-
Biochimica Et Biophysica Acta.... Jun 2024Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is...
Human DNA polymerase ι (Polι) belongs to the Y-family of specialized DNA polymerases engaged in the DNA damage tolerance pathway of translesion DNA synthesis that is crucial to the maintenance of genome integrity. The extreme infidelity of Polι and the fact that both its up- and down-regulation correlate with various cancers indicate that Polι expression and access to the replication fork should be strictly controlled. Here, we identify RNF2, an E3 ubiquitin ligase, as a new interacting partner of Polι that is responsible for Polι stabilization in vivo. Interestingly, while we report that RNF2 does not directly ubiquitinate Polι, inhibition of the E3 ubiquitin ligase activity of RNF2 affects the cellular level of Polι thereby protecting it from destabilization. Additionally, we indicate that this mechanism is more general, as DNA polymerase η, another Y-family polymerase and the closest paralogue of Polι, share similar features.
Topics: Humans; Ubiquitin-Protein Ligases; DNA-Directed DNA Polymerase; DNA Polymerase iota; Ubiquitination; DNA Replication; DNA Damage; HEK293 Cells; Enzyme Stability; Polycomb Repressive Complex 1
PubMed: 38705361
DOI: 10.1016/j.bbamcr.2024.119743 -
Molecular Cell May 2024In a recent publication in Nature, Xu et al. discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which...
In a recent publication in Nature, Xu et al. discovered a role of CRL5-SPSB3 ubiquitin ligase in promoting ubiquitination and degradation of nuclear cGAS, which prevents aberrant cGAS activation by genomic DNA and contributes to the maintenance of immune homeostasis.
Topics: Nucleotidyltransferases; Humans; Homeostasis; Ubiquitination; Ubiquitin-Protein Ligases; Cell Nucleus; Proteolysis; Animals
PubMed: 38701740
DOI: 10.1016/j.molcel.2024.04.003 -
Biomolecular NMR Assignments Jun 2024The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic...
The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains.
Topics: Mycobacterium tuberculosis; Protein Domains; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; DNA Ligase ATP; DNA Ligases
PubMed: 38689205
DOI: 10.1007/s12104-024-10175-5 -
Journal of Experimental & Clinical... Apr 2024TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes...
BACKGROUND
TP53, the most frequently mutated gene in human cancers, orchestrates a complex transcriptional program crucial for cancer prevention. While certain TP53-dependent genes have been extensively studied, others, like the recently identified RNF144B, remained poorly understood. This E3 ubiquitin ligase has shown potent tumor suppressor activity in murine Eμ Myc-driven lymphoma, emphasizing its significance in the TP53 network. However, little is known about its targets and its role in cancer development, requiring further exploration. In this work, we investigate RNF144B's impact on tumor suppression beyond the hematopoietic compartment in human cancers.
METHODS
Employing TP53 wild-type cells, we generated models lacking RNF144B in both non-transformed and cancerous cells of human and mouse origin. By using proteomics, transcriptomics, and functional analysis, we assessed RNF144B's impact in cellular proliferation and transformation. Through in vitro and in vivo experiments, we explored proliferation, DNA repair, cell cycle control, mitotic progression, and treatment resistance. Findings were contrasted with clinical datasets and bioinformatics analysis.
RESULTS
Our research underscores RNF144B's pivotal role as a tumor suppressor, particularly in lung adenocarcinoma. In both human and mouse oncogene-expressing cells, RNF144B deficiency heightened cellular proliferation and transformation. Proteomic and transcriptomic analysis revealed RNF144B's novel function in mediating protein degradation associated with cell cycle progression, DNA damage response and genomic stability. RNF144B deficiency induced chromosomal instability, mitotic defects, and correlated with elevated aneuploidy and worse prognosis in human tumors. Furthermore, RNF144B-deficient lung adenocarcinoma cells exhibited resistance to cell cycle inhibitors that induce chromosomal instability.
CONCLUSIONS
Supported by clinical data, our study suggests that RNF144B plays a pivotal role in maintaining genomic stability during tumor suppression.
Topics: Animals; Humans; Mice; Cell Line, Tumor; Cell Proliferation; Genomic Instability; Lung Neoplasms; Tumor Suppressor Protein p53; Ubiquitin-Protein Ligases
PubMed: 38685100
DOI: 10.1186/s13046-024-03045-4 -
Mikrobiyoloji Bulteni Apr 2024The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases...
The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.
Topics: Vancomycin-Resistant Enterococci; Carbon-Oxygen Ligases; RNA, Antisense; Bacterial Proteins; Humans; Vancomycin; Plasmids; Vancomycin Resistance; Microbial Sensitivity Tests; Anti-Bacterial Agents; Gene Silencing
PubMed: 38676581
DOI: 10.5578/mb.202498191 -
International Journal of Molecular... Apr 2024Base excision repair (BER), which involves the sequential activity of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases, is one of...
Base excision repair (BER), which involves the sequential activity of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases, is one of the enzymatic systems that preserve the integrity of the genome. Normal BER is effective, but due to single-nucleotide polymorphisms (SNPs), the enzymes themselves-whose main function is to identify and eliminate damaged bases-can undergo amino acid changes. One of the enzymes in BER is DNA polymerase β (Polβ), whose function is to fill gaps in DNA. SNPs can significantly affect the catalytic activity of an enzyme by causing an amino acid substitution. In this work, pre-steady-state kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring variants of Polβ that have the substitutions L19P and G66R in the dRP-lyase domain. Despite the substantial distance between the dRP-lyase domain and the nucleotidyltransferase active site, it was found that the capacity to form a complex with DNA and with an incoming dNTP is significantly altered by these substitutions. Therefore, the lower activity of the tested polymorphic variants may be associated with a greater number of unrepaired DNA lesions.
Topics: DNA Polymerase beta; Humans; Amino Acid Substitution; Polymorphism, Single Nucleotide; Molecular Dynamics Simulation; DNA Repair; Kinetics; Catalytic Domain; DNA; Protein Domains
PubMed: 38673769
DOI: 10.3390/ijms25084182 -
Functional Plant Biology : FPB Apr 2024Plants regularly encounter various environmental stresses such as salt, drought, cold, heat, heavy metals and pathogens, leading to changes in their proteome. Of these,... (Review)
Review
Plants regularly encounter various environmental stresses such as salt, drought, cold, heat, heavy metals and pathogens, leading to changes in their proteome. Of these, a post-translational modification, SUMOylation is particularly significant for its extensive involvement in regulating various plant molecular processes to counteract these external stressors. Small ubiquitin-like modifiers (SUMO) protein modification significantly contributes to various plant functions, encompassing growth, development and response to environmental stresses. The SUMO system has a limited number of ligases even in fully sequenced plant genomes but SUMO E3 ligases are pivotal in recognising substrates during the process of SUMOylation. E3 ligases play pivotal roles in numerous biological and developmental processes in plants, including DNA repair, photomorphogenesis, phytohormone signalling and responses to abiotic and biotic stress. A considerable number of targets for E3 ligases are proteins implicated in reactions to abiotic and biotic stressors. This review sheds light on how plants respond to environmental stresses by focusing on recent findings on the role of SUMO E3 ligases, contributing to a better understanding of how plants react at a molecular level to such stressors.
Topics: Ubiquitin-Protein Ligases; Stress, Physiological; Plants; Sumoylation; Plant Proteins; Small Ubiquitin-Related Modifier Proteins
PubMed: 38669463
DOI: 10.1071/FP24032 -
Cells Apr 2024Cervical cancer is a major cause of death in women despite the advancement of current treatment modalities. The conventional therapeutic agent, cisplatin (CCDP), is the...
Cervical cancer is a major cause of death in women despite the advancement of current treatment modalities. The conventional therapeutic agent, cisplatin (CCDP), is the standard treatment for CC; however, resistance often develops due to the cancer's heterogeneity. Therefore, a detailed elucidation of the specific molecular mechanisms driving CC is crucial for the development of targeted therapeutic strategies. Retinoblastoma binding protein 6 () is a potential biomarker associated with cell proliferation and is upregulated in cervical cancer sites, exhibiting apoptosis and dysregulated expression. Furthermore, has been demonstrated to sensitize cancer cells to radiation and certain chemotherapeutic agents by regulating the gene, thus suggesting a crosstalk among / oncogenic signatures. The present study, therefore, investigated the relationship between cisplatin and expression in CC cells. Herein, we first explored bioinformatics simulations and identified that the signaling pathway is overexpressed and correlated with CC. For further analysis, we explored the Genomics of Drug Sensitivity in Cancer (GDSC) and found that most of the CC cell lines are sensitive to CCDP. To validate these findings, was silenced in HeLa and Vero cells using RNAi technology, followed by measurement of wild-type and at the mRNA level using qPCR. Cells co-treated with cisplatin and siRBBP6 were subsequently analyzed for apoptosis induction and real-time growth monitoring using flow cytometry and the xCELLigence system, respectively. Cancer cells in the co-treatment group showed a reduction in apoptosis compared to the cisplatin-treated group. Moreover, the real-time growth monitoring revealed a reduced growth rate in knockdown cells treated with cisplatin. Although wild-type remained unchanged in the co-treatment group of cancer cells, was completely repressed, suggesting that is necessary for sensitizing cervical cancer cells to cisplatin treatment by downregulating . The Vero cell population, which served as a non-cancerous control cell line in this study, remained viable following treatment with both siRBBP6 and cisplatin. Findings from this study suggest that expression promotes cisplatin sensitivity in HeLa cells through downregulation. Knockdown of limits apoptosis induction and delays cell growth inhibition in response to cisplatin. The knowledge obtained here has the potential to help improve cisplatin efficacy through personalized administration based on the expression profile of among individual patients.
Topics: Humans; Cisplatin; Female; Uterine Cervical Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Tumor Suppressor Protein p53; Gene Expression Regulation, Neoplastic; Proto-Oncogene Proteins c-bcl-2; Apoptosis; Gene Knockdown Techniques; Cell Proliferation; Antineoplastic Agents; Signal Transduction; HeLa Cells; DNA-Binding Proteins; Ubiquitin-Protein Ligases
PubMed: 38667315
DOI: 10.3390/cells13080700 -
Discovery Medicine Apr 2024Pneumonia is a prevalent respiratory ailment involving complex physiological and pathological mechanisms. The tripartite motif containing 27 (TRIM27) plays a crucial...
BACKGROUND
Pneumonia is a prevalent respiratory ailment involving complex physiological and pathological mechanisms. The tripartite motif containing 27 (TRIM27) plays a crucial role in regulating inflammation mechanisms. Therefore, the purpose of this study is to further explore the therapeutic potential of TRIM27 in pneumonia, based on its regulatory mechanisms in inflammation and autophagy.
METHODS
This study established a mouse pneumonia animal model through lipopolysaccharide (LPS) administration, designating it as the LPS model group. Subsequently, adenovirus-mediated TRIM27 overexpression was implemented in the animals of the LPS model group, creating the TRIM27 treatment group. After a 7-day treatment period, lung tissues from the mice were collected. Various techniques, including immunohistochemistry, quantitative reverse transcription PCR (RT-qPCR), western blot, enzyme-linked immunosorbent assay (ELISA), and electron microscopy were utilized to analyze the impact of TRIM27 overexpression on inflammatory factors, oxidative stress, autophagy, and inflammatory processes in pulmonary tissues. Finally, an LPS cell model was established, and the effects of TRIM27 overexpression and autophagy inhibition on inflammatory cytokines and autophagosomes in LPS-induced inflammatory cells were examined through RT-qPCR and immunofluorescence techniques.
RESULTS
The research findings demonstrate a significant reduction in the elevated levels of interleukin-6 (IL-6), IL-1β, and Tumor necrosis factor-alpha (TNF-α) induced by LPS with TRIM27 overexpression ( < 0.01). Conversely, the autophagy inhibitor 3-Methyladenine (3-MA) diminished the effects induced by TRIM27 overexpression. Moreover, TRIM27 overexpression enhanced the expression of Microtubule-associated protein 1A/1B light chain 3 (LC3) II/I and Beclin-1 proteins in mice subjected to LPS stimulation ( < 0.01), while reducing the expression of the p62 protein ( < 0.01). The addition of 3-MA, however, decreased Beclin-1 expression and inhibited autophagy ( < 0.01). Additionally, TRIM27 overexpression decreased the expression of NOD-like receptor thermal protein domain associated protein 3 (), cleaved caspase-1, β, and Gasdermin D N-terminal fragment (GSDMD-N) proteins in LPS-stimulated mice ( < 0.05). TRIM27 overexpression also decreased the levels of malondialdehyde (MDA), Activating Transcription Factor 6 (6), and C/EBP-homologous protein (), while increasing the levels of superoxide dismutase (SOD) and glutathione (GSH) in mice exposed to LPS ( < 0.01).
CONCLUSION
The induction of TRIM27 overexpression emerges as a potential and effective pneumonia treatment. The underlying mechanism may involve inducing protective autophagy, thereby reducing oxidative stress and cell pyroptosis.
Topics: Animals; Male; Mice; Adenine; Autophagy; Beclin-1; Disease Models, Animal; DNA-Binding Proteins; Lipopolysaccharides; Lung; Mice, Inbred C57BL; Oxidative Stress; Pneumonia; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 38665029
DOI: 10.24976/Discov.Med.202436183.76 -
Zhongguo Dang Dai Er Ke Za Zhi =... Apr 2024The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait...
The first patient, a 10-year-old girl, presented with pancytopenia and recurrent epistaxis, along with a history of repeated upper respiratory infections, café-au-lait spots, and microcephaly. Genetic testing revealed compound heterozygous mutations in the DNA ligase IV () gene, leading to a diagnosis of LIG4 syndrome. The second patient, a 6-year-old girl, was seen for persistent thrombocytopenia lasting over two years and was noted to have short stature, hyperpigmented skin, and hand malformations. She had a positive result from chromosome breakage test. She was diagnosed with Fanconi anemia complementation group A. Despite similar clinical presentations, the two children were diagnosed with different disorders, suggesting that children with hemocytopenia and malformations should not only be evaluated for hematological diseases but also be screened for other potential underlying conditions such as immune system disorders.
Topics: Humans; Female; Child; Abnormalities, Multiple; Pancytopenia; DNA Ligase ATP; Thrombocytopenia; Cytopenia
PubMed: 38660906
DOI: 10.7499/j.issn.1008-8830.2311058