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Nature Communications May 2024The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The...
The master DNA damage repair histone protein, H2AX, is essential for orchestrating the recruitment of downstream mediator and effector proteins at damaged chromatin. The phosphorylation of H2AX at S139, γH2AX, is well-studied for its DNA repair function. However, the extended C-terminal tail is not characterized. Here, we define the minimal motif on H2AX for the canonical function in activating the MDC1-RNF8-RNF168 phosphorylation-ubiquitination pathway that is important for recruiting repair proteins, such as 53BP1 and BRCA1. Interestingly, H2AX recruits 53BP1 independently from the MDC1-RNF8-RNF168 pathway through its evolved C-terminal linker region with S139 phosphorylation. Mechanistically, 53BP1 recruitment to damaged chromatin is mediated by the interaction between the H2AX C-terminal tail and the 53BP1 Oligomerization-Tudor domains. Moreover, γH2AX-linker mediated 53BP1 recruitment leads to camptothecin resistance in H2AX knockout cells. Overall, our study uncovers an evolved mechanism within the H2AX C-terminal tail for regulating DNA repair proteins at damaged chromatin.
Topics: Tumor Suppressor p53-Binding Protein 1; Histones; Humans; Chromatin; Phosphorylation; DNA Repair; DNA Damage; Ubiquitination; Ubiquitin-Protein Ligases; Camptothecin; HEK293 Cells; BRCA1 Protein; Cell Cycle Proteins; Adaptor Proteins, Signal Transducing
PubMed: 38821984
DOI: 10.1038/s41467-024-49071-w -
Journal of Biomedical Science May 2024Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in...
BACKGROUND
Radioresistance is a key clinical constraint on the efficacy of radiotherapy in lung cancer patients. REV1 DNA directed polymerase (REV1) plays an important role in repairing DNA damage and maintaining genomic stability. However, its role in the resistance to radiotherapy in lung cancer is not clear. This study aims to clarify the role of REV1 in lung cancer radioresistance, identify the intrinsic mechanisms involved, and provide a theoretical basis for the clinical translation of this new target for lung cancer treatment.
METHODS
The effect of targeting REV1 on the radiosensitivity was verified by in vivo and in vitro experiments. RNA sequencing (RNA-seq) combined with nontargeted metabolomics analysis was used to explore the downstream targets of REV1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to quantify the content of specific amino acids. The coimmunoprecipitation (co-IP) and GST pull-down assays were used to validate the interaction between proteins. A ubiquitination library screening system was constructed to investigate the regulatory proteins upstream of REV1.
RESULTS
Targeting REV1 could enhance the radiosensitivity in vivo, while this effect was not obvious in vitro. RNA sequencing combined with nontargeted metabolomics revealed that the difference result was related to metabolism, and that the expression of glycine, serine, and threonine (Gly/Ser/Thr) metabolism signaling pathways was downregulated following REV1 knockdown. LC-MS/MS demonstrated that REV1 knockdown results in reduced levels of these three amino acids and that cystathionine γ-lyase (CTH) was the key to its function. REV1 enhances the interaction of CTH with the E3 ubiquitin ligase Rad18 and promotes ubiquitination degradation of CTH by Rad18. Screening of the ubiquitination compound library revealed that the ubiquitin-specific peptidase 9 X-linked (USP9X) is the upstream regulatory protein of REV1 by the ubiquitin-proteasome system, which remodels the intracellular Gly/Ser/Thr metabolism.
CONCLUSION
USP9X mediates the deubiquitination of REV1, and aberrantly expressed REV1 acts as a scaffolding protein to assist Rad18 in interacting with CTH, promoting the ubiquitination and degradation of CTH and inducing remodeling of the Gly/Ser/Thr metabolism, which leads to radioresistance. A novel inhibitor of REV1, JH-RE-06, was shown to enhance lung cancer cell radiosensitivity, with good prospects for clinical translation.
Topics: Humans; Lung Neoplasms; Ubiquitin-Protein Ligases; Nucleotidyltransferases; Radiation Tolerance; Ubiquitination; DNA-Binding Proteins; Ubiquitin Thiolesterase; Cell Line, Tumor; Mice; Animals; DNA-Directed DNA Polymerase
PubMed: 38802791
DOI: 10.1186/s12929-024-01044-3 -
Nucleic Acids Research Jun 2024TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression....
TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.
Topics: Humans; Cell Cycle Proteins; DNA; DNA Ligase ATP; DNA Repair; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Flap Endonucleases; Intracellular Signaling Peptides and Proteins; Poly (ADP-Ribose) Polymerase-1; X-ray Repair Cross Complementing Protein 1
PubMed: 38801073
DOI: 10.1093/nar/gkae445 -
Scientific Reports May 2024The E3 ubiquitin-ligase UHRF1 is an epigenetic regulator coordinating DNA methylation and histone modifications. However, little is known about how it regulates...
The E3 ubiquitin-ligase UHRF1 is an epigenetic regulator coordinating DNA methylation and histone modifications. However, little is known about how it regulates adipogenesis or metabolism. In this study, we discovered that UHRF1 is a key regulatory factor for adipogenesis, and we identified the altered molecular pathways that UHRF1 targets. Using CRISPR/Cas9-based knockout strategies, we discovered the whole transcriptomic changes upon UHRF1 deletion. Bioinformatics analyses revealed that key adipogenesis regulators such PPAR-γ and C/EBP-α were suppressed, whereas TGF-β signaling and fibrosis markers were upregulated in UHRF1-depleted differentiating adipocytes. Furthermore, UHRF1-depleted cells showed upregulated expression and secretion of TGF-β1, as well as the glycoprotein GPNMB. Treating differentiating preadipocytes with recombinant GPNMB led to an increase in TGF-β protein and secretion levels, which was accompanied by an increase in secretion of fibrosis markers such as MMP13 and a reduction in adipogenic conversion potential. Conversely, UHRF1 overexpression studies in human cells demonstrated downregulated levels of GPNMB and TGF-β, and enhanced adipogenic potential. In conclusion, our data show that UHRF1 positively regulates 3T3-L1 adipogenesis and limits fibrosis by suppressing GPNMB and TGF-β signaling cascade, highlighting the potential relevance of UHRF1 and its targets to the clinical management of obesity and linked metabolic disorders.
Topics: Animals; Humans; Mice; 3T3-L1 Cells; Adipocytes; Adipogenesis; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Eye Proteins; Fibrosis; Membrane Glycoproteins; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Protein Ligases
PubMed: 38789534
DOI: 10.1038/s41598-024-62508-y -
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