RESUMO
Oxidative stress is crucial in ulcerative colitis (UC) and colitis-associated colorectal cancer (CAC). Intestinal epithelial cells (IECs) are an important component of the intestinal barrier. In previous studies, we have demonstrated that suppressing microRNA-222-3p (miR-222-3p) can protect against oxidative stress in IECs, which ameliorates colonic injuries in UC mice and prevents the conversion of UC to CAC. In this case, we hope to explore whether moxibustion can alleviate UC and CAC by inhibiting miR-222-3p based on mouse models of UC and CAC. After herb-partitioned moxibustion (HPM) intervention, the disease activity index (DAI) and colon macroscopic damage index (CMDI) were significantly reduced in UC mice, and the number and volume of intestinal tumors were decreased considerably in CAC mice. Meanwhile, we found that HPM suppressed miR-222-3p expression and upregulated the mRNA and protein expression of Brahma-related gene 1 (BRG1), nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), while inhibiting Kelch-like ECH-associated protein 1 (Keap1) expression in IECs of UC and CAC mice. With changes in reactive oxygen species (ROS), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor (TNF)-α), we verified that HPM protects against oxidative stress and inflammation in IECs of UC and CAC mice. The effect of HPM was inhibited in miR-222-3p overexpression mice, further demonstrating that the protective effect of HPM on UC and CAC mice was through inhibiting miR-222-3p. In summary, HPM regulates the BRG1/Nrf2/HO-1 pathway by inhibiting miR-222-3p to attenuate oxidative stress in IECs in UC and CAC.
Assuntos
Colite Ulcerativa , Modelos Animais de Doenças , Heme Oxigenase-1 , MicroRNAs , Moxibustão , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Fatores de Transcrição , Animais , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Colite Ulcerativa/terapia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/genética , Camundongos , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , DNA Helicases/metabolismo , DNA Helicases/genética , Neoplasias Associadas a Colite/etiologia , Neoplasias Associadas a Colite/patologia , Neoplasias Associadas a Colite/metabolismo , Neoplasias Associadas a Colite/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , HumanosRESUMO
The Regulator of Telomere Helicase 1 (RTEL1) gene encodes a critical DNA helicase intricately involved in the maintenance of telomeric structures and the preservation of genomic stability. Germline mutations in the RTEL1 gene have been clinically associated with Hoyeraal-Hreidarsson syndrome, a more severe version of Dyskeratosis Congenita. Although various research has sought to link RTEL1 mutations to specific disorders, no comprehensive investigation has yet been conducted on missense mutations. In this study, we attempted to investigate the functionally and structurally deleterious coding and non-coding SNPs of the RTEL1 gene using an in silico approach. Initially, out of 1392 nsSNPs, 43 nsSNPs were filtered out through ten web-based bioinformatics tools. With subsequent analysis using nine in silico tools, these 43 nsSNPs were further shortened to 11 most deleterious nsSNPs. Furthermore, analyses of mutated protein structures, evolutionary conservancy, surface accessibility, domains & PTM sites, cancer susceptibility, and interatomic interaction revealed the detrimental effect of these 11 nsSNPs on RTEL1 protein. An in-depth investigation through molecular docking with the DNA binding sequence demonstrated a striking change in the interaction pattern for F15L, M25V, and G706R mutant proteins, suggesting the more severe consequences of these mutations on protein structure and functionality. Among the non-coding variants, two had the highest likelihood of being regulatory variants, whereas one variant was predicted to affect the target region of a miRNA. Thus, this study lays the groundwork for extensive analysis of RTEL1 gene variants in the future, along with the advancement of precision medicine and other treatment modalities.
Assuntos
Simulação por Computador , DNA Helicases , Polimorfismo de Nucleotídeo Único , Humanos , DNA Helicases/genética , DNA Helicases/química , Simulação de Acoplamento Molecular , Mutação de Sentido Incorreto , Biologia Computacional/métodos , Disceratose Congênita/genética , Predisposição Genética para DoençaRESUMO
DdmDE is a novel plasmid defense system that was discovered in the seventh pandemic Vibrio cholerae strain of the biotype O1 EI Tor. In this issue of Cell, Yang and coworkers reveal the mechanisms underlying the assembly and activation of the DdmDE defense system.
Assuntos
Plasmídeos , Vibrio cholerae , Plasmídeos/metabolismo , Plasmídeos/genética , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio cholerae/enzimologia , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , DNA Helicases/metabolismo , DNA Helicases/genéticaRESUMO
Mammalian DNA replication relies on various DNA helicase and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN-FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks.
Assuntos
DNA Helicases , Replicação do DNA , RecQ Helicases , Helicase da Síndrome de Werner , Humanos , Cromatina/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , Células HEK293 , Células HeLa , RecQ Helicases/metabolismo , RecQ Helicases/genética , Telômero/metabolismo , Telômero/genética , Homeostase do Telômero/efeitos dos fármacos , Proteases Específicas de Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/genética , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/genéticaRESUMO
In this issue of Molecular Cell, Engeholm et al.1 present cryo-EM structures of the chromatin remodeler Chd1 bound to a hexasome-nucleosome complex, an intermediate state during transcription either with or without FACT to restore the missing H2A-H2B dimer. These two binding modes reveal how Chd1 and FACT cooperate in nucleosome re-establishment during transcription.
Assuntos
Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Nucleossomos , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Montagem e Desmontagem da Cromatina , Histonas/metabolismo , Histonas/genética , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Ligação Proteica , Transcrição Gênica , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/químicaRESUMO
BACKGROUND: Müllerian duct anomalies (MDAs) are congenital developmental disorders that present as a series of abnormalities within the reproductive tracts of females. Genetic factors are linked to MDAs and recent advancements in whole-exome sequencing (WES) provide innovative perspectives in this field. However, relevant mechanism has only been investigated in a restricted manner without clear elucidation of respective observations. METHODS: Our previous study reported that 2 of 12 patients with MDAs harbored the CHD1L variant c.348-1G>C. Subsequently, an additional 85 MDAs patients were recruited. Variants in CHD1L were screened through the in-house database of WES performed in the cohort and two cases were identified. One presented with partial septate uterus with left renal agenesis and the other with complete septate uterus, duplicated cervices and longitudinal vaginal septum. The pathogenicity of the discovered variants was further assessed by molecular dynamics simulation and various functional assays. RESULTS: Ultimately, two novel heterozygous CHD1L variants, including a missense variant c.956G>A (p.R319Q) and a nonsense variant c.1831C>T (p.R611*) were observed. The variants were absent in 100 controls. Altogether, the contribution yield of CHD1L to MDAs was calculated as 4.12% (4/97). All three variants were assessed as pathogenic through various functional analysis. The splice-site variant c.348-1G>C resulted in a 11 bp sequence skipping in exon 4 of CHD1L and led to nonsense mediated decay of its transcripts. Unlike WT CHD1L, the truncated R611* protein mislocalized to the cytoplasm, abolish the ability of CHD1L to promote cell migration and failed to interact with PARP1 owing to the loss of macro domain. The R319Q variant exhibited conformational disparities and showed abnormal protein recruitment behavior through laser microirradiation comparing with the WT CHD1L. All these variants impaired the CHD1L function in DNA damage repair, thus participating in MDAs. CONCLUSIONS: The current study not only expands the mutational spectrum of CHD1L in MDAs but determines three variants as pathogenic according to ACMG guidelines with reliable functional evidence. Additionally, the impairment in DNA damage repair is an underlying mechanism involved in MDAs.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Ductos Paramesonéfricos , Feminino , Humanos , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Sequenciamento do Exoma , Ductos Paramesonéfricos/anormalidades , Mutação , Mutação de Sentido IncorretoRESUMO
DNA double strand breaks (DSBs) are critical for the efficacy of radiotherapy as they lead to cell death if not repaired. DSBs caused by ionizing radiation (IR) initiate histone modifications and accumulate DNA repair proteins, including 53BP1, which forms distinct foci at damage sites and serves as a marker for DSBs. DSB repair primarily occurs through Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR). NHEJ directly ligates DNA ends, employing proteins such as DNA-PKcs, while HR, involving proteins such as Rad54, uses a sister chromatid template for accurate repair and functions in the S and G2 phases of the cell cycle. Both pathways are crucial, as illustrated by the IR sensitivity in cells lacking DNA-PKcs or Rad54. We generated mouse embryonic stem (mES) cells which are knockout (KO) for DNA-PKcs and Rad54 to explore the combined role of HR and NHEJ in DSB repair. We found that cells lacking both DNA-PKcs and Rad54 are hypersensitive to X-ray radiation, coinciding with impaired 53BP1 focus resolution and a more persistent G2 phase cell cycle block. Additionally, mES cells deficient in DNA-PKcs or both DNA-PKcs and Rad54 exhibit an increased nuclear size approximately 18-24 h post-irradiation. To further explore the role of Rad54 in the absence of DNA-PKcs, we generated DNA-PKcs KO mES cells expressing GFP-tagged wild-type (WT) or ATPase-defective Rad54 to track the Rad54 foci over time post-irradiation. Cells lacking DNA-PKcs and expressing ATPase-defective Rad54 exhibited a similar phenotypic response to IR as those lacking both DNA-PKcs and Rad54. Despite a strong G2 phase arrest, live-cell imaging showed these cells eventually progress through mitosis, forming micronuclei. Additionally, mES cells lacking DNA-PKcs showed increased Rad54 foci over time post-irradiation, indicating an enhanced reliance on HR for DSB repair without DNA-PKcs. Our findings underscore the essential roles of HR and NHEJ in maintaining genomic stability post-IR in mES cells. The interplay between these pathways is crucial for effective DSB repair and cell cycle progression, highlighting potential targets for enhancing radiotherapy outcomes.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Recombinação Homóloga , Células-Tronco Embrionárias Murinas , Radiação Ionizante , Animais , Camundongos , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Embrionárias Murinas/efeitos da radiação , Células-Tronco Embrionárias Murinas/citologia , Recombinação Homóloga/efeitos da radiação , Proteína Quinase Ativada por DNA/metabolismo , Proteína Quinase Ativada por DNA/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteínas NuclearesRESUMO
Despite their prevalent cancer implications, the in vivo dynamics of SWI/SNF chromatin remodelers and how misregulation of such dynamics underpins cancer remain poorly understood. Using live-cell single-molecule tracking, we quantify the intranuclear diffusion and chromatin-binding of three key subunits common to all major human SWI/SNF remodeler complexes (BAF57, BAF155 and BRG1), and resolve two temporally distinct stable binding modes for the fully assembled complex. Super-resolved density mapping reveals heterogeneous, nanoscale remodeler binding "hotspots" across the nucleoplasm where multiple binding events (especially longer-lived ones) preferentially cluster. Importantly, we uncover distinct roles of the bromodomain in modulating chromatin binding/targeting in a DNA-accessibility-dependent manner, pointing to a model where successive longer-lived binding within "hotspots" leads to sustained productive remodeling. Finally, systematic comparison of six common BRG1 mutants implicated in various cancers unveils alterations in chromatin-binding dynamics unique to each mutant, shedding insight into a multi-modal landscape regulating the spatio-temporal organizational dynamics of SWI/SNF remodelers.
Assuntos
Montagem e Desmontagem da Cromatina , Cromatina , Proteínas Cromossômicas não Histona , DNA Helicases , Neoplasias , Proteínas Nucleares , Imagem Individual de Molécula , Fatores de Transcrição , Humanos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Imagem Individual de Molécula/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromatina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/patologia , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ligação Proteica , Mutação , Linhagem Celular Tumoral , Domínios Proteicos , Adenosina TrifosfatasesRESUMO
BACKGROUND: Bivalent regions of chromatin (BvCR) are characterized by trimethylated lysine 4 (H3K4me3) and lysine 27 on histone H3 (H3K27me3) deposition which aid gene expression control during cell differentiation. The role of BvCR in post-transcriptional DNA damage response remains unidentified. Oncoprotein survivin binds chromatin and mediates IFNγ effects in CD4+ cells. In this study, we explored the role of BvCR in DNA damage response of autoimmune CD4+ cells in rheumatoid arthritis (RA). METHODS: We performed deep sequencing of the chromatin bound to survivin, H3K4me3, H3K27me3, and H3K27ac, in human CD4+ cells and identified BvCR, which possessed all three histone H3 modifications. Protein partners of survivin on chromatin were predicted by integration of motif enrichment analysis, computational machine-learning, and structural modeling, and validated experimentally by mass spectrometry and peptide binding array. Survivin-dependent change in BvCR and transcription of genes controlled by the BvCR was studied in CD4+ cells treated with survivin inhibitor, which revealed survivin-dependent biological processes. Finally, the survivin-dependent processes were mapped to the transcriptome of CD4+ cells in blood and in synovial tissue of RA patients and the effect of modern immunomodulating drugs on these processes was explored. RESULTS: We identified that BvCR dominated by H3K4me3 (H3K4me3-BvCR) accommodated survivin within cis-regulatory elements of the genes controlling DNA damage. Inhibition of survivin or JAK-STAT signaling enhanced H3K4me3-BvCR dominance, which improved DNA damage recognition and arrested cell cycle progression in cultured CD4+ cells. Specifically, BvCR accommodating survivin aided sequence-specific anchoring of the BRG1/SWI chromatin-remodeling complex coordinating DNA damage response. Mapping survivin interactome to BRG1/SWI complex demonstrated interaction of survivin with the subunits anchoring the complex to chromatin. Co-expression of BRG1, survivin and IFNγ in CD4+ cells rendered complete deregulation of DNA damage response in RA. Such cells possessed strong ability of homing to RA joints. Immunomodulating drugs inhibited the anchoring subunits of BRG1/SWI complex, which affected arthritogenic profile of CD4+ cells. CONCLUSIONS: BvCR execute DNA damage control to maintain genome fidelity in IFN-activated CD4+ cells. Survivin anchors the BRG1/SWI complex to BvCR to repress DNA damage response. These results offer a platform for therapeutic interventions targeting survivin and BRG1/SWI complex in autoimmunity.
Assuntos
Linfócitos T CD4-Positivos , Cromatina , Dano ao DNA , DNA Helicases , Proteínas Nucleares , Survivina , Fatores de Transcrição , Humanos , Survivina/metabolismo , Survivina/genética , Linfócitos T CD4-Positivos/metabolismo , Cromatina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Histonas/metabolismo , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genéticaRESUMO
OBJECTIVE: To explore the clinical phenotype and genetic etiology for seven children with CHARGE syndrome (CS). METHODS: Clinical data of seven children with CS diagnosed between March 2020 and December 2022 at the Children's Hospital Affiliated to Zhengzhou University were analyzed. Genomic DNA was extracted from peripheral blood samples from the children and their parents, and subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. RESULTS: The ages of the children had ranged from 1 day after birth to 12 years old, and all of them had shown growth retardation. The reasons for their admission had included postnatal breathing, swallowing and feeding difficulties in five cases. One child was found to have abnormal external genitalia in conjunct with hearing impairment, whilst another child had shown no secondary sexual characteristics during puberty. All of the children were found to harbor CHD7 gene variants, which included 3 nonsense variants, 2 frameshifting variants and 2 missense variants, i.e., c.6292C>T (p.R2098*), c.2754G>A (p.W918*), c.469C>T (p.R157*), c.3308T>A (p.V1103D), c.7111delC (p.Q2371Kfs), c.6023delA (p.D2008Vfs) and c.3565C>T (p.R1189C). All of the variants were de novo in origin. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.3308T>A (p.V1103D) and c.3565C>T (p.R1189C) variants were rated as likely pathogenic (PS2+PM2_Supporting+PP3), whilst the remainders were rated as pathogenic (PVS1+PS2+PM2_Supporting). CONCLUSION: There is strong clinical and genetic heterogeneity in CS. Early genetic testing may facilitate accurate diagnosis. The detection of novel variants has expanded the phenotypic spectrum of CS and the mutational spectrum of the CHD7 gene.
Assuntos
Síndrome CHARGE , DNA Helicases , Proteínas de Ligação a DNA , Sequenciamento do Exoma , Fenótipo , Humanos , Síndrome CHARGE/genética , Criança , Masculino , Pré-Escolar , Feminino , Lactente , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Recém-Nascido , MutaçãoRESUMO
Telomeres protect chromosome ends from damage and their length is linked with human disease and aging. We developed a joint telomere length metric, combining quantitative PCR and whole-genome sequencing measurements from 462,666 UK Biobank participants. This metric increased SNP heritability, suggesting that it better captures genetic regulation of telomere length. Exome-wide rare-variant and gene-level collapsing association studies identified 64 variants and 30 genes significantly associated with telomere length, including allelic series in ACD and RTEL1. Notably, 16% of these genes are known drivers of clonal hematopoiesis-an age-related somatic mosaicism associated with myeloid cancers and several nonmalignant diseases. Somatic variant analyses revealed gene-specific associations with telomere length, including lengthened telomeres in individuals with large SRSF2-mutant clones, compared with shortened telomeres in individuals with clonal expansions driven by other genes. Collectively, our findings demonstrate the impact of rare variants on telomere length, with larger effects observed among genes also associated with clonal hematopoiesis.
Assuntos
Bancos de Espécimes Biológicos , Polimorfismo de Nucleotídeo Único , Telômero , Sequenciamento Completo do Genoma , Humanos , Telômero/genética , Reino Unido , Sequenciamento Completo do Genoma/métodos , Homeostase do Telômero/genética , Masculino , Feminino , Hematopoiese Clonal/genética , Estudo de Associação Genômica Ampla/métodos , Idoso , DNA Helicases/genética , Pessoa de Meia-Idade , Biobanco do Reino UnidoRESUMO
Strategies beyond hormone-related therapy need to be developed to improve prostate cancer mortality. Here, we show that FUBP1 and its methylation were essential for prostate cancer progression, and a competitive peptide interfering with FUBP1 methylation suppressed the development of prostate cancer. FUBP1 accelerated prostate cancer development in various preclinical models. PRMT5-mediated FUBP1 methylation, regulated by BRD4, was crucial for its oncogenic effect and correlated with earlier biochemical recurrence in our patient cohort. Suppressed prostate cancer progression was observed in various genetic mouse models expressing the FUBP1 mutant deficient in PRMT5-mediated methylation. A competitive peptide, which was delivered through nanocomplexes, disrupted the interaction of FUBP1 with PRMT5, blocked FUBP1 methylation, and inhibited prostate cancer development in various preclinical models. Overall, our findings suggest that targeting FUBP1 methylation provides a potential therapeutic strategy for prostate cancer management.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Neoplasias da Próstata , Proteína-Arginina N-Metiltransferases , Proteínas de Ligação a RNA , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias da Próstata/metabolismo , Humanos , Animais , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Metilação , DNA Helicases/genética , DNA Helicases/metabolismo , Progressão da Doença , Linhagem Celular Tumoral , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoAssuntos
Biomarcadores Tumorais , Carcinoma de Células Pequenas , DNA Helicases , Hipercalcemia , Proteínas Nucleares , Neoplasias Ovarianas , Fatores de Transcrição , Humanos , Feminino , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análiseAssuntos
Biomarcadores Tumorais , Carcinoma de Células Pequenas , DNA Helicases , Hipercalcemia , Proteínas Nucleares , Neoplasias Ovarianas , Fatores de Transcrição , Humanos , Feminino , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/genética , DNA Helicases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Carcinoma de Células Pequenas/patologia , Carcinoma de Células Pequenas/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/análise , Imuno-HistoquímicaRESUMO
DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
Assuntos
Microscopia Crioeletrônica , DNA Helicases , DNA Polimerase teta , DNA Polimerase Dirigida por DNA , Humanos , DNA Helicases/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/antagonistas & inibidores , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/química , Piperazinas/farmacologia , Piperazinas/química , Linhagem Celular Tumoral , Ftalazinas/farmacologia , Ftalazinas/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Ligação ProteicaRESUMO
Chromodomain helicase DNA-binding protein (CHD) gene family, an ATP (adenosine triphosphate) -dependent chromatin remodeler family, is involved in multiple developmental process and tumor development. However, there have been none pan-cancer analyses of this family. The expression levels, survival profiles, mutation profiles and immune infiltration of the CHD family genes from TCGA and TARGET database were analyzed using online tools or R packages. Interestingly, all types of CHD gene expressions were associated with the prognosis of Neuroblastoma, Acute lymphoblastic leukemia-Phase 3 and Acute Myeloid Leukemia (All P < 0.05). Knock down of CHD7 and CHD9 in K562 (human erythromyeloblastoid leukemia) and HEC-1-B (human endometrial adenocarcinoma) cells significantly inhibit cell proliferation and migration (P < 0.05). Proliferation, colony formation and migration assays were performed in CHD7 and CHD9 knockdown K562 and HBC-1-B cell lines. Mechanisms were also analyzed by PPI and GO ontology for our experiments. Histone modification, especially the methylation of H3K4, might be involved in CHD7 and CHD9 related oncogenesis. Through bioinformatic analysis, we showed CHD genes significantly affected the prognosis of different tumor types, including childhood tumor. Our findings provide new insights into the function and mechanism of CHD gene family, especially in CHD7 and CHD9.
Assuntos
Biologia Computacional , DNA Helicases , Proteínas de Ligação a DNA , Neoplasias , Humanos , Biologia Computacional/métodos , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Movimento Celular/genética , Prognóstico , Linhagem Celular Tumoral , MutaçãoRESUMO
DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation.
Assuntos
Tolerância ao Dano no DNA , Animais , Humanos , DNA/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Reparo do DNA , Instabilidade GenômicaRESUMO
Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT , Metilação de DNA , Nucleossomos , Ubiquitina-Proteína Ligases , Humanos , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Nucleossomos/metabolismo , Nucleossomos/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/genética , Microscopia Crioeletrônica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Ilhas de CpG , Ubiquitinação , Evolução Molecular , DNA/metabolismo , DNA/química , DNA/genética , Dedos de Zinco , Cromatina/metabolismo , Cromatina/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/química , Eucariotos/genética , Eucariotos/metabolismo , Ligação Proteica , Histonas/metabolismo , Histonas/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/químicaRESUMO
Liquid-liquid phase separation (LLPS) mediated by G3BP1/2 proteins and non-translating mRNAs mediates stress granule (SG) assembly. We investigated the phylogenetic evolution of G3BP orthologs from unicellular yeast to mammals and identified both conserved and divergent features. The modular domain organization of G3BP orthologs is generally conserved. However, invertebrate orthologs displayed reduced capacity for SG assembly in human cells compared to vertebrate orthologs. We demonstrated that the protein-interaction network facilitated by the NTF2L domain is a crucial determinant of this specificity. The evolution of the G3BP1 network coincided with its exploitation by certain viruses, as evident from the interaction between viral proteins and G3BP orthologs in insects and vertebrates. We revealed the importance and divergence of the G3BP interaction network in human SG formation. Leveraging this network, we established a 7-component in vitro SG reconstitution system for quantitative studies. These findings highlight the significance of G3BP network divergence in the evolution of biological processes.
Assuntos
DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , Mapas de Interação de Proteínas , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Humanos , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Grânulos de Estresse/metabolismo , Animais , DNA Helicases/metabolismo , DNA Helicases/genética , Filogenia , Células HeLa , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas de Ligação a RNA , Proteínas Adaptadoras de Transdução de SinalRESUMO
Cutaneous squamous cell carcinoma (cSCC) comprises 20% of cases of nonmelanoma skin cancers in the United States. In total, 3% to 5% of squamous cell carcinoma (SCC) are metastatic at the time of presentation, associated with significant mortality due to a lack of standardized treatment options. In total, 95% of these tumors are amenable to the initial standard of treatment, which is surgical resection. However, a small percentage of them require systemic therapy as they are either locally advanced to regional lymph nodes or have distant metastasis. The common sites of presentation of cSCC are the scalp and the face with predictable spread to the intra-parotid, upper jugular, and perifacial lymph nodes. In our case report, however, our patient had a large lump lesion on the upper back, an unusual site of presentation of cSCC, with locally advanced metastasis to the left axillary lymph nodes. Subsequently, the tumor marker study revealed a positive SMARCA4 variant (the essential ATPase subunit of the Switch (SWI)/Sucrose Nonfermenting (SNF) chromatin-remodeling complex) that is even rarer in the context of cSCC. Furthermore, abnormalities in SWI/SNF chromatin-remodeling complex subunits have shown promising results as a target therapy for immune checkpoint inhibitor (ICI) therapy. We present an atypical presentation site of locally advanced rare variant SMARCA4-positive cSCC in a patient who received treatment with chemoradiation and systemic therapy with ICI after primary surgical resection. To date, only 2 cases of SMARCA4-positive cSCC were found in the literature with no details of the treatment received. Our case is unique in its atypical site of presentation as well as showing partial response to radiotherapy (RT) and systemic therapy with ICI.