquitylation of nucleic acids by DTX3L could block cleavage by 3′–5′ nuclease and represents a reversible process. Nucleic acids are a novel type of ubiquitylation substrate. [ABSTRACT FROM AUTHOR]

The transcription factor E2F1 serves as a regulator of the cell cycle and promotes cell proliferation. It is highly expressed in cancer tissues and contributes to their malignant transformation. Degradation by the ubiquitin-proteasome system may help to prevent such overexpression of E2F1 and thereby to suppress carcinogenesis. A detailed understanding of the mechanisms underlying E2F1 degradation may therefore inform the development of new cancer treatments. We here identified SCFFBXW7 as a ubiquitin ligase for E2F1 by comprehensive analysis. We found that phosphorylation of E2F1 at serine-403 promotes its binding to FBXW7 (F-box/WD repeat-containing protein 7) followed by its ubiquitination and degradation. Furthermore, calcineurin, a Ca2+/calmodulin-dependent serine-threonine phosphatase, was shown to stabilize E2F1 by mediating its dephosphorylation at serine-403 and thereby preventing FBXW7 binding. Treatment of cells with Ca2+ channel blockers resulted in downregulation of both E2F1 protein and the expression of E2F1 target genes, whereas treatment with the Ca2+ ionophore ionomycin induced upregulation of E2F1. Finally, the calcineurin inhibitor FK506 attenuated xenograft tumor growth in mice in association with downregulation of E2F1 in the tumor tissue. Impairment of the balance between the opposing actions of FBXW7 and calcineurin in the regulation of E2F1 abundance may therefore play an important role in carcinogenesis. [ABSTRACT FROM AUTHOR]

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post‐translational modifications (PTMs). In this study, we developed an expedient site‐specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys‐aminoethylation reaction with the process of ubiquitin‐activating enzyme UBA1‐assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63‐linked di‐ and tri‐ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63‐linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo‐EM structural analysis of the RNF168/UbcH5c–Ub/H1.0‐K63‐Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63‐linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0–K63‐Ub3 di‐nucleosome substrate, revealing a comparable stimulation effect of both the inter‐ and intra‐nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in‐depth mechanistic investigations of in‐trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation. [ABSTRACT FROM AUTHOR]

We aimed to determine if testican-1 and ubiquitin can serve as early indicators for diagnosing worsening clinical course (presence of intraparenchymal pathology) and mortality in patients with moderate traumatic brain injury (TBI).In this study, we conducted an observational and prospective study in the Emergency Department (ED) of a tertiary care hospital. The department admitted an average of 25,000 patients per month between October 2020 and March 2021. We focused on patients over 18 years old with moderate traumatic brain injury (Glasgow coma score (GCS): 9-13). We compared the prognostic values of blood testican and ubiquitin levels with Receiver Operating Characteristic (ROC) analysis for intracranial pathologies resulting from head trauma. Additionally, we used logistic regression analysis to compare the effectiveness of these markers in diagnosing intraparenchymal injury. The study included a total of 89 patients, with 45 in the case group (moderate TBI) and 44 in the healthy control group. It was found that levels of ubiquitin and testican levels were significantly higher in patients with intraparenchymal pathology (p = 0.048, p = 0.046, respectively). The cut-off point for detecting intraparenchymal pathology was 161.3 pg/mL for testican (Area under curve (AUC): 0.810; min: 0.654; max: 0.965, p = 0.002) and 44.42 ng/mL for Ubiquitin (AUC: 0.863; min: 0.727; max: 1.000, p < 0.001). High testican-1 and ubiquitin levels were independent markers for intraparenchymal pathology in moderate head trauma. [ABSTRACT FROM AUTHOR]

Damaged mitochondria are selectively eliminated in a process called mitophagy. PINK1 and Parkin amplify ubiquitin signals on damaged mitochondria, which are then recognized by autophagy adaptors to induce local autophagosome formation. NDP52 and OPTN, two essential mitophagy adaptors, facilitate de novo synthesis of pre-autophagosomal membranes near damaged mitochondria by linking ubiquitinated mitochondria and ATG8 family proteins and by recruiting core autophagy initiation components. The multifunctional serine/threonine kinase TBK1 also plays an important role in mitophagy. OPTN directly binds TBK1 to form a positive feedback loop for isolation membrane expansion. TBK1 is also thought to indirectly interact with NDP52; however, its role in NDP52-driven mitophagy remains largely unknown. Here, we focused on two TBK1 adaptors, AZI2/NAP1 and TBKBP1/SINTBAD, that are thought to mediate the TBK1-NDP52 interaction. We found that both AZI2 and TBKBP1 are recruited to damaged mitochondria during Parkin-mediated mitophagy. Further, a series of AZI2 and TBKBP1 knockout constructs combined with an OPTN knockout showed that AZI2, but not TBKBP1, impacts NDP52-driven mitophagy. In addition, we found that AZI2 at S318 is phosphorylated during mitophagy, the impairment of which slightly inhibits mitochondrial degradation. These results suggest that AZI2, in concert with TBK1, plays an important role in NDP52-driven mitophagy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The Herpesviridae include the Epstein–Barr Virus (EBV) and the Kaposi Sarcoma-associated Herpesvirus (KSHV), both of which are oncogenic gamma-herpesviruses. These viruses manipulate host cellular mechanisms, including through ubiquitin-mediated pathways, to promote viral replication and oncogenesis. Ubiquitin, a regulatory protein which tags substrates for degradation or alters their function, is manipulated by both EBV and KSHV to facilitate viral persistence and cancer development. EBV infects approximately 90% of the global population and is implicated in malignancies including Burkitt lymphoma (BL), Hodgkin lymphoma (HL), post-transplant lymphoproliferative disorder (PTLD), and nasopharyngeal carcinoma. EBV latency proteins, notably LMP1 and EBNA3C, use ubiquitin-mediated mechanisms to inhibit apoptosis, promote cell proliferation, and interfere with DNA repair, contributing to tumorigenesis. EBV's lytic proteins, including BZLF1 and BPLF1, further disrupt cellular processes to favor oncogenesis. Similarly, KSHV, a causative agent of Kaposi's Sarcoma and lymphoproliferative disorders, has a latency-associated nuclear antigen (LANA) and other latency proteins that manipulate ubiquitin pathways to degrade tumor suppressors, stabilize oncogenic proteins, and evade immune responses. KSHV's lytic cycle proteins, such as RTA and Orf64, also use ubiquitin-mediated strategies to impair immune functions and promote oncogenesis. This review explores the ubiquitin-mediated interactions of EBV and KSHV proteins, elucidating their roles in viral oncogenesis. Understanding these mechanisms offers insights into the similarities between the viruses, as well as provoking thought about potential therapeutic targets for herpesvirus-associated cancers. [ABSTRACT FROM AUTHOR]

Ubiquitin modification of viral proteins to degrade or regulate their function is one of the strategies of the host to resist viral infection. Here, we report that ubiquitin protein ligase E3C (UBE3C), an E3 ubiquitin ligase, displayed inhibitory effects on EV-A71 replication. UBE3C knockdown resulted in increased viral protein levels and virus titers, whereas overexpression of UBE3C reduced EV-A71 replication. To explore the mechanism by which UBE3C affected EV-A71 infection, we found that the C-terminal of UBE3C bound to 2C protein and facilitated K33/K48-linked ubiquitination degradation of 2C K268. Moreover, UBE3C lost its ability to degrade 2C K268R and had a diminished inhibitory impact against the replication of recombinant EV-A71-FY-2C K268R. In addition, UBE3C also promoted ubiquitination degradation of the 2C protein of CVB3 and CVA16 and inhibited viral replication. Thus, our findings reveal a novel mechanism that UBE3C acts as an enterovirus host restriction factor, including EV-A71, by targeting the 2C protein. [ABSTRACT FROM AUTHOR]

The ubiquitin system regulates protein homeostasis and is critical for neurophysiology. Protein-coding mutations in ubiquitin ligases and deubiquitylating enzymes (DUBs) underlie numerous neurological diseases. Substrate profiling for ubiquitin ligases and DUBs in neurons has begun to help identify the mechanism behind some neurodevelopmental and neurodegenerative diseases. Targeting the ubiquitin system may offer therapeutic potential for neurological disease. Neuronal ubiquitin balance impacts the fate of countless cellular proteins, and its disruption is associated with various neurological disorders. The ubiquitin system is critical for proper neuronal cell state transitions and the clearance of misfolded or aggregated proteins that threaten cellular integrity. This article reviews the state of and recent advancements in our understanding of the disruptions to components of the ubiquitin system, in particular E3 ligases and deubiquitylases, in neurodevelopmental and neurodegenerative diseases. Specific focus is on enzymes with recent progress in their characterization, including identifying enzyme-substrate pairs, the use of stem cell and animal models, and the development of therapeutics for ubiquitin-related diseases. [ABSTRACT FROM AUTHOR]

Many bacteria possess proteasomes and a tagging system that is functionally analogous to the ubiquitin system. In this system, Pup, the tagging protein, marks protein targets for proteasomal degradation. Despite the analogy to the ubiquitin system, where the ubiquitin tag is recycled, it remained unclear whether Pup is similarly recycled, given how the bacterial proteasome does not include a depupylase. We previously showed in vitro that as Pup lacks effective proteasome degradation sites, it is released from the proteasome following target degradation, remaining conjugated to a degradation fragment that can be later depupylated. Here, we tested this model in Mycobacterium smegmatis, using a Pup mutant that is effectively degraded by the proteasome. Our findings indicate that Pup recycling not only occurs in vivo but is also essential to maintain normal pupylome levels and to support bacterial survival under starvation conditions. Accordingly, Pup recycling is an essential process in the mycobacterial Pup‐proteasome system. [ABSTRACT FROM AUTHOR]

Autophagy is essential for cell survival and cellular homeostasis under various stress conditions. Therefore, autophagy dysfunction is associated with the pathogenesis of various human diseases. We explored the regulatory role of RhoBTB3 in autophagy and its interaction with activating molecules in AMBRA1. RhoBTB3 deficiency was found to induce autophagy, while its overexpression inhibited autophagy induction. Through immunoprecipitation and mass spectrometry, AMBRA1 was identified as a substrate of RhoBTB3. The study revealed that RhoBTB3 regulates AMBRA1 stability by influencing its protein levels without affecting its mRNA levels. RhoBTB3 induced the ubiquitination of AMBRA1, leading to proteasome-mediated degradation, with the ubiquitination occurring at K45 on AMBRA1 through a K27-linked ubiquitin chain. The knockdown of AMBRA1 blocked RhoBTB3 knockdown-induced autophagy, indicating the dependency of autophagy on AMBRA1. Thus, RhoBTB3 negatively regulates autophagy by mediating AMBRA1 ubiquitination and degradation, suggesting RhoBTB3 as a potential therapeutic target for autophagy-related diseases. [ABSTRACT FROM AUTHOR]

Cell–cell adhesion in endothelial monolayers is tightly controlled and crucial for vascular integrity. Recently, we reported on the importance of fast protein turnover for maintenance of endothelial barrier function. Specifically, continuous ubiquitination and degradation of the Rho GTPase RhoB is crucial to preserve quiescent endothelial integrity. Here, we sought to identify other barrier regulators, which are characterized by a short half-life, using a proteomics approach. Following short-term inhibition of ubiquitination with E1 ligase inhibitor MLN7243 or Cullin E3 ligase inhibitor MLN4924 in primary human endothelial cells, we identified sixty significantly differentially expressed proteins. Intriguingly, our data showed that AAMP and MTSS1 are novel negative regulators of endothelial barrier function and that their turnover is tightly controlled by ubiquitination. Mechanistically, AAMP regulates the stability and activity of RhoA and RhoB, and colocalizes with F-actin and cortactin at membrane ruffles, possibly regulating F-actin dynamics. Taken together, these findings demonstrate the critical role of protein turnover of specific proteins in the regulation of endothelial barrier function, contributing to our options to target dysregulation of vascular permeability. [ABSTRACT FROM AUTHOR]

The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The SAC prevents the APC/C from recognising Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live-cell imaging, in vitro reconstitution biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross-correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an 'arginine-anchor' in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the arginine anchor on Cyclin B1 reduces its interaction with the APC/C and delays its degradation: cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes promote the efficient interaction between Cyclin B1 and the APC/C to ensure the timely degradation of Cyclin B1 and genomic stability. Synopsis: How the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase initiates Cyclin B1 degradation as soon as the spindle assembly checkpoint is switched off remains mysterious. This work reveals chromosomes as the crucial platform for inactivating the major mitotic kinase Cyclin B1-CDK1. Cyclin B1 binds to nucleosomes through an 'arginine anchor' motif in its amino terminus. APC/C binds to nucleosomes through an arginine anchor in its APC3 subunit. Fluorescence cross-correlation shows that the APC/C primarily binds Cyclin B1 at chromosomes. Preventing Cyclin B1 chromosome binding by arginine anchor mutations interferes with its binding to the APC/C and delays its degradation by several minutes, leading to aneuploidy. Chromosomes are the crucial platform for timely inactivation of CDK1-Cyclin B1, the major kinase that keeps cells in mitosis. [ABSTRACT FROM AUTHOR]

Although the ubiquitin ligase U-box protein family is a vital component of plant development and stress response, the exact role of kiwifruit U-box genes family is not known. The study identified 73 putative U-box genes from the kiwifruit genome. Chromosomal localisation, phylogenetic analysis, physicochemical properties, gene structure, motifs, and promoters of these genes were analysed. Distribution of Actinidia chinensis PUB (AcPUB) genes exhibited varying densities across 27 chromosomes. The study found that U-box clusters were classified into groups I–VII in phylogenetic analyses of a variety of plants, including kiwifruit, Arabidopsis thaliana, and rice, with most of the genes in kiwifruit being related to rice. The conserved motif analyses revealed that all the AcPUB genes have typical U-box structural domains. Analysis of the cis-acting elements suggested that AcPUBs expression may be responsive to several hormones and stresses. RNA-Seq analysis showed that six kiwifruit U-box genes were differentially expressed after induction by Wickerhamomyces anomalus, suggesting a potential involvement of these U-box genes in the kiwifruit's response to W. anomalus, which might include mechanisms related to resistance. Overall, our findings provide valuable insights into the function and structure of U-box genes that will help develop new genomic technologies and study plant resistance. [ABSTRACT FROM AUTHOR]

Ubiquitin ligases are important regulators of nervous system development, function and disease. To date, numerous ubiquitin ligases have been discovered that regulate presynaptic biology. Here, we discuss recent findings on presynaptic ubiquitin ligases that include members from the three major ubiquitin ligase classes: RING, RBR and HECT. Several themes emerge based on findings across a range of model systems. A cadre of ubiquitin ligases is required presynaptically to orchestrate development and transmission at synapses. Multiple ubiquitin ligases deploy both enzymatic and non‐enzymatic mechanisms, and act as hubs for signalling networks at the synapse. Both excitatory and inhibitory presynaptic terminals are influenced by ligase activity. Finally, there are several neurodevelopmental disorders and neurodegenerative diseases associated with presynaptic ubiquitin ligases. These findings highlight the growing prominence and biomedical relevance of the presynaptic ubiquitin ligase network. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) is an age-related chronic neurological condition characterized by progressive degeneration of dopaminergic neurons and the presence of Lewy bodies, primarily composed of alpha-synuclein and ubiquitin. The pathophysiology of PD encompasses alpha-synuclein aggregation, oxidative stress, neuroinflammation, mitochondrial dysfunction, and impaired autophagy and ubiquitin–proteasome systems. Among these, the Keap1–Nrf2 pathway is a key regulator of antioxidant defense mechanisms. Nrf2 has emerged as a crucial factor in managing oxidative stress and inflammation, and it also influences ubiquitination through p62 expression. Keap1 negatively regulates Nrf2 by targeting it for degradation via the ubiquitin–proteasome system. Disruption of the Nrf2–Keap1 pathway in PD affects cellular responses to oxidative stress and inflammation, thereby playing a critical role in disease progression. In addition, the role of neuroinflammation in PD has gained significant attention, highlighting the interplay between immune responses and neurodegeneration. This review discusses the various mechanisms responsible for neuronal degeneration in PD, with a special emphasis on the neuroprotective role of the Nrf2–Keap1 pathway. Furthermore, it explores the implications of inflammopharmacology in modulating these pathways to provide therapeutic insights for PD. [ABSTRACT FROM AUTHOR]

The noncanonical ubiquitin-like conjugation cascade involving the E1 (Atg7), E2 (Atg3, Atg10), and E3 (Atg12–Atg5-Atg16 complex) enzymes is essential for incorporation of Atg8 into the growing phagophore via covalent linkage to PE. This process is an indispensable step in autophagy. Atg8 and E1-E3 enzymes are the first subset from the core autophagy protein machinery structures that were investigated in earlier studies by crystallographic analyses of globular domains. However, research over the past decade shows that many important functions in the conjugation machinery are mediated by intrinsically disordered protein regions (IDPRs) – parts of the protein that do not adopt a stable secondary or tertiary structure, which are inherently dynamic and well suited for protein-membrane interactions but are invisible in protein crystals. Here, we summarize earlier and recent findings on the autophagy conjugation machinery by focusing on the IDPRs. This summary reveals that IDPRs, originally considered dispensable, are in fact major players and a driving force in the function of the autophagy conjugation system. Abbreviation: AD, activation domain of Atg7; AH, amphipathic helix; AIM, Atg8-family interacting motif; CL, catalytic loop (of Atg7); CTD, C-terminal domain; FR, flexible region (of Atg3 or Atg10); GUV, giant unilammelar vesicles; HR, handle region (of Atg3); IDPR, intrinsically disordered protein region; IDPs: intrinsically disordered proteins; LIR, LC3-interacting region; NHD: N-terminal helical domain; NMR, nuclear magnetic resonance; PE, phosphatidylethanolamine; UBL, ubiquitin like. [ABSTRACT FROM AUTHOR]

Ubiquitination is an enzymatic process characterized by the covalent attachment of ubiquitin to target proteins, thereby modulating their degradation, transportation, and signal transduction. By precisely regulating protein quality and quantity, ubiquitination is essential for maintaining protein homeostasis, DNA repair, cell cycle regulation, and immune responses. Nevertheless, the diversity of ubiquitin enzymes and their extensive involvement in numerous biological processes contribute to the complexity and variety of diseases resulting from their dysregulation. The ubiquitination process relies on a sophisticated enzymatic system, ubiquitin domains, and ubiquitin receptors, which collectively impart versatility to the ubiquitination pathway. The widespread presence of ubiquitin highlights its potential to induce pathological conditions. Ubiquitinated proteins are predominantly degraded through the proteasomal system, which also plays a key role in regulating protein localization and transport, as well as involvement in inflammatory pathways. This review systematically delineates the roles of ubiquitination in maintaining protein homeostasis, DNA repair, genomic stability, cell cycle regulation, cellular proliferation, and immune and inflammatory responses. Furthermore, the mechanisms by which ubiquitination is implicated in various pathologies, alongside current modulators of ubiquitination are discussed. Enhancing our comprehension of ubiquitination aims to provide novel insights into diseases involving ubiquitination and to propose innovative therapeutic strategies for clinical conditions. [ABSTRACT FROM AUTHOR]

An N-degron is a degradation signal whose main determinant is a "destabilizing" N-terminal residue of a protein. Specific N-degrons, discovered in 1986, were the first identified degradation signals in short-lived intracellular proteins. These N-degrons are recognized by a ubiquitin-dependent proteolytic system called the Arg/N-degron pathway. Although bacteria lack the ubiquitin system, they also have N-degron pathways. Studies after 1986 have shown that all 20 amino acids of the genetic code can act, in specific sequence contexts, as destabilizing N-terminal residues. Eukaryotic proteins are targeted for the conditional or constitutive degradation by at least five N-degron systems that differ both functionally and mechanistically: the Arg/N-degron pathway, the Ac/N-degron pathway, the Pro/N-degron pathway, the fMet/N-degron pathway, and the newly named, in this perspective, GASTC/N-degron pathway (GASTC = Gly, Ala, Ser, Thr, Cys). I discuss these systems and the expanded terminology that now encompasses the entire gamut of known N-degron pathways. [ABSTRACT FROM AUTHOR]

The dynamic process of membrane shaping and remodeling plays a vital role in cellular functions, with proteins and cellular membranes interacting intricately to adapt to various cellular needs and environmental cues. Ubiquitination—a posttranslational modification—was shown to be essential in regulating membrane structure and shape. It influences virtually all pathways relying on cellular membranes, such as endocytosis and autophagy by directing protein degradation, sorting, and oligomerization. Ubiquitin is mostly known as a protein modifier; however, it was reported that ubiquitin and ubiquitin-like proteins can associate directly with lipids, affecting membrane curvature and dynamics. In this review, we summarize some of the current knowledge on ubiquitin-mediated membrane remodeling in the context of endocytosis, autophagy, and ER-phagy. [Display omitted] In their review, Jacomin and Dikic discuss the role of ubiquitination in regulating membrane structure and its impact on pathways such as endocytosis and autophagy. Besides protein modification, ubiquitin also interacts with lipids, directly influencing membrane dynamics. The review covers ubiquitin-mediated membrane remodeling in endocytosis, autophagy, and ER-phagy. [ABSTRACT FROM AUTHOR]

Cellular trafficking between organelles is typically assured by short motifs that contact carrier proteins to transport them to their destination. The ubiquitin E3 ligase RING finger protein 13 (RNF13), a regulator of proliferation, apoptosis and protein trafficking, localizes to endolysosomal compartments through the binding of a dileucine motif to clathrin adaptor protein complex AP-3. Mutations within this motif reduce the ability ofRNF13 to interactwithAP-3. Here, our study shows the discovery of a glutamine-based motif that resembles a tyrosinebased motif within the C-terminal region of RNF13 that binds to the clathrin adaptor protein complex AP-1, notably without a functional interaction with AP-3. Using biochemical, molecular and cellular approaches in HeLa cells, our study demonstrates that a RNF13 dileucine variant uses anAP-1-dependent pathway to be exported from the Golgi towards the endosomal compartment. Overall, this study provides mechanistic insights into the alternate route used by this variant of the dileucine sorting motif of RNF13. [ABSTRACT FROM AUTHOR]

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN. [ABSTRACT FROM AUTHOR]

The cytosolic peptide:N‐glycanase (PNGase) is involved in the quality control of N‐glycoproteins via the endoplasmic reticulum‐associated degradation (ERAD) pathway. Mutations in the gene encoding cytosolic PNGase (NGLY1 in humans) cause NGLY1 deficiency. Recent findings indicate that the F‐box protein FBS2 of the SCFFBS2 ubiquitin ligase complex can be a promising drug target for NGLY1 deficiency. Here, we determined the crystal structure of bovine FBS2 complexed with the adaptor protein SKP1 and a sugar ligand, Man3GlcNAc2, which corresponds to the core pentasaccharide of N‐glycan. Our crystallographic data together with NMR data revealed the structural basis of disparate sugar‐binding specificities in homologous FBS proteins and identified a potential druggable pocket for in silico docking studies. Our results provide a potential basis for the development of selective inhibitors against FBS2 in NGLY1 deficiency. [ABSTRACT FROM AUTHOR]

Lys48‐linked ubiquitin chains, regulating proteasomal protein degradation, are known to include cyclized forms. This cyclization hinders recognition by many downstream proteins by occluding the Ile44‐centered patch. In contrast, the A20‐like Znf domain of ZNF216 (a ubiquitin‐binding protein, A20 Znf) is expected to bind to cyclic ubiquitin chains via constitutively solvent‐exposed surfaces. However, the underlying interaction mechanism remains unclear. Here, our ITC and NMR experiments collectively showed that cyclization did not interfere with and even slightly enhance the molecular recognition of diubiquitin by A20 Znf. This effect is explained by the cyclization‐induced repression of conformational dynamics in diubiquitin and an enlarged molecular interface in the complex. Thus, these results suggest that cyclic ubiquitin chains can be involved in regulation of ZNF216‐dependent proteasomal protein degradation. [ABSTRACT FROM AUTHOR]

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, β-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. [ABSTRACT FROM AUTHOR]

Rare mutations in CARD14 promote psoriasis by inducing CARD14-BCL10-MALT1 complexes that activate NF-κB and MAP kinases. Here, the downstream signalling mechanism of the highly penetrant CARD14E138A alteration is described. In addition to BCL10 and MALT1, CARD14E138A associated with several proteins important in innate immune signalling. Interactions with M1-specific ubiquitin E3 ligase HOIP, and K63-specific ubiquitin E3 ligase TRAF6 promoted BCL10 ubiquitination and were essential for NF-κB and MAP kinase activation. In contrast, the ubiquitin binding proteins A20 and ABIN1, both genetically associated with psoriasis development, negatively regulated signalling by inducing CARD14E138A turnover. CARD14E138A localized to early endosomes and was associated with the AP2 adaptor complex. AP2 function was required for CARD14E138A activation of mTOR complex 1 (mTORC1), which stimulated keratinocyte metabolism, but not for NF-κB nor MAP kinase activation. Furthermore, rapamycin ameliorated CARD14E138A-induced keratinocyte proliferation and epidermal acanthosis in mice, suggesting that blocking mTORC1 may be therapeutically beneficial in CARD14-dependent psoriasis. [ABSTRACT FROM AUTHOR]

Ubiquitination is one of the most conserved post-translational modifications and together with mRNA translation contributes to cellular protein homeostasis (proteostasis). Temporal and spatial regulation of proteostasis is particularly important during synaptic plasticity, when translation of specificmRNAs requires tight regulation. Mutations in genes encoding regulators of mRNA translation and in ubiquitin ligases have been associated with several neurodevelopmental disorders. RNA metabolism and translation are regulated by RNA-binding proteins, critical for the spatial and temporal control of translation in neurons. Several ubiquitin ligases also regulate RNA-dependent mechanisms in neurons, with numerous ubiquitination events described in splicing factors and ribosomal proteins. Here we will explore how ubiquitination regulates translation in neurons, from RNA biogenesis to alternative splicing and how dysregulation of ubiquitin signaling can be the underlying cause of pathology in neurodevelopmental disorders, such as Fragile X syndrome. Finally we propose that targeting ubiquitin signaling is an attractive novel therapeutic strategy for neurodevelopmental disorders where mRNA translation and ubiquitin signaling are disrupted. [ABSTRACT FROM AUTHOR]

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation. [ABSTRACT FROM AUTHOR]