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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 -
The Journal of Clinical Investigation Mar 2024The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been...
The mammalian SUMO-targeted E3 ubiquitin ligase Rnf4 has been reported to act as a regulator of DNA repair, but the importance of RNF4 as a tumor suppressor has not been tested. Using a conditional-knockout mouse model, we deleted Rnf4 in the B cell lineage to test the importance of RNF4 for growth of somatic cells. Although Rnf4-conditional-knockout B cells exhibited substantial genomic instability, Rnf4 deletion caused no increase in tumor susceptibility. In contrast, Rnf4 deletion extended the healthy lifespan of mice expressing an oncogenic c-myc transgene. Rnf4 activity is essential for normal DNA replication, and in its absence, there was a failure in ATR-CHK1 signaling of replication stress. Factors that normally mediate replication fork stability, including members of the Fanconi anemia gene family and the helicases PIF1 and RECQL5, showed reduced accumulation at replication forks in the absence of RNF4. RNF4 deficiency also resulted in an accumulation of hyper-SUMOylated proteins in chromatin, including members of the SMC5/6 complex, which contributes to replication failure by a mechanism dependent on RAD51. These findings indicate that RNF4, which shows increased expression in multiple human tumor types, is a potential target for anticancer therapy, especially in tumors expressing c-myc.
Topics: Animals; Humans; Mice; Ataxia Telangiectasia Mutated Proteins; B-Lymphocytes; Carcinogenesis; Checkpoint Kinase 1; DNA Replication; Genomic Instability; Mice, Knockout; Nuclear Proteins; Proto-Oncogene Proteins c-myc; Signal Transduction; Sumoylation; Ubiquitin-Protein Ligases
PubMed: 38530355
DOI: 10.1172/JCI167419 -
Scientific Reports Mar 2024Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for...
Maintenance of genome integrity is instrumental in preventing cancer. In addition to DNA repair pathways that prevent damage to DNA, damage tolerance pathways allow for the survival of cells that encounter DNA damage during replication. The Rad6/18 pathway is instrumental in this process, mediating damage bypass by ubiquitination of proliferating cell nuclear antigen. Previous studies have shown different roles of Rad18 in vivo and in tumorigenesis. Here, we show that B cells induce Rad18 expression upon proliferation induction. We have therefore analysed the role of Rad18 in B cell activation as well as in B cell lymphomagenesis mediated by an Eµ-Myc transgene. We find no activation defects or survival differences between Rad18 WT mice and two different models of Rad18 deficient tumour mice. Also, tumour subtypes do not differ between the mouse models. Accordingly, functions of Rad18 in B cell activation and tumorigenesis may be compensated for by other pathways in B cells.
Topics: Animals; Mice; Carcinogenesis; DNA Damage; DNA Repair; DNA Replication; Neoplasms; Proliferating Cell Nuclear Antigen; Ubiquitin-Protein Ligases; Ubiquitination; DNA-Binding Proteins; B-Lymphocytes; Lymphocyte Activation
PubMed: 38528023
DOI: 10.1038/s41598-024-57018-w -
Advanced Science (Weinheim,... Jun 2024Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4...
Bladder cancer (BC) is one of the most common tumors characterized by a high rate of relapse and a lack of targeted therapy. Here, YEATS domain-containing protein 4 (YEATS4) is an essential gene for BC cell viability using CRISPR-Cas9 library screening is reported, and that HUWE1 is an E3 ligase responsible for YEATS4 ubiquitination and proteasomal degradation by the Protein Stability Regulators Screening Assay. KAT8-mediated acetylation of YEATS4 impaired its interaction with HUWE1 and consequently prevented its ubiquitination and degradation. The protein levels of YEATS4 and KAT8 are positively correlated and high levels of these two proteins are associated with poor overall survival in BC patients. Importantly, suppression of YEATS4 acetylation with the KAT8 inhibitor MG149 decreased YEATS4 acetylation, reduced cell viability, and sensitized BC cells to cisplatin treatment. The findings reveal a critical role of the KAT8/YEATS4 axis in both tumor growth and cisplatin sensitivity in BC cells, potentially generating a novel therapeutic strategy for BC patients.
Topics: Urinary Bladder Neoplasms; Humans; Cisplatin; Cell Line, Tumor; Mice; Histone Acetyltransferases; Animals; Ubiquitin-Protein Ligases; Antineoplastic Agents; Tumor Suppressor Proteins; Acetylation; Disease Models, Animal; Drug Resistance, Neoplasm
PubMed: 38526153
DOI: 10.1002/advs.202310146 -
The Journal of Biological Chemistry May 2024Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of...
Human DNA ligase 1 (LIG1) is the main replicative ligase that seals Okazaki fragments during nuclear replication and finalizes DNA repair pathways by joining DNA ends of the broken strand breaks in the three steps of the ligation reaction. LIG1 can tolerate the RNA strand upstream of the nick, yet an atomic insight into the sugar discrimination mechanism by LIG1 against a ribonucleotide at the 3'-terminus of nick DNA is unknown. Here, we determined X-ray structures of LIG1/3'-RNA-DNA hybrids and captured the ligase during pre- and post-step 3 the ligation reaction. Furthermore, the overlays of 3'-rA:T and 3'-rG:C step 3 structures with step 2 structures of canonical 3'-dA:T and 3'-dG:C uncover a network of LIG1/DNA interactions through Asp570 and Arg871 side chains with 2'-OH of the ribose at nick showing a final phosphodiester bond formation and the other ligase active site residues surrounding the AMP site. Finally, we demonstrated that LIG1 can ligate the nick DNA substrates with pre-inserted 3'-ribonucleotides as efficiently as Watson-Crick base-paired ends in vitro. Together, our findings uncover a novel atomic insight into a lack of sugar discrimination by LIG1 and the impact of improper sugar on the nick sealing of ribonucleotides at the last step of DNA replication and repair.
Topics: Humans; DNA Ligase ATP; DNA; Ribonucleotides; Crystallography, X-Ray; DNA Repair
PubMed: 38522520
DOI: 10.1016/j.jbc.2024.107216 -
Nature Communications Mar 2024Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the...
Histone H2B monoubiquitination (at Lys120 in humans) regulates transcription elongation and DNA repair. In humans, H2B monoubiquitination is catalyzed by the heterodimeric Bre1 complex composed of Bre1A/RNF20 and Bre1B/RNF40. The Bre1 proteins generally function as tumor suppressors, while in certain cancers, they facilitate cancer cell proliferation. To obtain structural insights of H2BK120 ubiquitination and its regulation, we report the cryo-electron microscopy structure of the human Bre1 complex bound to the nucleosome. The two RING domains of Bre1A and Bre1B recognize the acidic patch and the nucleosomal DNA phosphates around SHL 6.0-6.5, which are ideally located to recruit the E2 enzyme and ubiquitin for H2BK120-specific ubiquitination. Mutational experiments suggest that the two RING domains bind in two orientations and that ubiquitination occurs when Bre1A binds to the acidic patch. Our results provide insights into the H2BK120-specific ubiquitination by the Bre1 proteins and suggest that H2B monoubiquitination can be regulated by nuclesomal DNA flexibility.
Topics: Humans; Cryoelectron Microscopy; DNA; Histones; Neoplasms; Nucleosomes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38519511
DOI: 10.1038/s41467-024-46910-8 -
Cell Reports Apr 2024Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying...
Hypoxia-inducible factor 1 (HIF-1) is a transcriptional activator that mediates cellular adaptation to decreased oxygen availability. HIF-1 recruits chromatin-modifying enzymes leading to changes in histone acetylation, citrullination, and methylation at target genes. Here, we demonstrate that hypoxia-inducible gene expression in estrogen receptor (ER)-positive MCF7 and ER-negative SUM159 human breast cancer cells requires the histone H2A/H2B chaperone facilitates chromatin transcription (FACT) and the H2B ubiquitin ligase RING finger protein 20/40 (RNF20/40). Knockdown of FACT or RNF20/40 expression leads to decreased transcription initiation and elongation at HIF-1 target genes. Mechanistically, FACT and RNF20/40 are recruited to hypoxia response elements (HREs) by HIF-1 and stabilize binding of HIF-1 (and each other) at HREs. Hypoxia induces the monoubiquitination of histone H2B at lysine 120 at HIF-1 target genes in an HIF-1-dependent manner. Together, these findings delineate a cooperative molecular mechanism by which FACT and RNF20/40 stabilize multiprotein complex formation at HREs and mediate histone ubiquitination to facilitate HIF-1 transcriptional activity.
Topics: Humans; Cell Hypoxia; Cell Line, Tumor; DNA-Binding Proteins; Histones; Hypoxia-Inducible Factor 1; MCF-7 Cells; Protein Binding; Response Elements; Transcription Factors; Transcriptional Activation; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 38517892
DOI: 10.1016/j.celrep.2024.113972 -
Genes & Development Apr 2024The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to...
The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9-NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.
Topics: Cell Cycle Proteins; DNA Damage; Sumoylation; Ubiquitin-Protein Ligases; Humans; Genomic Instability
PubMed: 38503515
DOI: 10.1101/gad.350914.123