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Asia-Pacific Journal of Ophthalmology... 2024Retinoblastoma (RB), originating from the developing retina, is an aggressive intraocular malignant neoplasm in childhood. Biallelic loss of RB1 is conventionally... (Review)
Review
Retinoblastoma (RB), originating from the developing retina, is an aggressive intraocular malignant neoplasm in childhood. Biallelic loss of RB1 is conventionally considered a prerequisite for initiating RB development in most RB cases. Additional genetic mutations arising from genome instability following RB1 mutations are proposed to be required to promote RB development. Recent advancements in high throughput sequencing technologies allow a deeper and more comprehensive understanding of the etiology of RB that additional genetic alterations following RB1 biallelic loss are rare, yet epigenetic changes driven by RB1 loss emerge as a critical contributor promoting RB tumorigenesis. Multiple epigenetic regulators have been found to be dysregulated and to contribute to RB development, including noncoding RNAs, DNA methylations, RNA modifications, chromatin conformations, and histone modifications. A full understanding of the roles of genetic and epigenetic alterations in RB formation is crucial in facilitating the translation of these findings into effective treatment strategies for RB. In this review, we summarize current knowledge concerning genetic defects and epigenetic dysregulations in RB, aiming to help understand their links and roles in RB tumorigenesis.
Topics: Retinoblastoma; Humans; Retinal Neoplasms; Epigenesis, Genetic; Mutation; DNA Methylation; Retinoblastoma Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 38789041
DOI: 10.1016/j.apjo.2024.100072 -
Science Advances May 2024Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for...
Resistance to therapy commonly develops in patients with high-grade serous ovarian carcinoma (HGSC) and triple-negative breast cancer (TNBC), urging the search for improved therapeutic combinations and their predictive biomarkers. Starting from a CRISPR knockout screen, we identified that loss of RB1 in TNBC or HGSC cells generates a synthetic lethal dependency on casein kinase 2 (CK2) for surviving the treatment with replication-perturbing therapeutics such as carboplatin, gemcitabine, or PARP inhibitors. CK2 inhibition in RB1-deficient cells resulted in the degradation of another RB family cell cycle regulator, p130, which led to S phase accumulation, micronuclei formation, and accelerated PARP inhibition-induced aneuploidy and mitotic cell death. CK2 inhibition was also effective in primary patient-derived cells. It selectively prevented the regrowth of RB1-deficient patient HGSC organoids after treatment with carboplatin or niraparib. As about 25% of HGSCs and 40% of TNBCs have lost RB1 expression, CK2 inhibition is a promising approach to overcome resistance to standard therapeutics in large strata of patients.
Topics: Humans; Casein Kinase II; Retinoblastoma Binding Proteins; Female; Cell Line, Tumor; Triple Negative Breast Neoplasms; Ovarian Neoplasms; Ubiquitin-Protein Ligases; Carboplatin; Synthetic Lethal Mutations; DNA Replication; Drug Resistance, Neoplasm; Poly(ADP-ribose) Polymerase Inhibitors; Antineoplastic Agents
PubMed: 38781347
DOI: 10.1126/sciadv.adj1564 -
Frontiers in Cellular and Infection... 2024Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of...
Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of . Expression of was found to be upregulated in the Δ strain, and the addition of exogenous competence-stimulating peptide repressed the transcription in the wild-type strain but not the Δ mutant, indicating that ComE exerted a negative regulatory effect on the transcription of . Mutation in the JH2 region of upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.
Topics: Teichoic Acids; Bacterial Proteins; Lipopolysaccharides; Gene Expression Regulation, Bacterial; Streptococcus pneumoniae; Transformation, Bacterial; Transcription, Genetic; Promoter Regions, Genetic; DNA Transformation Competence; Mutation; Protein Binding; Ligases
PubMed: 38779562
DOI: 10.3389/fcimb.2024.1375312 -
Retrovirology May 2024Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral...
BACKGROUND
Detection of viruses by host pattern recognition receptors induces the expression of type I interferon (IFN) and IFN-stimulated genes (ISGs), which suppress viral replication. Numerous studies have described HIV-1 as a poor activator of innate immunity in vitro. The exact role that the viral capsid plays in this immune evasion is not fully understood.
RESULTS
To better understand the role of the HIV-1 capsid in sensing we tested the effect of making HIV-1 by co-expressing a truncated Gag that encodes the first 107 amino acids of capsid fused with luciferase or GFP, alongside wild type Gag-pol. We found that unlike wild type HIV-1, viral particles produced with a mixture of wild type and truncated Gag fused to luciferase or GFP induced a potent IFN response in THP-1 cells and macrophages. Innate immune activation by Gag-fusion HIV-1 was dependent on reverse transcription and DNA sensor cGAS, suggesting activation of an IFN response by viral DNA. Further investigation revealed incorporation of the Gag-luciferase/GFP fusion proteins into viral particles that correlated with subtle defects in wild type Gag cleavage and a diminished capacity to saturate restriction factor TRIM5α, likely due to aberrant particle formation. We propose that expression of the Gag fusion protein disturbs the correct cleavage and maturation of wild type Gag, yielding viral particles that are unable to effectively shield viral DNA from detection by innate sensors including cGAS.
CONCLUSIONS
These data highlight the crucial role of capsid in innate evasion and support growing literature that disruption of Gag cleavage and capsid formation induces a viral DNA- and cGAS-dependent innate immune response. Together these data demonstrate a protective role for capsid and suggest that antiviral activity of capsid-targeting antivirals may benefit from enhanced innate and adaptive immunity in vivo.
Topics: HIV-1; Humans; gag Gene Products, Human Immunodeficiency Virus; Immunity, Innate; Nucleotidyltransferases; Antiviral Restriction Factors; Macrophages; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; THP-1 Cells; Carrier Proteins; Immune Evasion; Capsid; Virus Replication; Virion; Host-Pathogen Interactions; DNA, Viral; Cell Line
PubMed: 38778414
DOI: 10.1186/s12977-024-00643-0 -
Gut Microbes 2024Emerging evidence has revealed the novel role of gut microbiota in the development of cancer. The characteristics of function and composition in the gut microbiota of...
Emerging evidence has revealed the novel role of gut microbiota in the development of cancer. The characteristics of function and composition in the gut microbiota of patients with breast cancer patients has been reported, however the detailed causation between gut microbiota and breast cancer remains uncertain. In the present study, 16S rRNA sequencing revealed that , particularly the dominant species , is significantly enriched and prevalent in gut microbiota of breast cancer patients. Prior-oral administration of could promote breast cancer growth in specific pathogen-free mice and germ-free mice, accompanied with sharp reduction of indole-3-pyruvic acid (IPyA). Mechanistically, the present of excessive consumed a large amount of tryptophan (Trp), thus hampering the physiological accumulation of IPyA in the host. Our results revealed that IPyA is an intrinsic anti-cancer reagent in the host at physiological level. Briefly, IPyA directly suppressed the transcription of UHRF1, following by the declined UHRF1 and PP2A C in nucleus, thus inhibiting the phosphorylation of AMPK, which is just opposite to the cancer promoting effect of . Therefore, the exhaustion of IPyA by excessive strengthens the UHRF1-mediated negative control to inactivated the energy-controlling AMPK signaling pathway to promote tumor growth, which was indicated by the alternation in pattern of protein expression and DNA methylation. Our findings, for the first time, highlighted as a risk factor for the progression of breast cancer.
Topics: Breast Neoplasms; Animals; Female; Humans; Mice; Gastrointestinal Microbiome; AMP-Activated Protein Kinases; Indoles; Prevotella; Ubiquitin-Protein Ligases; CCAAT-Enhancer-Binding Proteins; Disease Progression; Mice, Inbred BALB C; Tryptophan; Cell Line, Tumor
PubMed: 38773738
DOI: 10.1080/19490976.2024.2347757 -
Nature Communications May 2024Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair...
Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial. Here, we observe that the BRCA1/BARD1 ubiquitin E3 activity is not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identify substrates for BRCA1/BARD1. We find that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results provide additional insight about the importance of BRCA1/BARD1 E3 activity in Homologous Recombination.
Topics: Humans; BRCA1 Protein; Ubiquitin-Protein Ligases; Proliferating Cell Nuclear Antigen; DNA Replication; Ubiquitination; Tumor Suppressor Proteins; Phthalazines; Piperazines; Homologous Recombination; Female; HEK293 Cells; Cell Line, Tumor; DNA
PubMed: 38769345
DOI: 10.1038/s41467-024-48427-6 -
Journal of Molecular Modeling May 2024Ubiquitin-like with PHD and RING finger domain containing protein 1 (UHRF1) is responsible for preserving the stability of genomic methylation through the recruitment of...
CONTEXT
Ubiquitin-like with PHD and RING finger domain containing protein 1 (UHRF1) is responsible for preserving the stability of genomic methylation through the recruitment of DNA methyltransferase 1 (DNMT1). However, the interaction between Developmental pluripotency associated 3 (DPPA3) and the pre-PHD-PHD (PPHD) domain of UHRF1 hinders the nuclear localization of UHRF1. This disruption has implications for potential cancer treatment strategies. Drugs that mimic the binding pattern between DPPA3 and PPHD could offer a promising approach to cancer treatment. Our study reveals that DPPA3 undergoes dissociation from the C-terminal through three different modes of helix unfolding. Furthermore, we have identified key residue pairs involved in this dissociation process and potential drug-targeting residues. These findings offer valuable insights into the dissociation mechanism of DPPA3 from PPHD and have the potential to inform the design of novel drugs targeting UHRF1 for cancer therapy.
METHODS
To comprehend the dissociation process and binding patterns of PPHD-DPPA3, we employed enhanced sampling techniques, including steered molecular dynamics (SMD) and conventional molecular dynamics (cMD). Additionally, we utilized self-organizing maps (SOM) and time-resolved force distribution analysis (TRFDA) methodologies. The Gromacs software was used for performing molecular dynamics simulations, and the AMBER FF14SB force field was applied to the protein.
Topics: Ubiquitin-Protein Ligases; Molecular Dynamics Simulation; CCAAT-Enhancer-Binding Proteins; Protein Binding; Humans; Binding Sites
PubMed: 38767734
DOI: 10.1007/s00894-024-05946-9 -
Environmental and Molecular Mutagenesis 2024As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a...
As final process of every DNA repair pathway, DNA ligation is crucial for maintaining genomic stability and preventing DNA strand breaks to accumulate. Therefore, a method reliably assessing DNA ligation capacity in protein extracts from murine tissues was aimed to establish. To optimize applicability, the use of radioactively labeled substrates was avoided and replaced by fluorescently labeled oligonucleotides. Briefly, tissue extracts were incubated with those complementary oligonucleotides so that in an ensuing gel electrophoresis ligated strands could be separated from unconnected molecules. Originally, the method was intended for use in cerebellum tissue to further elucidate possible mechanisms of neurodegenerative diseases. However, due to its inhomogeneous anatomy, DNA ligation efficiency varied strongly between different cerebellar areas, illuminating the established assay to be suitable only for homogenous organs. Thus, for murine liver tissue sufficient intra- and interday repeatability was shown during validation. In further experiments, the established assay was applied to an animal study comprising young and old (24 and 110 weeks) mice which showed that DNA ligation efficiency was affected by neither sex nor age. Finally, the impact of in vitro addition of the trace elements copper, iron, and zinc on DNA ligation in tissue extracts was investigated. While all three metals inhibited DNA ligation, variations in their potency became evident. In conclusion, the established method can be reliably used for investigation of DNA ligation efficiency in homogenous murine tissues.
Topics: Animals; Mice; DNA; Male; Female; Liver; Cerebellum; Mice, Inbred C57BL; DNA Ligases; DNA Repair
PubMed: 38767089
DOI: 10.1002/em.22602 -
BioRxiv : the Preprint Server For... May 2024DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair...
DNA ligase 1 (LIG1) joins broken strand-breaks in the phosphodiester backbone to finalize DNA repair pathways. We previously reported that LIG1 fails on nick repair intermediate with 3'-oxidative damage incorporated by DNA polymerase (pol) β at the downstream steps of base excision repair (BER) pathway. Here, we determined X-ray structures of LIG1/nick DNA complexes containing 3'-8oxodG and 3'-8oxorG opposite either a templating Cytosine or Adenine and demonstrated that the ligase active site engages with mutagenic repair intermediates during steps 2 and 3 of the ligation reaction referring to the formation of DNA-AMP intermediate and a final phosphodiester bond, respectively. Furthermore, we showed the mutagenic nick sealing of DNA substrates with 3'-8oxodG:A and 3'-8oxorG:A by LIG1 wild-type, immunodeficiency disease-associated variants, and DNA ligase 3α (LIG3α) . Finally, we observed that LIG1 and LIG3α seal resulting nick after an incorporation of 8oxorGTP:A by polβ and AP-Endonuclease 1 (APE1) can clean oxidatively damaged ends at the final steps. Overall, our findings uncover a mechanistic insight into how LIG1 discriminates DNA or DNA/RNA junctions including oxidative damage and a functional coordination between the downstream enzymes, polβ, APE1, and BER ligases, to process mutagenic repair intermediates to maintain repair efficiency.
PubMed: 38766188
DOI: 10.1101/2024.05.06.592774 -
Acta Biochimica Et Biophysica Sinica Jun 2024Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of...
Bacteriophages have been used across various fields, and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages. However, some bacteriophages can escape from the cleavage of Cas protein, such as Cas9, and decrease the efficiency of genome editing. This study focuses on the bacteriophage T7, which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated. First, we test the escape rates of T7 phage at different cleavage sites, ranging from 10 to 10 . The sequencing results show that DNA point mutations and microhomology-mediated end joining (MMEJ) at the target sites are the main causes. Next, we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region. Moreover, we also knock out the ATP-dependent DNA ligase . gene, which may be involved in the MMEJ event, and the frequency of MMEJ at . is reduced from 83% to 18%. Finally, the genome editing efficiency in T7 Δ . increases from 20% to 100%. This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene . can reduce MMEJ events and enhance gene editing efficiency. These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.
Topics: CRISPR-Cas Systems; Gene Editing; Bacteriophage T7; DNA Ligases; Viral Proteins; Genome, Viral
PubMed: 38761011
DOI: 10.3724/abbs.2024030