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1.
Cell Rep ; 43(4): 114064, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38578830

RESUMO

Assembly of TopBP1 biomolecular condensates triggers activation of the ataxia telangiectasia-mutated and Rad3-related (ATR)/Chk1 signaling pathway, which coordinates cell responses to impaired DNA replication. Here, we used optogenetics and reverse genetics to investigate the role of sequence-specific motifs in the formation and functions of TopBP1 condensates. We propose that BACH1/FANCJ is involved in the partitioning of BRCA1 within TopBP1 compartments. We show that Chk1 is activated at the interface of TopBP1 condensates and provide evidence that these structures arise at sites of DNA damage and in primary human fibroblasts. Chk1 phosphorylation depends on the integrity of a conserved arginine motif within TopBP1's ATR activation domain (AAD). Its mutation uncouples Chk1 activation from TopBP1 condensation, revealing that optogenetically induced Chk1 phosphorylation triggers cell cycle checkpoints and slows down replication forks in the absence of DNA damage. Together with previous work, these data suggest that the intrinsically disordered AAD encodes distinct molecular steps in the ATR/Chk1 pathway.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica , Quinase 1 do Ponto de Checagem , Proteínas de Ligação a DNA , Humanos , Quinase 1 do Ponto de Checagem/metabolismo , Fosforilação , Proteínas de Ligação a DNA/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Proteínas de Transporte/metabolismo , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Proteína BRCA1/metabolismo , Transdução de Sinais , Proteínas Nucleares/metabolismo , Fibroblastos/metabolismo , Pontos de Checagem do Ciclo Celular
2.
CNS Neurosci Ther ; 30(4): e14696, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38668740

RESUMO

AIMS: Excessive neuroinflammation mediated mainly by microglia plays a crucial role in ischemic stroke. AZD1390, an ataxia telangiectasia mutated (ATM) specific inhibitor, has been shown to promote radio-sensitization and survival in central nervous system malignancies, while the role of AZD1390 in ischemic stroke remains unknown. METHODS: Real-time PCR, western blot, immunofluorescence staining, flow cytometry and enzyme-linked immunosorbent assays were used to assess the activation of microglia and the release of inflammatory cytokines. Behavioral tests were performed to measure neurological deficits. 2,3,5-Triphenyltetrazolium chloride staining was conducted to assess the infarct volume. The activation of NF-κB signaling pathway was explored through immunofluorescence staining, western blot, co-immunoprecipitation and proximity ligation assay. RESULTS: The level of pro-inflammation cytokines and activation of NF-κB signaling pathway was suppressed by AZD1390 in vitro and in vivo. The behavior deficits and infarct size were partially restored with AZD1390 treatment in experimental stroke. AZD1390 restrict ubiquitylation and sumoylation of the essential regulatory subunit of NF-κB (NEMO) in an ATM-dependent and ATM-independent way respectively, which reduced the activation of the NF-κB pathway. CONCLUSION: AZD1390 suppressed NF-κB signaling pathway to alleviate ischemic brain injury in experimental stroke, and attenuated microglia activation and neuroinflammation, which indicated that AZD1390 might be an attractive agent for the treatment of ischemic stroke.


Assuntos
Microglia , Doenças Neuroinflamatórias , Piridinas , Quinolonas , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Citocinas/metabolismo , Transdução de Sinais/efeitos dos fármacos
3.
Cell Syst ; 15(4): 339-361.e8, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38593799

RESUMO

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-ß expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.


Assuntos
Ataxia Telangiectasia , Infecções por Herpesviridae , Humanos , Fosforilação , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Citocinas/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA
4.
JCO Precis Oncol ; 8: e2300635, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38635934

RESUMO

PURPOSE: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS: We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS: At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION: The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.


Assuntos
Gencitabina , Isoxazóis , Neoplasias Ovarianas , Pirazinas , Humanos , Feminino , Desoxicitidina/uso terapêutico , Carcinoma Epitelial do Ovário/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Proteínas Mutadas de Ataxia Telangiectasia/genética
5.
Cell Syst ; 15(4): 305-306, 2024 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-38636456

RESUMO

The cellular DNA damage response pathway can have vastly different outcomes depending on the source of its activation. Justice and colleagues apply phosphoproteomics to uncover a divergence in DNA-PK and ATM kinase activities in the contexts of DNA damage and DNA virus infection.


Assuntos
Infecções por Vírus de DNA , Transdução de Sinais , Humanos , Transdução de Sinais/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética
7.
World J Gastroenterol ; 30(10): 1377-1392, 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38596500

RESUMO

BACKGROUND: Crohn's disease (CD) is often misdiagnosed as intestinal tuberculosis (ITB). However, the treatment and prognosis of these two diseases are dramatically different. Therefore, it is important to develop a method to identify CD and ITB with high accuracy, specificity, and speed. AIM: To develop a method to identify CD and ITB with high accuracy, specificity, and speed. METHODS: A total of 72 paraffin wax-embedded tissue sections were pathologically and clinically diagnosed as CD or ITB. Paraffin wax-embedded tissue sections were attached to a metal coating and measured using attenuated total reflectance fourier transform infrared spectroscopy at mid-infrared wavelengths combined with XGBoost for differential diagnosis. RESULTS: The results showed that the paraffin wax-embedded specimens of CD and ITB were significantly different in their spectral signals at 1074 cm-1 and 1234 cm-1 bands, and the differential diagnosis model based on spectral characteristics combined with machine learning showed accuracy, specificity, and sensitivity of 91.84%, 92.59%, and 90.90%, respectively, for the differential diagnosis of CD and ITB. CONCLUSION: Information on the mid-infrared region can reveal the different histological components of CD and ITB at the molecular level, and spectral analysis combined with machine learning to establish a diagnostic model is expected to become a new method for the differential diagnosis of CD and ITB.


Assuntos
Doença de Crohn , Enterite , Tuberculose Gastrointestinal , Humanos , Doença de Crohn/diagnóstico , Doença de Crohn/patologia , Espectroscopia de Infravermelho com Transformada de Fourier , Diagnóstico Diferencial , Parafina , Tuberculose Gastrointestinal/diagnóstico , Tuberculose Gastrointestinal/patologia , Enterite/diagnóstico , Aprendizado de Máquina , Proteínas Mutadas de Ataxia Telangiectasia
8.
Stem Cell Res Ther ; 15(1): 97, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38581065

RESUMO

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.


Assuntos
Células-Tronco Mesenquimais , Insuficiência Ovariana Primária , Animais , Feminino , Humanos , Camundongos , Antioxidantes/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Cofator PQQ/farmacologia , Insuficiência Ovariana Primária/patologia , RNA Interferente Pequeno/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Cytokine ; 178: 156592, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38574505

RESUMO

The severity of COVID-19 has been reported to differ among SARS-CoV-2 mutant variants. The overactivation of macrophages is involved in severe COVID-19, yet the effects of SARS-CoV-2 mutations on macrophages remain poorly understood. To clarify the effects, we examined whether mutations of spike proteins (S-proteins) affect macrophage activation. CD14+ monocyte-derived macrophages were stimulated with the recombinant S-protein of the wild-type, Delta, and Omicron strains or live viral particles of individual strains. Regarding IL-6 and TNF-α, Delta or Omicron S-protein had stronger or weaker pro­inflammatory ability, respectively, than the wild-type. Similar trends were observed between S-proteins and viral particles. S-protein mutations could be related to the diversity in macrophage activation and severity rates in COVID-19 caused by various SARS-CoV-2 strains.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Glicoproteína da Espícula de Coronavírus/genética , Proteínas Mutadas de Ataxia Telangiectasia
10.
J Exp Clin Cancer Res ; 43(1): 77, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38475864

RESUMO

BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.


Assuntos
Glioma , Peixe-Zebra , Camundongos , Animais , Linhagem Celular Tumoral , Reparo do DNA , Dano ao DNA , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
11.
J Cancer Res Ther ; 20(1): 126-132, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-38554309

RESUMO

BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.


Assuntos
Proteínas de Ciclo Celular , Radiação Ionizante , Animais , Humanos , Células HeLa , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Reparo do DNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fosforilação , Mamíferos/genética , Mamíferos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
EMBO J ; 43(7): 1301-1324, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38467834

RESUMO

Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.


Assuntos
Telomerase , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Telomerase/genética , Ubiquitinação , Replicação do DNA , Telômero/genética , Telômero/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA
13.
Radiother Oncol ; 194: 110198, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38438016

RESUMO

BACKGROUND AND PURPOSE: Ionizing radiation (IR) induces DNA double-strand breaks (DSBs), leading to micronuclei formation, which has emerged as a key mediator of inflammatory responses after IR. This study aimed to investigate the signaling cascade in inflammatory gene expression using fibroblasts harboring DNA damage response deficiency after exposure to IR. MATERIALS AND METHODS: Micronuclei formation was examined in human dermal fibroblasts derived from patients with deficiencies in ATM, ATR, MRE11, XLF, Artemis, or BRCA2 after IR. RNA-sequencing analysis was performed to assess gene expression, pathway mapping, and the balance of transcriptional activity using the transcription factor-based downstream gene expression mapping (TDEM) method developed in this study. RESULTS: Deficiencies in ATM, ATR, or MRE11 led to increased micronuclei formation after IR compared to normal cells. RNA-seq analysis revealed significant upregulation of inflammatory expression in cells deficient in ATM, ATR, or MRE11 following IR. Pathway mapping analysis identified the upregulation of RIG-I, MDA-5, IRF7, IL6, and interferon stimulated gene expression after IR. These changes were pronounced in cells deficient in ATM, ATR, or MRE11. TDEM analysis suggested the differential activation of STAT1/3-pathway between ATM and ATR deficiency. CONCLUSION: Enhanced micronuclei formation upon ATM, ATR, or MRE11 deficiency activated the cGAS/STING, RIG-I-MDA-5-IRF7-IL6 pathway, resulting in its downstream interferon stimulated gene expression following exposure to IR. Our study provides comprehensive information regarding the status of inflammation-related gene expression under DSB repair deficiency after IR. The generated dataset may be useful in developing functional biomarkers to accurately identify patients sensitive to radiotherapy.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Fibroblastos , Radiação Ionizante , Transdução de Sinais , Humanos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína Homóloga a MRE11/genética , Inflamação/etiologia , Quebras de DNA de Cadeia Dupla
14.
Cell Rep ; 43(3): 113896, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38442018

RESUMO

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.


Assuntos
Ataxia Telangiectasia , Poli Adenosina Difosfato Ribose , Humanos , RNA , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA , Ataxia Telangiectasia/genética , Reparo do DNA , Dano ao DNA , Proteínas de Ciclo Celular/metabolismo
15.
Angew Chem Int Ed Engl ; 63(17): e202318568, 2024 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-38433368

RESUMO

ATR has emerged as a promising target for anti-cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase-independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis-targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53-mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti-proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti-AML activity is regulated by the kinase-independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.


Assuntos
Antineoplásicos , Leucemia Mieloide Aguda , Humanos , Proteína Supressora de Tumor p53/metabolismo , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Linhagem Celular Tumoral , Proteólise , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/uso terapêutico
16.
Front Cell Infect Microbiol ; 14: 1374659, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38524184

RESUMO

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.


Assuntos
Toxoplasma , Toxoplasmose , Humanos , Animais , Toxoplasmose/parasitologia , DNA , Dano ao DNA , Instabilidade Cromossômica , Recombinação Homóloga , Proteínas Mutadas de Ataxia Telangiectasia/genética
17.
Nat Commun ; 15(1): 2210, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472229

RESUMO

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.


Assuntos
Proteínas Quinases , Transdução de Sinais , Proteínas Quinases/metabolismo , Fosforilação , Proteína de Replicação A/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Replicação do DNA , Dano ao DNA , DNA de Cadeia Simples , Reparo do DNA
18.
BMJ Case Rep ; 17(3)2024 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-38453233

RESUMO

Ataxia telangiectasia (A-T) (OMIM 208900) is an autosomal recessive multisystem disorder characterised by progressive cerebellar ataxia, telangiectasias, immunodeficiency and a predisposition to malignancy. 'Variant' A-T has later onset of neurological symptoms and slower progression compared with the 'classic' form. A woman presented with short stature in late childhood. Karyotype revealed rearrangements involving chromosomes 7 and 14. A chromosomal breakage disorder gene panel demonstrated compound heterozygote mutations in her ATM gene including one mutation c.7271T>G with residual ATM function, confirming the diagnosis of variant A-T. Since diagnosis, she has developed progressive cerebellar ataxia and telangiectasias. Long-standing restrictive and aversive feeding behaviours presented challenges for her management and necessitated gastrostomy.


Assuntos
Ataxia Telangiectasia , Ataxia Cerebelar , Degenerações Espinocerebelares , Feminino , Humanos , Ataxia Telangiectasia/complicações , Ataxia Telangiectasia/diagnóstico , Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Mutação , Adolescente
19.
Langmuir ; 40(11): 5858-5868, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38445553

RESUMO

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular ß-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.


Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Humanos , Anticorpos Monoclonais/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Estrutura Secundária de Proteína , Imunoglobulina G/química , Desdobramento de Proteína , Proteínas Mutadas de Ataxia Telangiectasia
20.
J Investig Med High Impact Case Rep ; 12: 23247096241240176, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38504422

RESUMO

Gastric cancer ranks as the fifth leading cause of global cancer incidences, exhibiting varied prevalence influenced by geographical, ethnic, and lifestyle factors, as well as Helicobacter pylori infection. The ATM gene on chromosome 11q22 is vital for genomic stability as an initiator of the DNA damage response, and mutations in this gene have been associated with various cancers. Poly ADP-ribose polymerase (PARP) inhibitors, such as olaparib, have shown efficacy in cancers with homologous recombination repair deficiencies, notably in those with ATM mutations. Here, we present a case of a 66-year-old patient with germline ATM-mutated metastatic gastric cancer with very high CA 19-9 (48 000 units/mL) who demonstrated an exceptional response to the addition of olaparib to chemo-immunotherapy and subsequent olaparib maintenance monotherapy for 12 months. CA 19-9 was maintained at low level for 18 months. Despite the failure of a phase II clinical trial on olaparib in gastric cancer (NCT01063517) to meet its primary endpoint, intriguing findings emerged in the subset of ATM-mutated patients, who exhibited notable improvements in overall survival. Our case underscores the potential clinical utility of olaparib in germline ATM-mutated gastric cancer and emphasizes the need for further exploration through larger clinical trials. Ongoing research and clinical trials are essential for optimizing the use of PARP inhibitors, identifying biomarkers, and advancing personalized treatment strategies for gastric cancer.


Assuntos
Infecções por Helicobacter , Helicobacter pylori , Ftalazinas , Piperazinas , Neoplasias Gástricas , Humanos , Idoso , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Helicobacter pylori/metabolismo , Células Germinativas/metabolismo , Células Germinativas/patologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo
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