Search Results (719)

Malaria remains a major global health burden, and the emergence of resistance to blood stage antimalarials underscores the need for new interventions targeting earlier stages of the parasite’s life cycle. The pre-erythrocytic liver stage represents a critical bottleneck and an attractive target for chemotherapeutic and prophylactic interventions. In this study, we functionally characterized the putative E3 ubiquitin ligase Trophozoite Exported Protein 1 (TEX1; PBANKA_0102200) in Plasmodium berghei using gene knockout, tagging, and imaging approaches across the mosquito and liver stages. TEX1 knockout parasites (PbTEX1-KO) showed impaired development during mosquito-stage transitions, with significant reductions in ookinete formation, oocyst numbers, and sporozoites reaching the salivary glands. In hepatic stages, TEX1-KO parasites displayed reduced growth, abnormal nuclear division, and impaired liver stage maturation, ultimately leading to a dramatic decline in detached cell formation and blood stage infectivity. Endogenous C-terminal tagging of TEX1 with GFP and 3×HA revealed a discrete subnuclear localization pattern, indicating a critical role in DNA synthesis and/or mitotic regulation. Our findings reveal that TEX1 is required for nuclear replication and division and successful development in both the mosquito and liver stages of Plasmodium. Given its pivotal role and nuclear localization during hepatic schizogony, TEX1 represents a promising target for the development of liver stage antimalarial interventions. Full article

(1) Background: The naked mole-rat (Heterocephalus glaber) survives hypoxia–reoxygenation stresses by utilizing metabolic rate depression, achieved in part by downregulating nonessential genes and processes to conserve endogenous cellular resources and prevent buildup of toxic waste byproducts. Tight molecular control of protein degradation (specifically the ubiquitin–proteasome system) is a potent regulatory tool for maintaining muscle integrity during hypoxia, but how this system is regulated in the heart of hypoxia-tolerant species is poorly understood. (2) Methods: The protein expression levels of cullin-RING E3 ligases (specifically CRL4 architecture), deubiquitinating enzymes, and proteasomal activity were assayed in cardiac tissues from H. glaber exposed to 24 h of normoxia or hypoxia in vivo. (3) Results: Overall, the protein expression of E3 ligases decreased, whereas expression of deubiquitinating enzymes increased during hypoxia, all of which play roles in themes of oxidative stress, heightened DNA damage repair, and the HIF-1-VHL-NFκB axis. Proteasomal activity was elevated during hypoxia, which conceivably links to the oxidative stress theory of aging and longevity of H. glaber. (4) Conclusions: Taken together, our results expand current research into protein degradation and extreme environmental stress responses, with a specific focus on cardiac mechanisms related to oxidative stress resistance along the hypoxia-longevity axis. Full article

Accumulation of misfolded proteins is implicated in neurodegenerative diseases. One of these is Huntington’s disease, which is caused by an expansion of trinucleotide (CAG) repeats in exon 1 of huntingtin gene (HTT). This expansion results in the production of mutant huntingtin exon1 protein (mHttEx1) containing polyglutamine tracks that is prone to cytotoxic aggregation. These mHttEx1 aggregates range from small soluble aggregates to large insoluble inclusion bodies. The mechanisms to clear mHttEx1 aggregates include ubiquitin-dependent proteasomal degradation and autophagy. For the proteasomal degradation of mHttEx1, ubiquitinated protein is first recognized by the Cdc48 complex for extraction and unfolding. For autophagy, mHttEx1 inclusion bodies are engulfed by an autophagosome, which fuses with the vacuole/lysosome and delivers cargo for vacuolar degradation. We name this autophagy IBophagy. In this study, we further show that the ubiquitination of mHttEx1 by the E3 ligase San1, its extraction and unfolding by the Cdc48 complex, and subsequent proteasomal degradation are all essential steps for mHttEx1 IBophagy in budding yeast, revealing a new layer of autophagy regulation and mHttEx1 cytotoxicity. Full article

Atrial fibrillation (AF) is the most common sustained arrhythmia, and its initiation and progression involve multiple mechanisms, including electrical remodeling, structural remodeling, inflammatory responses, and oxidative stress. In recent years, the ubiquitin–proteasome system (UPS), a central pathway for maintaining intracellular protein homeostasis, has attracted increasing attention in the pathogenesis of AF. By regulating the degradation and expression of ion channel proteins, Ca²⁺-handling molecules, and pro-fibrotic signaling factors, the UPS plays a pivotal role in key pathological processes such as electrical and structural remodeling. Several E3 ubiquitin ligases (e.g., NEDD4-1/2, MuRF1, WWP1/2, TRAF6), deubiquitinating enzymes (e.g., JOSD2), and immunoproteasome subunits (e.g., β5i) have been shown to exert critical regulatory effects on atrial electrophysiological disturbances, interstitial remodeling, and inflammation. This review provides a comprehensive summary of the regulatory mechanisms of the UPS in AF-associated pathological processes, outlines potential therapeutic targets, and highlights current intervention strategies, including proteasome inhibitors, selective E3 ligase modulators, and natural compounds. Moreover, we discuss the latest advances and future perspectives regarding the application of UPS-based interventions in AF, aiming to provide theoretical foundations and research insights for the mechanistic exploration and innovative therapeutic development of AF. Full article

Ring Box Protein-1 (RBX1) is an essential component of the Skp1-cullin-F-box protein (SCF) E3 ubiquitin ligase, which is involved in the regulation of oocyte maturation in the form of ubiquitination substrate modification. In this study, a sequence of RBX1 (Sp-RBX1) was identified and analyzed using bioinformatics methods from the transcriptome data of Scylla paramamosain. The length of Sp-RBX1 cDNA sequence was 1247 bp, consisting of a 336 bp open reading frame (ORF). Sequence analysis revealed that the protein contained a C-terminal modified RING-H2 finger domain, with two zinc binding sites and a Cullin binding site, classifying it as a member of the RBX1 superfamily. The results of real-time fluorescence quantitative PCR (RT-qPCR) showed that Sp-RBX1 expression in the ovary was low at stages I and II, then significantly increased from stage III to V (p < 0.05), which indicated that it might be closely related to the maturation of oocytes. It also peaked at stage II in the hepatopancreas, then sharply declined from stages III to V. The expression pattern might be related to the accumulation of fat in the early development of hepatopancreas. Furthermore, we characterized the expression of Sp-RBX1 induced by follicle-stimulating hormone (FSH) and estradiol (E2) hormones. The results showed that the expression in the ovary was up-regulated by FSH and significantly inhibited by E2. The expression in the hepatopancreas increased only at 0.5 µmol/L concentration of FSH, and decreased in other groups. Conversely, it was up-regulated by E2. Thus, the expression of Sp-RBX1 was influenced by FSH in a concentration-dependent manner. These findings could offer valuable insights for further research on ovarian maturation in crustaceans. Full article

Decreased skeletal muscle mass and function are a serious complication of long-term diabetes, often leading to numerous adverse outcomes. The primary pathological features of diabetic sarcopenia include muscle fiber atrophy and interstitial fibrosis. Although resistance exercise (RE) has been reported to mitigate skeletal muscle atrophy in type 2 diabetes mellitus (T2DM), the underlying mechanisms remain unclear. Fibroblast growth factor 21 (FGF21), an exercise-induced cytokine, has been shown to protect against skeletal muscle atrophy at elevated levels. In this study, a T2DM mouse model was established through 12 weeks of high-fat diet feeding and intraperitoneal injection of streptozotocin (STZ) to investigate the effect and mechanism of RE on skeletal muscle atrophy in T2DM mice. Our results demonstrated that 8 weeks of RE significantly decreased body weight, fat mass, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), fasting blood glucose (FBG), and serum insulin levels in T2DM mice. RE also improved lean mass, glucose tolerance (IPGTT), and insulin tolerance (ITT). Additionally, RE increased skeletal muscle mass cross-sectional area (CSA) while attenuating fibrosis and inflammatory responses in skeletal muscle. Notably, RE upregulated FGF21 expression and activated the PI3K/Akt signaling pathway in diabetic skeletal muscle. RE promoted the phosphorylation of mTOR, 4EBP1, and p70S6K while suppressing the expression of the atrophy-related E3 ubiquitin ligases MuRF1 and MAFbx/Atrogin-1. Furthermore, RE inhibited lipid synthesis and enhanced both lipid oxidation and glucose utilization in skeletal muscle of T2DM mice. RE also improved mitochondrial biogenesis and dynamics in skeletal muscle of T2DM mice. In summary, 8 weeks of RE alleviated skeletal muscle atrophy in T2DM mice via activation of the FGF21/PI3K/Akt signaling pathway, which enhanced protein synthesis, improved glycolipid metabolism and mitochondrial quality control, and attenuated fibrosis and inflammation. Full article

Ovarian cancer (OC) represents the most lethal gynecologic malignancy because the majority of patients with OC are diagnosed at advanced stages with peritoneal colonization of OC cells owing to subtle and nonspecific nature of symptoms. Thus, peritoneal colonization-directed therapeutic approaches are urgently needed for patients with advanced OC. Here, we investigated whether Ring-finger protein 126 (RNF126), an E3 ubiquitin ligase that is aberrantly upregulated in epithelial OC tissues, contributes to the peritoneal colonization of OC. RNF126-depleted OC cells showed comparable proliferation under normal culture conditions but displayed decreased growth under floating (anchorage-independent) conditions in vitro. Further analyses showed that RNF126 promoted anoikis resistance in vitro and increased peritoneal colonization in immunodeficient mice in a RING domain-dependent manner. Mechanistically, RNF126 activated the transcriptional factor NF-κB in OC cells under floating conditions in a RING domain-dependent manner, and this NF-κB activation was essential for anchorage-independent growth and peritoneal colonization of OC cells. Thus, RNF126 is a possible target for the prevention and/or therapy of peritoneally colonized OC. Full article

Background: In recent years, compounds known as Proteolysis Targeted Chimeras (PROTACs) have revitalized the field of bioactive molecule design. These compounds promote proteolysis of therapeutic targets by recruiting them to ubiquitin ligases. One of the most commonly used classes of compounds in the synthesis of PROTACs are immunomodulatory imides (IMiDs), such as thalidomide (TLD), which interact with the E3 ligase CRL4^CRBN via the CULT domain of the cereblon protein (CRBN). This domain has been identified in proteins across various phylogenetic groups, including trypanosomatids, leading to the hypothesis that IMiD-derived PROTACs should be active in these organisms. Methods: The trypanocidal activity of the PROTAC dBET1 and its separated components (JQ1 and TLD) were assayed using a T. cruzi strain expressing β-glalactosidase. Potential CRL4-E3L complexes from humans and trypanosomatids were assembled in silico with MultimerMapper. The IMiD-binding site of HsCRBN and its trypanosomatid homologs were analyzed using molecular dynamics and docking simulations. Results: We demonstrate that the compound dBET1 does not function as a PROTAC in Trypanosoma cruzi. In silico structural analysis of CRL4-E3L complex orthologs revealed that the trypanosomal CULT-containing protein is not part of such a complex. Molecular dynamics simulations showed that the pocket of this CULT domain is smaller than that of mammalian CRBN and cannot accommodate IMiDs within. Conclusions: We underscore the importance of functional and structural validation in drug discovery, particularly when extrapolating mechanisms between evolutionarily distant species. While PROTACs hold promise in human therapeutics, our work advocates for re-evaluating the rationale behind thalidomide-based PROTACs in trypanosomatid research. Full article

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the deficiency of the UBE3A gene that for a E3 ligase protein part of the ubiquitin–proteasome system (UPS). Autophagy and UPS systems remove abnormal proteins, but any dysfunction in these processes can affect neuronal development and wellbeing. Herein, the involvement of the UPS/autophagy system in the regulation of intracellular signaling pathways related to toxic protein accumulation was investigated in cellular/mice AS models, silenced for UB3A (UB3A⁻). The main findings are as follows: (i) autophagy was upregulated in UBE3A⁻ cells with respect to control cells; (ii) a dysregulation of the AKT/mTOR pathway, linked to autophagy/synaptic development, was evidenced in cellular/animal models of AS with respect to controls; (iii) the ubiquitin ligase MDM2 was downregulated, and the tumor suppressor p53, normally inhibited by MDM2, enhanced its expression and transcriptional activity in UB3A⁻ cells with respect to controls. Finally, UB3A⁻ cells presented a significant alteration in the levels of β-amyloids with respect to control cells, and a reduction of α-synuclein levels, typical of neurodevelopmental disorder. Nevertheless, UB3A⁻ cells do not show evident morphological abnormalities. Overall, these data suggest that AS models presented an altered signaling pathway related to autophagy/UPS systems, potentially leading to the accumulation of toxic proteins affecting synaptic development. Full article

Disuse-induced muscle atrophy (DMA), commonly resulting from immobilization, is driven by chronic inflammation and oxidative stress, which disrupts the balance between protein synthesis and degradation. Quinic acid (QA), a natural compound with known antioxidant and anti-inflammatory properties, was investigated for its potential to counteract muscle atrophy. Using a DMA-induced immobilization model in male C57BL/6N (8 weeks) mice, we found that oral QA administration significantly restored the weight and cross-sectional area of atrophic muscles and improved muscle function, as measured by grip strength and treadmill performance. QA also reduced the expression of pro-inflammatory cytokines (Tnf, Il6, and Myostatin) and E3 ubiquitin ligases (Trim63 and Fbxo32), while increasing antioxidant enzyme levels and serum IL-15 in DMA. In tumor necrosis factor-α-stimulated L6 myotubes, QA reversed inflammation- and oxidative stress-induced gene changes, suppressed NF-ĸB activation, and downregulated protein degradation pathways mediated by FoxO3α. Furthermore, QA restored the expression of myogenesis-related genes and reactivated PI3K/Akt and mTOR/p70S6K/4EBP1 signaling pathways, enhancing protein synthesis. Collectively, our findings demonstrate that QA mitigates immobilization-induced muscle atrophy by modulating inflammation, oxidative stress, and key anabolic and catabolic signaling pathways. These results suggest that QA is a promising functional compound for preserving skeletal muscle health under conditions of disuse. Full article

Crystallography plays a crucial role in understanding the functions of macromolecules by determining their three-dimensional structures at the atomic level. This review outlines the history of crystallization, explains the principles of crystallization, and provides a comprehensive retrospective on the role of crystallography in enzymology, with a particular focus on the seven Enzyme Commission (EC) classes. For each class, we highlight representative enzymes and the specific mechanistic insights enabled by crystal structures, oxidoreductases (the “yellow enzyme” lineage), transferases (phosphotransferase systems), hydrolases (RNase III and chymotrypsin), lyases (fumarase), isomerases (pseudouridine synthases), ligases (E3 ubiquitin ligases), and translocases (ATP synthase), emphasizing cofactor usage, conformational change, regulation, and implications for disease and drug discovery. We also compile EC-wide statistics from the Protein Data Bank (PDB) to quantify structural coverage. The limitations and challenges of current crystallization techniques are addressed, along with alternative experimental methods for structural elucidation. In addition, emerging computational tools and biomolecular design are also discussed. By reviewing the trajectory of enzymology and crystallography, we demonstrated their profound impact on biochemistry and therapeutic discovery. Full article

Background: Gastric cancer (GC) remains a leading cause of cancer mortality. E3 ubiquitin ligases, as central regulators of protein stability and signaling within the ubiquitin–proteasome system, have been implicated in tumor progression, but their functional roles in GC are not well established. Methods: We integrated bioinformatics analysis of TCGA and GEO datasets, in vitro experiments (including cell proliferation, migration, and apoptosis assays), and computational modeling to identify key prognostic factors in GC. Results: We established two molecular subtypes (E3GC1/E3GC2) with distinct clinical outcomes and developed a 10-gene prognostic signature. The model showed moderate predictive accuracy (AUC: 0.61–0.71) and was validated externally. MMRN1 was upregulated in GC cells and its knockdown significantly inhibited malignant phenotypes. Critically, drug sensitivity analysis revealed high-risk patients were more sensitive to proteasome inhibitors (bortezomib), while low-risk patients responded better to taxane-based chemotherapy (docetaxel). Molecular docking predicted a high-confidence interaction between MMRN1 and NEDD4L, suggesting potential ubiquitination regulation. Conclusions: MMRN1 drives GC cell proliferation and migration in vitro and may be regulated by NEDD4L-mediated ubiquitination. Our study provides a foundation for E3 ligase-based patient stratification and personalized therapy selection in GC. While this study provides comprehensive multi-omics evidence supporting the role of MMRN1 in GC progression, its clinical translation is limited by the lack of in vivo validation and direct experimental evidence of NEDD4L-MMRN1 physical interaction. Further studies using animal models and clinical specimens are warranted to confirm these findings. Full article

Background/Objectives: This review discusses the development and application of targeted protein degradation strategies, particularly focusing on the ubiquitin–proteasome pathway and PROteolysis-TArgeting Chimeras (PROTAC) technology, for antiviral therapies. Methods/Results: The synthesis of specific PROTACs exemplifies the potential of this approach to inhibit viral replication. The discussion also covers ongoing efforts to develop broad-spectrum antivirals and explores the limitations of small-molecule ligands, proposing antibody mimetics as a versatile alternative. The review details innovative strategies involving engineered antibody mimetics, termed ‘diving antibodies’ (DAbs), capable of intracellular delivery and targeting viral proteins within cells. These molecules are engineered using modular nanotransporters to facilitate intracellular delivery. The integration of E3 ligase-binding sites into DAbs enhances their capacity to induce targeted protein degradation, with experimental evidence supporting their efficacy. Conclusions: Overall, the review underscores the potential of combining targeted degradation technologies with innovative delivery systems to create effective antiviral therapies, especially for viruses with limited treatment options. Full article

Background: Spermatogenesis depends on precise cytoskeletal regulation, particularly the microtubule system; however, the mechanisms governing tubulin homeostasis during meiosis are poorly defined. While the E3 ubiquitin ligase Hyd (Hyperplastic discs), the Drosophila homolog of UBR5 (Ubiquitin Protein Ligase E3 Component N-Recognin 5), plays roles in diverse cellular processes, its precise role in male meiosis is unknown. This study aims to define the function and expression dynamics of Hyd during Drosophila spermatogenesis and elucidate whether it acts independently of its canonical ligase activity. Methods: Using Drosophila genetics, immunofluorescence, CRISPR/Cas9-mediated tagging, and mosaic clonal analysis, we characterized Hyd expression and function in the testis. Hyd knockdown and rescue experiments were performed with wild-type and catalytically inactive transgenes. β2-tubulin expression and microtubule organization were assessed in hyd mutant clones. Results: Hyd exhibits a dynamic, stage-specific expression pattern, localizing to nuclear and meiotic structures. Hyd loss led to meiotic arrest, disrupted spindle formation, aberrant centrosome behavior, and failure of spermatid elongation. Genetic rescue demonstrated that both wild-type and catalytically inactive Hyd partially restored spermatid elongation, indicating a catalysis-independent role. Furthermore, Hyd deficiency resulted in β2-tubulin overexpression, disrupted microtubule organization, and abnormal spermatocyte morphology. Conclusions: Hyd ensures meiotic fidelity in Drosophila by fine-tuning β2-tubulin expression independently of its E3 ubiquitin ligase activity. These findings reveal a non-proteolytic function for UBR5/Hyd in cytoskeletal regulation during male gametogenesis, providing new insights into tubulin homeostasis in meiosis. Full article

Pheochromocytomas and paragangliomas (PPGL) are classified as rare cancers but can be highly metastatic, particularly in individuals with inherited succinate dehydrogenase B (SDHB) mutations. As current therapies and the availability of SDHB-deficient animal models are both limited, we have previously constructed a nematode PPGL model, a transgenic worm carrying the R244H missense mutation equivalent to human R230H in the sdhb-1 gene. In this study, we show that R244H mutants display characteristics of PPGL tumors, such as pseudohypoxia activation and the accumulation of reactive oxygen species. The latter can be the result of compromised antioxidant machinery, as R244H mutants have reduced levels of cytosolic and mitochondrial superoxide dismutase enzymes. In addition, the expression of mitophagy markers pink-1 (PTEN-induced putative kinase) and pdr-1 (E3 ubiquitin-protein ligase parkin) were downregulated in R244H mutants, suggesting impaired mitophagy and reflecting the crucial role of mitochondrial health in PPGL pathology. Treatments by the SDH inhibitor fluopyram revealed that the SDH complex carrying the R244H mutation in subunit B displayed residual SDH activity, which was also confirmed by our structural analyses. We also observed a link between dopaminergic neuronal health and SDHB-1. Full article