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Advanced Science (Weinheim,... Jul 2024The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions....
The structural diversity of biological macromolecules in different environments contributes complexity to enzymological processes vital for cellular functions. Fluorescence resonance energy transfer and electron microscopy are used to investigate the enzymatic reaction of T4 DNA ligase catalyzing the ligation of nicked DNA. The data show that both the ligase-AMP complex and the ligase-AMP-DNA complex can have four conformations. This finding suggests the parallel occurrence of four ligation reaction pathways, each characterized by specific conformations of the ligase-AMP complex that persist in the ligase-AMP-DNA complex. Notably, these complexes have DNA bending angles of ≈0°, 20°, 60°, or 100°. The mechanism of parallel reactions challenges the conventional notion of simple sequential reaction steps occurring among multiple conformations. The results provide insights into the dynamic conformational changes and the versatile attributes of T4 DNA ligase and suggest that the parallel multiple reaction pathways may correspond to diverse T4 DNA ligase functions. This mechanism may potentially have evolved as an adaptive strategy across evolutionary history to navigate complex environments.
Topics: DNA Ligases; DNA; DNA Repair; Fluorescence Resonance Energy Transfer; Nucleic Acid Conformation; Bacteriophage T4; Microscopy, Electron
PubMed: 38582512
DOI: 10.1002/advs.202401150 -
Nature Communications Apr 2024DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely...
DNA methylation is an essential epigenetic chromatin modification, and its maintenance in mammals requires the protein UHRF1. It is yet unclear if UHRF1 functions solely by stimulating DNA methylation maintenance by DNMT1, or if it has important additional functions. Using degron alleles, we show that UHRF1 depletion causes a much greater loss of DNA methylation than DNMT1 depletion. This is not caused by passive demethylation as UHRF1-depleted cells proliferate more slowly than DNMT1-depleted cells. Instead, bioinformatics, proteomics and genetics experiments establish that UHRF1, besides activating DNMT1, interacts with DNMT3A and DNMT3B and promotes their activity. In addition, we show that UHRF1 antagonizes active DNA demethylation by TET2. Therefore, UHRF1 has non-canonical roles that contribute importantly to DNA methylation homeostasis; these findings have practical implications for epigenetics in health and disease.
Topics: Humans; CCAAT-Enhancer-Binding Proteins; Chromatin; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Neoplasms; Ubiquitin-Protein Ligases
PubMed: 38580649
DOI: 10.1038/s41467-024-47314-4 -
Cellular and Molecular Life Sciences :... Apr 2024The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding...
The DNA methylation is gradually acquired during oogenesis, a process sustained by successful follicle development. However, the functional roles of methyl-CpG-binding protein 2 (MeCP2), an epigenetic regulator displaying specifical binding with methylated DNA, remains unknown in oogenesis. In this study, we found MeCP2 protein was highly expressed in primordial and primary follicle, but was almost undetectable in secondary follicles. However, in aged ovary, MeCP2 protein is significantly increased in both oocyte and granulosa cells. Overexpression of MeCP2 in growing oocyte caused transcription dysregulation, DNA hypermethylation, and genome instability, ultimately leading to follicle growth arrest and apoptosis. MeCP2 is targeted by DCAF13, a substrate recognition adaptor of the Cullin 4-RING (CRL4) E3 ligase, and polyubiquitinated for degradation in both cells and oocytes. Dcaf13-null oocyte exhibited an accumulation of MeCP2 protein, and the partial rescue of follicle growth arrest induced by Dcaf13 deletion was observed following MeCP2 knockdown. The RNA-seq results revealed that large amounts of genes were regulated by the DCAF13-MeCP2 axis in growing oocytes. Our study demonstrated that CRL4 E3 ubiquitin ligase targets MeCP2 for degradation to ensure normal DNA methylome and transcription in growing oocytes. Moreover, in aged ovarian follicles, deceased DCAF13 and DDB1 protein were observed, indicating a potential novel mechanism that regulates ovary aging.
Topics: Female; Humans; Cullin Proteins; DNA; DNA Methylation; Methyl-CpG-Binding Protein 2; Oocytes; Ubiquitin-Protein Ligases
PubMed: 38578457
DOI: 10.1007/s00018-024-05185-4 -
Arthritis Research & Therapy Apr 2024Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such...
BACKGROUND
Transitioning from a genetic association signal to an effector gene and a targetable molecular mechanism requires the application of functional fine-mapping tools such as reporter assays and genome editing. In this report, we undertook such studies on the osteoarthritis (OA) risk that is marked by single nucleotide polymorphism (SNP) rs34195470 (A > G). The OA risk-conferring G allele of this SNP associates with increased DNA methylation (DNAm) at two CpG dinucleotides within WWP2. This gene encodes a ubiquitin ligase and is the host gene of microRNA-140 (miR-140). WWP2 and miR-140 are both regulators of TGFβ signaling.
METHODS
Nucleic acids were extracted from adult OA (arthroplasty) and foetal cartilage. Samples were genotyped and DNAm quantified by pyrosequencing at the two CpGs plus 14 flanking CpGs. CpGs were tested for transcriptional regulatory effects using a chondrocyte cell line and reporter gene assay. DNAm was altered using epigenetic editing, with the impact on gene expression determined using RT-qPCR. In silico analysis complemented laboratory experiments.
RESULTS
rs34195470 genotype associates with differential methylation at 14 of the 16 CpGs in OA cartilage, forming a methylation quantitative trait locus (mQTL). The mQTL is less pronounced in foetal cartilage (5/16 CpGs). The reporter assay revealed that the CpGs reside within a transcriptional regulator. Epigenetic editing to increase their DNAm resulted in altered expression of the full-length and N-terminal transcript isoforms of WWP2. No changes in expression were observed for the C-terminal isoform of WWP2 or for miR-140.
CONCLUSIONS
As far as we are aware, this is the first experimental demonstration of an OA association signal targeting specific transcript isoforms of a gene. The WWP2 isoforms encode proteins with varying substrate specificities for the components of the TGFβ signaling pathway. Future analysis should focus on the substrates regulated by the two WWP2 isoforms that are the targets of this genetic risk.
Topics: Adult; Humans; Base Sequence; Ubiquitin; Protein Isoforms; Ubiquitin-Protein Ligases; DNA Methylation; MicroRNAs; Osteoarthritis; Transforming Growth Factor beta
PubMed: 38570801
DOI: 10.1186/s13075-024-03315-8 -
Science Translational Medicine Apr 2024Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and...
Cancer-associated fibroblasts (CAFs) are abundant stromal cells in the tumor microenvironment that promote cancer progression and relapse. However, the heterogeneity and regulatory roles of CAFs underlying chemoresistance remain largely unclear. Here, we performed a single-cell analysis using high-dimensional flow cytometry analysis and identified a distinct senescence-like tetraspanin-8 (TSPAN8) myofibroblastic CAF (myCAF) subset, which is correlated with therapeutic resistance and poor survival in multiple cohorts of patients with breast cancer (BC). TSPAN8 myCAFs potentiate the stemness of the surrounding BC cells through secretion of senescence-associated secretory phenotype (SASP)-related factors IL-6 and IL-8 to counteract chemotherapy. NAD-dependent protein deacetylase sirtuin 6 (SIRT6) reduction was responsible for the senescence-like phenotype and tumor-promoting role of TSPAN8 myCAFs. Mechanistically, TSPAN8 promoted the phosphorylation of ubiquitin E3 ligase retinoblastoma binding protein 6 (RBBP6) at Ser by recruiting MAPK11, thereby inducing SIRT6 protein destruction. In turn, SIRT6 down-regulation up-regulated and , which caused TSPAN8 myCAFs to secrete aspartate and proline, and therefore proved a nutritional niche to support BC outgrowth. By demonstrating that TSPAN8SIRT6 myCAFs were tightly associated with unfavorable disease outcomes, we proposed that the combined regimen of anti-TSPAN8 antibody and SIRT6 activator MDL-800 is a promising approach to overcome chemoresistance. These findings highlight that senescence contributes to CAF heterogeneity and chemoresistance and suggest that targeting TSPAN8 myCAFs is a promising approach to circumvent chemoresistance.
Topics: Humans; Female; Breast Neoplasms; Cancer-Associated Fibroblasts; Drug Resistance, Neoplasm; Neoplasm Recurrence, Local; Sirtuins; Fibroblasts; Tumor Microenvironment; DNA-Binding Proteins; Ubiquitin-Protein Ligases; Tetraspanins
PubMed: 38569015
DOI: 10.1126/scitranslmed.adj5705 -
Zhongguo Dang Dai Er Ke Za Zhi =... Mar 2024Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly... (Review)
Review
Central precocious puberty (CPP) is a developmental disorder caused by early activation of the hypothalamic-pituitary-gonadal axis. The incidence of CPP is rapidly increasing, but the underlying mechanisms are not fully understood. Previous studies have shown that gain-of-function mutations in the and genes and loss-of-function mutations in the , , and genes may lead to early initiation of pubertal development. Recent research has also revealed the significant role of epigenetic factors such as DNA methylation and microRNAs in the regulation of gonadotropin-releasing hormone neurons, as well as the modulating effect of gene networks involving multiple variant genes on pubertal initiation. This review summarizes the genetic etiology and pathogenic mechanisms underlying CPP.
Topics: Humans; Puberty, Precocious; Gonadotropin-Releasing Hormone; Mutation; MicroRNAs; Puberty; Ubiquitin-Protein Ligases
PubMed: 38557384
DOI: 10.7499/j.issn.1008-8830.2309098 -
Circulation Jul 2024Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α),...
BACKGROUND
Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive.
METHODS
Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global -knockout and vascular smooth muscle cell-specific -knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl; 0.5 mol/L; 42 days).
RESULTS
Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II- or CaCl-treated mice. Global or vascular smooth muscle cell-specific knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. , an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II- and CaCl-induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process.
CONCLUSIONS
Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.
Topics: Aortic Aneurysm, Abdominal; Animals; Liver X Receptors; Epigenesis, Genetic; MicroRNAs; Humans; CCAAT-Enhancer-Binding Proteins; Mice; Mice, Knockout; Ubiquitin-Protein Ligases; Male; Disease Models, Animal; Mice, Inbred C57BL; DNA Methylation; Muscle, Smooth, Vascular; Angiotensin II
PubMed: 38557060
DOI: 10.1161/CIRCULATIONAHA.123.065202 -
Antiviral Research May 2024Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long...
Following acute human alphaherpesvirus 1 (HSV-1) infection of oral-facial mucosal surfaces, sensory neurons in trigeminal ganglia (TG) are important sites for life-long latency. Neurons in the central nervous system, including brainstem, also harbor viral genomes during latency. Periodically, certain cellular stressors trigger reactivation from latency, which can lead to recurrent HSV-1 disease: herpes labialis, herpes stromal keratitis, and encephalitis for example. Activation of the glucocorticoid receptor (GR) by stressful stimuli enhances HSV-1 gene expression, replication, and explant-induced reactivation. GR and certain stress-induced Krüppel like factors (KLF) cooperatively transactivate cis-regulatory modules (CRM) that drive expression of viral transcriptional regulatory proteins (ICP0, ICP4, and ICP27). These CRMs lack GR response elements (GRE); however, specificity protein 1 (Sp1) binding sites are crucial for GR and KLF15 or KLF4 mediated transactivation. Hence, we tested whether Sp1 or Sp3 regulate viral replication and transactivation of the ICP0 promoter. During early stages of explant-induced reactivation from latency, the number of Sp3+ TG neurons were significantly higher relative to TG from latently infected mice. Conversely, Sp1+ TG neurons were only increased in females, but not male mice, during explant-induced reactivation. Sp1 siRNA significantly reduced HSV-1 replication in cultured mouse (Neuro-2A) and monkey (CV-1) cells. Mithramycin A, an antibiotic that has anti-tumor activity preferentially interacts with GC-rich DNA, including Sp1 binding sites, significantly reduced HSV-1 replication indicating it has antiviral activity. GR and Sp1 or Sp3 transactivated the HSV-1 ICP0 promoter in Neuro-2A and CV-1 cells confirming these transcription factors enhance viral replication and gene expression.
Topics: Female; Humans; Mice; Animals; Herpesvirus 1, Human; Receptors, Glucocorticoid; Virus Activation; Virus Latency; Immediate-Early Proteins; Anti-Bacterial Agents; Ubiquitin-Protein Ligases; Herpes Simplex; Plicamycin
PubMed: 38556059
DOI: 10.1016/j.antiviral.2024.105870 -
The Journal of Biological Chemistry May 2024Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation leading to atherosclerosis. Here, we report that tripartite motif-containing protein...
Impaired cholesterol efflux and/or uptake can influence arterial lipid accumulation leading to atherosclerosis. Here, we report that tripartite motif-containing protein 13 (TRIM13), a RING-type E3 ubiquitin ligase, plays a role in arterial lipid accumulation leading to atherosclerosis. Using molecular approaches and KO mouse model, we found that TRIM13 expression was induced both in the aorta and peritoneal macrophages (pMφ) of ApoE mice in response to Western diet (WD) in vivo. Furthermore, proatherogenic cytokine interleukin-1β also induced TRIM13 expression both in pMφ and vascular smooth muscle cells. Furthermore, we found that TRIM13 via ubiquitination and degradation of liver X receptor (LXR)α/β downregulates the expression of their target genes ABCA1/G1 and thereby inhibits cholesterol efflux. In addition, TRIM13 by ubiquitinating and degrading suppressor of cytokine signaling 1/3 (SOCS1/3) mediates signal transducer and activator of transcription 1 (STAT1) activation, CD36 expression, and foam cell formation. In line with these observations, genetic deletion of TRIM13 by rescuing cholesterol efflux and inhibiting foam cell formation protects against diet-induced atherosclerosis. We also found that while TRIM13 and CD36 levels were increased, LXRα/β, ABCA1/G1, and SOCS3 levels were decreased both in Mφ and smooth muscle cells of stenotic human coronary arteries as compared to nonstenotic arteries. More intriguingly, the expression levels of TRIM13 and its downstream signaling molecules were correlated with the severity of stenotic lesions. Together, these observations reveal for the first time that TRIM13 plays a crucial role in diet-induced atherosclerosis, and that it could be a potential drug target against this vascular lesion.
Topics: Animals; Humans; Male; Mice; Atherosclerosis; ATP Binding Cassette Transporter 1; ATP Binding Cassette Transporter, Subfamily G, Member 1; Cholesterol; Diet, Western; DNA-Binding Proteins; Foam Cells; Lipoproteins, LDL; Liver X Receptors; Mice, Knockout, ApoE; RAW 264.7 Cells; STAT1 Transcription Factor; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38537695
DOI: 10.1016/j.jbc.2024.107224 -
Clinical Cancer Research : An Official... Jun 2024Shallow whole-genome sequencing (sWGS) can detect copy-number (CN) aberrations. In high-grade serous ovarian cancer (HGSOC) sWGS identified CN signatures such as...
PURPOSE
Shallow whole-genome sequencing (sWGS) can detect copy-number (CN) aberrations. In high-grade serous ovarian cancer (HGSOC) sWGS identified CN signatures such as homologous recombination deficiency (HRD) to direct therapy. We applied sWGS with targeted sequencing to p53abn endometrial cancers to identify additional prognostic stratification and therapeutic opportunities.
EXPERIMENTAL DESIGN
sWGS and targeted panel sequencing was performed on formalin-fixed, paraffin-embedded p53abn endometrial cancers. CN alterations, mutational data and CN signatures were derived, and associations to clinicopathologic and outcomes data were assessed.
RESULTS
In 187 p53abn endometrial cancers, 5 distinct CN signatures were identified. Signature 5 was associated with BRCA1/2 CN loss with features similar to HGSOC HRD signature. Twenty-two percent of potential HRD cases were identified, 35 patients with signature 5, and 8 patients with BRCA1/2 somatic mutations. Signatures 3 and 4 were associated with a high ploidy state, and CCNE1, ERBB2, and MYC amplifications, with mutations in PIK3CA enriched in signature 3. We observed improved overall survival (OS) for patients with signature 2 and worse OS for signatures 1 and 3. Twenty-eight percent of patients had CCNE1 amplification and this subset was enriched with carcinosarcoma histotype. Thirty-four percent of patients, across all histotypes, had ERBB2 amplification and/or HER2 overexpression on IHC, which was associated with worse outcomes. Mutations in PPP2R1A (29%) and FBXW7 (16%) were among the top 5 most common mutations.
CONCLUSIONS
sWGS and targeted sequencing identified therapeutic opportunities in 75% of patients with p53abn endometrial cancer. Further research is needed to determine the efficacy of treatments targeting these identified pathways within p53abn endometrial cancers.
Topics: Humans; Female; Endometrial Neoplasms; Whole Genome Sequencing; DNA Copy Number Variations; Tumor Suppressor Protein p53; Mutation; F-Box-WD Repeat-Containing Protein 7; Middle Aged; Aged; BRCA2 Protein; BRCA1 Protein; Prognosis; Class I Phosphatidylinositol 3-Kinases; Cyclin E; Adult; Ubiquitin-Protein Ligases; Biomarkers, Tumor; Cystadenocarcinoma, Serous; Aged, 80 and over; Oncogene Proteins
PubMed: 38536067
DOI: 10.1158/1078-0432.CCR-23-3689