DNA Repair-related Proteins Research Articles

Multitargeted docking approach reveals droxidopa against DNA replication and repair-related protein of cervical cancer

Cervical cancer begins in the cells lining the cervix and is caused by persistent infection with certain types of human papillomavirus (HPV). Initially, it has no symptoms, and later it causes pelvic pain, abnormal vaginal bleeding, and pain during intercourse. It is the fourth-ranked cancer among women, and many women die from cervical cancer every year, particularly in low-income countries and the majority could be prevented with early detection and treatment. In this study, we have taken Cervical Cancer DNA Replication and Repair-Related Protein with the PDBID- 3H15, 5VBN, and 6NT9 and performed the multitargeted molecular docking with the FDA-approved drug library using HTVS, SP and XP docking. Then, the poses were filtered with MM\\GBSA for proper computations of free energy, identified a multitargeted inhibitor Droxidopa with docking and MM\\GBSA scores ranging from − 5.559 to − 6.835 Kcal/mol and − 26.04 to − 37.33 Kcal/mol, respectively. We also performed interaction fingerprints revealing 2VAL, 2LYS, 1ALA, 1ARG, 1ASN, 1CYS, 1GLN, 1GLU, 1ILE, 1MET, 1PHE, 1PRO, 1SER, and 1THR were most interacted residues and computed the ADMET properties with QikProp and DFT with Jaguar, which supported the study and compounds’ suitability. Moreover, we performed the 100ns MD simulation in water, showing the controlled deviation and fluctuations of the residues with many interactions, and MM\\GBSA was performed with the same trajectories, showing a better understanding of each frame’s total complex and binding-free energy. The whole study favours droxidopa as an inhibitor of cervical cancer DNA Replication and Repair-Related Proteins—however, experimental studies are needed before use.

Matrine alkaloids modulating DNA damage repair in chemoresistant non-small cell lung cancer cells

BackgroundNon-small cell lung cancer (NSCLC) presents a significant challenge in the medical field due to its high incidence and resistance to chemotherapy. Chemoresistance in NSCLC diminishes treatment efficacy and contributes to poor patient outcomes. Matrine alkaloids have shown promise in reversing chemotherapy resistance in NSCLC by targeting DNA repair mechanisms.MethodsUtilizing molecular dynamics simulations, we explored the interactions between Matrine alkaloids and DNA repair-related proteins to elucidate their impact on NSCLC cells. In vitro experiments involved treating A549/DDP cells with Matrine alkaloids to evaluate their sensitizing effects on lung cancer cells. Additionally, animal model experiments were conducted to validate the therapeutic potential of Matrine alkaloids in NSCLC treatment.ResultsOur findings demonstrate that Matrine alkaloids disrupt DNA damage repair processes in NSCLC cells, leading to increased sensitivity to chemotherapy. Molecular docking studies revealed the intricate mechanisms by which Matrine alkaloids interact with DNA repair proteins, impacting cell survival and proliferation. Both cell experiments and animal models confirmed the chemosensitizing effects of Matrine alkaloids in NSCLC treatment.ConclusionMatrine alkaloids offer a promising avenue for overcoming chemotherapy resistance in NSCLC by interfering with DNA repair pathways. This study lays a solid foundation for future clinical investigations into the potential of Matrine alkaloids as effective therapeutic agents for enhancing NSCLC treatment outcomes.

Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor

Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.

MST1 interactomes profiling across cell death in esophageal squamous cell carcinoma

Abstract Objectives Resistance to apoptosis in esophageal squamous cell carcinoma (ESCC) constitutes a significant impediment to treatment efficacy. Exploring alternative cell death pathways and their regulatory factors beyond apoptosis is crucial for overcoming drug resistance and enhancing therapeutic outcomes in ESCC. Methods Mammalian Ste 20-like kinase 1 (MST1) is implicated in regulating various cell deaths, including apoptosis, autophagy, and pyroptosis. Employing enhanced ascorbate peroxidase 2 (APEX2) proximity labeling coupled with immunoprecipitation-mass spectrometry (IP-MS), we elucidated the interactomes of MST1 across these three cell death paradigms. Results Proteomic profiling unveiled the functional roles and subcellular localization of MST1 and its interacting proteins during normal proliferation and various cell death processes. Notably, MST1 exhibited an expanded interactome during cell death compared to normal proliferation and chromosome remodeling functions consistently. In apoptosis, there was a notable increase of mitosis-associated proteins such as INCENP, ANLN, KIF23, SHCBP1 and SUPT16H, which interacted with MST1, alongside decreased expression of the pre-apoptotic protein STK3. During autophagy, the bindings of DNA repair-related proteins CBX8 and m6A reader YTHDC1 to MST1 were enhanced. In pyroptosis, LRRFIP2 and FLII which can inhibit pyroptosis increasingly binding to MST1. Conclusions Our findings delineate potential mechanisms through which MST1 and its interactomes regulate cell death, paving the way for further investigation to validate and consolidate these observations.

Apigenin protects against ischemic stroke by increasing DNA repair.

Oxidative stress is an important pathological process in ischemic stroke (IS). Apigenin (APG) is a natural product with favorable antioxidative effects, and some studies have already demonstrated the antioxidative mechanism of APG in the treatment of IS. However, the mechanism of APG on DNA damage and repair after IS is not clear. The aim of this study was to investigate the mechanism of APG on DNA repair after IS. Male Sprague-Dawley rats were used to establish a model of permanent middle cerebral artery occlusion (pMCAO) on one side, and were pre-treated with gavage of APG (30, 60, or 120mg/kg) for 7 days. One day after pMCAO, the brain tissues were collected. Cerebral infarct volume, brain water content, HE staining and antioxidant index were analyzed to evaluated the brain damage. Molecular Docking, molecular dynamics (MD) simulation, immunohistochemistry, and Western blot were used to explore the potential proteins related to DNA damage repair. APG has a low binding score with DNA repair-related proteins. APG treatment has improved the volume of cerebral infarction and neurological deficits, reduced brain edema, and decreased parthanatos and apoptosis by inhibiting PARP1/AIF pathway. In addition, APG improved the antioxidative capacity through reducing reactive oxygen species and malondialdehyde, and increasing glutathione and superoxide dismutase. Also, APG has reduced DNA damage- and cell death-related proteins such as PARP1, γH2A.X, 53BP1, AIF, cleaved caspase3, Cytochrome c, and increased DNA repair by BRCA1 and RAD51 through homologous recombination repair, and reduced non-homologous end link repair by KU70. APG can improve nerve damage after IS, and these protective effects were realized by reducing oxidative stress and DNA damage, and improving DNA repair.

Surviving in a multistressor world: Gene expression changes in earthworms exposed to heat, desiccation, and chemicals

An investigation of the effects of anthropogenic stress on terrestrial ecosystems is urgently needed. In this work, we explored how exposure to heat, desiccation, and chemical stress alters the expression of genes that encode heat shock proteins (HSPs), an enzyme that responds to oxidative stress (CAT), hypoxia-related proteins (HIF1 and HYOU), and a DNA repair–related protein (PARP1) in the earthworm Eisenia fetida. Exposure to heat (31°C) for 24 h upregulated HSPs and hypoxia-related genes, suggesting possible acquired thermotolerance. Desiccation showed a similar expression profile; however, the HSP response was activated to a lesser extent. Heat and desiccation activated the small HSP at 24 h, suggesting that they may play a role in adaptation. Simultaneous exposure to endosulfan and temperature for 7 h upregulated all of the evaluated genes, implicating a coordinated response involving multiple biological processes to ensure survival and acclimation. These results highlight the relevance of multistress analysis in terrestrial invertebrates.

Transcriptome analysis reveals the important roles of a two-component system, flagellar assembly, active efflux system and outer membrane proteins in the anti-quinolone ability of Vibrio harveyi from orange-spotted grouper (Epinephelus coioides)

The drug resistance of Vibrio harveyi in aquaculture became more severe because the control of Vibriosis depends majorly on the current antibiotics. Transcriptomes of a wild-type strain (VS) and its quinolone-resistant mutants (VR) of V. harveyi were respectively sequenced by RNA-seq technology. A total of 2,082 unigenes were obtained after de novo splicing and assembly. 129 genes were identified with significant differential expression in strain VR compared to strain VS, among which 65 were up-regulated and 64 down-regulated. Then, functional annotation and enrichment analysis of these differentially expressed genes (DEGs) were performed. GO enrichment results showed that DEGs focused mainly on cell structure, substance metabolism, and transporter. COG classification of the DEGs mainly focused on amino acid transport and metabolism, cell wall/membrane biosynthesis, carbohydrate transport and metabolism, ribosomal structure, and biosynthesis. KEGG pathways related to a two-component system, ABC transport system and flagellar assembly (ko02040) were enriched significantly, and 9 genes associated with quinolone-resistance ability, including genes for resistance-related transport proteins, outer membrane proteins, and DNA repair-related proteins were discovered through analysis of the drug-resistance related genes. Ten DEGs (including the above part genes of 9 drug resistance-related genes) in the transcriptome data were taken to analyze their expression with real-time qPCR. The results were the same as the changes of the above transcriptome analysis, further confirming the reliability of the transcriptome sequencing and data analysis. In a word, genes from a two-component system, flagellar assembly, active efflux system and outer membrane proteins take great roles in the quinolone-resistance of V. harveyi. These results provide enough information for further study on the molecular mechanism of quinolone-resistance and give a helpful transcriptomic resource to unravel the contact between quinolone-resistance and metabolic pathways in Vibrios.

Altered DNA repair related proteins in Parkinson’s disease model VMAT2 Lo mice

Altered DNA repair related proteins in Parkinson’s disease model VMAT2 Lo mice

A Photoactivated Ru (II) Polypyridine Complex Induced Oncotic Necrosis of A549 Cells by Activating Oxidative Phosphorylation and Inhibiting DNA Synthesis as Revealed by Quantitative Proteomics.

The ruthenium polypyridine complex [Ru(dppa)2(pytp)] (PF6)2 (termed as ZQX-1), where dppa = 4,7-diphenyl-1,10-phenanthroline and pytp = 4'-pyrene-2,2':6',2''-terpyridine, has been shown a high and selective cytotoxicity to hypoxic and cisplatin-resistant cancer cells either under irradiation with blue light or upon two-photon excitation. The IC50 values of ZQX-1 towards A549 cancer cells and HEK293 health cells are 0.16 ± 0.09 µM and >100 µM under irradiation at 420 nm, respectively. However, the mechanism of action of ZQX-1 remains unclear. In this work, using the quantitative proteomics method we identified 84 differentially expressed proteins (DEPs) with |fold-change| ≥ 1.2 in A549 cancer cells exposed to ZQX-1 under irradiation at 420 nm. Bioinformatics analysis of the DEPs revealed that photoactivated ZQX-1 generated reactive oxygen species (ROS) to activate oxidative phosphorylation signaling to overproduce ATP; it also released ROS and pyrene derivative to damage DNA and arrest A549 cells at S-phase, which synergistically led to oncotic necrosis and apoptosis of A549 cells to deplete excess ATP, evidenced by the elevated level of PRAP1 and cleaved capase-3. Moreover, the DNA damage inhibited the expression of DNA repair-related proteins, such as RBX1 and GPS1, enhancing photocytotoxicity of ZQX-1, which was reflected in the inhibition of integrin signaling and disruption of ribosome assembly. Importantly, the photoactivated ZQX-1 was shown to activate hypoxia-inducible factor 1A (HIF1A) survival signaling, implying that combining use of ZQX-1 with HIF1A signaling inhibitors may further promote the photocytotoxicity of the prodrug.

Co-Expression of Chromatin Assembly Factor 1 Subunit A and Proliferating Cell Nuclear Antigen Is a Prognostic Biomarker of Esophageal Cancer

(1) Background: Esophageal cancer (EC) is an important global health challenge. Due to the lack of necessary biomarkers and therapeutic targets, the survival of EC patients is poor. The EC proteomic data of 124 patients recently published by our group provides a database for research in this field. (2) Methods: Bioinformatics analysis was used to identify DNA replication and repair-related proteins in EC. Proximity ligation assay, colony formation assay, DNA fiber assay, and flow cytometry were used to study the effects of related proteins on EC cells. Kaplan–Meier survival analysis was used to evaluate the relationship between gene expression and the survival time of EC patients. (3) Results: Chromatin assembly factor 1 subunit A (CHAF1A) was highly correlated with proliferating cell nuclear antigen (PCNA) expression in EC. CHAF1A and PCNA colocalized in the nucleus of EC cells. Compared with the knockdown of CHAF1A or PCNA alone, the double knockdown of CHAF1A and PCNA could significantly inhibit EC cell proliferation. Mechanistically, CHAF1A and PCNA synergistically accelerated DNA replication and promoted S-phase progression. EC patients with high expression of both CHAF1A and PCNA had a worse survival rate. (4) Conclusion: we identify CHAF1A and PCNA as key cell cycle-related proteins leading to the malignant progression of EC, and these proteins could serve as important prognostic biomarkers and targets for EC.

Polymorphisms in DNA Repair Genes and Association with Rheumatoid Arthritis in a Pilot Study on a Central European Population.

Rheumatoid arthritis (RA) is a chronic, multifactorial autoimmune disease characterized by chronic arthritis, a tendency to develop joint deformities, and involvement of extra-articular tissues. The risk of malignant neoplasms among patients with RA is the subject of ongoing research due to the autoimmune pathogenesis that underlies RA, the common etiology of rheumatic disease and malignancies, and the use of immunomodulatory therapy, which can alter immune system function and thus increase the risk of malignant neoplasms. This risk can also be increased by impaired DNA repair efficiency in individuals with RA, as reported in our recent study. Impaired DNA repair may reflect the variability in the genes that encode DNA repair proteins. The aim of our study was to evaluate the genetic variation in RA within the genes of the DNA damage repair system through base excision repair (BER), nucleotide excision repair (NER), and the double strand break repair system by homologous recombination (HR) and non-homologous end joining (NHEJ). We genotyped a total of 28 polymorphisms in 19 genes encoding DNA repair-related proteins in 100 age- and sex-matched RA patients and healthy subjects from Central Europe (Poland). Polymorphism genotypes were determined using the Taq-man SNP Genotyping Assay. We found an association between the RA occurrence and rs25487/XRCC1, rs7180135/RAD51, rs1801321/RAD51, rs963917/RAD51B, rs963918/RAD51B, rs2735383/NBS1, rs132774/XRCC6, rs207906/XRCC5, and rs861539/XRCC3 polymorphisms. Our results suggest that polymorphisms of DNA damage repair genes may play a role in RA pathogenesis and may be considered as potential markers of RA.

Topoisomerase IIβ binding protein 1 serves as a novel prognostic biomarker for stage II-III colorectal cancer patients

Colorectal cancer (CRC) is a commonly diagnosed human malignancy worldwide. Accumulating evidence has suggested DNA repair related proteins widely participate in CRC initiation and development. TOPBP1 is recently identified as a novel regulator for DNA repair, however, its biological role in CRC remains unknown. Firstly, the bioinformatics analysis was utilized to investigate the expression and clinical significance of TOPBP1 in CRC patients. Then, a retrospective study enrolling 129 stage II/III CRC patients was performed for validation. The CCK-8, colony formation, transwell assay and xenograft model were used to clarify the biological impact of TOPBP1 on CRC cells. Finally, transcriptome sequencing was performed to investigate the potential oncogenic mechanisms regulated by TOPBP1 in CRC development. The expression of TOPBP1 was significantly higher in CRC tissues than that in normal tissues. High TOPBP1 expression was an independent unfavorable prognostic factor for overall and disease-free survival in II/III CRC patients. Knockdown of TOPBP1 not only significantly inhibited the proliferation, colony formation, invasion, migration and epithelial-mesenchymal transition (EMT) molecular phenotype of CRC cells, while the opposite was for TOPBP1 expression. Moreover, knockdown of TOPBP1 slowed down the growth speed of xenografts. The transcriptome sequencing identified MAP3K3 as a downstream gene of TOPBP1 and MAP3K3 knockdown inhibited the EMT molecular phenotype in CRC cells. Finally, the rescue assay indicated MAP3K3 overexpression counteracted the inhibitory effect of TOPBP1 knockdown on the proliferation, colony formation, invasion, migration and EMT phenotype of CRC cells. TOPBP1 promotes the malignant progression of CRC through MAP3K3 induced EMT. TOPBP1 is a promising clinical biomarker or therapeutical target for CRC patients.

Levels of base excision repair proteins in CD4+ T cells in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multi-systemic disease with the unknown pathogenic mechanism. DNA demethylation is involved in SLE pathogenesis. Growth arrest and DNA damage inducible 45 alpha (Gadd45a) takes part in the process of DNA demethylation. Gadd45a is a DNA repair-related protein. This study aims to investigate the expressions of some proteins [including activation-induced cytidine deaminase (AID), thymine DNA glycosylase (TDG), and methyl-CpG-binding domain protein 4 (MBD4)] involving in base excision repair (BER) process in CD4+ T cells in patients with SLE, and to analyze the correlations between the above BER proteins and lupus disease. From January 2019 to September 2020, 12 SLE patients and 12 healthy controls were recruited from Second Xiangya Hospital of Central South University. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hypaque density gradient centrifugation and then CD4+ T cells were isolated via positive selection using Miltenyi beads. We measured the messenger RNA (mRNA) and protein expressions of AID, TDG, and MBD4 by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, respectively. In contrast to controls, in SLE CD4+ T cells, the mRNA and protein expressions of AID were elevated (P=0.003, P=0.022, respectively); TDG protein expression was increased (P=0.017); and MBD4 protein level was reduced (P<0.001). No visible distinctions was found in the mRNA expressions of either TDG or MBD4 between the 2 groups (both P>0.05). The mRNA and protein expressions of AID and the protein levels of TDG were positively correlated with SLE disease activity index (SLEDAI). And the mRNA and protein expressions of MBD4 were negatively correlated with SLEDAI. In SLE CD4+ T cells, the increased expressions of AID and TDG and the decreased MBD4 expression may participate in SLE pathogenic mechanism.

HMG20A was identified as a key enhancer driver associated with DNA damage repair in oral squamous cell carcinomas

BackgroundOral squamous cell carcinoma (OSCC) is the main type of oral cancer. Disturbing DNA repair is an invaluable way to improve the effectiveness of tumor treatment. Here, we aimed to explore the key enhancer drivers associated with DNA damage repair in OSCC cells.MethodsGene Set Enrichment Analysis (GSEA), Gene Set Variation Analysis (GSVA) and Kaplan-Meier analysis were applied to explore the relationship among DNA repair-related genes expression and clinical phenotypes based on The Cancer Genome Atlas (TCGA) database. HOMER software and Integrative Genomics Viewer were applied to identify and visualize enhancers using GSE120634. Toolkit for Cistrome Data Browser was applied to predict transcription factors. Human Protein Atlas Database was used to analyze the protein levels of transcription factors in OSCC and control tissues. Seventy-two OSCC patients were included in this study. qRT-PCR was used to detect transcription factor expression in OSCC and adjacent control tissues collected in this study. qRT-PCR and ChIP-qPCR were used to verify the binding of transcription factors to enhancers, and regulation of target genes transcription. Transcription factor knockdown and control cells were treated with cisplatin. CCK8 was used to detect cell viability and proliferation. Western blotting was implemented to detect the levels of DNA repair-related proteins. Transwell assay was used to detect cell invasion.ResultsDNA repair was positively associated with the OSCC metastatic phenotype. Patients in the cluster with high expression of DNA repair-related genes had a worse prognosis and a higher proportion of advanced stage, low-differentiation, alcohol consumption and smoking compared to the cluster with low DNA repair-related gene expression. Seventeen metastasis-specific enhancer-controlled upregulated DNA repair-related genes, with the top two upregulated genes being ADRM1 26 S proteasome ubiquitin receptor (ADRM1) and solute carrier family 12 member 7 (SLC12A7) were screened. High mobility group 20 A (HMG20A) was the key prognostic enhancer driver regulating metastasis-specific DNA repair-related genes, with higher expression in OSCC tissues than normal control tissues, and higher expression in metastatic OSCC tissues than non-metastatic OSCC tissues. HMG20A bound to the metastasis-specific enhancers of ADRM1 and SLC12A7, thereby promoting ADRM1 and SLC12A7 expression. Knockdown of HMG20A enhanced cisplatin sensitivity of cells, and inhibited OSCC cells from repairing DNA damage caused by cisplatin, as well as proliferation and invasion of OSCC cells.ConclusionHMG20A was identified as the key prognostic enhancer driver regulating DNA repair in OSCC cells, providing a new therapeutic target for OSCC.

EGFR Mutant Lung Adenocarcinoma Showed Reduced Radiosensitivity after Acquired Resistance to EGFR TKIs

<h3>Purpose/Objective(s)</h3> Though earlier application of local radiotherapy for the primary lesion may improve the survival of patients, the timing of radiotherapy intervention for advanced EGFR mutant lung adenocarcinoma is still controversial. This study aimed to detect the difference of radiosensitivity before and after resistance and explore the related mechanism by constructing EGFR mutant lung adenocarcinoma cell lines with acquired resistance to gefitinib in vitro, so as to provide theoretical basis for determining the optimal intervention timing of radiotherapy. <h3>Materials/Methods</h3> Two lung adenocarcinoma cell lines harboring EGFR mutation-PC9 and HCC827, were used for establishment of acquired resistant cells to gefitinib. The radiosensitivity was detected using a cell counting kit. Flow cytometry was introduced to explore the cell cycle distribution and Western blot was applied to further test cell cycle and DNA repair related proteins. Soft agar assay and Western blot were performed to determine the cancer stem cell (CSC) characteristics. RT-PCR detected the expression of CSC markers. Finally, the relevant signal pathways were explored. <h3>Results</h3> The cell counting kit showed that the inhibitory activity of cells with irradiation after gefitinib resistance is weaker than that before resistance. Flow cytometry revealed that the G2/M phase proportion of PC9 and HCC827 were significantly higher than that of PC9/GR and HCC827/GR under the same irradiation dose, which is consistent with the results of western blot that the increased expression of G2/M regulatory protein, including p21, Cyclin B1, CyclinA2, pCDC2 and Myt1 were more significant increased in parental cells than resistant cells. The phosphorylated level of RAD17, ATM and CHK2 which were DNA damage repair protein were more upregulated in PC9GR and HCC827GR cells. Furthermore, GR had strong capability of sphere-forming and EMT phenotype. RT-PCR showed that the expression of many genes regulating CSC characteristics increased obviously after gefitinib resistance, including SOX2, OCT4, Nanog, Notch1, Notch3, ALDH1A1, ALDH1A3 and BMI. But the up-regulated genes in the two drug-resistant cells were not completely same. Western blot and RT-PCR showed that BMP/SMAD signal pathway was more activated in both resistant cells than that in parental cells. <h3>Conclusion</h3> Lung adenocarcinoma with EGFR mutations showed reduced radiosensitivity after acquired resistance to EGFR TKIs through obtaining CSC characteristics, which may be mediated by BMP/SMAD signal pathway.

HMCES modulates the transcriptional regulation of nodal/activin and BMP signaling in mESCs.

Despite the fundamental roles of TGF-β family signaling in cell fate determination in all metazoans, the mechanism by which these signals are spatially and temporally interpreted remains elusive. The cell-context-dependent function of TGF-β signaling largely relies on transcriptional regulation by SMAD proteins. Here, we discover that the DNA repair-related protein, HMCES, contributes to early development by maintaining nodal/activin- or BMP-signaling-regulated transcriptional network. HMCES binds with R-SMAD proteins, co-localizing at active histone marks. However, HMCES chromatin occupancy is independent on nodal/activin or BMP signaling. Mechanistically, HMCES competitively binds chromatin to limit binding by R-SMAD proteins, thereby forcing their dissociation and resulting in repression of their regulatory effects. In Xenopus laevis embryo, hmces KD causes dramatic development defects with abnormal left-right axis asymmetry along with increasing expression of lefty1. These findings reveal HMCES transcriptional regulatory function in the context of TGF-β family signaling.

Catechins protect against acrylamide- and glycidamide-induced cellular toxicity via rescuing cellular apoptosis and DNA damage

Acrylamide (AA) occurs in both various environmental and dietary sources and has raised widespread concern as a probable carcinogen. Glycidamide (GA) is the main genotoxic metabolite through P450 2E1 (CYP2E1). In the present study, we investigated the protective effect of (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin (EC) against AA- and GA-induced hepatotoxicity in HepG2 cells. The results demonstrated that EC and EGCG inhibited AA- and GA-induced cytotoxicity and mitochondria-mediated cellular apoptosis. Moreover, exposure to AA (100 μg/mL) and GA (50 μg/mL) caused cell cycle arrest and DNA damage, while EC and EGCG ranging from 12.5 to 50 μg/mL rescued cell cycle arrest and inhibited DNA damage. Furthermore, EC and EGCG down-regulated pro-apoptotic protein Bax and Caspase 3 after a 24-h treatment in HepG2 cells exposed to AA (100 μg/mL) or GA (50 μg/mL). Also, the intervention with EC or EGCG up-regulated the expression of DNA repair related protein PARP and down-regulated the expression of Cleaved-PARP. Besides, EC exerted better protective effect than EGCG against AA- and GA-induced cytotoxicity in HepG2 cells. Altogether, EC and EGCG were effective in protecting AA- and GA-induced hepatotoxicity via rescuing cellular apoptosis and DNA damage, as well as promoting cell cycle progression in HepG2 cells.

HGG-50. Specific sensitivity of pediatric high-grade glioma with ATRX inactivation to PARP inhibitor combinations

Pediatric high-grade gliomas (pHGG) account for approximately 12% of pediatric brain tumors. Despite advances in molecular diagnosis and identification of discrete molecular subtypes, pHGG are the leading cause of cancer-related death in children. Thus, current research focuses on identifying novel therapeutic targets. Sequencing analyses across pediatric cancer types identified DNA repair perturbations as potentially targetable events in certain types of pediatric brain tumors. Herein, we investigated the potential of PARP inhibitors (PARPi), impeding the central role of PARP in DNA damage repair, in pHGG. We screened a patient-derived primary pHGG cell line panel (n=7) for their sensitivity towards 6 different PARPi (niraparib, olaparib, pamiparib, rucaparib, talazoparib, veliparib) using cell viability assays. Basal expression of DNA repair related proteins was assessed by immunoblot, and propidium iodide-based flow cytometry was used for cell cycle analysis. All pHGG were resistant towards single compound PARP inhibition. Interestingly, two H3F3A-G34R mutant pHGG models harboring inactivating ATRX mutations were characterized by elevated basal levels of pH2AX, suggesting increased stress resulting from DNA damage. Consequently, simultaneous targeting of PARP and other components of DNA repair in the respective models showed strong synergistic effects on cell viability, which was not observed to a comparable extent in other models such as BRAFV600E/TERT promotor mutant pHGG. Combination of talazoparib and irinotecan resulted in S-phase arrest. Within a precision oncology approach, we treated a 11-year-old child suffering from H3F3A-G34R mutant pHGG with ATRX mutation, that progressed during radiation, with niraparib and topotecan. The patient achieved partial remission and disease stabilization for 1 year. Taken together, PARPi combinations show potential for the treatment of pHGG with ATRX mutations. Currently, all cell models are characterized for DNA repair signatures by DNA sequencing. Further, in depth characterization of DNA damage responses upon concomitant PARP and topoisomerase inhibition in ATRX-mutated pHGG are ongoing.

DNA repair-related genes and adipogenesis: Lessons from congenital lipodystrophies.

Classical and progeroid congenital lipodystrophies are a collection of rare diseases displaying a large genetic heterogeneity. They occur due to pathogenic variants in genes associated with adipogenesis, DNA repair pathways, and genome stability. Subjects with lipodystrophy exhibit an impairment in the homeostasis of subcutaneous white adipose tissue (sWAT), resulting in low leptin and adiponectin levels, insulin resistance (IR), diabetes, dyslipidemia, ectopic fat deposition, inflammation, mitochondrial and endoplasmic reticulum commitments, among others. However, how pathogenic variants in adipogenesis-related genes modulate DNA repair in some classical congenital lipodystrophies has not been elucidated. In the same way, no data is clarifying how pathogenic variants in DNA repair genes result in sWAT loss in different types of progeroid lipodystrophies. This review will concentrate on the main molecular findings to understand the link between DNA damage/repair and adipogenesis in human and animal models of congenital lipodystrophies. We will focus on classical and progeroid congenital lipodystrophies directly or indirectly related to DNA repair pathways, highlighting the role of DNA repair-related proteins in maintaining sWAT homeostasis.

Novel Ubiquitinated Proteins Downstream of the Fanconi Anemia Core Complex

The Fanconi anemia (FA) pathway is a major player in the control of DNA replication integrity in response to replication stress. Germline defect in the pathway results in the FA syndrome characterized by developmental abnormalities, bone marrow (BM) failure, and genome instability which greatly elevates the incidence of cancers. A pivotal step in the activation of the FA DNA repair pathway is the monoubiquitination of the FANCD2 and FANCI proteins (ID2) by the FA core complex, a unique ubiquitin ligase complex which includes eight proteins (FANCA-FANCG, FANCL, and FAAP100) and UBE2T/FANCT. This monoubiquitination event enables the recruitment of the ID2 complex to chromatin and nuclear foci at sites of DNA damage. Cells with mutations in any of the FA core complex proteins lack the ability to monoubiquitinated ID2, making ID2 ubiquitination a convergence point in the pathway, with an estimation of>90% FA patients defective in this step. Additionally, somatic mutations In FA genes render tumor cells sensitive to DNA crosslinking agents, so identification of FA pathway defects provides an opportunity for therapeutic targeting. In search for additional potential target/substrate of this unique FA core ubiquitin ligase complex, we performed a high throughput genome-wide ubiquitin-specific proteomics (UbiScan) screen and found, in addition to the ID2 complex, many ubiquitinated proteins are dysregulated (mostly downregulated) in FA deficient cells compared with that of FA proficient cells.We used a Ubiquitin Remnant Motif (K- ∑-GG) Antibody Bead Conjugate (Cell Signaling Technology), a proprietary ubiquitin branch (“K- ∑-GG”) antibody with specificity for a di-glycine tag that is the remnant of ubiquitin left on protein substrates after trypsin digestion, to enrich ubiquitinated peptides from trypsin digested cell samples (shNT vs shFANCA). This enrichment is followed by LC-MS/MS analysis for quantitative profiles of hundreds to over a thousand nonredundant ubiquitinated sequences. We were successful in demonstrating that under steady-state conditions (without proteasome inhibitor treatment), the ubiquitinated forms of both FANCD2 and FANCI proteins are much higher in control (shNT) HeLa cells compared with that of the cells depleted of FANCA (shFANCA).We then collaborated with the Cell signaling technology to perform a high throughput UbiScan® analysis of total ubiquitinated proteins both in total nuclei and chromatin fractions under replicative stress conditions. UbiScan® enables researchers to isolate, identify and quantitate large numbers of ubiquitin-modified cellular peptides with a high degree of specificity and sensitivity, providing a global overview of the ubiquitination sites in cellular proteins in cell and tissue samples without preconceived biases about where these modified sites occur. A total of 16,249 redundant modified peptide assignments to 7,856 modified sites for the Ubiquitin K-GG Remnant Motif Antibody were obtained. As expected, the amount of monoubiquitinated FANCD2 (at K651) and FANCI (at K523) were highly reduced in both the nuclear and chromatin fractions of Hela cells depleted of FANCA (shA). Consistent with the earlier findings, the amount of ubiquitinated ID2 proteins were extremely low in the chromatin fraction of the Hela cells depleted of FANCA.Since there are numerous ubiquitinated proteins found to be dysregulated in our UbiScan analyses, we used the following criteria to select the target proteins based on; a) -fold changes, and b) proteins that are known to participate in the DNA repair signaling pathways. We validated our UbiScan results by using an assay system to detect endogenous protein ubiquitination. We also found a significant reduction in the ubiquitination of several DNA repair-related proteins (found in our UbiScan analysis) in FANCA deficient cells. To assess FA pathway functions, we generated HAP1 and appropriate cells knock out of these select ubiquitinated target proteins by using CRISPR-Cas9 system. Then, the KO cells were examined for FA pathway functions. These results will be discussed.In conclusion, our findings reveal that the FA core ubiquitin ligase complex regulates (directly or indirectly) the ubiquitinated levels of many novel proteins outside of the ID2 complex, and these novel target proteins may provide important additional mechanistic insights into the FA DNA repair pathway. DisclosuresNo relevant conflicts of interest to declare.