RESUMO
Malignant rhabdoid tumors (MRTs) are rare but lethal solid neoplasms that overwhelmingly affect infants and young children. While the central nervous system is the most common site of occurrence, tumors can develop at other sites, including the kidneys and soft tissues throughout the body. The anatomic site of involvement dictates tumor nomenclature and nosology. While the clinical and imaging manifestations of MRTs and other more common entities may overlap, there are some site-specific distinctive imaging characteristics. Irrespective of the site of occurrence, somatic and germline mutations in SMARCB1, and rarely in SMARCA4, underlie the entire spectrum of rhabdoid tumors. MRTs have a simple and remarkably stable genome but can demonstrate considerable molecular and biologic heterogeneity. Related neoplasms encompass an expanding category of phenotypically dissimilar (nonrhabdoid tumors driven by SMARC-related alterations) entities. US, CT, MRI, and fluorodeoxyglucose PET/CT or PET/MRI facilitate diagnosis, initial staging, and follow-up, thus informing therapeutic decision making. Multifocal synchronous or metachronous rhabdoid tumors occur predominantly in the context of underlying rhabdoid tumor predisposition syndromes (RTPSs). These autosomal dominant disorders are driven in most cases by pathogenic variants in SMARCB1 (RTPS type 1) and rarely by pathogenic variants in SMARCA4 (RTPS type 2). Genetic testing and counseling are imperative in RTPS. Guidelines for imaging surveillance in cases of RTPS are based on age at diagnosis. ©RSNA, 2024 Supplemental material is available for this article.
Assuntos
Imagem Multimodal , Tumor Rabdoide , Humanos , Tumor Rabdoide/diagnóstico por imagem , Tumor Rabdoide/genética , Imagem Multimodal/métodos , Criança , Lactente , Proteína SMARCB1/genética , Pré-Escolar , Diagnóstico Diferencial , DNA Helicases , Proteínas Nucleares , Fatores de TranscriçãoRESUMO
Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.
Assuntos
Dano ao DNA , Histonas , Nucleossomos , Histonas/metabolismo , Histonas/química , Nucleossomos/metabolismo , Humanos , Ligação Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte/química , Modelos Moleculares , ATPases Associadas a Diversas Atividades Celulares , DNA HelicasesRESUMO
BACKGROUND: The global outbreak of COVID-19 caused by the SARS-CoV-2 has led to millions of deaths. This unanticipated emergency has prompted virologists across the globe to delve deeper into the intricate dynamicity of the host-virus interface with an aim to identify antiviral targets and elucidate host and viral determinants of severe disease. AIM: The present study was undertaken to analyse the role of histone deacetylase 6 (HDAC6) in regulating SARS-CoV-2 infection. RESULTS: Gradual increase in HDAC6 expression was observed in different SARS-CoV-2-permissive cell lines following SARS-CoV-2 infection. The SARS-CoV-2 nucleocapsid protein (N protein) was identified as the primary viral factor responsible for upregulating HDAC6 expression. Downregulation of HDAC6 using shRNA or a specific inhibitor tubacin resulted in reduced viral replication suggesting proviral role of its deacetylase activity. Further investigations uncovered the interaction of HDAC6 with stress granule protein G3BP1 and N protein during infection. HDAC6-mediated deacetylation of SARS-CoV-2 N protein was found to be crucial for its association with G3BP1. CONCLUSION: This study provides valuable insights into the molecular mechanisms underlying the disruption of cytoplasmic stress granules during SARS-CoV-2 infection and highlights the significance of HDAC6 in the process.
Assuntos
COVID-19 , Proteínas do Nucleocapsídeo de Coronavírus , Desacetilase 6 de Histona , SARS-CoV-2 , Replicação Viral , Desacetilase 6 de Histona/metabolismo , Desacetilase 6 de Histona/genética , Humanos , SARS-CoV-2/fisiologia , Proteínas do Nucleocapsídeo de Coronavírus/metabolismo , Proteínas do Nucleocapsídeo de Coronavírus/genética , COVID-19/virologia , COVID-19/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Acetilação , Linhagem Celular , Chlorocebus aethiops , Fosfoproteínas/metabolismo , Fosfoproteínas/genética , Células Vero , Animais , Interações Hospedeiro-Patógeno , Proteínas de Ligação a Poli-ADP-Ribose , DNA Helicases , RNA HelicasesRESUMO
The transmembrane protein ß-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The ß-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic ß-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.
Assuntos
Precursor de Proteína beta-Amiloide , Proteínas de Drosophila , Junção Neuromuscular , Proteínas rab de Ligação ao GTP , Animais , Junção Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rab de Ligação ao GTP/genética , Precursor de Proteína beta-Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Transmissão Sináptica , Sinapses/metabolismo , Receptores de Glutamato/metabolismo , Receptores de Glutamato/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Humanos , DNA Helicases/metabolismo , DNA Helicases/genética , Proteínas de Membrana , Proteínas do Tecido Nervoso , Proteínas de Homeodomínio , Receptores Ionotrópicos de GlutamatoRESUMO
Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.
Assuntos
Autofagia , Dano ao DNA , DNA Helicases , Inflamação , Enzimas Multifuncionais , Nucleotidiltransferases , Estruturas R-Loop , RNA Helicases , Autofagia/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , DNA Helicases/metabolismo , DNA Helicases/genética , DNA Helicases/deficiência , RNA Helicases/metabolismo , RNA Helicases/genética , Humanos , Animais , Inflamação/patologia , Inflamação/metabolismo , Inflamação/genética , Camundongos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiência , Exodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/genética , Imunidade Inata , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/deficiência , FosfoproteínasRESUMO
OBJECTIVE: To explore the genetic basis for child with CHARGE syndrome. METHODS: A child who was diagnosed at Ningbo Women and Children's Hospital on September 29, 2022 was selected as the study subject. Relevant clinical data were collected. The child and her parents were subjected to whole exome sequencing (WES), and candidate variant was verified by Sanger sequencing and bioinformatic analysis. RESULTS: The child was found to harbor a de novo c.2972T>C (p.L991S) missense variant of the CHD7 gene, which was detected in neither of her parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was predicted to be likely pathogenic (PM6+PM2_Supporting+PP2+PP3+PP4). Bioinformatic analysis predicted that amino acid 991 is highly conserved among various species, and a hydrogen bond has formed between Asp993 and the mutant Ser991. CONCLUSION: The heterozygous c.2972T>C (p.L991S) missense variant of the CHD7 gene probably underlay the pathogenesis of CHARGE syndrome in this child. Above finding has also enriched the mutational spectrum for CHARGE syndrome.
Assuntos
Síndrome CHARGE , DNA Helicases , Proteínas de Ligação a DNA , Mutação de Sentido Incorreto , Humanos , Síndrome CHARGE/genética , DNA Helicases/genética , Feminino , Proteínas de Ligação a DNA/genética , Sequenciamento do Exoma , Lactente , Sequência de AminoácidosRESUMO
RATIONALE: SMARCA4-deficient undifferentiated tumor (SMARCA4-UT) is a recently reported rare malignancy that can rapidly metastasize to tissues and organs throughout the body. The tumor is characterized by a lower response to platinum-based chemotherapy. More regrettably, the mean survival time of patients with this disease after diagnosis is only 4 to 7 months. PATIENT CONCERNS: A 58-year-old man was admitted to a hospital for fatigue, sudden syncope, and a mass-like shadow of his left upper lobe demonstrated by a pulmonary computed tomographic. Based on his subsequent clinical and pathological features, he was highly suspected of SMARCA4-UT. DIAGNOSES: Combined with next-generation sequencing genetic testing and immunohistochemical examination results, the patient was diagnosed with SMARCA4-UT. INTERVENTIONS: The patient received a left upper lobectomy and lymph node dissection, four-course chemotherapy divided into 8 sessions with the use of paclitaxel simply, and a proper post-discharge self-care. OUTCOMES: The patient's operation and chemotherapy were all successful and he maintained a high quality of life after surgery that far exceeded his predicted survival. LESSONS: Early diagnosis, higher education level, attention to the disease and complications, reducing chemotherapy damage, adequate nutrient intake, relieving symptoms, controlling depression, and maintaining immunity and the ability to perform activities of daily living may all be the positive factors that can prolong the survival of patients with SMARCA4-UT.
Assuntos
DNA Helicases , Neoplasias Pulmonares , Qualidade de Vida , Fatores de Transcrição , Humanos , Masculino , Pessoa de Meia-Idade , DNA Helicases/genética , DNA Helicases/deficiência , Fatores de Transcrição/genética , Proteínas Nucleares/genética , Proteínas Nucleares/deficiência , PneumonectomiaRESUMO
Objective: Infertility affects approximately one-sixth of the people of childbearing age worldwide, causing not only economic burdens of treatment for families with fertility problems but also psychological stress for patients and presenting challenges to societal and economic development. Premature ovarian insufficiency (POI) refers to the loss of ovarian function in women before the age of 40 due to the depletion of follicles or decreased quality of remaining follicles, constituting a significant cause of female infertility. In recent years, with the help of the rapid development in genetic sequencing technology, it has been demonstrated that genetic factors play a crucial role in the onset of POI. Among the population suffering from POI, genetic studies have revealed that genes involved in processes such as meiosis, DNA damage repair, and mitosis account for approximately 37.4% of all pathogenic and potentially pathogenic genes identified. FA complementation group M (FANCM) is a group of genes involved in the damage repair of DNA interstrand crosslinks (ICLs), including FANCA-FANCW. Abnormalities in the FANCM genes are associated with female infertility and FANCM gene knockout mice also exhibit phenotypes similar to those of POI. During the genetic screening of POI patients, this study identified a suspicious variant in FANCM. This study aims to explore the pathogenic mechanisms of the FANCM genes of the FA pathway and their variants in the development of POI. We hope to help shed light on potential diagnostic and therapeutic strategies for the affected individuals. Methods: One POI patient was included in the study. The inclusion criteria for POI patients were as follows: women under 40 years old exhibiting two or more instances of basal serum follicle-stimulating hormone levels>25 IU/L (with a minimum interval of 4 weeks inbetween tests), alongside clinical symptoms of menstrual disorders, normal chromosomal karyotype analysis results, and exclusion of other known diseases that can lead to ovarian dysfunction. We conducted whole-exome sequencing for the POI patient and identified pathogenic genes by classifying variants according to the standards and guidelines established by the American College of Medical Genetics and Genomics (ACMG). Subsequently, the identified variants were validated through Sanger sequencing and subjected to bioinformatics analysis. Plasmids containing wild-type and mutant FANCM genes were constructed and introduced into 293T cells. The 293T cells transfected with wild-type and mutant human FANCM plasmids and pEGFP-C1 empty vector plasmids were designated as the EGFP FANCM-WT group, the EGFP FANCM-MUT group, and the EGFP group, respectively. To validate the production of truncated proteins, cell proteins were extracted 48 hours post-transfection from the three groups and confirmed using GFP antibody. In order to investigate the impact on DNA damage repair, immunofluorescence experiments were conducted 48 hours post-transfection in the EGFP FANCM-WT group and the EGFP FANCM-MUT group to examine whether the variant affected FANCM's ability to localize on chromatin. Mitomycin C was used to induce ICLs damage in vitro in both the EGFP FANCM-WT group and the EGFP FANCM-MUT group, which was followed by verification of its effect on ICLs damage repair using γ-H2AX antibody. Results: In a POI patient from a consanguineous family, we identified a homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10. The patient presented with primary infertility, experiencing irregular menstruation since menarche at the age of 16. Hormonal evaluation revealed an FSH level of 26.79 IU/L and an anti-Müllerian hormone (AMH) level of 0.07 ng/mL. Vaginal ultrasound indicated unsatisfactory visualization of the ovaries on both sides and uterine dysplasia. The patient's parents were a consanguineous couple, with the mother having regular menstrual cycles. The patient had two sisters, one of whom passed away due to osteosarcoma, while the other exhibited irregular menstruation, had been diagnosed with ovarian insufficiency, and remained childless. Bioinformatics analysis revealed a deletion of four nucleotides (c.1152-1155del) in the exon 6 of the patient's FANCM gene. This variant resulted in a frameshift at codon 386, introducing a premature stop codon at codon 396, which ultimately led to the production of a truncated protein consisting of 395 amino acids. In vitro experiments demonstrated that this variant led to the production of a truncated FANCM protein of approximately 43 kDa and caused a defect in its nuclear localization, with the protein being present only in the cytoplasm. Following treatment with mitomycin C, there was a significant increase in γ-H2AX levels in 293T cells transfected with the mutant plasmid (P<0.01), indicating a statistically significant impairment of DNA damage repair capability caused by this variant. Conclusions: The homozygous variant in the FANCM gene, c.1152-1155del:p.Leu386Valfs*10, results in the production of a truncated FANCM protein. This truncation leads to the loss of its interaction site with the MHF1-MHF2 complex, preventing its entry into the nucleus and the subsequent recognition of DNA damage. Consequently, the localization of the FA core complex on chromatin is disrupted, impeding the normal activation of the FA pathway and reducing the cell's ability to repair damaged ICLs. By disrupting the rapid proliferation and meiotic division processes of primordial germ cells, the reserve of oocytes is depleted, thereby triggering premature ovarian insufficiency in females.
Assuntos
Insuficiência Ovariana Primária , Feminino , Insuficiência Ovariana Primária/genética , Humanos , Mutação , Anemia de Fanconi/genética , Adulto , Infertilidade Feminina/genética , Infertilidade Feminina/etiologia , DNA HelicasesRESUMO
The 5th WHO classification of thoracic tumours includes thoracic SMARCA4-deficient undifferentiated tumour (SMARCA4-UT) among the "other epithelial tumours of the lung" chapter. Herein, we present a case of undifferentiated thoracic neoplasm with retention of SMARCA4 expression, lack of NUT fusion protein and loss of SMARCB1/INI1 expression. After presenting the clinical and pathological features of the tumour, we carried out a review of the literature on the same topic. Albeit very rare, we believe this entity should be included in the heterogeneous group of undifferentiated neoplasms of the thorax.
Assuntos
DNA Helicases , Proteína SMARCB1 , Neoplasias Torácicas , Fatores de Transcrição , Humanos , Proteína SMARCB1/deficiência , Proteína SMARCB1/genética , Fatores de Transcrição/genética , Fatores de Transcrição/deficiência , Neoplasias Torácicas/patologia , Neoplasias Torácicas/genética , DNA Helicases/deficiência , DNA Helicases/genética , Proteínas Nucleares/genética , Proteínas Nucleares/deficiência , Masculino , Feminino , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Pessoa de Meia-Idade , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/diagnósticoRESUMO
Poly(ADP-ribose) polymerase inhibitors (PARPis) exhibit remarkable anticancer activity in tumors with homologous recombination (HR) gene mutations. However, the role of other DNA repair proteins in PARPi-induced lethality remains elusive. Here, we reveal that FANCM promotes PARPi resistance independent of the core Fanconi anemia (FA) complex. FANCM-depleted cells retain HR proficiency, acting independently of BRCA1 in response to PARPis. FANCM depletion leads to increased DNA damage in the second S phase after PARPi exposure, driven by elevated single-strand DNA (ssDNA) gap formation behind replication forks in the first S phase. These gaps arise from both 53BP1- and primase and DNA directed polymerase (PRIMPOL)-dependent mechanisms. Notably, FANCM-depleted cells also exhibit reduced resection of collapsed forks, while 53BP1 deletion restores resection and mitigates PARPi sensitivity. Our results suggest that FANCM counteracts 53BP1 to repair PARPi-induced DNA damage. Furthermore, FANCM depletion leads to increased chromatin bridges and micronuclei formation after PARPi treatment, elucidating the mechanism underlying extensive cell death in FANCM-depleted cells.
Assuntos
DNA de Cadeia Simples , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Humanos , DNA de Cadeia Simples/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Linhagem Celular TumoralRESUMO
Cell fate is likely regulated by a common machinery, while components of this machine remain to be identified. Here we report the design and testing of engineered cell fate controller NanogBiD, fusing BiD or BRG1 interacting domain of SS18 with Nanog. NanogBiD promotes mouse somatic cell reprogramming efficiently in contrast to the ineffective native protein under multiple testing conditions. Mechanistic studies further reveal that it facilitates cell fate transition by recruiting the intended Brg/Brahma-associated factor (BAF) complex to modulate chromatin accessibility and reorganize cell state specific enhancers known to be occupied by canonical Nanog, resulting in precocious activation of multiple genes including Sall4, miR-302, Dppa5a and Sox15 towards pluripotency. Although we have yet to test our approach in other species, our findings suggest that engineered chromatin regulators may provide much needed tools to engineer cell fate in the cells as drugs era.
Assuntos
Proteína Homeobox Nanog , Fatores de Transcrição , Animais , Camundongos , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteína Homeobox Nanog/metabolismo , Proteína Homeobox Nanog/genética , Reprogramação Celular/genética , Cromatina/metabolismo , Cromatina/genética , DNA Helicases/metabolismo , DNA Helicases/genética , Diferenciação Celular , Engenharia Celular/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genéticaRESUMO
G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5' single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5' most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.
Assuntos
DNA Helicases , Quadruplex G , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , DNA Helicases/metabolismo , DNA Helicases/química , DNA Helicases/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA/metabolismo , DNA/química , DNA/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/química , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Replicação do DNA , Instabilidade GenômicaRESUMO
Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumors worldwide. Brahma-related gene 1 (BRG1), as a catalytic ATPase, is a major regulator of gene expression and is known to mutate and overexpress in HCC. The purpose of this study was to investigate the mechanism of action of BRG1 in HCC cells. In our study, BRG1 was silenced or overexpressed in human HCC cell lines. Transwell and wound healing assays were used to analyze cell invasiveness and migration. Mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore (mPTP) detection were used to evaluate mitochondrial function in HCC cells. Colony formation and cell apoptosis assays were used to evaluate the effect of BRG1/TOMM40/ATP5A1 on HCC cell proliferation and apoptosis/death. Immunocytochemistry (ICC), immunofluorescence (IF) staining and western blot analysis were used to determine the effect of BRG1 on TOMM40, ATP5A1 pathway in HCC cells. As a result, knockdown of BRG1 significantly inhibited cell proliferation and invasion, promoted apoptosis in HCC cells, whereas BRG1 overexpression reversed the above effects. Overexpression of BRG1 can up-regulate MMP level, inhibit mPTP opening and activate TOMM40, ATP5A1 expression. Our results suggest that BRG1, as an oncogene, promotes HCC progression by regulating TOMM40 affecting mitochondrial function and ATP5A1 synthesis. Targeting BRG1 may represent a new and effective way to prevent HCC development.
Assuntos
Apoptose , Carcinoma Hepatocelular , Proliferação de Células , DNA Helicases , Neoplasias Hepáticas , Mitocôndrias , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Nucleares , Fatores de Transcrição , Humanos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Movimento Celular , DNA Helicases/metabolismo , DNA Helicases/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Metástase Neoplásica , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Most of the human cancers are dependent on telomerase to extend the telomeres. But â¼10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to the poor prognosis, ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available to date for ALT positive cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homologous recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led toward the increased localization of PML (promyelocytic leukemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukemia bodies) in ALT positive cell lines, increasing telomere replication stress and DNA damage at a genomic level. Induction of replication stress and hyper-recombinogenic phenotype in ALT positive cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT positive tumors. In accordance with this, our study will also provide a valuable insight toward the development of G4-based ALT therapeutics targeting SMARCAL1.
Assuntos
DNA Helicases , Quadruplex G , Neoplasias , Regiões Promotoras Genéticas , Telômero , Humanos , Telômero/genética , Telômero/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Neoplasias/genética , Linhagem Celular Tumoral , Replicação do DNA , Homeostase do TelômeroRESUMO
Multiple Sclerosis (MS) is a heterogeneous inflammatory and neurodegenerative disease with an unpredictable course towards progressive disability. Treating progressive MS is challenging due to limited insights into the underlying mechanisms. We examined the molecular changes associated with primary progressive MS (PPMS) using a cross-tissue (blood and post-mortem brain) and multilayered data (genetic, epigenetic, transcriptomic) from independent cohorts. In PPMS, we found hypermethylation of the 1q21.1 locus, controlled by PPMS-specific genetic variations and influencing the expression of proximal genes (CHD1L, PRKAB2) in the brain. Evidence from reporter assay and CRISPR/dCas9 experiments supports a causal link between methylation and expression and correlation network analysis further implicates these genes in PPMS brain processes. Knock-down of CHD1L in human iPSC-derived neurons and knock-out of chd1l in zebrafish led to developmental and functional deficits of neurons. Thus, several lines of evidence suggest a distinct genetic-epigenetic-transcriptional interplay in the 1q21.1 locus potentially contributing to PPMS pathogenesis.
Assuntos
Encéfalo , Cromossomos Humanos Par 1 , Metilação de DNA , Proteínas de Ligação a DNA , Epigênese Genética , Peixe-Zebra , Humanos , Peixe-Zebra/genética , Animais , Metilação de DNA/genética , Cromossomos Humanos Par 1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , DNA Helicases/genética , DNA Helicases/metabolismo , Neurônios/metabolismo , Esclerose Múltipla Crônica Progressiva/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Predisposição Genética para Doença , AdultoRESUMO
Stress triggers the formation of two distinct cytoplasmic biomolecular condensates: stress granules (SGs) and processing bodies (PBs), both of which may contribute to stress-responsive translation regulation. Though PBs can be present constitutively, stress can increase their number and size and lead to their interaction with stress-induced SGs. The mechanism of such interaction, however, is largely unknown. Formation of canonical SGs requires the RNA binding protein Ubiquitin-Associated Protein 2-Like (UBAP2L), which is a central SG node protein in the RNA-protein interaction network of SGs and PBs. UBAP2L binds to the essential SG and PB proteins G3BP and DDX6, respectively. Research on UBAP2L has mostly focused on its role in SGs, but not its connection to PBs. We find that UBAP2L is not solely an SG protein but also localizes to PBs in certain conditions, contributes to PB biogenesis and SG-PB interactions, and can nucleate hybrid granules containing SG and PB components in cells. These findings inform a new model for SG and PB formation in the context of UBAP2L's role.
Assuntos
RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Humanos , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Corpos de Processamento/metabolismo , Corpos de Processamento/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Grânulos Citoplasmáticos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Células HeLa , DNA Helicases/metabolismo , DNA Helicases/genética , Células HEK293 , Ligação Proteica , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genética , Proteínas Proto-OncogênicasRESUMO
Mutations in the Cockayne Syndrome group B (CSB) gene cause cancer in mice, but premature aging and severe neurodevelopmental defects in humans. CSB, a member of the SWI/SNF family of chromatin remodelers, plays diverse roles in regulating gene expression and transcription-coupled nucleotide excision repair (TC-NER); however, these functions do not explain the distinct phenotypic differences observed between CSB-deficient mice and humans. During investigating Cockayne Syndrome-associated genome instability, we uncover an intrinsic mechanism that involves elongating RNA polymerase II (RNAPII) undergoing transient pauses at internal T-runs where CSB is required to propel RNAPII forward. Consequently, CSB deficiency retards RNAPII elongation in these regions, and when coupled with G-rich sequences upstream, exacerbates genome instability by promoting R-loop formation. These R-loop prone motifs are notably abundant in relatively long genes related to neuronal functions in the human genome, but less prevalent in the mouse genome. These findings provide mechanistic insights into differential impacts of CSB deficiency on mice versus humans and suggest that the manifestation of the Cockayne Syndrome phenotype in humans results from the progressive evolution of mammalian genomes.
Assuntos
Síndrome de Cockayne , DNA Helicases , Enzimas Reparadoras do DNA , Instabilidade Genômica , Proteínas de Ligação a Poli-ADP-Ribose , Estruturas R-Loop , RNA Polimerase II , Síndrome de Cockayne/genética , Síndrome de Cockayne/patologia , Síndrome de Cockayne/metabolismo , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Camundongos , DNA Helicases/metabolismo , DNA Helicases/genética , Estruturas R-Loop/genética , Reparo do DNA , Elongação da Transcrição Genética , Camundongos KnockoutRESUMO
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:GâT:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
Assuntos
Corynebacterium glutamicum , Genoma Bacteriano , Corynebacterium glutamicum/genética , Engenharia Genética/métodos , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , Taxa de Mutação , Evolução Molecular , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/genética , MutaçãoRESUMO
The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' â 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.
Assuntos
DNA Helicases , SARS-CoV-2 , Proteínas não Estruturais Virais , SARS-CoV-2/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/química , DNA Helicases/metabolismo , DNA Helicases/química , DNA/metabolismo , DNA/química , Humanos , Dano ao DNA , COVID-19/virologia , Cinética , Metiltransferases , RNA HelicasesRESUMO
Germ cells mutant for bam or bgcn are locked in a germline stem cell (GSC)-like state, leading to tumor-like overgrowth in Drosophila ovaries. Our previous studies have demonstrated that germline overgrowth in bam mutants can be suppressed by defects in the miRNA pathway but enhanced by a null mutation in hippo. However, the genetic epistasis between the miRNA and Hippo pathways still remains unknown. Here, we determined that the miRNA pathway acts downstream of the Hippo pathway in regulating this process. Germ cells mutant for bam or bgcn and defective in both pathways divide very slowly, phenocopying those defective only in the miRNA pathway. In addition, we found that Yki, a key oncoprotein in the Hippo pathway, promotes the growth of both wild-type germ cells and bam mutant GSC-like cells. Like wild-type GSCs, bam mutant GSC-like cells predominantly stay in the G2 phase. Remarkably, many of those defective in the miRNA pathway are arrested before entering this phase. Furthermore, our studies identified bantam as a critical miRNA promoting germline overgrowth in bam or bgcn mutants. Taken together, these findings establish a genetic circuitry controlling Drosophila female germline overgrowth.