Ubiquitin Ligase Research Articles

Unraveling the interaction between a glycolytic regulator protein EhPpdk and an anaphase promoting complex protein EhApc10: yeast two hybrid screening, in vitro binding assays and molecular simulation study.

The anaphase promoting complex (APC or cyclosome) is a major ubiquitin ligase that coordinates mitotic and G1 progression, acting as a major regulator of chromosome segregation. While the human APC contains fourteen subunits, it is yet to be explored in the pathogen Entamoeba histolytica. Our study reveals the existence of a single functional Apc10 homolog in E. histolytica, which acts as a processivity factor of ubiquitin ligase activity in human. A cDNA library generated from HM1:IMSS strain of E. histolytica was screened for interaction partners of EhApc10 in yeast two hybrid study. The novel interactor, a glycolytic enzyme, pyruvate phosphate dikinase (Ppdk) was found to interact with EhApc10 and further validated by in vitro assay. A comprehensive in silico study has emphasized the structural and functional aspects, encompassing physicochemical traits, predictive 3D structure modelling, validation of EhApc10-EhPpdk interaction through molecular docking and simulation. The interplay between a cell cycle protein and a glycolytic enzyme highlights the connection between cellular metabolism and the cell cycle regulatory mechanism. The study serves as the groundwork for future research on the non-mitotic role of APC beyond cell cycle.

NEDD4L Inhibits the Proliferation and Migration of Keloid Fibroblasts by Regulating YY1 Ubiquitination-Mediated Glycolytic Metabolic Reprogramming.

Keloid scarring is a complex fibroproliferative disorder characterised by excessive fibroblast proliferation. Inhibition of cellular glycolysis effectively suppresses the proliferation of keloid fibroblasts (KFs). Neural precursor cell-expressed developmentally downregulated gene 4-like (NEDD4L), a ubiquitin ligase, regulates cell proliferation in different diseases. This study investigated the effects of NEDD4L on glucose metabolism, proliferation and migration in KFs. Primary KFs were isolated from keloid skin tissues obtained from patients with active-stage keloids. Cell transfection was used to upregulate or downregulate NEDD4L and Yin Yang 1 (YY1) in KFs. Protein expression was assessed by immunohistochemistry and western blotting. The viability, proliferative capacity and migration ability of KFs were evaluated using the MTT method and the EdU and wound healing assays, respectively. The regulatory effect of NEDD4L on YY1 ubiquitination was examined by coimmunoprecipitation. The interaction between YY1 and hexokinase 2 (HK2) was confirmed by a dual-luciferase reporter assay. NEDD4L was downregulated, whereas YY1 and HK2 were highly expressed in keloid tissues compared with normal skin. Overexpression of NEDD4L inhibited the proliferation and migration of KFs. NEDD4L promoted YY1 degradation in KFs by inducing its ubiquitination. Upregulation of YY1 induced glucose consumption and lactate production in KFs via the transcriptional regulation of HK2. Increased expression of YY1 reversed the reduced viability, proliferation, and migration of KFs overexpressing NEDD4L. YY1 also reversed the NEDD4L-induced inhibition of glucose consumption and lactate production in KFs. Additionally, an invivo study confirmed the inhibitory roles of NEDD4L overexpression and YY1 knockdown in keloid formation. NEDD4L suppressed the viability, proliferation and migration of KFs by regulating YY1 ubiquitination-mediated glycolysis through HK2. These findings suggest a novel regulatory axis, NEDD4L/YY1/HK2, that mediates glucose metabolism in keloid formation.

Siling Decoction Ameliorates Adenine-Induced Renal Fibrosis in Rats by the AKT/IKKβ/NFκB Signaling Pathway

ObjectiveInvestigating how Siling decoction (SLD) mitigates fibrosis in rats with chronic kidney disease CKD (chronic kidney disease) through network pharmacology analysis and experimental verification. MethodsInitially, the primary active components and their target actions of SLD (Fuling, Zhuling, Zexie, and Baizhu) were identified by the TCMSP database and liquid chromatography mass spectrometry (LC-MS). Treatment targets for renal fibrosis were screened through databases such as GeneCard, OMIM, PharmGkb, and GEO. Subsequently, a drug-disease-target network was constructed and subjected to PPI analysis. Intersecting targets underwent GO and KEGG pathway enrichment analyses. Renal fibrosis model was induced by adenine gavage, then treat with SLD. Masson, Sirius red, immunohistochemistry, and Western blot were used to detect renal function and fibrosis-related indicators. The mechanism was further validated in vitro experiments. ResultsNetwork pharmacology analysis identified 100 common targets associated with the active components of SLD, including core genes such as AKT1 and CCND1. GO enrichment analysis revealed that the top three biological processes impacted include response to xenobiotic stimulus, response to nutrient levels and response to oxidative stress. These processes involved cellular components such as membrane raft, membrane microdomain and synaptic membrane, with molecular functions predominantly associated with ubiquitin-like protein ligase binding, ubiquitin protein ligase binding, DNA-binding transcription factor binding, and RNA polymerase II-specific DNA binding transcription factor binding. KEGG pathway enrichment analysis indicated potential involvement of pathways like Lipid and atherosclerosis, PI3K-AKT signaling pathway, and prostate cancer are likely involved in the anti-fibrotic effect of SLD. Notably, the highlighted was the AKT/IKKβ/NFκB signaling pathway as a key mechanism. These findings were further confirmed in vivo and in vitro. ConclusionThe SLD effectively ameliorates adenine-induced chronic kidney disease fibrosis in rats, potentially by inhibiting the AKT/IKKβ/NFκB signaling pathway.

Role of angiopoietin-like 4 in neovascularization associated with retinopathy of prematurity

To elucidate the mechanisms of angiopoietin-like 4 (ANGTPL4) in neovascularization (NV) in retinopathy of prematurity (ROP). We compared ANGPTL4 expression levels of aqueous humor and vitreous fluid samples in infants with acute-phase ROP and control group. ANGPTL4-/- mice and WT mice were used to constructed oxygen-induced retinopathy (OIR) mouse models, with retinal tissues collected on postnatal days 12 (P12), 15 (P15) and 17 (P17). Analysis of retinal vessels and transcriptomics were performed to explore the role of ANGTPL4 in NV. The results showed ANGPTL4 level was significantly higher in the aqueous humour and vitreous fluid of children with ROP than that of control group. At P15 and P17, the vascular indices in the ANGPTL4-/--CON group were lower than those in the WT-CON group. The central non-perfused area of the retina and number of neovascular nuclei were also smaller in the ANGPTL4-/--OIR group than in the WT-OIR group. Immunofluorescence results showed the overexpression of ANGPTL4 protein in the WT-OIR group than in the WT-CON group, especially at P17. Furthermore, extracellular matrix (ECM) organisation was one of the key involved pathways based on gene ontology (GO) enrichment analyses. ANGPTL4 was one of the core genes involved in ECM organization, and neuralized E3 ubiquitin protein ligase 1B (NEURL1B), cd36 Molecule (CD36), matrix metallopeptidase 3 (MMP3) and collagen type III alpha 1 chain (COL3A1) were the first nodes interacting with ANGPTL4.In conclusion, ANGPTL4 is involved in the pathological NV by regulating NEURL1B, CD36, MMP3, and COL3A1. Thus, ANGPTL4 is a potential therapeutic target for ROP.

Vitamin C Alleviates Heat-Stress-Induced Damages in Pig Thoracic Vertebral Chondrocytes via the Ubiquitin-Mediated Proteolysis Pathway

Heat stress can impair organismal growth by inducing ubiquitination, proteasome-mediated degradation, and subsequent cellular damage. Vitamin C (VC) has been shown to potentially mitigate the detrimental effects of abiotic stresses on cells. Nevertheless, the impact of heat stress on growth plate chondrocytes remains unclear, and the underlying protective mechanisms of VC in these cells warrant further investigation. In this study, we focused on pig thoracic vertebral chondrocytes (PTVCs) that are crucial for promoting the body’s longitudinal elongation and treated them with 41 °C heat stress for 24 h, under varying concentrations of VC. Our findings reveal that, while oxidative stress induced by heat triggers apoptosis and inhibits the ubiquitin-mediated proteolysis pathway, the addition of VC alleviates heat-stress-induced oxidative stress and apoptosis, mitigates cell cycle arrest, and promotes cellular viability. Furthermore, we demonstrate that VC enhances the ubiquitin-proteasome proteolysis pathway by promoting the expression of ubiquitin protein ligase E3A, which thereby stabilizes the ubiquitin-mediated degradation machinery, alleviates the apoptosis, and enhances cell proliferation. Our results suggest the involvement of the ubiquitin-mediated proteolysis pathway in the effects of VC on PTVCs under heat stress, and offer a potential strategy to make use of VC to ensure the skeletal growth of animals under high temperature pressures in summer or in tropical regions.

Transcriptome signature for multiple biotic and abiotic stress in barley (Hordeum vulgare L.) identifies using machine learning approach

Barley (Hordeum vulgare L.) is exposed to various biotic and abiotic stresses, making it crucial to fully understand the gene signatures that respond to stress. This study utilizes machine learning to analyze transcriptomic data from 515 RNA-seq profiles across 18 independent studies, covering eleven abiotic and three biotic stress types. Through meticulous data preprocessing, including quality assessment and batch effect correction, we have identified 4,311 genes for further analysis. Feature selection was performed using five weighting algorithms, resulting in the prioritization of 400 core genes. Machine learning models, specifically Random Forest and C4.5, were optimized and evaluated using a 10-fold cross-validation approach. The C4.5 algorithm demonstrated superior accuracy in predicting stress-responsive signatures. Key genes, such as bHLH119 and E3 ubiquitin protein ligase DRIP2, were identified as potential biomarkers. Functional enrichment analysis, conducted through protein-protein interaction networks and Gene Ontology/KEGG pathway analysis, has revealed significant involvement in lipid biosynthesis, signal transduction, and defense response processes. These findings highlight the crucial roles of the identified biomarkers genes in barley's resilience to stress and provide potential targets for genetic improvement. Future research should focus on validating these biomarkers in different barley cultivars and under field conditions to enhance crop resilience against stressors.

FBXO22 inhibits colitis and colorectal carcinogenesis by regulating the degradation of the S2448-phosphorylated form of mTOR

Inflammatory bowel disease (IBD) is a considerable threat to human health with a significant risk for colorectal cancer (CRC). However, currently, both the molecular pathogenesis and therapeutic treatment of IBD remain limited. In this report, using both systemic and intestinal epithelium-specific gene knockout mouse models, we demonstrate that FBXO22, a substrate receptor within the SKP1-Cullin 1-F-box family of E3 ubiquitin ligases, plays an inhibitory role in the Azoxymethane/Dextran Sodium Sulfate-induced colorectal inflammatory responses and CRC. FBXO22 targets the serine 2448-phosphorylated form of mammalian mechanistic target of rapamycin (pS2448-mTOR) for ubiquitin-dependent degradation. This proteolytic targeting effect is established based on multiple lines of evidence including the results of colon tissue immunoblots, analysis of cultured cells with altered abundance of FBXO22 by depletion or overexpression, comparison of protein decay rate, effects on mTOR substrates S6K1 and 4E-BP1, analysis of protein-protein interactions, phosphor-peptide binding and competition, as well as reconstituted and cellular ubiquitination. Finally, we have shown that mTOR inhibitor rapamycin (RAPA) was able to alleviate the effects of fbxo22 deletion on colorectal inflammatory response and CRC. These RAPA effects are correlated with the ability of RAPA to inhibit pS2448-mTOR, pS6K1, and p4E-BP1. Collectively, our data support a suppressive role for FBXO22 in colorectal inflammation signaling and CRC initiation by targeting pS2448-mTOR for degradation.

Insights into degradation and targeting of the photoreceptor channelrhodopsin-1.

In Chlamydomonas, the directly light-gated, plasma membrane-localized cation channels channelrhodopsins ChR1 and ChR2 are the primary photoreceptors for phototaxis. Their targeting and abundance is essential for optimal movement responses. However, our knowledge how Chlamydomonas achieves this is still at its infancy. Here we show that ChR1 internalization occurs via light-stimulated endocytosis. Prior or during endocytosis ChR1 is modified and forms high molecular mass complexes. These are the solely detectable ChR1 forms in extracellular vesicles and their abundance therein dynamically changes upon illumination. The ChR1-containing extracellular vesicles are secreted via the plasma membrane and/or the ciliary base. In line with this, ciliogenesis mutants exhibit increased ChR1 degradation rates. Further, we establish involvement of the cysteine protease CEP1, a member of the papain-type C1A subfamily. ΔCEP1-knockout strains lack light-induced ChR1 degradation, whereas ChR2 degradation was unaffected. Low light stimulates CEP1 expression, which is regulated via phototropin, a SPA1 E3 ubiquitin ligase and cyclic AMP. Further, mutant and inhibitor analyses revealed involvement of the small GTPase ARL11 and SUMOylation in ChR1 targeting to the eyespot and cilia. Our study thus defines the degradation pathway of this central photoreceptor of Chlamydomonas and identifies novel elements involved in its homoeostasis and targeting.

E3 ubiquitin ligase DTX2 fosters ferroptosis resistance via suppressing NCOA4-mediated ferritinophagy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) remains the foremost contributor to cancer-related fatalities globally, with limited effective therapeutic modalities. Recent research has shed light on the role of ferroptosis in various types of cancers, offering a potential avenue for improving cancer therapy. Herein, we identified E3 ubiquitin ligase deltex 2 (DTX2) as a potential therapeutic target candidate implicated in promoting NSCLC cell growth by inhibiting ferroptosis. Our investigation revealed a significant upregulation of DTX2 in NSCLC cells and tissues, which was correlated with poor prognosis. Downregulation of DTX2 suppressed NSCLC cell growth both in vitro and in vivo, while its overexpression accelerated cell proliferation. Moreover, knockdown of DTX2 promoted ferroptosis in NSCLC cells, which was mitigated by DTX2 overexpression. Mechanistically, we uncovered that DTX2 binds to nuclear receptor coactivator 4 (NCOA4), facilitating its ubiquitination and degradation via the K48 chain, which subsequently dampens NCOA4-driven ferritinophagy and ferroptosis in NSCLC cells. Notably, DTX2 knockdown promotes cisplatin-induced ferroptosis and overcomes drug resistance of NSCLC cells. These findings underscore the critical role of DTX2 in regulating ferroptosis and NCOA4-mediated ferritinophagy, suggesting its potential as a novel therapeutic target for NSCLC.

PIAS4 regulates pluripotency exit and cell fate commitment in porcine embryonic stem cells

Post-translational modifications (PTMs) are dynamic processes that regulate cell states by enhancing proteome diversity. However, the overall impact of PTMs on pluripotency exit in porcine embryonic stem cells (pESCs) remains largely unknown. Here, we present a systematic assay to identify E3 ubiquitin ligases for pluripotency exit by using CRISPR/Cas9 pooled screening and identified PIAS4 as a major regulator of pluripotency exit, as the cell differentiation was significantly impaired upon PIAS4 depletion in pESCs. PIAS4 shows a high degree of genomic occupation in promoter regions, particularly in key pluripotency maintenance genes. Moreover, we found that PIAS4 was recruited to the gene promoter marked by H3K4me3 and interacted with lysine demethylase KDM5B via SUMOylation, thereby affecting the stability of KDM5B and further facilitating the regulation of H3K4me3-mediated lineage-specific genes. Together, our findings reveal a regulatory mechanism by which PIAS4 modulates H3K4me3 modification on development-related genes, subsequently influencing pluripotency exit and cell fate commitment by interacting with KDM5B in pESCs.

CRBN-PROTACs in Cancer Therapy: From Mechanistic Insights to Clinical Applications.

Cereblon (CRBN), a member of the E3 ubiquitin ligase complex, has gained significant attention as a therapeutic target in cancer. CRBN regulates the degradation of various proteins in cancer progression, including transcription factors and signaling molecules. PROTACs (proteolysis-targeting chimeras) are a novel approach that uses the cell's degradation system to remove disease-causing proteins selectively. CRBN-dependent PROTACs work by tagging harmful proteins for destruction through the ubiquitin-proteasome system. This strategy offers several advantages over traditional protein inhibition methods, including the potential to overcome drug resistance. Recent progress in developing CRBN-based PROTACs has shown promising preclinical results in both hematologic malignancies and solid tumors. Additionally, CRBN-based PROTACs have enhanced our understanding of CRBN's role in cancer, potentially serving as biomarkers for patient stratification and predicting therapeutic responses. In this review, we delineate the mechanisms of action for CRBN-dependent PROTACs (CRBN-PROTACs), summarize recent advances in preclinical and clinical applications, and provide our perspective on future development.

ARMC5 selectively degrades SCAP-free SREBF1 and is essential for fatty acid desaturation in adipocytes

SREBF1 plays the central role in lipid metabolism. It has been known that full-length SREBF1 that did not associate with SCAP (SCAP-free SREBF1) is actively degraded, but its molecular mechanism and its biological meaning remain unclear. ARMC5-CUL3 complex was recently identified as E3 ubiquitin ligase of full-length SREBF. Although ARMC5 was involved in SREBF pathway in adrenocortical cells, the role of ARMC5 in adipocytes has not been investigated. In this study, adipocyte-specific Armc5 knockout mice were generated. In the white adipose tissue (WAT) of these mice, all the stearoyl-CoA desaturase (Scd) were drastically downregulated. Consistently, unsaturated fatty acids were decreased and saturated fatty acids were increased. The protein amount of full-length SREBF1 were increased, but ATAC-Seq peaks at the SREBF1-binding sites were markedly diminished around the Scd1 locus in the WAT of Armc5 knockout mice. Armc5-deficient 3T3-L1 adipocytes also exhibited downregulation of Scd. Mechanistically, disruption of Armc5 restored decreased full-length SREBF1 in CHO cells deficient for Scap. Overexpression of Scap inhibited ARMC5-mediated degradation of full-length SREBF1, and overexpression of Armc5 increased nuclear SREBF1/full-length SREBF1 ratio and SREBF1 transcriptional activity in the presence of exogenous SCAP. These results demonstrated that ARMC5 selectively removes SCAP-free SREBF1 and stimulates SCAP-mediated SREBF1 processing, hence is essential for fatty acid desaturation in vivo.

PHLDA1 protects intestinal barrier function via restricting intestinal epithelial cells apoptosis in inflammatory bowel disease

The current approach to treating inflammatory bowel disease (IBD) primarily focuses on managing inflammation rather than maintaining the integrity of the intestinal barrier. In our study, we sought to investigate the potential role of PHLDA1 in preserving intestinal barrier function as a promising strategy for treating IBD. We observed a significant decrease in PHLDA1 expression in intestinal epithelial cells (IECs) of both IBD patients and mice with chemically induced colitis. This deficiency of PHLDA1 led to increased apoptosis of IECs, resulting in a compromised epithelial barrier and the invasion of commensal bacteria into the mucosa. Consequently, this microbial invasion substantially exacerbated colonic inflammation in mice with the specific knockout of PHLDA1 in IECs (Phlda1IEC-KO) compared to their control littermates. Mechanistically, we found evidence of PHLDA1 interacting with MCL1 to protect against K48-linked polyubiquitylation at the K40 lysine residue, thus preventing ubiquitin-proteasome degradation through the MCL1 ubiquitin ligase E3 (Mule). We further confirmed that the PHLDA1-MCL1-Mule signaling pathway plays a critical role in the development of IBD. Notably, our study demonstrated that enhancing MCL1 levels or reducing Mule expression using adeno-associated virus (AAV) attenuated experimental colitis in Phlda1IEC-KO mice. Collectively, our findings emphasize the significance of PHLDA1 in the pathogenesis of IBD and propose that targeting the PHLDA1-MCL1-Mule signaling pathway could be a viable approach for combating IBD.

The E3 ligase OsHel2 impedes readthrough of stalled mRNAs to regulate male fertility in thermo-sensitive genic male sterile rice.

Heterosis is extensively utilized in the two-line hybrid breeding system. Photo-/thermo-sensitive genic male sterile (P/TGMS) lines are key components of two-line hybrid rice. TGMS lines containing tms5 have significantly advanced two-line hybrid rice breeding. We cloned the TMS5 gene and found that TMS5 is a tRNA cyclic phosphatase that can remove 2',3'-cyclic phosphate (cP) from cP-ΔCCA-tRNAs for efficient repair to ensure maintenance of mature tRNA levels. tms5 mutation causes increased cP-ΔCCA-tRNAs and reduced mature tRNAs, leading to male sterility at the restrictive temperatures. However, the regulatory network of tms5-mediated TGMS remains to be elucidated. Here, we identified that an E3 ligase OsHel2 cooperates with TMS5 to regulate TGMS at the restrictive temperatures. Consistently, both the accumulation of 2',3'-cP-ΔCCA-tRNAs and insufficiency of mature tRNAs in tms5 mutant were largely recovered in the tms5 oshel2-1 mutant. A lesion in OsHel2 results in partial readthrough of the stalled sequences, thereby evading ribosome-associated protein quality control (RQC) surveillance. Our findings reveal a mechanism by which the OsHel2 impede readthrough of stalled mRNA sequences to regulate male fertility in TGMS rice, thus providing a paradigm for investigating how disorders in the components of the RQC pathway impair cellular functions and lead to diseases or defects in other organisms.

Redundant pathways for removal of defective RNA polymerase II complexes at a promoter-proximal pause checkpoint.

The biological purpose of Integrator and RNA polymerase II (RNAPII) promoter-proximal pausing remains uncertain. Here, we show that loss of INTS6 in human cells results in increased interaction of RNAPII with proteins that can mediate its dissociation from the DNA template, including the CRL3ARMC5 E3 ligase, which ubiquitylates CTD serine5-phosphorylated RPB1 for degradation. ARMC5-dependent RNAPII ubiquitylation is activated by defects in factors acting at the promoter-proximal pause, including Integrator, DSIF, and capping enzyme. This ARMC5 checkpoint normally curtails a sizeable fraction of RNAPII transcription, and ARMC5 knockout cells produce more uncapped transcripts. When both the Integrator and CRL3ARMC5 turnover mechanisms are compromised, cell growth ceases and RNAPII with high pausing propensity disperses from the promoter-proximal pause site into the gene body. These data support a model in which CRL3ARMC5 functions alongside Integrator in a checkpoint mechanism that removes faulty RNAPII complexes at promoter-proximal pause sites to safeguard transcription integrity.

Female sex hormones inversely regulate acute kidney disease susceptibility throughout life.

While epidemiological and experimental studies have demonstrated kidney-protective effects of estrogen and female sex in adulthood, some epidemiological data showed deterioration of kidney function during puberty when estrogen production increases. However, molecular mechanisms explaining these conflicting phenomena remain unknown. Here, we showed that the pubertal sex hormone surge in female mice increases susceptibility to kidney ischemia reperfusion injury partly via downregulation of insulin-like growth factor 1 receptor (IGF-1R) expression in proximal tubules. Adult mice ovariectomized pre-pubertally (at postnatal day 21) showed strong tolerance to kidney ischemia, which was partly reversed by the administration of 17β-estradiol, while adult mice ovariectomized post-pubertally (at 8 weeks of age) were vulnerable to kidney ischemia. Kidney tubular IGF-1R protein expression decreased during postnatal growth but was highly expressed in adult mice ovariectomized pre-pubertally and in infant mice, which might be partly explained by different expression of an E3 ligase (MDM2) of IGF-1R. Mice deficient of Igf-1r in proximal tubules (iIGF-1RKO mice) during postnatal kidney growth showed increased susceptibility to ischemic injury. RNA-seq and western blotting analysis using proximal tubular cells from pre-pubertally ovariectomized iIGF-1RKO and control mice revealed altered expression of cell cycle-associated molecules such as cyclin D1. These results suggest that Igf-1r deletion during postnatal growth renders proximal tubular cells susceptible to ischemia possibly via altered cell cycle regulation. Thus, our findings provide evidence that exposure to pubertal sex hormones leads to increased susceptibility to kidney ischemia, which is partly mediated by modulation of IGF-1R signaling.

CUL3-Related Neurodevelopmental Disorder: Clinical Phenotype of 20 New Individuals and Identification of a Potential Phenotype-Associated Episignature.

Neurodevelopmental disorder with or without autism or seizures (NEDAUS; OMIM #619239) is a neurodevelopmental disorder characterized by global developmental delay, speech delay, seizures, autistic features and/or behavior abnormalities. It is caused by CUL3 (Cullin-3 ubiquitin ligase; OMIM #603136) haploinsufficiency. We collected clinical and molecular data from twenty-six individuals carrying pathogenic variants and variants of uncertain significance (VUS) in the CUL3 gene, including twenty previously unreported cases. By comparing their DNA methylation (DNAm) classifiers with those of healthy controls and other neurodevelopmental disorders characterized by established episignatures, we aimed to create a diagnostic biomarker (episignature) and gain more knowledge into the molecular pathophysiology. We discovered a sensitive and specific DNAm episignature for patients with pathogenic variants in CUL3 and utilized it to reclassify patients carrying a VUS in the CUL3 gene. Comparative epigenomic analysis revealed similarities between NEDAUS and several other rare genetic neurodevelopmental disorders with previously identified episignatures, highlighting the broader implication of our findings. In addition, we preformed genotype-phenotype correlation studies to explain the variety in clinical presentation between the cases. We discovered a highly accurate DNAm episignature serving as a robust diagnostic biomarker for NEDAUS. Furthermore, we broadened the phenotypic spectrum by identifying twenty new individuals and confirming five previously reported cases of NEDAUS.

Manganese inhibits HBV transcription and promotes HBsAg degradation at non-toxic levels

Chronic hepatitis B virus (HBV) infection continues to pose a significant global health challenge. However, therapeutic measures for a cure are lacking in clinical practice. Manganese, an essential trace element, has garnered attention due to its potential to activate innate immune pathways and its significant role in antiviral and antitumor immunity. Yet, the specific impact of manganese on chronic hepatitis B has been largely unexplored. Our research reveals that manganese substantially inhibits HBV replication in hepatocellular carcinoma cells at non-toxic levels. This suppression occurs independently of well-known anti-HBV innate immune pathways, such as the cGAS-STING pathway. Mechanistically, manganese decreases HBV transcription by diminishing the levels of liver-specific transcription factors. Furthermore, it activates the mTOR pathway, enhancing HBsAg ubiquitination through the upregulation of the ubiquitin ligase β-TrCP and increasing proteasome activity via the augmentation of its subunits, leading to a ubiquitin-dependent degradation of HBsAg. Significantly, our study also uncovers a notable clinical correlation between manganese levels and chronic hepatitis B infection. These findings position manganese as a critical element in diminishing HBV replication, offering a new direction in the management of chronic hepatitis B.

STK19 drives transcription-coupled repair by stimulating repair complex stability, RNA Pol II ubiquitylation, and TFIIH recruitment.

Transcription-coupled nucleotide excision repair (TC-NER) efficiently eliminates DNA damage that impedes gene transcription by RNA polymerase II (RNA Pol II). TC-NER is initiated by the recognition of lesion-stalled RNA Pol II by CSB, which recruits the CRL4CSA ubiquitin ligase and UVSSA. RNA Pol II ubiquitylation at RPB1-K1268 by CRL4CSA serves as a critical TC-NER checkpoint, governing RNA Pol II stability and initiating DNA damage excision by TFIIH recruitment. However, the precise regulatory mechanisms of CRL4CSA activity and TFIIHrecruitment remain elusive. Here, we reveal human serine/threonine-protein kinase 19 (STK19) as a TC-NER factor, which is essential for correct DNA damage removal and subsequent transcription restart. Cryogenic electron microscopy (cryo-EM) studies demonstrate that STK19 is an integral part of the RNA Pol II-TC-NER complex, bridging CSA, UVSSA, RNA Pol II, and downstream DNA. STK19 stimulates TC-NER complex stability and CRL4CSA activity, resulting in efficient RNA Pol II ubiquitylation and correct UVSSA and TFIIH binding. These findings underscore the crucial role of STK19 as a core TC-NER component.

Role and therapeutic potential of E3s in the tumor microenvironment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a high-incidence, poor-prognosis malignancy worldwide, requiring new strategies for treatment. Ubiquitination, especially ubiquitination through E3 ubiquitin ligases, plays an indispensable role in the development and progression of HCC. E3 ubiquitin ligases are crucial enzymes in ubiquitination, controlling the degradation of specific substrate proteins and influencing various cellular functions, such as tumor cell proliferation, apoptosis, migration, and immune evasion. In this review, we systematically summarize the mechanisms of E3 ubiquitin ligases in HCC, with a focus on the significance of RING, HECT, and RBR types in HCC progression. The review also looks at the potential for targeting E3 ligases to modulate the tumor microenvironment (TME) and increase immunotherapy efficacy. Future studies will optimize HCC treatment by formulating specific inhibitors or approaches that will be based on gene therapy targeting E3 ligases in order to overcome resistance issues with present treatments and create optimism in the journey of treatment for HCC patients.