Search Results (889)

Nearly all eukaryotic proteins are turned over by the ubiquitin (Ub)-26S proteasome system (UPS). Despite its broad cellular roles, only a handful of UPS members, particularly the Ub E3 ligases that specifically recognize a protein for ubiquitylation, have been characterized in plants to date. The challenge arises from the transient recognition and rapid degradation of ubiquitylation substrates by the UPS. To tackle this challenge, the emerging biotinylation-based proximity labeling (PL) offers an exciting tool for enriching transient interactors of Ub E3 ligases. In this study, we examined the efficacy of TurboID in identifying substrates of Arabidopsis Skp1-cullin1-F-box (SCF) ligases. We demonstrate that the Arabidopsis Skp1 Like (ASK)1-TurboID is not fully functioning in planta, which led us to discover a novel antagonism between biotinylation and ubiquitylation in regulating protein stability in vivo. This discovery lowers the effectiveness of PL in ubiquitylome studies. However, using one long-known SCF substrate, phytochrome A, we succeeded to apply its TurboID fusion for complementing the far-red-light response of the phyA-211 null mutant allele, suggesting an efficacy of PL in characterizing single ubiquitylation pathways. This study highlighted a limitation of PL in ubiquitylome studies, discovered a new antagonistic pathway of biotinylation, and developed a theoretical guidance for future PL-based characterization of ubiquitylation pathways. Full article

Glucose functions as both an essential energy source and a critical signaling molecule, playing pivotal roles in regulating plant growth, development, and stress responses. Here, we report that AtCHYR1, a previously characterized RING-type ubiquitin E3 ligase involved in drought tolerance, also participates in glucose signaling. Exposure to high glucose levels significantly inhibits AtCHYR1 expression, particularly in root tips, while low glucose conditions, such as osmotic stress, sugar starvation, and dark conditions, induce its expression. Importantly, analysis of chyr1 mutants and plants overexpressing AtCHYR1 revealed that AtCHYR1 positively regulated the high glucose-mediated inhibition of germination and root growth, as well as starvation-induced growth retardation, through enhanced reactive oxygen species (ROS) accumulation in root tips. Additionally, transcriptional levels of glucose-activated pathogenesis-related (PR) and defense-related genes were reduced, while hypoxia-associated and ROS-inducing genes were significantly upregulated in AtCHYR1-overexpressing plants. Collectively, our findings provide novel insights into the role of AtCHYR1 in plant responses to fluctuating sugar availability and its control of ROS homeostasis during seed germination and plant growth. Full article

Wheat (Triticum aestivum L.), a crucial global food crop, provides approximately 20% of daily protein and caloric intake globally. However, its production is increasingly threatened by abiotic stressors, especially drought and salinity, which are exacerbated by climate change. These stressors adversely affect plant growth, development, and yield, necessitating the development of stress-tolerant varieties. This study investigated the role of TaUBX57, a U-box E3 ubiquitin ligase, in enhancing stress tolerance in wheat. Functional domain analysis of TaUBX57 confirmed the presence of a conserved U-box and a protein kinase domain, suggesting its involvement in protein ubiquitination and stress responses. TaUBX57-overexpressing transgenic Arabidopsis lines exhibited significantly improved germination rates, root growth, and survival under drought and salt stress conditions. TaUBX57 overexpression enhanced antioxidant enzyme activities and reduced the expression of oxidative stress markers, such as malondialdehyde. These findings highlight the potential role of TaUBX57 in modulating stress-responsive pathways and enhancing abiotic stress tolerance, offering a promising avenue for developing more resilient wheat varieties through genetic engineering. Full article

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), represents a major global threat to wheat (Triticum aestivum. L). Planting varieties with adult-plant resistance (APR) is an effective approach for long-term management of this disease. The Chinese winter wheat variety Lantian 25 exhibits moderate-to-high APR against stripe rust under field conditions. To investigate the genetic basis of APR in Lantian 25, a set of 219 F₆ recombinant inbred lines (RILs) was created from a cross between Lantian 25 (resistant parent) and Huixianhong (susceptible parent). These RILs were assessed for maximum disease severity (MDS) in Pixian of Sichuan and Qingshui of Gansu over the 2020–2021 and 2021–2022 growing seasons, resulting in data from four different environments. Genotyping was performed on these lines and their parents using the wheat Illumina 50K single-nucleotide polymorphism (SNP) arrays. Composite interval mapping (CIM) identified six quantitative trait loci (QTL), named QYr.gaas-2BS, QYr.gaas-2BL, QYr.gaas-2DS, QYr.gaas-2DL, QYr.gaas-3BS and QYr.gaas-4BL, which were consistently found across two or more environments and explained 4.8–12.0% of the phenotypic variation. Of these, QYr.gaas-2BL, QYr.gaas-2DS, and QYr.gaas-3BS overlapped with previous studies, whereas QYr.gaas-2BS, QYr.gaas-2DS, and QYr.gaas-4BL might be novel. All the resistance alleles for these QTL originated from Lantian 25. Furthermore, four kompetitive allele-specific PCR (KASP) markers, Kasp_2BS_YR (QYr.gaas-2BS), Kasp_2BL_YR (QYr.gaas-2BL), Kasp_2DS_YR (QYr.gaas-2DS) and Kasp_2DL_YR (QYr.gaas-2DL), were developed and validated in 110 wheat diverse accessions. Additionally, we identified seven candidate genes linked to stripe rust resistance, including disease resistance protein RGA2, serine/threonine-protein kinase, F-box family proteins, leucine-rich repeat family proteins, and E3 ubiquitin-protein ligases. These QTL, along with their associated KASP markers, hold promise for enhancing stripe rust resistance in wheat breeding programs. Full article

Mammalian circadian rhythms, governing ~24 h oscillations in behavior, physiology, and hormone levels, are orchestrated by transcriptional–translational feedback loops centered around the core clock protein cryptochrome 1 (CRY1). While CRY1 ubiquitination is known to regulate clock function, the roles of specific ubiquitination sites remain unclear. Here, we identify lysine 151 (K151) as a critical residue modulating the circadian period through non-canonical mechanisms. Using site-directed mutagenesis, we generated CRY1-K151Q/R mutants mimicking constitutive deubiquitination. Circadian rescue assays in Cry1/2-deficient cells revealed period shortening (K151Q: −2.25 h; K151R: −1.4 h; n = 3, p < 0.01, Student’s t-test), demonstrating K151’s functional importance. Despite normal nuclear localization kinetics, K151Q/R mutants exhibited reduced transcriptional repression in luciferase assays, a weakened interaction with BMAL1 by the luciferase complementation assay, and enhanced binding to E3 ligase FBXL12 (but not FBXL3) while showing more stability than wild-type CRY1. Notably, the absence of ubiquitination-linked degradation or altered FBXL3 engagement suggests a ubiquitination-independent mechanism. We propose that CRY1-K151 serves as a structural hub fine-tuning circadian periodicity by modulating core clock protein interactions rather than through traditional ubiquitin-mediated turnover. These findings redefine the mechanistic landscape of post-translational clock regulation and offer new therapeutic avenues for circadian disorders. Full article

Proteolysis-targeting chimeras (PROTACs) selectively degrade target proteins by recruiting intracellular E3 ubiquitin ligases, overcoming the limitations of traditional small-molecule inhibitors that merely block protein function. This approach has garnered significant interest in precision cancer therapy. However, the clinical translation of PROTACs is hindered by their typically high molecular weight, poor membrane permeability, and suboptimal pharmacokinetic properties. Nanodrug delivery technologies represent a promising approach to overcome the limitations of PROTACs. By encapsulating, conjugating, or integrating PROTACs into functionalized nanocarriers, these systems can substantially enhance solubility and biostability, enable tumor-targeted and stimuli-responsive delivery, and thereby effectively alleviate the “hook effect” and minimize off-target toxicity. This review systematically outlines the primary design strategies for current nano-PROTAC delivery systems, including physical encapsulation, chemical conjugation, carrier-free self-assembly systems, and intelligent “split-and-mix” delivery platforms. We provide an overview and evaluation of recent advances in diverse nanomaterial carriers—such as lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, biological carriers, and hybrid nanoparticles—highlighting their synergistic therapeutic potential for PROTACs delivery. The clinical translation prospects of these innovative systems are also discussed. This comprehensive analysis aims to deepen the understanding of this rapidly evolving field, address current challenges and opportunities, promote the advancement of nano-PROTACs, and offer insights into their future development. Full article

Soil salinization constitutes a major constraint on global agricultural production, with marked divergence in salt adaptation strategies between salt-tolerant barley (Hordeum vulgare) and salt-sensitive rice (Oryza sativa). This study systematically investigated the evolution and functional specialization of the C3HC4-type RING zinc finger gene family, known to mediate abiotic stress responses through E3 ubiquitin ligase activity, in these contrasting cereal species. Through comparative genomics, we identified 123 HvC3HC4 genes and 90 OsC3HC4 genes, phylogenetically classified into four conserved subgroups. Differences in C3HC4 genes in phylogenetic relationships, chromosomal distribution, gene structure, motif composition, gene duplication events, and cis-elements in the promoter region were observed between barley and rice. Moreover, HvC3HC4s in barley tissues preferentially adopted an energy-conserving strategy, which may be a key mechanism for barley’s higher salt tolerance. Additionally, we found that C3HC4 genes were evolutionarily conserved in salt-tolerant species. The current results reveal striking differences in salt tolerance between barley and rice mediated by the C3HC4 gene family and offer valuable insight for potential genetic engineering applications in improving crop resilience to salinity stress. Full article

Cadmium (Cd) pollution poses a serious threat to freshwater ecosystems. The freshwater mussel Anodonta woodiana is increasingly used as a bioindicator for monitoring Cd pollution in aquatic environments. However, the primary routes of Cd accumulation in A. woodiana remain unclear, and the molecular regulatory mechanisms underlying Cd accumulation are poorly understood. To address these gaps, this study employed a novel stable isotope dual-tracer technique to trace Cd from water (waterborne ¹¹²Cd) and the green alga Chlorella vulgaris (dietary ¹¹³Cd) during the simultaneous exposure experiment. Comparative transcriptomic analysis was then conducted to characterize molecular responses in A. woodiana following Cd exposure. The results showed that although newly accumulated ¹¹²Cd and ¹¹³Cd increased with exposure concentration and duration, the relative importance of ¹¹²Cd (91.6 ± 2.8%) was significantly higher than that of ¹¹³Cd (8.4 ± 2.8%) (p < 0.05). Cd exposure induced differentially expressed genes primarily enriched in the metabolic processes, cellular processes, and/or the ubiquitin-mediated proteolysis pathway. Within the ubiquitin-mediated proteolysis pathway, TRIP12 (E3 ubiquitin-protein ligase TRIP12) and Cul5 (cullin-5) were significantly upregulated. The findings will provide critical insights for interpreting Cd biomonitoring data in freshwater environments using mussels as bioindicators. Full article

Background: Vitiligo, a chronic depigmentation disorder driven by oxidative stress and immune dysregulation, remains poorly understood mechanistically. The Keap1/NRF2/ARE pathway is critical for melanocyte protection against oxidative damage; however, the role of Cullin-3 (CUL3), a scaffold for E3 ubiquitin ligases that regulate NRF2 degradation, and its interplay with inflammatory mediators in vitiligo pathogenesis are underexplored. This study investigates CUL3, NRF2, and the associated regulatory networks in vitiligo, integrating clinical profiling and computational docking to identify therapeutic targets. Methods: A case-control study compared non-segmental vitiligo patients with age-/sex-matched controls. Lesional skin biopsies were analyzed by qRT-PCR for the expression of CUL3, NRF2, miRNA-146a, FOXP3, NF-κB, IL-6, TNF-α, and P53. Molecular docking was used to evaluate vitexin’s binding affinity to Keap1, validated by root mean square deviation (RMSD) calculations. Results: Patients with vitiligo exhibited significant downregulation of CUL3 (0.27 ± 0.03 vs. 1 ± 0.58; p = 0.013), NRF2 (0.37 ± 0.26 vs. 1 ± 0.8; p = 0.001), and FOXP3 (0.09 ± 0.2 vs. 1 ± 0.3; p = 0.001), alongside the upregulation of miRNA-146a (4.7 ± 1.9 vs. 1 ± 0.8; p = 0.001), NF-κB (4.7 ± 1.9 vs. 1 ± 0.5; p = 0.001), IL-6 (2.8 ± 1.5 vs. 1 ± 0.4; p = 0.001), and TNF-α (2.2 ± 1.1 vs. 1 ± 0.3; p = 0.001). P53 showed no differential expression (p > 0.05). Docking revealed a strong binding of vitexin to Keap1 (RMSD: 0.23 Å), mirroring the binding of the control ligand CDDO-Im. Conclusions: Dysregulation of the CUL3/Keap1/NRF2 axis and elevated miRNA-146a levels correlate with vitiligo progression, suggesting a role for oxidative stress and immune imbalance. Vitexin’s high-affinity docking to Keap1 positions it as a potential modulator of the NRF2 pathway, offering novel therapeutic avenues. This study highlights the translational potential of targeting the ubiquitin–proteasome and antioxidant pathways in the management of vitiligo. Full article

Skeletal muscle atrophy is a debilitating condition characterized by the loss of muscle mass and function. It is commonly associated with aging, chronic diseases, disuse, and prolonged glucocorticoid therapy. Oxidative stress and catabolic signaling pathways play significant roles in the progression of muscle degradation. Despite its clinical relevance, few effective therapeutic options are currently available. In this study, we investigated the protective effects of an ethanolic extract of Glycine Semen Preparata (GSP), i.e., fermented black soybeans, using in vitro and in vivo models of dexamethasone (Dexa)-induced muscle atrophy. In C2C12 myoblasts and myotubes, GSP significantly attenuated both oxidative stress-induced and Dexa-induced damages by reducing reactive oxygen species levels and by suppressing the expression of the muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1. Moreover, GSP upregulated key genes involved in muscle regeneration (Myod1 and Myog) and mitochondrial biogenesis (PGC1α), indicating its dual role in muscle protection and regeneration. Oral administration of GSP to mice with Dexa-induced muscle atrophy resulted in improved muscle fiber integrity, increased proportion of large cross-sectional area fibers, and partial recovery of motor function. Isoflavone aglycones, such as daidzein and genistein, were identified as active compounds that contribute to the beneficial effects of GSP through antioxidant activity and gene promoter enhancement. Thus, GSP is a promising nutraceutical that prevents or mitigates muscle atrophy by targeting oxidative stress and promoting myogenesis and mitochondrial function. Further studies are warranted to standardize the bioactive components and explore their clinical applications. Full article

The temporary or permanent occlusion of cerebral blood vessels results in ischemic stroke (IS). Ischemia per se causes focal neuronal damage, and the subsequent ischemia–reperfusion injury that occurs after blood flow restoration further compromises brain tissue and cells in the neurovascular unit, significantly contributing to poor patient outcomes and functional impairments. Current research indicates that the ubiquitin–proteasome system (UPS) plays a crucial role in the pathological processes associated with cerebral ischemia–reperfusion injury (CIRI). Notably, E3 ubiquitin (Ub) ligases, which are essential in the UPS, have garnered increasing attention as potential novel therapeutic targets for treating ischemia–reperfusion damage in the brain. This review focuses primarily on the background of E3 Ub ligases and explores their intricate relationships with the pathological processes of CIRI. Full article

Ubiquitination is a dynamic and reversible post-translational modification mediated by ubiquitination regulators (UBRs), which plays an essential role in protein stability, cell differentiation and immunity. Dysregulation of UBRs can lead to destabilization of biological processes and may induce serious human diseases, including cancer. Many UBRs, such as E3 ubiquitin ligases and deubiquitinases (DUBs), have been identified as potential drug targets for cancer therapy. However, the potential clinical value of UBRs in lung adenocarcinoma (LUAD) remains to be elucidated. Here, we identified 17 hub UBRs from high-confidence protein–protein interaction networks of UBRs correlated with cancer hallmark-related pathways using four topological algorithms. The expression of hub UBRs is affected by copy number variation and post-transcriptional regulation, and their high expression is often detrimental to patient survival. Based on the expression profiles of hub UBRs, patients can be classified into two ubiquitination subtypes with different characteristics. These subtypes exhibit significant differences across multiple dimensions, including survival, expression level, mutation burden, female predominance, infiltration level, immune profile, and drug response. In addition, we established a scoring system for evaluating the ubiquitination status of individual LUAD patients, called the ubiquitination-related risk (UB_risk) score, and found that patients with low scores are more likely to gain advantages from immunotherapy. The results of this study emphasize the critical role of ubiquitination in the classification, tumor microenvironment and immunotherapy of LUAD. The construction of the UB_risk scoring system lays a research foundation for evaluating the ubiquitination status of individual LUAD patients and formulating precise treatment strategies from the ubiquitination level. Full article

The global burden of respiratory viral infections is notable, which is attributed to their higher transmissibility compared to other viral diseases. Respiratory viruses are seen to have evolved resistance to available treatment options. Although vaccines and antiviral drugs control some respiratory viruses, this control is limited due to unexpected events, such as mutations and the development of antiviral resistance. The technology of proteolysis-targeting chimeras (PROTACs) has been emerging as a novel technology in viral therapeutics. These are small molecules that can selectively degrade target proteins via the ubiquitin–proteasome pathway. PROTACs as a therapy were initially developed against cancer, but they have recently shown promising results in their antiviral mechanisms by targeting viral and/or host proteins involved in the pathogenesis of viral infections. In this review, we elaborate on the antiviral potential of PROTACs as therapeutic agents and their potential as vaccine components against important respiratory viral pathogens, including influenza viruses, coronaviruses (SARS-CoV-2), and respiratory syncytial virus. Advanced applications of PROTAC antiviral strategies, such as hemagglutinin and neuraminidase degraders for influenza and spike proteins of SARS-CoV-2, are detailed in this review. Additionally, the role of PROTACs in targeting cellular mechanisms within the host, thereby preventing viral pathogenesis and eliciting an antiviral effect, is discussed. The potential of PROTACs as vaccines, utilizing proteasome-based virus attenuation to achieve a robust protective immune response, while ensuring safety and enhancing efficient production, is also presented. With the promises exhibited by PROTACs, this technology faces significant challenges, including the emergence of novel viral strains, tissue-specific expression of E3 ligases, and pharmacokinetic constraints. With advanced computational design in molecular platforms, PROTAC-based antiviral development offers an alternative, transformative path in tackling respiratory viruses. Full article

Background/Objectives: Myogenesis, the process by which myoblasts differentiate into multinucleated muscle fibers, is tightly regulated by transcription factors, signaling pathways, and metabolic cues. Among these, fatty acids have emerged as key regulators beyond their traditional role as energy substrates. Oleic acid, a monounsaturated fatty acid, has been shown to modulate muscle differentiation, potentially influencing myogenic pathways. This study examines the role of oleic acid in promoting C2C12 myoblast differentiation and its associated molecular mechanisms, comparing it to standard horse serum (HS)-based differentiation protocols. Methods: C2C12 murine myoblasts were cultured under proliferative conditions and differentiated using DMEM supplemented with either 2% HS or oleic acid (C18:1, n-9). The molecular signaling pathway was evaluated by measuring the expression of p38 MAPK, β-catenin, GLUT4, and NDRG1. Results: Oleic acid promoted the differentiation of C2C12 cells, as evidenced by a progressively elongated morphology, as well as the induction of muscle-specific myogenin, myosin heavy chain (MHC), and MyoD. Moreover, oleic acid reduced the expression of Atrogin-1 and MuRF1 ubiquitin E3 ligase. BODIPY staining revealed the enhanced accumulation of lipid droplets in oleic acid-treated cells. The Western blot analysis demonstrated robust activation of p38 MAPK and β-catenin pathways in response to oleic acid, compared with HS. Additionally, oleic acid upregulated GLUT4 expression and increased the phosphorylation of insulin receptor and NDRG1, indicating an enhanced glucose uptake capacity. Conclusions: These findings demonstrate that oleic acid promotes C2C12 myoblast differentiation and improves glucose uptake via GLUT4. Oleic acid emerges as a promising metabolic regulator of myogenesis, offering potential therapeutic applications for muscle regeneration in muscle-related pathologies. Full article

Aberrant activation of the mitogen-activated protein kinase (MAPK) cascade promotes oncogenic transcriptomes. Despite efforts to inhibit oncogenic kinases, such as BRAFV600E, tumor responses in patients can be heterogeneous and limited by drug resistance mechanisms. Here, we describe patient tumors that acquired COP1 or DET1 mutations after treatment with the BRAF^V600E inhibitor vemurafenib. COP1 and DET1 constitute the substrate adaptor of the E3 ubiquitin ligase CRL4^COP1/DET1, which targets transcription factors, including ETV1, ETV4, and ETV5, for proteasomal degradation. MAPK-MEK-ERK signaling prevents CRL4^COP1/DET1 from ubiquitinating ETV1, ETV4, and ETV5, but the mechanistic details are still being elucidated. We found that patient mutations in COP1 or DET1 inactivated CRL4^COP1/DET1 in melanoma cells, stabilized ETV1, ETV4, and ETV5, and conferred resistance to inhibitors of the MAPK pathway. ETV5, in particular, enhanced cell survival and was found to promote the expression of the pro-survival gene BCL2A1. Indeed, the deletion of pro-survival BCL2A1 re-sensitized COP1 mutant cells to vemurafenib treatment. These observations indicate that the post-translational regulation of ETV5 by CRL4^COP1/DET1 modulates transcriptional outputs in ERK-dependent cancers, and its inactivation contributes to therapeutic resistance. Full article